RU2774820C2 - Hla-based methods and compositions and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular a composition and methods for isolating HLA-peptides from a cell are presented. In the present disclosure, a universal platform and methods for obtaining HLA-peptide profile, providing identification of endogenously presented HLA peptides from cell lines expressing any possible structure of I or II class, are presented.

EFFECT: obtaining HLA-based compositions and methods.

19 cl, 22 dwg, 4 tbl, 7 ex

Description

Cross-reference to related applications

[1] This application claims priority under U.S. Provisional Application No. 62/457,978, filed February 12, 2017, and U.S. Provisional Application No. 62/461,162, filed February 20, 2017, each of which incorporated herein by reference in its entirety.

Prior Art

[2] The major histocompatibility complex (MHC) is a gene complex encoding human leukocyte antigen (HLA) genes. HLA genes are expressed as protein heterodimers that are present on the surface of human cells for circulating T cells. The HLA genes are highly polymorphic, allowing them to fine-tune the adaptive immune system. Adaptive immune responses depend in part on the ability of T cells to identify and kill cells that display disease-associated peptide antigens associated with human leukocyte antigen (HLA) heterodimers.

[3] In humans, endogenous and exogenous proteins can be processed into peptides by the proteasome and by cytosolic and endosomal/lysosomal proteases and peptidases and are represented by two classes of cell surface proteins encoded by MHC. These cell surface proteins are called human leukocyte antigens (HLA class I and class II), and the group of peptides that bind them and elicit immune responses are called HLA epitopes. HLA epitopes are a key component that allows the immune system to detect danger signals such as pathogen infection and self-transformation. Circulating CD8+ T cells recognize MHC class I epitopes (HLA-A, HLA-B, and HLA-C) derived from endogenous processing pathways and present on almost all nucleated cells. CD4+ T cells recognize MHC class II epitopes (HLA-DR, HLA-DQ and HLA-DP) presented on antigen presenting cells (APCs) such as dendritic cells and macrophages. Presentation of the HLA class II peptide activates T helpers, subsequently promoting B cell differentiation and antibody production, as well as CTL responses. Activated helper T cells also secrete cytokines and chemokines that activate and induce the differentiation of other T cells.

[4] The genes encoding HLA heterodimers are highly polymorphic, with more than 12,000 class I and 4,000 class II allele variants identified in the human population. From maternal and paternal HLA haplotypes, an individual may inherit different alleles for each of the class I and class II HLA loci. HLA class I molecules are heterodimers consisting of the heavy α-chain encoded by HLA class I genes and β-2-microglobulin (B2M). HLA class II molecules are heterodimers of α- and β-chains, which are encoded by HLA class II genes. Because of the combinations of α- and β-strand pairings, the population of HLA heterodimers is very complex. In addition, each HLA heterodimer is estimated to bind thousands of peptides with allele-specific binding preferences. In fact, each HLA allele is estimated to bind and present ~1000-10000 unique peptides to T cells; ≤0.1% of ~10 million potential 9-mer peptides from genes encoding human proteins. Given this diversity in HLA binding, accurately predicting whether a peptide can bind to a particular HLA allele is a very difficult task. Less is known about the allele-specific peptide-binding characteristics of HLA class II molecules due to heterogeneity in α and β chain pairing, data complexity limiting the ability to confidently assign core-binding epitopes, and lack of gradation of immunoprecipitation, allele-specific antibodies required for biochemical assays. high resolution. In addition, analysis of peptide epitopes derived from a given HLA allele increases the ambiguity in the presentation of multiple HLA alleles on the cell surface.

[5] Understanding the binding preferences of each HLA heterodimer is key to successfully predicting which neoantigens might elicit tumor-specific T cell responses. Clearly, there is a need for methods for identifying and isolating specific HLA-associated class I and class II peptides (eg, neoantigenic peptides). Such a methodology and isolated molecules are useful, for example, for the study of HLA-associated peptides, as well as for the development of therapeutic agents, including, but not limited to, immunity-based therapeutic agents.

Inclusion by reference

[6] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually designated for incorporation by reference. .

Essence of the Invention

[7] The methods and compositions described herein find use in a wide range of applications. For example, the methods and compositions described herein can be used to identify immunogenic antigenic peptides and can be used to develop drugs such as personalized drugs.

[8] This document provides a method for characterizing HLA-peptide complexes, comprising: providing a cell population, wherein one or more cells of the cell population contain a polynucleic acid containing a sequence encoding an allele of an affinity-tagged HLA class I or class II acceptor, wherein the sequence , encoding HLA labeled with an affinity acceptor, contains a sequence encoding an allele of recombinant HLA class I or class II, functionally linked to a sequence encoding an affinity acceptor peptide; expressing the affinity acceptor-labeled HLA in at least one cell of one or more cells of the cell population, thereby complexing the affinity acceptor-labeled HLA peptides in at least one cell; enrichment of complexes labeled with an affinity acceptor HLA-peptides; and characterization of HLA-peptide complexes. In some embodiments, the encoded allele of class I or class II affinity-acceptor-labelled HLA is a soluble allele of class I or class II affinity-acceptor-labelled HLA.

[9] In some embodiments, characterization includes characterization of the peptide associated with the HLA affinity acceptor-tagged peptide complex as a result of enrichment. In some embodiments, the implementation of the method includes performing the steps of the method for two or more alleles of HLA class I and/or class II. In some embodiments, the two or more HLA class I and/or class II alleles include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 HLA class I and/or class II alleles. In some embodiments, the affinity acceptor-tagged HLA peptide complexes comprise a transmembrane domain. In some embodiments, the affinity acceptor-tagged HLA peptide complexes comprise an intracellular domain. In some embodiments, the affinity acceptor-tagged HLA peptide complexes are not secreted. In some embodiments, the affinity acceptor-tagged HLA peptide complexes are incorporated into the cell membrane upon expression. In some embodiments, the affinity acceptor-tagged HLA peptide complexes are soluble complexes of the affinity-acceptor-tagged HLA peptides. In some embodiments, the HLA acceptor affinity tagged peptide complexes are insoluble complexes of the HLA acceptor affinity tagged peptides. In some embodiments, the method further comprises creating a database of HLA allele-specific peptides. In some embodiments, the recombinant HLA Class I or Class II allele is a single recombinant HLA Class I or Class II allele.

[10] In some embodiments, the implementation of the method includes: providing a population of cells, each of which contains one or more cells containing affinity-acceptor-labeled HLA, and affinity-acceptor-labeled HLA contains another recombinant polypeptide encoded by a different HLA allele operably linked to the affinity peptide - acceptor; enrichment of complexes labeled with an affinity acceptor HLA-peptides; and characterizing the peptide, or part thereof, associated with the HLA affinity acceptor-labeled peptide complex as a result of enrichment.

[11] In some embodiments, the implementation of the method includes the introduction of one or more peptides in a population of cells. In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more peptides or the expression of one or more peptides in a population of cells. In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more nucleic acids encoding one or more peptides. In some embodiments, one or more nucleic acids encoding one or more peptides is DNA. In some embodiments, one or more nucleic acids encoding one or more peptides is RNA, optionally the RNA is mRNA. In some embodiments, enrichment does not include the use of a tetrameric reagent.

[12] In some embodiments, the characterization includes determining the sequence of the peptide or portion thereof associated with the HLA affinity acceptor-tagged peptide complex as a result of enrichment, optionally determining whether the peptide or portion thereof is modified. In some embodiments, the determination includes biochemical analysis, mass spectrometric analysis, MS analysis, MS/MS analysis, LC-MS/MS analysis, or a combination thereof. In some embodiments, the characterization includes evaluating the binding affinity or stability of the peptide, or portion thereof, associated with the enriched HLA affinity acceptor-labeled peptide complex. In some embodiments, characterization includes determining whether a peptide, or portion thereof, associated with an enriched HLA affinity acceptor-tagged peptide contains one or more mutations. In some embodiments, the characterization includes evaluating the binding of peptides to HLA molecules in complexes with affinity acceptor-labeled HLA peptides.

[13] In some embodiments, the implementation of the method includes the expression of the library of peptides in a population of cells, thereby forming a library of complexes labeled with an affinity acceptor HLA peptides. In some embodiments, the method includes contacting the cell population with a peptide library or a library of peptide coding sequences, thereby forming a library of HLA affinity acceptor-labeled peptide complexes. In some embodiments, the library is a library of peptides associated with a disease or condition. In some embodiments, the library is a library of peptides derived from a polypeptide drug, such as a biological drug (eg, an antibody containing drug).

[14] In some embodiments, the disease or condition is cancer, infection with an infectious agent, or an autoimmune reaction. In some embodiments, the method includes introducing the infectious agent or parts thereof into one or more cells of the cell population. In some embodiments, the method includes administering a polypeptide drug, such as a biological drug (eg, an antibody-containing drug), or portions thereof, to one or more cells of a cell population. In some embodiments, the method includes characterizing one or more peptides from HLA-peptide complexes, optionally the peptides are derived from one or more target proteins of the infectious agent or polypeptide drug. In some embodiments, the method includes characterizing one or more regions of peptides from one or more target proteins of the infectious agent or drug polypeptide.

[15] In some embodiments, the method includes identifying peptides from HLA-peptide complexes derived from the infectious agent. In some embodiments, the cell population is derived from a biological sample in a subject with a disease or condition. In some embodiments, the cell population is a cell line. In some embodiments, the cell population is a primary cell population. In some embodiments, the recombinant HLA Class I or Class II allele corresponds to a subject with a disease or condition.

[16] In some embodiments, a peptide from the HLA affinity acceptor-tagged peptide complex is capable of activating a T cell in a subject when presented by an antigen presenting cell. In some embodiments, characterization includes comparing HLA-peptide complexes from cancer cells with HLA-peptide complexes from non-cancerous cells. In some embodiments, the cell population includes a plurality of cell populations, with each cell population expressing a different recombinant class I or class II HLA allele. In some embodiments, each cell population of the plurality is in the same or separate container.

[17] In some embodiments, the method further comprises isolating peptides from complexes of HLA affinity acceptor-labeled peptides prior to characterization. In some embodiments, the HLA-peptide complex is isolated using an anti-HLA antibody. In some cases, an HLA-peptide complex with or without an affinity tag is isolated using an anti-HLA antibody. In some cases, soluble HLA (sHLA) with or without an affinity tag is isolated from the cell culture medium. In some cases, soluble HLA (sHLA), with or without an affinity tag, is isolated using an anti-HLA antibody. For example, HLA, such as soluble HLA (sHLA) with or without an affinity tag, can be isolated using a bead or column containing an anti-HLA antibody. In some embodiments, the peptides are isolated using anti-HLA antibodies. In some cases, soluble HLA (sHLA) with or without an affinity tag is isolated using anti-HLA antibodies. In some cases, soluble HLA (sHLA) with or without an affinity tag is isolated using a column containing an anti-HLA antibody. In some embodiments, the method further comprises removing one or more amino acids at the end of the peptide associated with the HLA affinity acceptor-tagged peptide complex.

[18] In some embodiments, the implementation of the population of cells is a population of cells with low expression of class I or class II cell surface HLA. In some embodiments, a population of cells expresses one or more endogenous HLA alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles and endogenous HLA class II alleles. In some embodiments, the cell population has a knockout of one or more HLA class I alleles. In some embodiments, the cell population has a knockout of one or more HLA class II alleles. In some embodiments, a population of cells has a knockout of all HLA class I alleles. In some embodiments, a population of cells has a knockout of all HLA class II alleles. In some embodiments, the cell population has a knockout of all HLA class I alleles and a knockout of all HLA class II alleles. In some embodiments, a sequence encoding an allele of a recombinant HLA class I or class II encodes a class I HLA. In some embodiments, the Class I HLA is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the sequence encoding the allele of the recombinant HLA class I or class II encodes HLA class II. In some embodiments, the HLA class II is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP. In some embodiments, the HLA class II comprises an HLA class II α chain, an HLA class II β chain, or a combination thereof. In some embodiments, each sequence encodes for at least two different HLA class I and/or class II alleles.

[19] In some embodiments, at least two different HLA class I and/or class II alleles are each operably linked to a sequence encoding an affinity acceptor peptide. In some embodiments, at least two different HLA class I and/or class II alleles are each operably linked to a sequence encoding a different acceptor affinity peptide. In some embodiments, at least two different HLA class I and/or class II alleles are each operably linked to a sequence encoding an affinity acceptor peptide. In some embodiments, one or more of at least two different HLA class I and/or class II alleles is operably linked to a sequence encoding a first acceptor affinity peptide and one or more of at least two different HLA class I and/or alleles. or class II is operably linked to a sequence encoding a second acceptor affinity peptide. In some embodiments, at least two different HLA class I and/or class II alleles are each operably linked to a sequence encoding a different acceptor affinity peptide. In some embodiments, each of at least two different HLA class I and/or class II alleles is operably linked to a sequence encoding a different acceptor affinity peptide. In some embodiments, at least two different HLA class I and/or class II alleles are each operably linked to an affinity tag encoding sequence. In some embodiments, the method includes administering at least a second polynucleic acid containing a sequence encoding a different recombinant HLA allele operably linked to the same or a different acceptor affinity peptide.

[20] In some embodiments, the sequence encoding the acceptor affinity peptide is operably linked to a sequence that encodes the extracellular portion of the recombinant HLA class I or class II allele. In some embodiments, the encoded acceptor affinity peptide is expressed extracellularly. In some embodiments, the encoded acceptor affinity peptide is located on the extracellular portion of the recombinant HLA class I or class II allele. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to the N-terminus of a sequence encoding a recombinant HLA Class I or Class II allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence that encodes the intracellular portion of the recombinant HLA class I or class II allele. In some embodiments, the encoded acceptor affinity peptide is expressed intracellularly. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to the C-terminus of a sequence encoding a recombinant HLA Class I or Class II allele. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to an internal sequence of a sequence encoding a recombinant HLA Class I or Class II allele, such as a flexible loop sequence. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to a sequence encoding a recombinant HLA Class I or Class II allele via a linker. In some embodiments, enrichment includes enrichment of intact cells expressing complexes of affinity acceptor-tagged HLA peptides. In some embodiments, the method does not include lysing the cells prior to enrichment. In some embodiments, the method further comprises lysing one or more cells prior to enrichment. In some embodiments, enrichment comprises contacting an acceptor affinity peptide binding molecule with the acceptor affinity-tagged HLA peptide complexes, wherein the acceptor affinity peptide binding molecule specifically binds to the acceptor affinity peptide.

[21] In some embodiments, the affinity acceptor peptide comprises a tag sequence including a biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine-glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 peptide tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep tag II, tag albumin-binding protein (ABP), alkaline phosphatase (AP) tag, bluetongue (B-tag), calmodulin-binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline binding domain (CBD) tag , chitin binding domain (CBD) tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose binding protein (GBP) tag, maltose binding protein (MBP), glutate S-ion transferase (GST), Glu-Glu tag (EE), human influenza virus hemagglutinin tag (HA), horseradish peroxidase (HA) tag, NE tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag , LacZ tag, Luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin tag, E tag, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, Xpress tag, Isopeptag, SpyTag , SnoopTag, Profinity eXact tag, protein C tag, S1 tag, S tag, carboxybiotin carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification tag (TAP), HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, sortase tag, covalent peptide bond tag, or a combination of them; optionally, the affinity acceptor peptide contains two or more repeats of the tag sequence.

[22] In some embodiments, the affinity acceptor peptide binding molecule is biotin or an antibody specific for the acceptor affinity peptide. In some embodiments, enrichment includes contacting an affinity molecule with complexes of affinity acceptor-labeled HLA peptides, wherein the affinity molecule specifically binds to the affinity acceptor peptide binding molecule.

[23] In some embodiments, the affinity molecule is a molecule that binds to biotin. For example, the affinity molecule may contain streptavidin, NeutrAvidin, including protein homologues from other organisms and derivatives thereof.

[24] In some embodiments, enrichment includes immunoprecipitation of complexes labeled with an affinity acceptor HLA peptides. In some embodiments, an acceptor affinity peptide binding molecule is attached to a solid surface. In some embodiments, the affinity molecule is attached to a solid surface. In some embodiments, the hard surface is a bead. In some embodiments, enrichment comprises immunoprecipitating complexes of the affinity acceptor-labeled HLA peptides with an acceptor affinity peptide binding molecule that specifically binds to the acceptor affinity peptide.

[25] In some embodiments, the acceptor affinity peptide binding molecule does not specifically interact with the amino acid sequence of the encoded recombinant HLA class I or class II. In some embodiments, enrichment includes contacting an affinity molecule specific for the extracellular portion of a recombinant HLA Class I or Class II allele. In some embodiments, enrichment includes contacting an affinity molecule specific for the N-terminal portion of a recombinant HLA Class I or Class II allele.

[26] In some embodiments, providing includes contacting a population of cells with a polynucleic acid. In some embodiments, the introduction into contact includes transfection or transduction. In some embodiments, providing includes contacting a population of cells with a vector containing a polynucleic acid. In some embodiments, the vector is a viral vector. In some embodiments, the polynucleic acid is stably inserted into the genome of a population of cells.

[27] In some embodiments, the implementation of the sequence encoding recombinant HLA class I or class II, contains a sequence encoding the α-chain of HLA class I. In some embodiments, the implementation of the method further includes the expression of a sequence encoding β2 microglobulin in one or more cells. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding an HLA class I α chain. In some embodiments, the β2 microglobulin coding sequence is linked to the HLA class I α chain coding sequence via a linker. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding a second acceptor affinity peptide. In some embodiments, a sequence encoding a recombinant HLA class I or class II contains a sequence encoding an HLA class II α chain. In some embodiments, the method further comprises expressing an HLA class II β chain coding sequence in one or more cells. In some embodiments, a sequence encoding an HLA class II β chain is linked to a sequence encoding an HLA class II α chain. In some embodiments, a sequence encoding an HLA class II β chain is linked to a sequence encoding an HLA class II α chain via a linker. In some embodiments, a sequence encoding an HLA class II β chain is linked to a sequence encoding a second acceptor affinity peptide.

[28] In some embodiments, the second acceptor affinity peptide is different from the first acceptor affinity peptide and is selected from the group consisting of a biotin acceptor peptide (BAP), a polyhistidine tag, a polyhistidine glycine tag, a polyarginine tag, a polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tags, Strep II tags, albumin-binding protein (ABP) tags, alkaline phosphatase (AP) tags, bluetongue virus tags (B-tags), calmodulin-binding peptide (CBP) tags, chloramphenicol acetyltransferase (CAT) tags ), choline-binding domain (CBD) tags, chitin-binding domain (CBD) tags, cellulose-binding domain (CBP) tags, dihydrofolate reductase (DHFR) tags, galactose-binding protein tags (GBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tags, human influenza virus hemagglutinin (HA) tags, horseradish peroxidase (HA) tags, NE-tags, HSV tags, ketosteroid isomerase (KSI) tags, KT3 tags, LacZ tags, luciferase tags, NusA tags, PDZ domain tags, AviTag, calmodulin tags, E-tags, S-tags, SBP-tags, Softag 1, Softag 3, TC tags, VSV tags, Xpress tags, Isopeptag, SpyTag, SnoopTag, Profinity eXact tags, protein C tags, S1 tags, S tags, carboxybiotin carrier protein (BCCP) tags, green fluorescent protein (GFP) tags, small ubiquitin tags similar modifier (SUMO), tandem affinity purification (TAP) tag, HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, and combinations thereof ; optionally, the first or second acceptor affinity peptide contains two or more repeats of the tag sequence.

[29] In some embodiments, the linker contains a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome escape site or internal ribosome entry site (IRES). In some embodiments, the ribosome escape site or IRES is cleaved when expressed in cells. In some embodiments, the ribosome exit site is selected from the group consisting of F2A, T2A, P2A, and E2A. In some embodiments, the IRES element is selected from common cellular or viral IRES sequences.

[30] In some embodiments, the determination includes performing a biochemical analysis or mass spectrometry, such as tandem mass spectrometry. In some embodiments, the determination includes obtaining a peptide sequence that corresponds to MS/MS spectra of one or more peptides isolated from enriched complexes of HLA affinity acceptor-labeled peptides from a peptide database; wherein one or more of the resulting sequences identifies the sequence of one or more peptides. In some embodiments, the peptide database is a peptide database with no enzyme specificity, such as an unmodified database or a modified database. In some embodiments, the method further comprises searching the peptide database using a flipped database search strategy. In some embodiments, the cell population is a cell line. In some embodiments, the cell population is a human cell line. In some embodiments, the cell population is a mouse cell line. In some embodiments, the cell population is a CHO cell line. In some embodiments, the cell population is a cell line selected from HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, Hep G2, SH-SY5Y, CACO-2, U937, U-2 OS, ExpiCHO, CHO and THP1.

[31] In some embodiments, the cell population is treated with one or more cytokines, checkpoint inhibitors, epigenetically active drugs, IFN-γ, agents that alter antigen processing (such as peptidase inhibitors, proteasome inhibitors, and TAP inhibitors), or a combination thereof. . In some embodiments, a population of cells is treated with one or more reagents that modulate the metabolic pathway or metabolic state of the cells. In some embodiments, a population of cells is treated with one or more reagents that modulate the cellular proteome of the cells. In some embodiments, the cell population is treated with one or more reagents that modulate or regulate cellular expression or transcription (eg, AIRE or CREB binding protein or modulators thereof) of the cells. In some embodiments, a population of cells is treated with one or more reagents that modulate or regulate a cell's transcription factor. In some embodiments, a population of cells is treated with one or more reagents that modulate or regulate cellular expression or transcription of HLA cells. In some embodiments, a population of cells is treated with one or more reagents that modulate or regulate cellular expression or transcription of a cell proteome.

[32] In some embodiments, the cell population includes at least 10 ⁵ cells, at least 10 ⁶ cells, or at least 10 ⁷ cells. In some embodiments, the cell population is a population of dendritic cells, macrophages, cancer cells, or B cells. In some embodiments, the cell population includes tumor cells. In some embodiments, a population of cells is contacted with an agent prior to isolating said HLA-peptide complexes from one or more cells. In some embodiments, said agent is an inflammatory cytokine, a chemical agent, an adjuvant, a therapeutic agent, or radiation.

[33] In some embodiments, the HLA allele is a mutant HLA allele. In some embodiments, the implementation of the sequence encoding the HLA allele is a barcoded sequence. In some embodiments, the method further comprises analyzing the expression of an allele tagged with an HLA class I or class II affinity acceptor. In some embodiments, the assay includes sequencing an allele tagged with an HLA class I or class II affinity acceptor, detecting an RNA allele tagged with an HLA class I or class II affinity acceptor, detecting a protein of the allele tagged with an HLA class I or class II affinity acceptor, or a combination thereof. In some embodiments, expression analysis may include Western blotting, fluorescence activated cell sorting (FACS), mass spectrometry (MS), microarray hybridization analysis, RNA sequencing analysis, polymerase chain reaction analysis, LAMP analysis, ligation analysis. chain reaction, southern blot, northern blot, or enzyme immunoassay (ELISA).

[34] In some embodiments, the method includes performing the steps of the method for different HLA alleles. In some embodiments, each HLA allele contains a unique barcoded sequence. In some embodiments, each polynucleic acid encoding a different HLA allele contains a unique barcoded sequence.

[35] This document provides a database of sequences of specifically binding alleles of HLA peptides obtained by performing the method described herein. Provided herein is a combination of two or more HLA allele-specific binding peptide sequence databases obtained by repeatedly performing the method described herein, each time using a different HLA allele. Provided herein is a method for generating a prediction algorithm for identifying HLA-specific allele-binding peptides, comprising training a machine with a sequence database of the peptides described herein or the combination described herein.

[36] In some embodiments, the machine combines one or more linear models, support vector machines, decision trees, and neural networks. In some embodiments, the variable used to train the machine includes one or more variables selected from the group consisting of peptide sequence, physical properties of amino acids, physical properties of peptides, expression level of the original peptide protein in the cell, protein stability, protein translation rate , ubiquitination sites, rate of protein degradation, translation efficiency from ribosomal profiling, protein cleavage, protein localization, host protein motifs that facilitate TAP transport, host protein undergoes autophagy, motifs that promote ribosome arrest, and protein features that promote NMD .

[37] In some embodiments, motifs that promote ribosome arrest contain polyproline or polylysine regions. In some embodiments, protein features that promote NMD are selected from the group consisting of a long 3' UTR, a stop codon of more than 50 nucleic acids before the last exon:exon junction, and cleavage of the peptide.

[38] Provided herein is a method for identifying HLA allele-specific binding peptides, comprising performing a peptide sequence analysis with a machine that has been trained with a peptide sequence database obtained by performing the method described herein for an HLA allele. In some embodiments, the implementation of the method includes determining the level of expression of the original protein of the peptide in the cell; and while the expression of the original protein is the prognostic variable used by the machine. In some embodiments, the level of expression is determined by measuring the amount of the parent protein or the amount of RNA encoding said parent protein.

[39] This document provides a composition containing a recombinant polynucleic acid containing two or more sequences, each of which encodes an affinity-acceptor-tagged HLA, while the sequences encoding the affinity-acceptor-tagged HLA contain a sequence encoding a different allele of the recombinant HLA α-chain class I, a sequence encoding an affinity acceptor peptide, and optionally, a sequence encoding β2 microglobulin; wherein sequences (a) and (b), and optionally (c), are operably linked.

[40] This document provides a composition containing a recombinant polynucleic acid containing two or more sequences, each of which contains a sequence encoding affinity-acceptor-labeled HLA, while sequences encoding affinity-acceptor-labeled HLA contain a sequence encoding an α-chain allele a recombinant HLA class II, a sequence encoding an affinity acceptor peptide, and optionally, a sequence encoding the β-chain of HLA class II; wherein sequences (a) and (b), and optionally (c), are operably linked. In some embodiments, a recombinant polynucleic acid is isolated. In some embodiments, Class I HLA is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the HLA class II is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP.

[41] In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence that encodes the extracellular portion of the recombinant HLA allele. In some embodiments, the sequence encoding the affinity acceptor molecule is operably linked to the N-terminus of the sequence encoding the recombinant HLA allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the sequence encoding the intracellular portion of the recombinant HLA allele. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to the C-terminus of a sequence encoding a recombinant HLA allele. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to a sequence encoding a recombinant HLA allele via a linker.

[42] In some embodiments, two or more affinity acceptor-tagged HLA encoding sequences are expressed from the same polynucleotide. In some embodiments, two or more affinity acceptor-tagged HLA encoding sequences are expressed from different polynucleotides. In some embodiments, the encoded acceptor affinity peptide specifically binds to an acceptor affinity peptide binding molecule. In some embodiments, two or more sequences encoding an affinity acceptor-tagged HLA contain two or more affinity acceptor peptides. In some embodiments, two or more sequences encoding an affinity-acceptor-tagged HLA contain three or more sequences encoding an affinity-acceptor-tagged HLA, wherein at least two of the three or more sequences encoding an affinity-acceptor-tagged HLA contain the same affinity acceptor peptide. In some embodiments, the two or more affinity acceptor peptides are unique for each of the two or more sequences encoding the affinity acceptor-tagged HLA.

[43] In some embodiments, the encoded affinity acceptor peptide is selected from the group consisting of a biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine-glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 peptide tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tags, Strep tags II, tags albumin-binding protein (ABP), alkaline phosphatase (AP) tags, bluetongue (B-tags), calmodulin-binding peptide (CBP) tags, chloramphenicol acetyltransferase (CAT) tags, choline binding domain (CBD) tags , chitin binding domain (CBD) tags, cellulose binding domain (CBP) tags, dihydrofolate reductase (DHFR) tags, galactose binding protein (GBP), maltose binding protein (MBP) tags, glutathione-S -transferases (GST), Glu-Glu tags (EE), human influenza virus hemagglutinin (HA) tags, horseradish peroxidase (HA) tags, NE tags, HSV tags, ketosteroid isomerase (KSI) tags, KT3 tags, LacZ tags , luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin tag, E tag, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity eXact tags, protein C tags, S1 tags, S tags, carboxybiotin carrier protein (BCCP) tags, green fluorescent protein (GFP) tags, small ubiquitin-like modifier (SUMO) tags, tandem affinity purification (TAP) tags , HaloTag, Nus tags, Thioredoxin tags, Fc tags, CYD tags, HPC tags, TrpE tags, ubiquitin tag, VSV-G epitope tag, V5 tag, and combinations thereof; optionally, the first or second acceptor affinity peptide contains two or more repeats of the tag sequence.

[44] In some embodiments, the affinity acceptor peptide binding molecule is biotin or an antibody specific for the acceptor affinity peptide. In some embodiments, the affinity acceptor peptide binding molecule specifically binds to the affinity molecule. In some embodiments, the affinity molecule is streptavidin, NeutrAvidin, or a derivative thereof. In some embodiments, the acceptor affinity peptide binding molecule does not specifically interact with the recombinant HLA class I or class II amino acid sequence. In some embodiments, for two or more recombinant polynucleic acids: a sequence encoding an affinity acceptor-tagged HLA is stably inserted into the cell's genome. In some embodiments, a sequence encoding a .beta.2 microglobulin or a sequence encoding the .beta. chain of an HLA class II is linked to a sequence encoding a second acceptor affinity peptide. In some embodiments, the second acceptor affinity peptide contains an HA tag. In some embodiments, a sequence encoding a .beta.2 microglobulin or a sequence encoding the .beta. chain of an HLA class II is linked to a sequence encoding a recombinant HLA and an acceptor affinity peptide via a linker.

[45] In some embodiments, the linker contains a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome escape site or internal ribosome entry site (IRES). In some embodiments, the ribosome escape site or IRES is cleaved when expressed in cells. In some embodiments, the ribosome exit site is selected from the group consisting of F2A, T2A, P2A, and E2A. In some embodiments, the IRES element is selected from common cellular or viral IRES sequences.

[46] Provided herein is a composition comprising two or more isolated polypeptide molecules encoded by a polynucleic acid of the composition described herein. Provided herein is a composition comprising a population of cells containing two or more polypeptide molecules encoded by a polynucleic acid of the composition described herein. This document provides a composition containing a population of cells containing the composition described in this document. This document provides a composition containing a population of cells containing one or more cells containing the composition described herein.

[47] In some embodiments, a population of cells expresses one or more endogenous HLA class I or class II alleles. In some embodiments, a population of cells is constructed that lacks one or more endogenous HLA class I alleles. In some embodiments, a population of cells is constructed that lack alleles for endogenous HLA class I. In some embodiments, a population of cells is constructed that lacks one or more endogenous HLA class II alleles. In some embodiments, a population of cells is constructed that lack alleles for endogenous HLA class II. In some embodiments, a population of cells is constructed that lacks one or more endogenous HLA class I alleles and one or more endogenous HLA class II alleles. In some embodiments, the cell population is a cell population with low cell surface HLA class I or class II expression. In some embodiments, a composition is formulated using peptides or polynucleic acids encoding peptides specific for the patient's HLA type. Provided herein is a method for producing a cell, comprising transduction or transfection of two or more cells with two or more polynucleic acids of the composition described herein.

[48] This document provides a peptide identified according to the method described in this document. Provided herein is a method for eliciting an antitumor response in a mammal, comprising administering to the mammal an effective amount of a polynucleic acid containing the peptide sequence described herein. Provided herein is a method for inducing an antitumor response in a mammal, comprising administering to the mammal an effective amount of a peptide comprising the peptide sequence described herein. Provided herein is a method for eliciting an antitumor response in a mammal, comprising administering to the mammal a cell containing a peptide comprising the peptide sequence described herein. Provided herein is a method for eliciting an antitumor response in a mammal, comprising administering to the mammal a cell containing an effective amount of a polynucleic acid containing a peptide coding sequence comprising the peptide sequence described herein. In some embodiments, the cell presents the peptide as an HLA-peptide complex. Provided herein is a method of eliciting an immune response in a mammal, comprising administering to the mammal an effective amount of a polynucleic acid containing a sequence encoding a peptide described herein. Provided herein is a method of eliciting an immune response in a mammal, comprising administering to the mammal an effective amount of a peptide comprising the peptide sequence described herein. Provided herein is a method of eliciting an immune response in a mammal, comprising administering to the mammal an effective amount of cells containing a peptide comprising the peptide sequence described herein. Provided herein is a method for eliciting an immune response in a mammal comprising administering to the mammal an effective amount of cells containing a polynucleic acid containing a peptide coding sequence comprising the peptide sequence described herein.

[49] In some embodiments, the implementation of the immune response is a T-cell immune response. In some embodiments, the immune response is a CD8 T cell response. In some embodiments, the immune response is a CD4 T cell response. In some embodiments, the immune response is a humoral immune response.

[50] Provided herein is a method for treating a mammal having a disease, comprising administering to the mammal an effective amount of a polynucleic acid containing a sequence encoding a peptide described herein. Provided herein is a method of treating a mammal having a disease comprising administering to the mammal an effective amount of a peptide comprising a peptide sequence as described herein. Provided herein is a method for treating a mammal having a disease, comprising administering to the mammal an effective amount of cells containing a peptide comprising the peptide sequence described herein. Provided herein is a method for treating a mammal having a disease comprising administering to the mammal an effective amount of cells containing a polynucleic acid containing a peptide coding sequence comprising the peptide sequence described herein. In some embodiments, the disease is cancer. In some embodiments, the disease is infection by an infectious agent. In some embodiments, the infectious agent is a pathogen, optionally a virus or bacterium, or a parasite.

[51] In some embodiments, the virus is selected from the group consisting of: BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4, DENV-5), cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), adenovirus, human immunodeficiency virus (HIV), human T-cell lymphotrophic virus (HTLV-1), influenza virus, RSV, HPV, rabies, mumps, rubella, poliovirus, yellow fever, hepatitis A, hepatitis B, rotavirus, varicella-zoster virus, human papillomavirus (HPV), smallpox, shingles, and any combination thereof.

[52] In some embodiments, the bacterium is selected from the group consisting of: Klebsiella species, Tropheryma whipplei, Mycobacterium leprae, Mycobacterium lepromatosis, and Mycobacterium tuberculosis, typhoid, pneumococcus, meningococcus, haemophilus B, anthrax, tetanus toxin, group B meningococcus, bcg , cholera, and any combination thereof.

[53] In some embodiments, the parasite is a helminth or a protozoan. In some embodiments, the parasitic organism is selected from the group consisting of: Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Schistosoma spp., and any combination thereof.

[54] Provided herein is a method for enriching immunogenic peptides, comprising: providing a cell population containing one or more cells expressing an affinity acceptor-tagged HLA, wherein the affinity-acceptor-tagged HLA contains an affinity acceptor peptide operably linked to a recombinant HLA encoded by an allele of the recombinant HLA; and enrichment of HLA-peptide complexes containing labeled HLA affinity acceptor. In some embodiments, the method further comprises determining the sequence of immunogenic peptides isolated from the HLA-peptide complexes. In some embodiments, the implementation of the definition includes the use of LC-MS/MS.

[55] Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a polynucleic acid containing a sequence encoding a peptide described herein. Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a peptide comprising the peptide sequence described herein. Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of cells containing a peptide comprising the peptide sequence described herein. Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject a cell containing an effective amount of a polynucleic acid containing a peptide coding sequence comprising the peptide sequence described herein.

[56] Provided herein is a method for developing a therapeutic agent for a subject with a disease or condition, comprising providing a population of cells derived from a subject with the disease or condition, expressing in one or more cells of the cell population an allele labeled with an HLA class I or class II affinity acceptor by introduction into one or more cells of a polynucleic acid encoding a sequence containing: a sequence encoding an allele of recombinant HLA class I or class II, operably linked to a sequence encoding an affinity acceptor peptide, thereby forming complexes of affinity acceptor-labeled HLA peptides in one or more cells; enrichment and characterization of complexes labeled with an affinity acceptor HLA-peptides; and, optionally, developing a therapeutic agent based on the resulting characteristic.

[57] Provided herein is a method for identifying at least one subject-specific immunogenic antigen and preparing a subject-specific immunogenic composition that contains at least one subject-specific immunogenic antigen wherein the subject has a disease and at least one subject-specific for a subject, the immunogenic antigen is specific for the subject and the subject's disease, said method comprising: providing a population of cells obtained from a subject with a disease or condition, expression in one or more cells of the cell population in the subject of an allele labeled with an HLA class I or class II affinity acceptor by introduction into one or more cells of a polynucleic acid encoding a sequence containing: a sequence encoding an allele of recombinant HLA class I or class II, functionally linked to a sequence encoding an affinity acceptor peptide, thereby forming complexes m affinity-acceptor-engineered HLA peptides in one or more cells; enrichment of complexes labeled with an affinity acceptor HLA peptides from one or more cells; identifying an immunogenic peptide from the enriched complexes labeled with an affinity acceptor HLA peptides that is specific for the subject and the subject's disease; and formulating a subject-specific immunogenic composition based on one or more subject-specific immunogenic peptides identified.

[58] In some embodiments, the therapeutic or subject-specific immunogenic composition comprises a peptide from enriched complexes of HLA affinity acceptor tagged peptides or a polynucleotide encoding a polypeptide from enriched complexes of HLA affinity acceptor tagged peptides. In some embodiments, the therapeutic or subject-specific immunogenic composition comprises a T cell expressing a T cell receptor (TCR) that specifically binds to a polypeptide from enriched complexes of HLA affinity acceptor tagged peptides. In some embodiments, the subject-specific immunogenic composition comprises a chimeric antigen receptor (CAR), a T cell expressing a receptor that specifically binds to a polypeptide from enriched complexes of HLA affinity acceptor tagged peptides.

[59] In some embodiments, the method further comprises administering to the subject another therapeutic agent, optionally an immune checkpoint inhibitor. In some embodiments, the method further comprises administering to the subject an adjuvant, optionally a poly-ICLC.

[60] In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, the disease or disorder is an infection. In some embodiments, the infection is infection with an infectious agent. In some embodiments, the infectious agent is a pathogen, virus, bacterium, or parasite.

[61] In some embodiments, the virus is selected from the group consisting of: BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4, DENV-5), cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), adenovirus, human immunodeficiency virus (HIV), human T-cell lymphotrophic virus (HTLV-1), influenza virus, RSV, HPV, rabies, mumps, rubella, poliovirus, yellow fever, hepatitis A, hepatitis B, rotavirus, varicella-zoster virus, human papillomavirus (HPV), smallpox, shingles, and any combination thereof.

[62] In some embodiments, the bacterium is selected from the group consisting of: Klebsiella species, Tropheryma whipplei, Mycobacterium leprae, Mycobacterium lepromatosis, and Mycobacterium tuberculosis, typhoid, pneumococcus, meningococcus, haemophilus B, anthrax, tetanus toxin, group B meningococcus, bcg , cholera, and combinations thereof.

[63] In some embodiments, the parasite is a helminth or a protozoan. In some embodiments, the parasitic organism is selected from the group consisting of: Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Schistosoma spp., and any combination thereof.

[64] Provided herein is a method for developing a therapeutic agent for a subject with a disease or condition, comprising: providing a cell population, wherein one or more cells of the cell population comprise a polynucleic acid containing a sequence encoding at least two alleles of an affine acceptor-tagged HLA class I or class II, wherein the sequence encoding at least two class I or class II HLA labeled with an affinity acceptor contains a first recombinant sequence containing a sequence encoding the first class I or class II HLA allele operably linked to the sequence encoding the first affinity peptide - acceptor; and a second recombinant sequence containing a sequence encoding a second HLA class I or class II allele operably linked to a sequence encoding a second acceptor affinity peptide; expressing at least two affinity acceptor-labeled HLA peptides in at least one cell of one or more cells of the cell population, thereby forming complexes of the affinity acceptor-labeled HLA peptides in at least one cell; enrichment of complexes labeled with an affinity acceptor HLA-peptides; and identification of the peptide from the enriched complexes labeled with an affinity acceptor HLA peptides; and formulating an immunogenic composition based on one or more of the identified peptides, wherein the alleles of the first and second recombinant HLA class I or class II match the HLA haplotype of the subject. In some embodiments, the subject has a disease or condition.

[65] In some embodiments, the first allele of the recombinant HLA class I or class II is different from the allele of the second recombinant HLA class I or class II. In some embodiments, the first acceptor affinity peptide is the same as the second acceptor affinity peptide. In some embodiments, the method includes characterizing the peptide associated with the first and/or second complexes of affinity acceptor-labeled HLA peptides by enrichment. In some embodiments, the at least two alleles of class I or class II affinity-acceptor-tagged HLA include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 HLA class I and/or class II alleles. In some embodiments, the implementation of the first and/or second complexes labeled with an affinity acceptor HLA peptides contain a transmembrane domain. In some embodiments, the implementation of the first and/or second complexes labeled with an affinity acceptor HLA peptides contain an intracellular domain. In some embodiments, the implementation of the first and/or second complexes labeled with an affinity acceptor HLA peptides are not excreted. In some embodiments, the first and/or second complexes of the affinity acceptor-tagged HLA peptides are incorporated into the cell membrane upon expression. In some embodiments, the first and/or second complexes of the HLA acceptor affinity labeled peptides are insoluble complexes of the HLA acceptor affinity labeled peptides.

[66] In some embodiments, the method further comprises creating a database of HLA allele-specific peptides. In some embodiments, the implementation of the method includes the introduction of one or more exogenous peptides in a population of cells. In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more exogenous peptides or the expression of one or more exogenous peptides in a population of cells. In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more nucleic acids encoding one or more exogenous peptides.

[67] In some embodiments, one or more nucleic acids encoding one or more peptides is DNA. In some embodiments, one or more nucleic acids encoding one or more peptides is RNA, optionally the RNA is mRNA.

[68] In some embodiments, enrichment does not include the use of a tetrameric reagent. In some embodiments, the method includes determining the sequence of the peptide or portion thereof associated with the first and/or second complex of the enriched HLA affinity acceptor-labeled peptide. In some embodiments, the determination includes biochemical analysis, mass spectrometric analysis, MS analysis, MS/MS analysis, LC-MS/MS analysis, or a combination thereof.

[69] In some embodiments, the method includes assessing the binding affinity or stability of the peptide or portion thereof associated with the first and/or second complex of the HLA affinity acceptor labeled peptide as a result of enrichment. In some embodiments, the method includes determining whether the peptide or portion thereof associated with the first and/or second complex enriched with an HLA acceptor affinity peptide has one or more mutations. In some embodiments, the method includes evaluating the binding of peptides to HLA molecules in the first and/or second complex of an HLA affinity acceptor-labeled peptide.

[70] In some embodiments, the implementation of the method includes the expression of the library of peptides in a population of cells, thereby forming a library of complexes labeled with an affinity acceptor HLA peptides. In some embodiments, the method includes contacting the cell population with a peptide library or a library of peptide coding sequences, thereby forming a library of HLA affinity acceptor-labeled peptide complexes. In some embodiments, the library is a library of peptides associated with a disease or condition.

[71] In some embodiments, the disease or condition is cancer or infection with an infectious agent. In some embodiments, the method includes introducing the infectious agent or parts thereof into one or more cells of the cell population. In some embodiments, the method includes characterizing one or more peptides from the first and/or second HLA-peptide complexes, optionally the peptides are derived from one or more target proteins of the infectious agent. In some embodiments, the method includes characterizing one or more peptide regions from one or more target proteins of the infectious agent. In some embodiments, the method includes identifying peptides from the first and/or second HLA-peptide complexes derived from the infectious agent.

[72] In some embodiments, the cell population is derived from a biological sample in a subject with a disease or condition. In some embodiments, the cell population is a cell line. In some embodiments, the cell population is a primary cell population. In some embodiments, a peptide from the first and/or second complex, the affinity acceptor-tagged HLA peptide, is capable of activating a T cell in a subject when presented by an antigen presenting cell. In some embodiments, the method includes comparing HLA-peptide complexes from diseased cells with HLA-peptide complexes from unaffected cells. In some embodiments, the method further comprises isolating peptides from the first and/or second complexes of HLA affinity acceptor-labeled peptides prior to identification. In some embodiments, the cell population is a cell population with low cell surface HLA class I or class II expression.

[73] In some embodiments, a population of cells expresses one or more endogenous HLA alleles. In some embodiments, the cell population expresses endogenous HLA alleles normally expressed by the cell population. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles and endogenous HLA class II alleles. In some embodiments, the cell population has a knockout of one or more HLA class I alleles. In some embodiments, the cell population has a knockout of one or more HLA class II alleles. In some embodiments, a population of cells has a knockout of all HLA class I alleles. In some embodiments, a population of cells has a knockout of all HLA class II alleles. In some embodiments, the cell population has a knockout of all HLA class I alleles and a knockout of all HLA class II alleles. In some embodiments, a sequence encoding at least two alleles of an affinity-acceptor-tagged class I or class II HLA encodes a class I HLA. In some embodiments, Class I HLA is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the first allele of the recombinant HLA class I or class II is the first allele of HLA class I, and the second allele of the recombinant HLA class I or class II is the second allele of HLA class I. In some embodiments, a sequence encoding at least two alleles of an affinity-acceptor-tagged class I or class II HLA encodes a class II HLA. In some embodiments, the HLA class II is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP. In some embodiments, the HLA class II comprises an HLA class II α chain, an HLA class II β chain, or a combination thereof. In some embodiments, the first allele of the recombinant HLA class I or class II is the first allele of HLA class II, and the second allele of the recombinant HLA class I or class II is the second allele of HLA class II.

[74] In some embodiments, each of the first sequence and the second sequence is operably linked. In some embodiments, the first sequence and the second sequence are in different polynucleotide molecules. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to a sequence that encodes the extracellular portion of the first and/or second HLA class I or class II allele. In some embodiments, the first and/or second encoded acceptor affinity peptide is extracellularly expressed. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to the N-terminus of the sequence encoding the first and/or second HLA class I or class II allele. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to a sequence that encodes the intracellular portion of the first and/or second HLA class I or class II allele. In some embodiments, the encoded first and/or second acceptor affinity peptide is expressed intracellularly. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to the C-terminus of the sequence encoding the first and/or second HLA class I or class II allele. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to the sequence encoding the first and/or second HLA class I or class II allele via a linker.

[75] In some embodiments, enrichment includes enrichment of intact cells expressing the first and/or second complexes of affinity acceptor-tagged HLA peptides. In some embodiments, the method does not include lysing the cells prior to enrichment. In some embodiments, the method further comprises lysing one or more cells prior to enrichment. In some embodiments, enrichment comprises contacting an acceptor affinity peptide binding molecule with a first and/or second complexes of affinity acceptor-labeled HLA peptides, wherein the acceptor affinity peptide binding molecule specifically binds to the first and/or second acceptor affinity peptide. .

[76] In some embodiments, the first and/or second affinity acceptor peptide comprises a tag sequence comprising a biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine-glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep -tag II, albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag), calmodulin-binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline- binding domain (CBD), chitin-binding domain (CBD) tag, cellulose-binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein (GBP) tag, maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu tag (EE), human influenza virus hemagglutinin (HA) tag, horseradish peroxidase (HA) tag, NE-tag, HSV tag, ketosteroid isomerase ( KSI), KT3 tag, LacZ tag, Luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin tag, E tag, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, tag Xpress, Isopepeptag, SpyTag, SnoopTag, Profinity eXact tag, protein C tag, S1 tag, S tag, carboxybiotin carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag , tandem affinity purification (TAP) tag, HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or a combination thereof; optionally, the first and/or second affinity acceptor peptide contains two or more repeats of the tag sequence.

[77] In some embodiments, the affinity acceptor peptide binding molecule is biotin or an antibody specific for the first and/or second acceptor affinity peptide. In some embodiments, enrichment includes contacting the affinity molecule with the first and/or second complexes of affinity acceptor-labeled HLA peptides, wherein the affinity molecule specifically binds to the affinity acceptor peptide binding molecule. In some embodiments, the affinity molecule is streptavidin, NeutrAvidin, or a derivative thereof. In some embodiments, enrichment includes immunoprecipitation of the first and/or second complexes of affinity acceptor-tagged HLA peptides.

[78] In some embodiments, an affinity acceptor peptide binding molecule is attached to a solid surface. In some embodiments, the affinity molecule is attached to a solid surface. In some embodiments, the hard surface is a bead.

[79] In some embodiments, enrichment comprises immunoprecipitating the first and/or second complexes of the affinity acceptor-labeled HLA peptides with an acceptor affinity peptide binding molecule that specifically binds to the first and/or second acceptor affinity peptide. In some embodiments, the affinity acceptor peptide binding molecule does not specifically interact with the amino acid sequence of the encoded first and/or second class I or class II HLA. In some embodiments, enrichment includes contacting an affinity molecule specific for the extracellular portion of the first and/or second HLA class I or class II allele. In some embodiments, enrichment includes contacting an affinity molecule specific for the N-terminal portion of the first and/or second HLA class I or class II allele.

[80] In some embodiments, providing includes contacting a population of cells with a polynucleic acid. In some embodiments, the introduction into contact includes transfection or transduction. In some embodiments, providing includes contacting a population of cells with a vector containing a polynucleic acid. In some embodiments, the vector is a viral vector. In some embodiments, the polynucleic acid is stably inserted into the genome of a population of cells.

[81] In some embodiments, the sequence encoding the first and/or second HLA class I or class II contains a sequence encoding the α-chain of HLA class I. In some embodiments, the first allele of the recombinant HLA class I or class II is the α chain of the first HLA class I, and the second allele of the recombinant HLA class I or II is the α chain of the second HLA class I.

[82] In some embodiments, the implementation of the method further includes the expression of a sequence encoding β2 microglobulin in one or more cells. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding a first and/or second class I or class II HLA. In some embodiments, the β2 microglobulin coding sequence is linked to the first and/or second class I or class II HLA coding sequence via a linker. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding a third acceptor affinity peptide.

[83] In some embodiments, the implementation of the third affinity acceptor peptide is different from the first and/or second affinity acceptor peptide. In some embodiments, the sequence encoding the first and/or second HLA class I or class II comprises a sequence encoding an HLA class II α chain and/or an HLA class II β chain. In some embodiments, the sequence encoding the first and/or second HLA class I or class II contains a sequence encoding the α chain of the first HLA class II and the α chain of the second HLA class II. In some embodiments, the method further comprises expressing an HLA class II β chain coding sequence in one or more cells. In some embodiments, a sequence encoding a first HLA class II α chain and a second HLA class II α chain are linked to a sequence encoding an HLA class II β chain. In some embodiments, the sequence encoding the first and/or second HLA class I or class II contains a sequence encoding the β chain of the first HLA class II and the β chain of the second HLA class II.

[84] In some embodiments, the implementation of the method further includes the expression of the sequence encoding the α-chain of HLA class II, in one or more cells. In some embodiments, a sequence encoding a first HLA class II β chain and a second HLA class II β chain are linked to a sequence encoding an HLA class II α chain via a linker. In some embodiments, a sequence encoding an HLA class II β chain or an HLA class II α chain is linked to a sequence encoding a third acceptor affinity peptide. In some embodiments, the third acceptor affinity peptide is different from the first and/or second acceptor affinity peptide.

[85] In some embodiments, the third acceptor affinity peptide is different from the first acceptor affinity peptide and is selected from the group consisting of a biotin acceptor peptide (BAP), a polyhistidine tag, a polyhistidine glycine tag, a polyarginine tag, a polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tags, Strep II tags, albumin-binding protein (ABP) tags, alkaline phosphatase (AP) tags, bluetongue virus tags (B-tags), calmodulin-binding peptide (CBP) tags, chloramphenicol acetyltransferase (CAT) tags ), choline-binding domain (CBD) tags, chitin-binding domain (CBD) tags, cellulose-binding domain (CBP) tags, dihydrofolate reductase (DHFR) tags, galactose-binding protein tags (GBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tags, human influenza virus hemagglutinin (HA) tags, horseradish peroxidase (HA) tags, NE-tags, HSV tags, ketosteroid isomerase (KSI) tags, KT3 tags, LacZ tags, luciferase tags, NusA tags, PDZ domain tags, AviTag, calmodulin tags, E-tags, S-tags, SBP-tags, Softag 1, Softag 3, TC tags, VSV tags, Xpress tags, Isopeptag, SpyTag, SnoopTag, Profinity eXact tags, protein C tags, S1 tags, S tags, carboxybiotin carrier protein (BCCP) tags, green fluorescent protein (GFP) tags, small ubiquitin tags similar modifier (SUMO), tandem affinity purification (TAP) tag, HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, and combinations thereof ; optionally, the first or second acceptor affinity peptide contains two or more repeats of the tag sequence.

[86] In some embodiments, the linker contains a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome escape site or internal ribosome entry site (IRES). In some embodiments, the ribosome escape site or IRES is cleaved when expressed in cells. In some embodiments, the ribosome exit site is selected from the group consisting of F2A, T2A, P2A, and E2A. In some embodiments, the IRES element is selected from common cellular or viral IRES sequences.

[87] In some embodiments, the implementation of the method includes conducting biochemical analysis or mass spectrometry, such as tandem mass spectrometry. In some embodiments, the method includes obtaining a peptide sequence that corresponds to MS/MS spectra of one or more peptides isolated from enriched complexes of HLA affinity acceptor-labeled peptides from a peptide database; wherein one or more of the resulting sequences identifies the sequence of one or more peptides.

[88] In some embodiments, the cell population is a cell line selected from HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, Hep G2, SH-SY5Y, CACO-2, U937, U-2 OS , ExpiCHO, CHO and THP1. In some embodiments, the cell line is treated with one or more cytokines, checkpoint inhibitors, epigenetically active drugs, IFN-γ, or a combination thereof. In some embodiments, the cell population includes at least 10 ⁵ cells, at least 10 ⁶ cells, or at least 10 ⁷ cells. In some embodiments, the cell population is a population of dendritic cells, macrophages, cancer cells, or B cells. In some embodiments, the cell population includes tumor cells.

[89] In some embodiments, a population of cells is contacted with an agent prior to isolation of the first and/or second HLA-peptide complexes from one or more cells. In some embodiments, the agent is an inflammatory cytokine, a chemical agent, an adjuvant, a therapeutic agent, or radiation.

[90] In some embodiments, the first and/or second HLA allele is a mutant HLA allele. In some embodiments, the sequence encoding the first and/or second HLA allele contains a barcoded sequence. In some embodiments, the method further comprises analyzing the expression of a first and/or second allele labeled with an HLA class I or class II affinity acceptor.

[91] In some embodiments, the analysis includes sequencing a first and/or second allele labeled with an HLA class I or class II affinity acceptor, detecting RNA encoding the first and/or second allele labeled with an HLA class I or class II affinity acceptor, detecting a protein of the first and/or second allele labeled with an HLA class I or class II affinity acceptor, or a combination thereof. In some embodiments, the first and second allele of an affinity-acceptor-tagged class I or class II HLA contains a unique barcoded sequence. In some embodiments, the first sequence and the second sequence comprise a unique barcoded sequence.

Brief description of the drawings

[92] The features of the present disclosure are detailed in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description, which sets forth illustrative embodiments in which the principles of the disclosure are used, and the accompanying drawings, in which:

[93] in FIG. 1A shows an exemplary pipeline for universal immunoaffinity purification and data generation. HLA class I and/or class II molecules are introduced into any cell, including a cell that does not express HLA class I or class II, in order to express the particular HLA class I or class II allele(s) in the cell. Populations of genetically engineered HLA-expressing cells are harvested, lysed, and their HLA-peptide complexes are labeled (eg, biotinylated) and immunoaffinity purified (eg, using biotin-streptavidin interaction). HLA-related peptides specific for a single HLA can be eluted from their labeled (eg, biotinylated) complexes and evaluated (eg, sequenced using high resolution LC-MS/MS).

[94] in FIG. 1B is a structural diagram of HLA class II molecules -DP, -DQ and -DR. HLA-DR molecules are heterodimers containing a constant α-chain and a variable β-chain. The HLA-DQ and HLA-DP molecules are heterodimers containing α variable chains and β variable chains.

[95] in FIG. 2 is a representative schematic of constructs designed to express HLA class I and II in cultured cell lines. The HLA-A*02:01 constructs are a class I HLA construct that implements biotin acceptor peptides (BAPs) for biotinylation and immunoaffinity purification. The HLA-DRB1*11:01 constructs are an HLA class II construct that implements biotin acceptor peptides (BAPs) for biotinylation and immunoaffinity purification.

[96] in FIG. 3 is a diagram of an exemplary lentiviral vector that can be used to generate stable cell lines expressing class I and class II HLA constructs.

[97] in FIG. 4A shows an exemplary transfection-based introduction of a class I or class II HLA construct for universal IP and sequencing of an HLA-related peptide by LC-MS/MS.

[98] in FIG. 4B shows an exemplary transfection-based introduction of class I or class II HLA constructs followed by a selection process such as inclusion of an antibiotic resistance gene. Selected cells can then be submitted for universal IP and HLA-related peptide sequencing by LC-MS/MS.

[99] in FIG. 5 is a generic immunoaffinity purification scheme for HLA class I and class II. Cells such as HEK293T (human primary kidney) are either transfected or transduced to express a single HLA class I or class II allele with an affinity tag for immunoaffinity purification. Labeled HLA expressing cells are harvested, lysed, and their HLA-peptide complexes are biotinylated and immunoaffinity purified using biotin-streptavidin interaction. HLA-bound peptides specific for a single HLA are eluted from their biotinylated complexes and analyzed (eg sequenced using high resolution LC-MS/MS).

[100] in FIG. 6A is a Western blot (antibiotinylation) comparing mimic, GFP, and empty plasmid transfections with biotinylation-based immunoprecipitation HLA-A*02:01 constructs demonstrating expression of HLA class I alleles in HEK293T cells.

[101] in FIG. 6B shows a stained Ponceau gel used as a transfer quality control for Western blotting.

[102] in FIG. 6C is a schematic representation of the HLA class I constructs used to generate the engineered HEK293T cells depicted in FIG. 6A and FIG. 6b .

[103] in FIG. 7A shows Western blotting (top) and transfer quality control (bottom) images of a biotinylation time-dependence experiment demonstrating that C- and N-terminal labeled HLA-BAP biotinylation is completed in 10 minutes for cells expressing HLA-BAP as Class I and Class II. The results show the optimization of transfection and biotinylation of class I and class II HLA-BAP alleles expressed by HEK293T cells.

[104] in FIG. 7B shows a Western blot against anti-BAP (top) and transfer quality control (bottom) from cells expressing HLA class I and class II constructs labeled with BAP and N- and C-termini.

[105] in FIG. 7C is a schematic representation of the (HLA-A*02:01) Class I and (HLA-DRβ*11:01) Class II constructs, labeled with BAP and N- and C-termini, used to optimize transfection and biotinylation.

[106] in FIG. 8A is a Western blot (anti-streptavidin for BAP tag and anti-HA for HA tag) and transfer quality control (Ponceau S) image showing expression of class I and class II biotinylated HLA constructs used for HLA immunoprecipitation in HEK293T cells. Lysates were analyzed before the addition of biotin (-Biotin), after the addition of biotin (+Introduction of Biotin) and after biotinylation and subsequent removal with streptavidin beads (+Biotin FT). a decrease in signal in the +Biotin FT band demonstrates that biotinylated MHC has been removed from the lysate and binding to the streptavidin beads.

[107] in FIG. 8B is a Western blot (anti-streptavidin for BAP tag and anti-HA for HA tag) and transfer quality control (Ponceau S) image showing expression of class I and class II biotinylated HLA constructs used for HLA immunoprecipitation in HeLa (cancer) cells. human cervix). The lysates were analyzed before the addition of biotin (-Biotin), after the addition of biotin (+Introduction of Biotin), and after biotinylation and subsequent removal with streptavidin beads (+Biotin FT). a decrease in signal in the +Biotin FT band demonstrates that biotinylated MHC has been removed from the lysate and binding to the streptavidin beads.

[108] in FIG. 8C is a Western blot (anti-streptavidin for BAP tag and anti-HA for HA tag) and transfer quality control (Ponceau S) image showing expression of class I and class II biotinylated HLA constructs used for HLA immunoprecipitation in A375 (malignant) cells. human melanoma). Lysates were analyzed before the addition of biotin (-Biotin), after the addition of biotin (+Introduction of Biotin) and after biotinylation and subsequent removal with streptavidin beads (+Biotin FT). a decrease in signal in the +Biotin FT band demonstrates that biotinylated MHC has been removed from the lysate and binding to the streptavidin beads.

[109] in FIG. 8D is a Western blot (anti-streptavidin for BAP tag and anti-HA for HA tag) and transfer quality control (Ponso S) image showing expression of class I and class II biotinylated HLA constructs used for HLA immunoprecipitation in Expi293 cells (primary human kidney genetically engineered for high density culture and protein expression). The lysates were analyzed before the addition of biotin (-Biotin), after the addition of biotin (+Introduction of Biotin), and after biotinylation and subsequent removal with streptavidin beads (+Biotin FT). a decrease in signal in the +Biotin FT band demonstrates that biotinylated MHC has been removed from the lysate and binding to the streptavidin beads.

[110] in FIG. 9A is a bar graph of an exemplary LC-MS/MS analysis of HLA-related peptides isolated using the Universal HLA Immunoprecipitation (Universal IP) pipeline. Shown as a bar graph of the total number of unique HLA-related peptides identified from a variety of cell types (A375; grey, HEK293T; orange, HeLa; blue) that express class I and class II HLA affinity tagged constructs used in the pipeline for universal IP.

[111] in FIG. 9B is a bar graph showing representative data from profiling a monoallelic HLA class I peptide by LC-MS/MS. Each bar represents the total number of unique HLA-associated peptides identified from class I monoallele experiments using HLA affinity tagged constructs.

[112] in FIG. 9C is a bar graph showing representative data from class II monoallelic HLA peptide profiling by LC-MS/MS. Each bar represents the total number of unique HLA-associated peptides identified from class II monoallele experiments using HLA affinity tagged constructs.

[113]in fig. 10Apresented illustrative characterization scheme of HLA-associated class I and class II peptides detected using the universal IP pipeline. Illustrative shown depiction of a sequence ligature of class I HLA-A*02:01 associated peptides and class II HLA-DRβ*11:01 associated peptides isolated and sequenced using the universal IP platform.

[114] in FIG. 10B is a bar graph showing a comparison of the length distribution of HLA class I (red; HLA-A*02:01) and class II (blue; HLA-DRβ*11:01) associated peptides, with HLA associated peptides identified using a pipeline. for generic IP. The length distributions of both HLA-associated peptides of both class I and class II, identified using the universal IP, are in line with the expected trends.

[115] in FIG. 11A is a schematic representation of HLA class II constructs that were designed to be expressed by different cell types for the universal IP pipeline.

[116] in FIG. 11B is a schematic representation of HLA class II complexes that can be formed upon expression of the construct shown in FIG. 11A in cell lines expressing the α-chain and β-chain subunits of endogenous HLA class II. Class II HLA complexes are formed by pairing the α-chain and β-chain, each of which is labeled with markers of different affinity.

[117] in FIG. 12A is a schematic representation of a serial universal IP strategy that can be used to deconvolve the α-chain and β-chain pairing of the HLA class II depicted in FIG. 11B and unambiguous peptide binding assignments to specific HLA class II complexes, and which demonstrates confirmation of the serial universal IP of class II HLA complexes containing multiple affinity tags. Cells expressing dual affinity tagged HLA class II constructs are lysed, biotinylated, and incubated with anti-HA antibody-coupled beads. HLA class II complexes with HA labeled subunits are isolated, washed and eluted using an HA peptide (eg YPYDVPDYA). The eluate is then incubated with either NeutrAvidin or streptavidin coupled beads to isolate HA labeled and biotin labeled HLA class II complexes. The peptides associated with double-labeled HLA class II complexes are then eluted and sequenced by LC-MS/MS.

[118] in FIG. 12B shows a Western blot validation of the serial universal IP strategy in HEK293T expressing double-labeled HLA-DRB*11:01 constructs. The anti-HA antibody was used for the batch enrichment process. Transfer quality control (Ponceau S stained gel) is shown.

[119] in FIG. 12C shows the results of an exemplary negative control experiment where cells expressing the HLA-DRB*11:01 dual affinity class II construct HLA-DRB*11:01 were lysed and incubated with anti-HA antibody-coupled beads without biotinylation. Western blotting and transfer quality control (Ponceau S stained gel) are shown to demonstrate the specificity of the pipeline for serial universal IP. When the biotinylation step was omitted from the Serial Universal IP protocol, no enrichment was observed.

[120] in FIG. 13 is a schematic representation of an overview of HLA class II pruning experiments that provide identification of core-binding epitopes. Class II HLA molecules bind nested sets of peptides, typically 12-18 amino acids in length, that are generated from the same parent protein. Longer peptides hang on the N- and C-terminal sides of HLA class II molecules, while the core epitope interacts most strongly with the peptide binding groove. Peptides bound to HLA class II molecules are truncated using a peptidase specific for the N- and C-terminal regions. After trimming, the core peptide epitopes are sequenced using LC-MS/MS.

[121] in FIG. 14A is a schematic representation of an approach to profiling a monoallelic HLA peptidome in which a biotin affinity tag is performed. In an exemplary embodiment of the present disclosure, a biotin acceptor peptide (BAP) is used that is biotinylated at a lysine (K) residue by the enzyme BirA. The BAP peptide sequence contains a lysine residue that is biotinylated with the addition of the BirA enzyme, biotin and ATP. The biotinylated product shows high affinity for streptavidin/NeutrAvidin. Streptavidin/NeutrAvidin beads can be used to enrich the biotinylated BAP peptide sequence.

[122] in FIG. 14B is a schematic representation of biotin-based immunoaffinity purification of genetically engineered HLA molecules. A particular HLA allele with a BAP sequence either at the N- or C-terminus of the HLA protein is introduced into the cell, for example, by transfection or transduction of a plasmid. It should be noted that the plasmid contains a DNA barcode, which allows PCR to control the cell line for each allele. the length of the barcode may be at least 5 base pairs, at least 10 base pairs, at least 15 base pairs, at least 20 base pairs, or more. Cells expressing HLA-BAP proteins are lysed and biotinylated. The HLA-BAP-peptide complexes are immunoaffinity purified from a mixture of complex lysates, which can be subjected to LC-MS/MS analysis to identify the peptides.

[123] in FIG. 15 is a schematic representation of an exemplary application of the Universal IP platform for target epitope screening and discovery. The cell line of interest is engineered to express an allele-specific tagged (eg, BAP) HLA construct. Cells expressing labeled (eg, BAP) HLA molecules are genetically engineered to express a single epitope or multiple epitopes. The epitope-expressing cells are lysed and the HLA-BAP-peptide complexes are purified by immunoaffinity. Isolated peptide antigens can be examined by any suitable means, eg sequenced by LC-MS/MS, and peptide fragments derived from introduced epitopes can be used as a high throughput read for processing and presentation of HLA allele-matched antigens.

[124] in FIG. 16 is a schematic representation of HLA allele multiplexing in the pipeline for Universal IP. Multiple class I and class II alleles can be expressed from a single HLA construct. For example, a class I construct may contain multiple heavy chains, and a class II construct may contain multiple β- and/or α-chains. By multiplexing HLA alleles in a single construct, multiple HLA molecules can be delivered and expressed in a cell line of interest. Allele multiplexing enables matching of patient HLA types and readings of personalized peptide antigens using a universal IP pipeline and subsequent analysis of complexes and/or peptides, such as LC-MS/MS readings.

[125] in FIG. 17 shows schematics of multi-allelic and mono-allelic approaches in HLA ligand profiling. In the multi-allelic approach, HLA ligands are co-immunoprecipitated with HLA heterodimers directly from patient material or cell lines (top). Because these cells naturally expressed multiple HLA alleles, the peptides identified from such multi-allelic approaches need to be deployed to assign binding to a specific HLA heterodimer if the HLA types are known. In the monoallelic approach, HLA ligands are co-immunoprecipitated with HLA heterodimers from cell lines genetically modified to express only one HLA allele (bottom). Thus, peptides identified from monoallelic approaches do not require deconvolution to assign binding to the HLA heterodimer.

[126] in FIG. 18A is a diagram showing the mutant neoantigenic peptide presented at the MHC.

[127] in FIG. 18B is a schematic of a method for developing a personalized neoantigen-targeted therapy as described herein.

[128] in FIG. 19 is a diagram showing different experimental approaches to profiling different HLA ligands. The peptide:MHC (p:MHC) binding biochemical assay is slow and low throughput and does not take into account processing. Multi-allelic mass spectrometry has a high throughput and the ability to learn processing rules; however, in silico substitution is required to assign peptides to alleles. Monoallelic mass spectrometry provides a fast, unbiased, and clean approach to determine peptide-binding motifs in various MHC alleles. Monoallelic mass spectrometry can quickly and systematically fill gaps in allele coverage and take advantage of the length of allele-specific peptides.

[129] in FIG. 20A is a table of exemplary HLA binding peptides for the A*01:01, B*51:01, A*29:02, and B*54:01 alleles disclosed using a monoallelic approach. The monoallelic approach reveals HLA binding peptides that are poorly assessed by NetMHCpan, but which are biochemically proven to be strong binders.

[130] in FIG. 20B is a histogram showing misassignment levels in 100 simulated deconvolutions. A genotype was generated from the patient's six random HLA alleles (2 alleles each of HLA-A, HLA-B, and HLA-C, taken at US allele frequency). For each allele, 500 peptides were sampled from the respective mono-allelic experiment and combined to create a simulated dataset of 3000 multi-allelic peptides. Each peptide was assigned to the allele that yielded the best NetMHCpan% ranking set to determine the proportion of peptides misassigned by NetMHCpan. This process was repeated 100 times.

[131] in FIG. 21 is a schematic illustration of an MHC presentation predictor for various individual MHC class I alleles using MS data. Model training and evaluation was performed on non-overlapping parent proteins. MS-observable peptides are assigned for training/testing depending on the starting protein. The evaluation approach uses an excess of 5000:1 traps to the true linkers.

[132] in FIG. 22 is a histogram showing significantly improved predictions for both processing and allele-specific binding.

Detailed description

[133] The following description and examples illustrate embodiments of the disclosure in detail. It should be understood that this disclosure is not limited to the specific embodiments described herein and is subject to change as such. Those skilled in the art will appreciate that there are many variations and modifications to this disclosure that fall within its scope.

[134] All terms should be understood as they would be understood by a person skilled in the art. Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning, which is usually understood by an ordinary specialist in the field to which the disclosure relates.

[135] The section headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter of the description.

[136] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although for clarity, the present disclosure may be described herein in the context of separate embodiments, the disclosure may also be implemented in one embodiment.

[137] The following definitions are in addition to the definitions in the art and are directed to the current application and should not refer to any related or unrelated case, such as any publicly available patent or application. Although any methods and materials similar or equivalent to those described herein may be used in practice to test the present disclosure, illustrative materials and methods are described here. Accordingly, the terminology used herein is only intended to describe specific embodiments and is not intended to be limiting.

Definitions

[138] In this application, the use of the singular includes the plural, unless specifically stated otherwise. It should be noted that, as used in the description, the singular forms include references to the plural, unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or", unless otherwise indicated. In addition, the use of the term "including", as well as other forms such as "include", "includes" and "included", is not limiting.

[139] The terms "one or more" or "at least one", such as one or more or at least one element(s) of a group of elements, are self-explanatory, by way of further examples, among other things, the term includes the reference to any one of the specified elements, or to any two or more of the specified elements, such as, for example, any ≥3, ≥4, ≥5, ≥6 or ≥7, etc. From the specified elements to all the specified elements.

[140] Reference in the description to “certain embodiments”, “an embodiment”, “one embodiment”, or “other embodiments” means that a feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments of the present disclosure.

[141] As used herein and in the claim(s), the words "comprising" (and any form containing, such as "comprise" and "comprises"), "having" (and any form having, such as "have " and "has"), "including" (and any form including, such as "includes" and "include"), or "enclosing" (and any form containing, such as "enclosing" and "enclosing") are inclusive or open and do not exclude additional, not listed, elements or steps of the method. It is contemplated that any embodiment discussed in this specification may be implemented with respect to any method or composition according to the disclosure, and vice versa. In addition, compositions according to the disclosure can be used to achieve the methods according to the disclosure.

[142] The term "about" or "approximately" in the context of the present invention, when referring to a measurable value such as a parameter, amount, duration of time, and the like, is intended to cover options of +/-20% or less, +/-10 % or less, +/-5% or less, or +/-1% or less of the specified value and from it, if such options are suitable for use in this disclosure. It should be understood that the meaning itself to which the modifier "about" or "approximately" refers is also specifically disclosed.

[143] The term "immune response" includes T cell mediated and/or B cell mediated immune responses that are affected by modulation of T cell costimulation. Exemplary immune responses include T cell responses, such as cytokine production, and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly affected by T cell activation, such as antibody production (humoral responses) and activation of cytokine-responsive cells, such as macrophages.

[144] "Receptor" should be understood to mean a biological molecule or group of molecules capable of binding a ligand. A receptor can serve to transmit information in a cell, cell formation, or organism. The receptor contains at least one receptor unit and may contain two or more receptor units, and each receptor unit may consist of a protein molecule, for example, a glycoprotein molecule. The receptor has a structure that complements that of the ligand and can complex the ligand as a binding partner. After binding to a ligand on the cell surface, conformational changes in the receptor can transmit signaling information. According to the present disclosure, a receptor may refer to MHC class I and II proteins capable of forming a receptor/ligand complex with a ligand, eg, a peptide or peptide fragment of suitable length.

[145] A "barcoded" sequence can be a nucleic acid sequence that can encode a sequence information element, such as the identity of the sequence to which the barcode is attached, or the identity of the sample from which the sequence is derived.

[146] By "ligand" is meant a molecule that is capable of complexing with a receptor. According to the present disclosure, a ligand should be understood to mean, for example, a peptide or peptide fragment that is of suitable length and suitable binding motifs in its amino acid sequence such that the peptide or peptide fragment is capable of complexing with MHC class I or MHC class II proteins.

[147] "Antigen" is a molecule capable of stimulating an immune response, which can be produced by cancer cells or infectious agents or an autoimmune disease. Antigens recognized by T cells, whether helper T lymphocytes (T helper ( _TH ) cells) or cytotoxic T lymphocytes (CTL), are not recognized as intact proteins, but rather as small peptides that bind to MHC I proteins. class or class II on the cell surface. In the course of a natural immune response, antigens that are recognized in association with MHC class II molecules on antigen presenting cells (APCs) are retrieved from outside the cell, internalized, and processed into small peptides that bind to MHC class II molecules. APCs can also cross-present peptide antigens by processing exogenous antigens and presenting processed antigens on class I MHC molecules. Antigens that elicit proteins that are recognized in association with MHC class I molecules are typically proteins that are produced in cells and these antigens are processed and associated with MHC class I molecules. It is now understood that peptides that bind to these MHC class I or class II molecules are characterized as having a common binding motif, and binding motifs have been identified for a wide variety of MHC class I and II molecules. It is also possible to synthesize synthetic peptides that match the amino acid sequence of a given antigen and that contain a binding motif for a given MHC class I or II molecule. These peptides can then be added to the appropriate APCs and the APCs can be used to stimulate a T helper cell or CTL response, either in vitro or in vivo. All binding motifs, methods for synthesizing peptides, and methods for stimulating a T helper cell or CTL response are known and readily available to one of ordinary skill in the art.

[148] the Term "peptide" in the present description is used interchangeably with "mutant peptide" and "neoantigenic peptide". Likewise, the term "polypeptide" is used interchangeably herein with "mutant polypeptide" and "neoantigenic polypeptide". By "neoantigen" or "neoepitope" is meant a class of tumor antigens or tumor epitopes that result from tumor-specific mutations in an expressed protein. The present disclosure further includes peptides that contain tumor-specific mutations, peptides that contain known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the method of the present disclosure. These peptides and polypeptides are referred to herein as "neoantigenic peptides" or "neoantigenic polypeptides". Polypeptides or peptides can be of various lengths, either in their neutral (uncharged) forms or in forms that are salts, and either do not contain modifications such as glycosylation, side chain oxidation, phosphorylation, or any post-translational modification, or contain these modifications , provided that the modification does not interfere with the biological activity of the polypeptides as described herein. In some embodiments, the neoantigenic peptides of the present disclosure may be: for MHC class I, 22 residues or less in length, eg, about 8 to about 22 residues, about 8 to about 15 residues, or 9 or 10 residues; for MHC class II, 40 residues or less in length, e.g., about 8 to about 40 residues in length, about 8 to about 24 residues in length, about 12 to about 19 residues in length, or about 14 to about 18 residues in length. In some embodiments, the neoantigenic peptide or neoantigenic polypeptide comprises a neoepitope.

[149] The term "epitope" includes any protein determinant capable of specifically binding to an antibody, antibody peptide, and/or antibody-like molecule (including, but not limited to, the T-cell receptor) as defined herein. Epitopic determinants usually consist of chemically active surface groups of molecules, such as amino acid or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.

[150] By "T-cell epitope" is meant a peptide sequence that MHC class I or II molecules can bind in the form of a peptide-presenting MHC molecule or MHC complex, and then, in this form, recognize and bind cytotoxic T-lymphocytes or T-helper cells, respectively.

[151] The term "antibody" as used herein includes IgG (including IgGl, IgG2, IgG3 and IgG4), IgA (including IgAl and IgA2), IgD, IgE or IgM, and IgY, and is intended to encompass whole antibodies, including single chain whole antibodies and their antigen-binding (Fab) fragments. Antigen-binding antibody fragments include, but are not limited to, Fab, Fab' and F(ab') ₂ , Fd (consisting of VH and CH1), single chain variable fragment (scFv), single chain antibodies, disulfide-linked variable fragment (dsFv), and fragments containing either VL or VH domain. The antibodies can be of any animal origin. Antigen binding fragments of antibodies, including single chain antibodies, may contain variable region(s), alone or in combination with all or part of the following: the hinge region, CH1, CH2 and CH3 domains. Also included are any combinations of variable region(s) and hinge region, CH1, CH2 and CH3 domains. The antibodies can be monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies that, for example, specifically bind an HLA-linked polypeptide or HLA-peptide complex. One skilled in the art will appreciate that a variety of immunoaffinity methods are suitable for enriching soluble proteins, such as soluble HLA-peptide complexes or membrane-bound HLA-associated polypeptides, for example, that have been proteolytically cleaved from the membrane. These include methods in which (1) one or more antibodies capable of specifically binding to a soluble protein are immobilized on a fixed or movable substrate (eg, plastic wells or resin, latex or paramagnetic beads), and (2) a solution containing soluble protein from the biological sample is passed over the antibody-coated substrate, allowing the soluble protein to bind to the antibodies. The antibody substrate and bound soluble protein are separated from the solution, and optionally the antibodies and soluble protein are dissociated, for example by changing the pH and/or ionic strength and/or ionic composition of the antibody solution. Alternatively, immunoprecipitation methods can be used in which the antibody and soluble protein are combined and can form macromolecular aggregates. Macromolecular aggregates can be isolated from solution by size exclusion methods or by centrifugation.

[152] The term "immunoaffinity purification (IP)" (or immunosorption purification or immunoprecipitation) is a process well known in the art and widely used to isolate the desired antigen from a sample. In general, the process involves contacting a sample containing the desired antigen with an affinity matrix containing an antibody to the antigen covalently attached to the solid phase. The antigen in the sample becomes bound to the affinity template via an immunochemical bond. The affinity matrix is then washed to remove any unbound components. The antigen is recovered from the affinity matrix by changing the chemical composition of the solution in contact with the affinity matrix. Immunoaffinity purification can be carried out on a column containing an affinity matrix, in which case the eluent is the solution. Alternatively, immunoaffinity purification is a batch process, in which case the affinity matrix is maintained as a suspension in solution. An important step in the process is the removal of the antigen from the matrix. This is usually achieved by increasing the ionic strength of the solution in contact with the affinity matrix, for example by adding an inorganic salt. Changing the pH can also be effective in dissociating the immunochemical bond between the antigen and the affinity matrix.

[153] By "agent" is meant any small chemical compound, antibody, nucleic acid molecule or polypeptide, or fragments thereof.

[154] By "replacement" or "change" is meant an increase or decrease. The change can be as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30% or 40%, 50%, 60%, or even 70%, 75%, 80% , 90% or 100%.

[155] By "biological sample" is meant any tissue, cell, fluid, or other material obtained from an organism. As used herein, the term "sample" includes a biological sample, such as any tissue, cell, fluid, or other material derived from an organism. By "specific binding" is meant a compound (eg, peptide) that recognizes and binds a molecule (eg, a polypeptide) but which does not substantially recognize and bind other molecules in a sample, eg, a biological sample.

[156] By "capture reagent" is meant a reagent that specifically binds a molecule (eg, nucleic acid molecule or polypeptide) to select or isolate the molecule (eg, nucleic acid molecule or polypeptide).

[157] For the purposes of the present invention, the terms "definition", "assessment", "analysis", "measurement", "detection" and their grammatical equivalents refer to both quantitative and qualitative definitions, and in this regard, the term "definition " is used in this document interchangeably with "analysis", "measurement" and the like. When quantification is meant, the phrase "quantitation" of the analyte and the like is used. Where qualitative and/or quantitative determination is implied, the phrase "level determination" of the analyte or "detection" of the analyte is used.

[158] By "fragment" is meant a portion of a protein or nucleic acid that is substantially identical to a reference protein or nucleic acid. In some embodiments, this portion retains at least 50%, 75%, or 80%, or 90%, 95%, or even 99% of the biological activity of the reference protein or nucleic acid described herein.

[159] The terms "isolated", "purified", "biologically pure" and their grammatical equivalents refer to material that is more or less free of the components that normally accompany it when it is in its natural state. "separate" means the degree of separation from the original source or environment. "Clear" means a degree of separation higher than extraction. A “purified” or “biologically pure” protein is sufficiently free of other substances such that any impurities do not significantly affect the biological properties of the protein or cause other adverse effects. That is, a nucleic acid or peptide of the present invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are usually determined using analytical chemistry methods such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may mean that the nucleic acid or protein gives essentially one band in the electrophoretic gel. For a protein that can be modified, such as phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be purified separately.

[160] By "isolated" polypeptide (e.g., a peptide from an HLA-peptide complex) or polypeptide complex (e.g., an HLA-peptide complex) is meant a polypeptide or polypeptide complex of the present invention that has been separated from the components that accompany it in its natural state. . Typically, a polypeptide or polypeptide complex is isolated when it is at least 60% by weight, free of proteins and naturally occurring organic molecules to which it is naturally associated. The drug may comprise at least 75%, at least 90%, or at least 99% by weight of the polypeptide or polypeptide complex of the present invention. An isolated polypeptide or polypeptide complex of the present invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide or one or more components of the polypeptide complex, or by chemical synthesis of the polypeptide or one or more components of the polypeptide complex. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[161] The term "vectors" refers to a nucleic acid molecule capable of transporting or mediating the expression of a heterologous nucleic acid. A plasmid is a species of the genus encompassed by the term "vector". A vector generally refers to a nucleic acid sequence containing an origin of replication and other fragments necessary for replication and/or maintenance in a host cell. Vectors capable of directing the expression of genes and/or nucleic acid sequences to which they are operatively linked are referred to herein as "expression vectors". Typically, the expression vectors used are often in the form of "plasmids" which refer to circular double-stranded DNA molecules which in their vector form are not associated with a chromosome and usually contain fragments for stable or transient expression, or encoded DNA. Other expression vectors that can be used in the methods described herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, or viral vectors, and such vectors can be integrated into the host genome or replicate autonomously in cell. The vector may be a DNA or RNA vector. Other forms of expression vectors known to those skilled in the art that perform equivalent functions can also be used, such as self-replicating extrachromosomal vectors or vectors capable of integrating into the host genome. Illustrative vectors are those capable of autonomous replication and/or expression of the nucleic acids to which they are associated.

[162] By "molecular profile" is meant a characteristic of the expression or level of expression of two or more markers (eg, polypeptides or polynucleotides).

[163] The terms "spacer" or "linker" as used in relation to a fusion protein refer to a peptide that is attached to proteins containing the fusion protein. Typically, a spacer has no specific biological activity other than to attach or maintain some minimum distance or other spatial relationship between proteins or RNA sequences. However, in some embodiments, the amino acid constituents of the spacer may be chosen to affect certain properties of the molecule, such as folding, net charge, or hydrophobicity of the molecule. Suitable linkers for use in an embodiment of the present invention are well known to those skilled in the art and include, but are not limited to, straight or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. The linker is used to separate the two antigenic peptides by a distance sufficient to ensure, in some embodiments, that each antigenic peptide folds correctly. Exemplary peptide linker sequences adopt a flexible extended conformation and do not tend to develop an ordered secondary structure. Representative amino acids in flexible protein regions include Gly, Asn and Ser. Almost any permutation of amino acid sequences containing Gly, Asn and Ser can be expected to satisfy the above criteria for a linker sequence. Other nearly neutral amino acids such as Thr and Ala can also be used in the linker sequence. Other amino acid sequences that can be used as linkers are described in Maratea et al. (1985), Gene 40: 39-46; Murphy et al. (1986) Proc. Nat'l. Acad. sci. USA 83: 8258-62; in US patent No. 4935233; and US Pat. No. 4,751,180.

[164] The term "neoplasm" refers to any disease that is caused by or results in disproportionately high levels of cell division, disproportionately low levels of apoptosis, or both. Glioblastoma is one non-limiting example of neoplasia or cancer. The terms "cancer" or "tumor" or "hyperproliferative disorder" refer to the presence of cells with characteristics typical of cancer cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and some characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone in the animal or may be a non-tumor cancer cell such as a leukemic cell. Cancer includes, but is not limited to, B cell cancers such as multiple myeloma, Waldenström macroglobulinemia, heavy chain diseases such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer (for example, metastatic, hormone-refractory prostate cancer), pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, cancer of the brain or central nervous system, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, hematological tissue cancer, and the like. Other non-limiting examples of cancers applicable to the methods covered by the present invention include human sarcomas and carcinomas, e.g. fibrosarcoma, malignant myxoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, tumor Ewing, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, fetal cancer, Wilms tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, cancer bladder, epithelial carcinoma noma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, for example, acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemic); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, cancer head and neck, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell cancer, cervical cancer, ovarian cancer (eg, serous ovarian cancer), or breast cancer. Epithelial cancer can be characterized in various other ways, including, but not limited to, serous, endometrioid, mucoid, clear cell, Brenner, or undifferentiated. In some embodiments, the present disclosure is used in the treatment, diagnosis, and/or prognosis of lymphoma or subtypes thereof, including, but not limited to, mantle cell lymphoma. Lymphoproliferative diseases are also considered proliferative diseases.

[165] the Term "vaccine" should be understood in the sense of a composition for creating immunity for the prevention and/or treatment of diseases (for example, neoplasms/tumors/infectious agents/autoimmune diseases). Accordingly, vaccines are drugs that contain antigens and are intended for use in humans or animals to create specific protective and protective substances by vaccination. A "vaccine composition" may contain a pharmaceutically acceptable excipient, carrier, or diluent. Aspects of the present disclosure relate to the use of technology in the preparation of an antigen-based vaccine. In these embodiments, the implementation of the vaccine is intended to refer to one or more disease-specific antigenic peptides (or corresponding nucleic acids encoding them). In some embodiments, the antigen vaccine contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30 or more antigenic peptides. In some embodiments, the antigen-based vaccine contains 2-100, 2-75, 2-50, 2-25, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2 -14, 2-13, 2-12, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 3-100, 3-75, 3-50 , 3-25, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-10, 3-9, 3 -8, 3-7, 3-6, 3-5, 4-100, 4-75, 4-50, 4-25, 4-20, 4-19, 4-18, 4-17, 4-16 , 4-15, 4-14, 4-13, 4-12, 4-10, 4-9, 4-8, 4-7, 4-6, 5-100, 5-75, 5-50, 5 -25, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-10, 5-9, 5-8 or 5-7 antigenic peptides. In some embodiments, the antigenic vaccine contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antigenic peptides. In some cases, the antigenic peptides are neoantigenic peptides. In some cases, antigenic peptides contain one or more neoepitopes.

[166] The term "pharmaceutically acceptable" refers to approved or warranted by a federal or state government regulatory agency, or listed in the USP or other generally accepted pharmacopoeia, for use in animals, including humans. A "pharmaceutically acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject with an agent and that does not interfere with its pharmacological activity and is non-toxic when administered in doses sufficient to deliver a therapeutic amount of the agent. A "pharmaceutically acceptable salt" of the combined disease-specific antigens that are listed herein may be an acidic or basic salt generally considered in the art to be suitable for use in contact with human or animal tissues without undue toxicity, irritation, allergic reaction. 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, hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzenesulfonic, ethanedisulfonic, 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-(CH2)n-COOH, where n is 0-4, and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those of ordinary skill in the art will appreciate from this disclosure and knowledge in the art that additional pharmaceutically acceptable salts for the combined disease-specific antigens provided herein include those listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company , Easton, PA, p. 1418 (1985). In general, a pharmaceutically acceptable acid or base salt may be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical means. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[167] Nucleic acid molecules used in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but will typically show substantial identity. Polynucleotides that have significant endogenous sequence identity are typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. By "hybridization" is meant a pair to form a double-stranded molecule between complementary polynucleotide sequences or portions thereof under different stringency conditions. (See, for example, Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). For example, a hard salt concentration may typically be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions typically may include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Various additional parameters such as hybridization time, detergent concentration such as sodium dodecyl sulfate (SDS) and inclusion or exclusion of carrier DNA are well known to those skilled in the art. Different levels of hardness are achieved by combining these different conditions as needed. In an exemplary embodiment, hybridization can occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another exemplary embodiment, hybridization can occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In another illustrative embodiment, hybridization can occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml sSDNA. Beneficial changes to these conditions will be apparent to those skilled in the art. For most applications, the washing steps following hybridization can also vary in severity. Rinse hardness conditions can be determined by salt concentration and temperature. As mentioned above, the washing hardness can be increased by decreasing the salt concentration or increasing the temperature. For example, the hard salt concentration in the washing steps may be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the washing steps may include a temperature of at least about 25°C, at least about 42°C, or at least about 68°C. In exemplary embodiments, the washing steps may occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In other exemplary embodiments, the washing steps may occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the washing steps may occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations to these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described in, for example, Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72: 3961, 1975); Ausubel et. al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[168] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity with a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or a nucleic acid sequence (e.g., any of nucleic acid sequences described herein). Such a sequence may be at least 60%, 80%, or 85%, 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid or nucleic acid level to the sequence used for comparison. Sequence identity is usually measured using sequence analysis software (e.g., sequence analysis software package provided by Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis., 53705, BLAST, BESTFIT, GAP, or PILEUP programs /PRETTYBOX). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and/or other modifications. Conservative substitutions usually include substitutions in the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program can be used with a probability score between e-3 and e-m° indicating a closely related sequence. By "reference" is meant the comparison standard.

[169] The term "subject" or "patient" refers to an animal that is the object of treatment, observation or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including but not limited to a human or non-human mammal such as a non-human primate, mouse, bovine, horse, dog, sheep, or feline.

[170] The terms "treat", "receiving treatment", "under treatment", "treatment", and the like are intended to mean the reduction, prevention, or amelioration of a disorder and/or associated symptoms (e.g., a neoplasm or tumor or an infectious agent or autoimmune disease). "Treatment" may refer to administering therapy to a subject after the onset or suspected onset of a disease (eg, cancer or infection from an infectious agent or an autoimmune disease). "Treatment" includes the terms "relief" which refers to reducing the incidence or recurrence or severity of any symptoms or other adverse effects associated with a disease and/or side effects associated with therapy. The term "treatment" also includes the concept of "management", which refers to reducing the severity of a disease or disorder in a patient, for example, prolonging the life or survival of a patient with a disease or delaying its recurrence, for example, prolonging the period of remission in a patient who suffers from the disease. It is understood that, although not excluded, treatment of a disorder or condition does not require complete elimination of the disorder, condition, or associated symptoms.

[171] The terms “preventing,” “preventing,” “preventing,” and their grammatical equivalents as used herein, mean preventing or delaying the onset of symptoms associated with a disease or condition in a subject who did not have such symptoms at the time of onset. administration of the agent or compound.

[172] The term "therapeutic effect" refers to some degree of amelioration of one or more symptoms of a disorder (eg, neoplasms, tumors, or infections from an infectious or autoimmune disease) or associated pathology. "A therapeutically effective amount" as used herein refers to an amount of an agent that is effective, when administered at one or more doses to a cell or subject, to prolong the survival of a patient with such a disorder, reduce one or more signs or symptoms of the disorder, prevent or delay, and the like, beyond what would be expected in the absence of such treatment. "Therapeutically effective amount" is intended to define the amount necessary to achieve a therapeutic effect. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the "therapeutically effective amount" (eg, ED50) of the desired pharmaceutical composition. For example, a physician or veterinarian may start doses of the compounds of the present invention used in a pharmaceutical composition at levels below those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Disease, condition, and disorder are used interchangeably here.

[173] In some embodiments, the nucleic acid sequence encoding the HLA allele further comprises a peptide tag, an affinity tag, an epitope tag, or an affinity acceptor tag that can be used for immunoaffinity purification of an HLA protein. Those skilled in the art will appreciate that the terms "peptide tag", "affinity tag", "epitope tag", or "affinity acceptor tag" are used interchangeably herein. The term "affinity acceptor tag" as used herein refers to an amino acid sequence that allows the labeled protein to be easily detected or purified, for example by affinity purification. The affinity acceptor tag is usually (but not necessarily) placed at or near the N- or C-terminus of the HLA allele. Various peptide labels are well known in the art. Non-limiting examples include a polyhistidine tag (eg 4-15 contiguous His residues, eg 8 contiguous His residues); polyhistidine-glycine label; an HA label (e.g., Field et al., Mol. Cell. Biol., 8: 2159, 1988); c-myc tag (e.g. Evans et al., Mol. Cell. Biol., 5: 3610, 1985); herpes simplex virus D glycoprotein (gD) tag (eg, Paborsky et al., Protein Engineering, 3: 547, 1990); a FLAG tag (eg, Hopp et al., BioTechnology, 6:1204, 1988; US Pat. Nos. 4,703,004 and 4,851,341); epitope tag KT3 (eg, Martine et al., Science, 255: 192, 1992); tubulin epitope tag (eg Skinner, Biol. Chem., 266:15173, 1991); a peptide tag for protein 10 of the T7 gene (e.g., Lutz-Freyemuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393, 1990); streptavidin tag (StrepTag™ or StrepTagII™); see, for example, Schmidt et al., J. Mol. Biol., 255(5): 753-766, 1996 or US Pat. No. 5,506,121; also commercially available from Sigma-Genosys); or a VSV-G epitope tag derived from a vesicular stomatitis viral glycoprotein; or a V5 tag derived from a small epitope (Pk) found on the P and V proteins of paramyxovirus simian virus 5 (SV5). In some embodiments, the acceptor affinity tag is an "epitope tag", which is a type of peptide tag that adds a recognizable epitope (antibody binding site) to an HLA protein to allow the corresponding antibody to bind, allowing identification or affinity purification of the tagged protein. A non-limiting example of an epitope tag is protein A or protein G that binds to IgG. In some embodiments, the fast flow IgG Sepharose 6 chromatography resin matrix is covalently linked to human IgG. This resin provides high throughput rates for fast and convenient purification of protein labeled with protein A. Many other labels known to those of ordinary skill in the art are provided herein for consideration. Any peptide tag may be used, provided that it is capable of being expressed as an element of the HLA affinity acceptor-labeled peptide complex.

[174] As used herein, the term "affinity molecule" refers to a molecule or ligand that binds chemically specific to an affinity acceptor peptide. Chemical specificity is the ability of a protein binding site to bind specific ligands. The fewer ligands a protein can bind, the greater its specificity. Specificity describes the strength of binding between a given protein and a ligand. This relationship can be described by the dissociation constant (K _D ), which characterizes the balance between bound and unbound states for the protein-ligand system.

[175] The term "HLA affinity-acceptor-labeled peptide complex" refers to a complex containing a peptide associated with HLA class I or class II, or a portion thereof, specifically associated with a single-allelic recombinant HLA class I or class II, wherein the peptide is affinity acceptor peptide.

[176] The terms "specific binding" or "specifically bound" when used in relation to the interaction of an affinity molecule and an affinity acceptor tag or an epitope and an HLA peptide means that the interaction depends on the presence of a specific structure (i.e., an antigenic determinant or epitope) on the protein; in other words, the affinity molecule recognizes and binds to the structure of a specific acceptor affinity peptide, and not to proteins in general.

[177] As used herein, the term “affinity” refers to the strength of binding between two elements of a binding pair, for example, an “affinity acceptor tag” and an “affinity molecule” and an HLA binding peptide and HLA class I or II. K _D is the dissociation constant and has units of molarity. The affinity constant is the reciprocal of the dissociation constant. The affinity constant is sometimes used as a general term to describe this chemical concept. This is a direct measure of bond energy. Affinity can be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available Biacore SPR elements. Affinity can also be expressed as an inhibitory concentration 50 (IC ₅₀ ), ie. The concentration at which 50% of the peptide is displaced. Similarly, ln(IC ₅₀ ) refers to the natural logarithm of IC ₅₀ . _Off refers to the dissociation rate constant, for example, for the dissociation of an affinity molecule from a complex affinity acceptor labeled HLA peptide.

[178] In some embodiments, the HLA acceptor affinity tagged peptide complex contains a biotin acceptor peptide (BAP) and is immunoaffinity purified from complex cell mixtures using streptavidin/NeutrAvidin beads. The binding of biotin-avidin/streptavidin is the strongest non-covalent interaction known in nature. This property is used as a biological tool for a wide range of applications such as immunoaffinity purification of a protein to which biotin is covalently attached. In an exemplary embodiment, a nucleic acid sequence encoding an HLA allele introduces a biotin acceptor peptide (BAP) as an affinity acceptor tag for immunoaffinity purification. BAP can be specifically biotinylated in vivo or in vitro at a single lysine residue in the tag (eg US Pat. Nos. 5,723,584; 5,874,239 and 5,932,433; US Pat. BAP is typically 15 amino acids long and contains one lysine as a biotin acceptor residue. In some embodiments, the BAP is located at or near the N- or C-terminus of a single-allelic HLA peptide. In some embodiments, the BAP is located between the heavy chain domain and the microglobulin β2 domain of an HLA class I peptide. In some embodiments, the BAP is located between the β chain domain and the α chain domain of an HLA class II peptide. In some embodiments, the BAP is placed in the loop region between the α1, α2 and α3 domains of the HLA class I heavy chain, or between the α1 and α2 and β1 and β2 domains of the α chain and β chain, respectively, of HLA class II. Exemplary constructs designed to express HLA class I and II using BAP for biotinylation and immunoprification are described in FIG. 2.

[179] As used herein, the term "biotin" refers to the biotin compound itself and its analogs, derivatives, and variants. Thus, the term "biotin" includes biotin (cis-hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic acid) and any of its derivatives and analogs, including biotin-like compounds. Such compounds include, for example, biotin-eN-lysine, biocytin hydrazide, amino or sulfhydryl derivatives of 2-iminobiotin and biotinyl-E-aminocaproic acid-N-hydroxysuccinimide ester, sulfosuccinimidiminobiotin, 3-(N-maleimidopropionyl)biocytin, dethiobiotin and the like. . The term "biotin" also includes biotin variants that can specifically bind to one or more fragments of rizavidin, avidin, streptavidin, tamavidin, or other avidin-like peptides.

HLA ligand profiling approaches

[180] A biochemical peptide-MHC binding assay to detect an HLA epitope was the basis for NetMHC, an allele-specific prediction tool using artificial neural networks; however, the slow biochemical analysis of p:MHC binding is a low throughput method (FIG. 19). Endogenously processed and presented HLA ligands profiled from cell lines and patient-derived materials are typically multi-allelic, meaning that LC-MS/MS data derived from these samples contain a mixed population of ligands that can bind to one of many co-expressed HLA alleles as shown in FIG. 17 and FIG. 19. Multi-allelic datasets require deconvolution to determine which peptides bind to the various HLA heterodimers presented by the individual. Thus, ligands from multi-allelic datasets must be assigned to their respective HLA heterodimers using (1) binding predictors trained with pre-existing data or (2) deconvolution algorithms that exploit the overlap of HLA alleles present in large ligand datasets. . It is important to note that only LC-MS/MS datasets with available HLA typing information can be deployed with confidence. In fact, nearly 40% of the naturally processed ligands associated with HLA class I complexes reported in multi-allelic studies in the immune epitope database (IEDB) do not have HLA allele-specific assignments, either due to a lack of HLA typing information. , or due to the inability of deconvolution, which makes it difficult to use this data subset for allele-specific epitope prediction. In addition, it is difficult to identify peptides associated with rare HLA class I heterodimers and many HLA class II heterodimers because there is insufficient annotated data for deconvolution. The multi-allelic data generation approach also limits the discovery of new binding motifs, as unfolding relies on pre-existing knowledge. While there are caveats regarding the use of multi-allelic datasets for allele-specific epitope predictions, they are extremely important for identifying ligand presentation patterns that require the co-expression of multiple alleles and for validating epitope prediction algorithms.

[181] An orthogonal approach to generating multi-allelic data and subsequent deconvolution is to create mono-allelic data sets from which peptide populations presented by a single HLA allele are identified (FIG. 17 and FIG. 19). One way to obtain monoallelic data is using cell lines that have deficient expression of HLA. These cells can be transfected or transduced with single HLA alleles, so ligand profiling can be done using LC-MS/MS to generate allele-specific ligand libraries. Also, to obtain monoallelic data from cellular media, soluble HLA (sHLA)-associated peptides can be isolated and profiled using LC-MS/MS. The main advantage of monoallelic datasets is that they do not require deconvolution and allow HLA alleles to be confidently assigned to a peptide without prior data. Monoallelic approaches also rapidly provide data for HLA alleles that have not previously been characterized, a task that multiallelic data can only accomplish if there is sufficient overlap in large datasets. In addition, novel peptide-binding motifs can be easily detected using monoallelic systems, since no prior knowledge is required to confidently assign HLA binding. Monoallelic data can even be used to assign ligands from multiallelic datasets when deconvolution methods do not allow this.

[182] A limiting factor to the currently available monoallelic approach is that it requires an HLA-deficient cell line. A key innovative feature of the present invention is that an HLA-deficient cell line is not required to generate monoallelic data. The affinity-tagged constructs provided herein can be placed in any cell line presenting endogenous HLA-peptide complexes to isolate the allele of interest using affinity tags. Another advantage of the present disclosure is that the same reagents can be used for any class I or class II allele in the library, as long as it has the same affinity tag, making the currently disclosed method scalable (automated). In some embodiments, the method comprises expressing a library of peptides in a population of cells, thereby generating a library of acceptor-labeled HLA-peptide acceptor complexes. In some embodiments, the method includes contacting a peptide library or a library of peptide coding sequences with a population of cells, thereby forming a library of HLA affinity acceptor-labeled peptide complexes. In some embodiments, the library includes a library of peptides associated with a disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cell population is derived from a biological sample from a subject with a disease or condition.

[183] In some embodiments, the method further comprises isolating peptides from complexes of affinity acceptor-labeled HLA peptides prior to characterization. In some embodiments, the peptides are isolated using anti-HLA antibodies. In some cases, affinity-tagged soluble HLA (sHLA) is isolated using anti-HLA antibodies. In some cases, affinity-tagged soluble HLA (sHLA) is isolated using a column containing an anti-HLA antibody.

Methods and Compositions

[184] Provided herein is a method for characterizing HLA-peptide complexes, comprising: providing a cell population, wherein one or more cells of the cell population contain a polynucleic acid containing a sequence encoding an allele of an affinity acceptor-tagged HLA class I or class II, wherein the sequence , encoding HLA labeled with an affinity acceptor, contains a sequence encoding an allele of recombinant HLA class I or class II, functionally linked to a sequence encoding an affinity acceptor peptide; expressing the affinity acceptor-labeled HLA in at least one cell of one or more cells of the cell population, thereby complexing the affinity acceptor-labeled HLA peptides in at least one cell; enrichment of complexes labeled with an affinity acceptor HLA-peptides; and characterization of HLA-peptide complexes.

[185] In some embodiments, characterization includes characterization of a peptide from a complex of an HLA affinity acceptor-labeled peptide. In some embodiments, the implementation of the method includes performing the steps of the method for different alleles of HLA class I and/or class II. In some embodiments, the implementation of the method includes the use of more than one allele of HLA class I and/or class II. In some embodiments, the cell population is obtained from a subject (eg, a patient with a disease). In some embodiments, the cell population is class I and/or class II negative cell lines. In some embodiments, the method further comprises creating a database of HLA allele-specific peptides.

[186] Provided herein is a method for generating a database of HLA allele-specific peptides, comprising: providing a first and a second population of cells each containing one or more cells containing an affinity-acceptor-tagged HLA, wherein the affinity-acceptor-tagged HLA sequence contains another recombinant a polypeptide encoded by another HLA allele operably linked to an affinity acceptor peptide; enrichment of complexes labeled with an affinity acceptor HLA-peptides; obtaining characteristics of the peptide or part thereof associated with the complex labeled with an affinity acceptor HLA peptide as a result of enrichment; and creating a database of HLA allele-specific peptides.

[187] In some embodiments, enrichment does not include the use of a tetrameric reagent.

[188] In some embodiments, characterization includes determining the sequence of the peptide, or portion thereof, associated with the enriched HLA affinity acceptor-labeled peptide complex. In some embodiments, characterization includes determining whether a peptide or portion thereof has been modified (eg, post-translational modification). In some embodiments, the determination includes biochemical analysis. In some embodiments, the determination includes mass spectrometric analysis. In some embodiments, the mass spectrometry is MS analysis, MS/MS analysis, LC-MS/MS analysis, or a combination thereof. In some embodiments, MS analysis is used to determine the mass of the intact peptide. For example, the determination may include determining the mass of the intact peptide (eg, MS analysis). In some embodiments, MS/MS analysis is used to determine the mass of peptide fragments. For example, the determination may include determining the mass of peptide fragments that can be used to determine the amino acid sequence of a peptide or portion thereof (eg, MS/MS analysis). In some embodiments, a mass of peptide fragments is used to determine the amino acid sequence of the peptide. In some embodiments, LC-MS/MS analysis is used to separate mixtures of peptide complexes. For example, the determination may include separating mixtures of peptide complexes, eg, by liquid chromatography, and determining intact peptide mass, peptide fragment mass, or a combination thereof (eg, LC-MS/MS analysis). This data can be used, for example, for peptide sequencing.

[189] In some embodiments, characterization includes evaluating the binding affinity or stability of a peptide, or portion thereof, associated with an enriched HLA affinity acceptor-labeled peptide complex. In some embodiments, characterization includes determining whether a peptide, or portion thereof, associated with an enriched HLA affinity acceptor-tagged peptide contains one or more mutations. In some embodiments, characterization includes determining whether a peptide or portion thereof has been modified (eg, post-translational modification). In some embodiments, characterization includes evaluating peptide associations of complexes of HLA acceptor affinity-labeled peptides with HLA alleles.

[190] In some embodiments, the implementation of the method includes the expression of the library of peptides in a population of cells, thereby forming a library of complexes labeled with an affinity acceptor HLA peptides. In some embodiments, the method includes contacting the cell population with a peptide library or a library of peptide coding sequences, thereby forming a library of HLA affinity acceptor tagged peptide complexes. In some embodiments, the library is a library of peptides associated with a disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cell population is derived from a biological sample in a subject with a disease or condition.

[191] In some embodiments, the cell population is a cell line. In some embodiments, the cell population is a primary cell population.

[192] In some embodiments, the recombinant HLA class I or class II allele corresponds to a subject with a disease or condition. In some embodiments, an antigen presenting cell comprising a peptide or mutant thereof bound to an HLA affinity acceptor-tagged peptide complex is reactive to a T cell expressing a T cell receptor in a subject. In some embodiments, characterization includes comparing HLA-peptide complexes from cancer cells with HLA-peptide complexes from non-cancerous cells.

[193] In some embodiments, a population of cells has a knockout of one or more HLA class I alleles. In some embodiments, the cell population has a knockout of one or more HLA class II alleles. In some embodiments, a population of cells has a knockout of all HLA class I alleles. In some embodiments, a population of cells has a knockout of all HLA class II alleles. In some embodiments, the cell population has a knockout of all HLA class I alleles and a knockout of all HLA class II alleles. In some embodiments, knocking out an HLA class I or class II allele includes eliminating the function of the class I or class II HLA allele. In some embodiments, knockout of an HLA class I or class II allele is achieved through gene editing. In some embodiments, gene editing is performed by introducing a nuclease to an individual in need, wherein the nuclease isolates the class I or class II allele to be knocked out. In some embodiments, the nuclease is a CRISPR-associated protein (eg, Cas proteins, eg, Cas9), zinc finger nuclease (ZFN), transcriptional activator-like effector nuclease (TALEN), or meganuclease. In some embodiments, gene editing is achieved by introducing the CRISPR-Cas9 system to an individual in need thereof. In some embodiments, any suitable nuclease is used that causes a break or double-strand break at the desired recognition site. In some embodiments, a natural or native nuclease is used. In some embodiments, a modified or engineered nuclease is used.

[194] In some embodiments, one or more HLA class I alleles are knocked down in a population of cells. In some embodiments, one or more HLA class II alleles are knocked down in a population of cells. In some embodiments, all HLA class I alleles are knocked down in a population of cells. In some embodiments, all HLA class II alleles are knocked down in a population of cells. In some embodiments, all HLA class I alleles are knocked down in a cell population and all HLA class II alleles are knocked out. In some embodiments, knocking down an HLA class I or class II allele includes reducing the expression of an HLA class I or class II allele. In some embodiments, knockdown of an HLA class I allele or a class II allele is achieved by administering to an individual in need thereof a therapeutically effective amount of a small double-stranded interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), wherein the siRNA, miRNA, shRNA are isolated HLA class I allele or class II allele for knockdown. In some embodiments, expression of an HLA class I or class II allele is reduced by about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, or about 20% compared to no HLA class I or class II allele knockdown.

[195] In some embodiments, the cell population includes cells that have been enriched or sorted for cell surface expression of an HLA class I allele, an HLA class II allele, or a combination thereof, e.g., by fluorescent activated cell sorting (FACS). In some embodiments, fluorescent activated cell sorting (FACS) is used to sort a population of cells. In some embodiments, fluorescent activated cell sorting (FACS) is used to sort a population of cells for cell surface expression of an HLA class I allele, an HLA class II allele, or a combination thereof. In some embodiments, FACS is used to enrich or sort cells with low HLA class I or class II cell surface expression.

[196] In some embodiments, the implementation of the population of cells includes multiple populations of cells, each of which expresses a different allele of recombinant HLA class I or class II. In some embodiments, each cell population of the plurality is in a separate container.

[197] In some embodiments, the method further comprises isolating peptides from complexes of affinity acceptor-labeled HLA peptides prior to characterization. In some embodiments, the method further comprises truncating the end of the peptide associated with the HLA-peptide complexes (FIG. 13 ).

[198] In some embodiments, the cell population expresses one or more endogenous HLA alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles and endogenous HLA class II alleles. In some embodiments, a sequence encoding an allele of a recombinant HLA class I or class II encodes a class I HLA. In some embodiments, the sequence encoding the allele of the recombinant HLA class I or class II encodes HLA class II. In some embodiments, the Class I HLA is selected from the group consisting of HLA-A, HLA-B, HLA-C. In some embodiments, the class I HLA is in the class-I-b non-classic group. In some embodiments, the Class I HLA is selected from the group consisting of HLA-E, HLA-F, and HLA-G. In some embodiments, the class I HLA refers to a class-I-b non-classical group selected from the group consisting of HLA-E, HLA-F, and HLA-G. In some embodiments, the HLA class II comprises an HLA class II α chain, an HLA class II β chain, or a combination thereof.

[199] In some embodiments, each sequence encoding a different HLA class I and/or class II allele is operably linked to a sequence encoding a different acceptor affinity peptide. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to a sequence encoding a recombinant HLA Class I or Class II allele that encodes the extracellular portion of the recombinant HLA Class I or Class II allele. In some embodiments, the encoded acceptor affinity peptide is expressed extracellularly. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to the N-terminus of a sequence encoding a recombinant HLA Class I or Class II allele. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to a sequence encoding a recombinant HLA Class I or Class II allele that encodes the intracellular portion of the recombinant HLA Class I or Class II allele. In some embodiments, the encoded acceptor affinity peptide is expressed intracellularly. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to the C-terminus of a sequence encoding a recombinant HLA Class I or Class II allele.

[200] In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to a sequence encoding a recombinant HLA Class I or Class II allele via a linker.

[201] In some embodiments, enrichment includes enrichment of intact cells expressing complexes labeled with an affinity acceptor HLA peptides.

[202] In some embodiments, the method does not include lysing one or more cells prior to enrichment. In some embodiments, the method further comprises lysing one or more cells prior to enrichment.

[203] In some embodiments, enrichment comprises contacting an acceptor affinity peptide binding molecule with complexes of affinity acceptor-labeled HLA peptides, wherein the acceptor affinity peptide binding molecule specifically binds to the acceptor affinity peptide. In some embodiments, the affinity acceptor peptide may include a biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine-glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, glycoprotein D (gD) tag herpes simplex virus, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 peptide tag, streptavidin tag, streptavidin binding peptide (SPB) tag, Strep tag, Strep tag II, albumin binding protein (ABP) tag ), alkaline phosphatase (AP) tag, bluetongue (B-tag), calmodulin-binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline-binding domain (CBD) tag, chitin-binding-domain tag (CBD), cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose binding protein (GBP) tag, maltose binding protein (MBP), glutathione-S-transferase (GST), Glu-G tag lu (EE), human influenza virus (HA) tag, horseradish peroxidase (HA) tag, NE tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, domain tag PDZ, AviTag, calmodulin tag, E tag, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, Xpress tag, Isopepeptag, SpyTag, SnoopTag, Profinity eXact tag, Protein C tag, S1 α-tag, S-tag, carboxybiotin carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus tag, Thioredoxin tag , Fc tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or a combination thereof; optionally, the affinity acceptor peptide contains two or more repeats of the tag sequence. In some embodiments, the affinity acceptor peptide binding molecule is biotin or an antibody specific for the acceptor affinity peptide.

[204] In some embodiments, enrichment includes contacting an affinity molecule with complexes of affinity acceptor-labeled HLA peptides, wherein the affinity molecule specifically binds to the affinity acceptor peptide binding molecule. In some embodiments, the affinity molecule is streptavidin, NeutrAvidin, or a derivative thereof. In some embodiments, enrichment includes immunoprecipitation of complexes labeled with an affinity acceptor HLA peptides. In some embodiments, an acceptor affinity peptide binding molecule is attached to a solid surface. In some embodiments, the affinity molecule is attached to a solid surface. In some embodiments, the hard surface is a bead.

[205] In some embodiments, enrichment comprises immunoprecipitating complexes of the affinity acceptor-labeled HLA peptides with an acceptor affinity peptide binding molecule that specifically binds to the acceptor affinity peptide. In some embodiments, the acceptor affinity peptide binding molecule does not specifically interact with the amino acid sequence of the encoded recombinant HLA class I or class II. In some embodiments, enrichment includes contacting an affinity molecule specific for the extracellular portion of the HLA-peptide complexes. In some embodiments, enrichment includes contacting an affinity molecule specific for the N-terminal portion of the HLA-peptide complexes.

[206] In some embodiments, providing includes contacting a population of cells with a polynucleic acid containing a sequence encoding an HLA affinity-tagged acceptor. In some embodiments, the introduction into contact includes transfection or transduction. In some embodiments, providing includes contacting a population of cells with a vector or plasmid containing a polynucleic acid containing a sequence encoding an HLA affinity-tagged acceptor. In some embodiments, the vector is a viral vector.

[207] Any suitable biochemical assay can be used to determine HLA expressed in a cell (eg, engineered cell line). Exemplary methods for determining the identity of an HLA allele expressed in a cell (e.g., engineered cell line) include Western blotting, e.g. to determine the class of the HLA allele (Class I or Class II), sequence analysis, e.g., sequencing of individual alleles (e.g. , using different primers to identify different alleles of a similar sequence). In some embodiments, the implementation of the polynucleic acid encoding the HLA allele contains a barcoded sequence. the barcoded sequence can be used to identify the HLA allele expressed in the cell. In some embodiments, the barcoded sequence is unique to a single HLA. In some embodiments, the barcoded sequence is unique for a single HLA class I or class II allele.

[208] In some embodiments, a polynucleic acid containing a sequence encoding an affinity-acceptor-tagged HLA is stably inserted into the genome of a cell population. In some embodiments, a sequence encoding a recombinant HLA class I or class II contains a sequence encoding an HLA class I α chain. In some embodiments, the implementation of the method further includes the expression of a sequence encoding β2 microglobulin in one or more cells. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding an HLA class I α chain. In some embodiments, the β2 microglobulin coding sequence is linked to the HLA class I α chain coding sequence via a linker. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding a second acceptor affinity peptide.

[209] In some embodiments, the implementation of the sequence encoding recombinant HLA class I or class II, contains a sequence encoding the α-chain of HLA class II. In some embodiments, the method further comprises expressing an HLA class II β chain coding sequence in one or more cells. In some embodiments, a sequence encoding an HLA class II β chain is linked to a sequence encoding an HLA class II α chain. In some embodiments, a sequence encoding an HLA class II β chain is linked to a sequence encoding an HLA class II α chain via a linker. In some embodiments, a sequence encoding an HLA class II β chain is linked to a sequence encoding a second acceptor affinity peptide.

[210] In some embodiments, the second acceptor affinity peptide is different from the first acceptor affinity peptide and may include a biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine-glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine , c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep II tag, albumin binding protein (ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus (B-tag), calmodulin binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline tag binding domain (CBD), chitin-binding domain (CBD) tag, cellulose-binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein (GBP) tag, maltose linging protein (MBP), glutathione-S-transferase (GST), Glu-Glu tag (EE), human influenza virus hemagglutinin tag (HA), horseradish peroxidase (HA) tag, NE tag, HSV tag, ketosteroid isomerase tag (KSI), KT3 tag, LacZ tag, Luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin tag, E tag, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, Xpress tag, Isopepeptag, SpyTag, SnoopTag, Profinity eXact tag, protein C tag, S1 tag, S tag, carboxybiotin carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag ), tandem affinity purification (TAP) tag, HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or a combination thereof.

[211] In some embodiments, the linker contains a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome escape site or internal ribosome entry site (IRES). In some embodiments, the ribosome escape site or IRES is cleaved when expressed in cells. In some embodiments, the ribosome exit site is selected from the group consisting of F2A, T2A, P2A, and E2A. In some embodiments, the IRES element is selected from common cellular or viral IRES sequences.

[212] In some embodiments, the determination includes performing mass spectrometry, such as tandem mass spectrometry. In some embodiments, the determination includes obtaining a peptide sequence that corresponds to MS/MS spectra of one or more peptides isolated from enriched complexes of HLA affinity acceptor-labeled peptides from a peptide database; wherein one or more of the resulting sequences identifies the sequence of one or more peptides.

[213] In some embodiments, the cell population is a cell line selected from HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, Hep G2, SH-SY5Y, CACO-2, U937, U-2 OS, ExpiCHO, CHO and THP1. In some embodiments, the cell line is treated with one or more cytokines, checkpoint inhibitors, epigenetically active drugs, IFN-γ, an agent that alters antigen processing (e.g., peptidase inhibitors, proteasome inhibitor, TAP inhibitor, etc.), or their combination. In some embodiments, the peptide database is a peptide database with no enzyme specificity, such as a database with no modification or a database with a modification (eg, phosphorylation or cysteinylation). In some embodiments, the peptide database is a polypeptide database. In some embodiments, the polypeptide database is a protein database. In some embodiments, the method further comprises searching the peptide database using a flipped database search strategy. In some embodiments, the method further comprises searching the protein database using a flipped database search strategy. In some embodiments, a de novo search is performed, for example, to discover new peptides that are not contained in the database of normal peptides or proteins.

[214] In some embodiments, the cell population includes at least 10 ⁵ cells, at least 10 ⁶ cells, or at least 10 ⁷ cells. In some embodiments, the cell population is a population of dendritic cells, macrophages, cancer cells, or B cells. In some embodiments, the cell population includes tumor cells or cells infected with an infectious agent or part thereof.

[215] In some embodiments, a population of cells is contacted with an agent prior to isolation of said HLA-peptide complexes from one or more cells. In some embodiments, said agent is an inflammatory cytokine, a chemical agent, an adjuvant, a therapeutic agent, or radiation.

[216] In some embodiments, the HLA allele is a mutant HLA allele.

[217] In some embodiments, the implementation of the method includes performing the steps of the method for different HLA alleles.

[218] This document provides a database of sequences specifically binding alleles of HLA peptides obtained by performing the methods described herein. Provided herein is a combination of two or more HLA allele-specific binding peptide sequence databases obtained by repeatedly performing the methods described herein, each time using a different HLA allele. Provided herein is a method for generating a prediction algorithm for identifying HLA-specific allele-binding peptides, comprising machine learning with the peptide sequence database described herein. In some embodiments, the machine combines one or more linear models, support vector machines, decision trees, and neural networks.

[219] Generating a prediction algorithm by machine learning is a well-known technique. The most important thing when training a machine is the quality of the database used for training. Typically, the machine integrates one or more linear models, support vector machines, decision trees, and/or neural networks.

[220] In some embodiments, the variable used to train the machine or algorithm includes one or more variables selected from the group consisting of peptide sequence, physical properties of amino acids, physical properties of peptides, level of expression of the original protein of the peptide in the cell, stability protein, protein translation rate, ubiquitination sites, rate of protein degradation, translation efficiency from ribosomal profiling, protein cleavage, protein localization, host protein motifs that facilitate TAP transport, host protein undergoes autophagy, motifs that promote ribosome arrest, (e.g. , polyproline or polylysine sites), protein features that promote NMD, (eg, long 3' UTR, >50 nt stop codon before last exon:exon junction, and cleavage of the peptide).

[221] Provided herein is a method for identifying HLA allele-specific binding peptides, comprising performing a peptide sequence analysis with a machine that has been trained with a peptide sequence database obtained by performing the method described herein for HLA alleles. In some embodiments, the implementation of the method includes determining the level of expression of the original protein of the peptide in the cell; and while the expression of the original protein is the prognostic variable used by the machine. In some embodiments, the level of expression is determined by measuring the amount of the parent protein or the amount of RNA encoding said parent protein.

[222] Provided herein is a composition comprising a first and a second recombinant polynucleic acid, each containing a sequence encoding an affinity acceptor-tagged HLA, wherein the sequence encoding an affinity-acceptor-tagged HLA contains (a) a sequence encoding a different recombinant α allele class I HLA chains, (b) a sequence encoding an affinity acceptor peptide, and optionally, (c) a sequence encoding β2 microglobulin; wherein sequences (a) and (b), and optionally (c), are operably linked.

[223] Provided herein is a composition comprising a first and a second recombinant polynucleic acid, each containing a sequence encoding an affinity-acceptor-tagged HLA, wherein the sequence encoding an affinity-acceptor-tagged HLA contains (a) a sequence encoding a recombinant α- chains of HLA class II, (b) a sequence encoding an affinity acceptor peptide, and optionally, (c) a sequence encoding a β-chain of HLA class II; wherein sequences (a) and (b), and optionally (c), are operably linked.

[224] In some embodiments, the first and second recombinant polynucleic acids are isolated.

[225] In some embodiments, the sequence encodes a recombinant HLA class I or class II allele. In some embodiments, the Class I HLA is selected from the group consisting of HLA-A, HLA-B, HLA-C. In some embodiments, the class I HLA is in the class-I-b non-classic group. In some embodiments, the Class I HLA is selected from the group consisting of HLA-E, HLA-F, and HLA-G. In some embodiments, Class I HLA refers to a class-I-b non-classic group selected from the group consisting of HLA-E, HLA-F, and HLA-G.

[226] In some embodiments, for both the first and second recombinant polynucleic acids: the sequence encoding the acceptor affinity peptide is operably linked to a sequence from a sequence encoding a different recombinant HLA allele that codes for the extracellular portion of the other recombinant HLA allele. In some embodiments, for both the first and second recombinant polynucleic acids: the sequence encoding the affinity acceptor molecule is operably linked to the N-terminus of the sequence encoding a different recombinant HLA allele. In some embodiments, for both the first and second recombinant polynucleic acids: a sequence encoding an affinity acceptor peptide is operably linked to a sequence from a sequence encoding a different recombinant HLA allele that encodes an intracellular portion of a different recombinant HLA allele. In some embodiments, for both the first and second recombinant polynucleic acids: the sequence encoding the acceptor affinity peptide is operably linked to the C-terminus of the sequence encoding a different recombinant HLA allele. In some embodiments, for both the first and second recombinant polynucleic acids: a sequence encoding an affinity acceptor peptide is operably linked to a sequence encoding a different recombinant HLA allele via a linker. In some embodiments, the encoded acceptor affinity peptide specifically binds to an acceptor affinity peptide binding molecule. In some embodiments, the affinity acceptor peptide of the first and second recombinant polynucleic acids is different.

[227] In some embodiments, the encoded acceptor affinity peptide may include a biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine-glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, glycoprotein D (gD) herpes simplex virus tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep II tag, albumin-tag binding protein (ABP), alkaline phosphatase (AP) tag, bluetongue (B-tag), calmodulin-binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline-binding domain (CBD) tag, chitin-binding domain (CBD), cellulose-binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein (GBP) tag, maltose-binding protein (MBP), glutathione-S-transferase ( GST), Glu-Glu tag (EE), human influenza virus hemagglutinin tag (HA), horseradish peroxidase (HA) tag, NE tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag , NusA tag, PDZ domain tag, AviTag, calmodulin tag, E tag, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity eXact tag , protein C tag, S1 tag, S tag, carboxybiotin carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or a combination thereof; optionally, the affinity acceptor peptide contains two or more repeats of the tag sequence. In some embodiments, the affinity acceptor peptide binding molecule is biotin or an antibody specific for the acceptor affinity peptide. In some embodiments, the affinity acceptor peptide binding molecule specifically binds to the affinity molecule. In some embodiments, the affinity molecule is streptavidin, NeutrAvidin, or a derivative thereof. In some embodiments, the acceptor affinity peptide binding molecule does not specifically interact with the amino acid sequence of the encoded recombinant HLA class I or class II. In some embodiments, for both the first and second recombinant polynucleic acids: the sequence encoding the affinity acceptor-tagged HLA is stably inserted into the cell's genome. In some embodiments, a sequence encoding a .beta.2 microglobulin or a sequence encoding the .beta. chain of an HLA class II is linked to a sequence encoding a second acceptor affinity peptide.

[228] In some embodiments, the second acceptor affinity peptide contains an HA tag. In some embodiments, the second acceptor affinity peptide may include a biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine-glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, glycoprotein D (gD) herpes simplex virus tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 peptide tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep tag II, albumin-binding protein tag ( ABP), alkaline phosphatase (AP) tag, bluetongue (B-tag), calmodulin-binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline-binding domain (CBD) tag, chitin-binding tag domain (CBD), cellulose-binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein (GBP) tag, maltose-binding protein (MBP), glutathione-S-transferase (GST), tag y Glu-Glu (EE), human influenza virus hemagglutinin (HA) tag, horseradish peroxidase (HA) tag, NE tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, Luciferase tag, NusA tag , PDZ domain tag, AviTag, calmodulin tag, E tag, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, Xpress tag, Isopepeptag, SpyTag, SnoopTag, Profinity eXact tag, protein tag C, S1 tag, S tag, carboxybiotin carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus tag , Thioredoxin tag, Fc tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or a combination thereof; optionally, the second acceptor affinity peptide contains two or more repeats of the tag sequence.

[229] In some embodiments, for both the first and second recombinant polynucleic acids: a sequence encoding a β2 microglobulin or a sequence encoding an HLA class II β chain is linked to a sequence encoding a different recombinant HLA and an affinity acceptor peptide via linker. In some embodiments, the linker contains a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome escape site or internal ribosome entry site (IRES). In some embodiments, the ribosome escape site or IRES is cleaved when expressed in cells. In some embodiments, the ribosome exit site is selected from the group consisting of F2A, T2A, P2A, and E2A. In some embodiments, the IRES element is selected from common cellular or viral IRES sequences.

[230] Provided herein is a composition comprising first and second isolated polypeptide molecules encoded by the first and second polynucleic acids, respectively, of the composition described herein. This document provides a composition containing the first and second cells containing the first and second polypeptide molecules encoded by the first and second polynucleic acids, respectively, of the composition described herein. This document provides a composition containing the first and second cells containing the first and second polynucleic acids, respectively, of the composition described herein. This document provides a composition containing the first and second population of cells containing one or more cells containing the first and second polynucleic acids, respectively, the composition described herein.

[231] In some embodiments, the implementation of the first and second populations of cells express one or more alleles of endogenous HLA class I or class II. In some embodiments, a first and second population of cells are constructed that lack one or more endogenous HLA class I alleles. In some embodiments, a first and second population of cells are constructed that lack endogenous HLA class I alleles. In some embodiments, a first and second population of cells are constructed that lack one or more endogenous HLA class II alleles. In some embodiments, the first and second populations of cells are constructed that lack endogenous HLA class II alleles. In some embodiments, the first and second populations of cells are constructed that lack endogenous HLA class I alleles and endogenous HLA class II alleles.

[232] This document provides a method for obtaining a cell, including transduction or transfection of the first and second cells with the first and second polynucleic acids, respectively, of the composition described herein.

[233] Provided herein is a peptide identified according to the method described herein.

[234] Provided herein is a method for enriching immunogenic peptides, comprising: providing a cell population comprising one or more cells expressing an affinity acceptor-tagged HLA, wherein the affinity-acceptor-tagged HLA contains an affinity acceptor peptide operably linked to a recombinant HLA encoded by an allele of the recombinant HLA; and enrichment of HLA-peptide complexes containing labeled HLA affinity acceptor. In some embodiments, the method further comprises determining the sequence of immunogenic peptides isolated from the HLA-peptide complexes. In some embodiments, the implementation of the definition includes the use of LC-MS/MS.

Human leukocyte antigen (HLA) system

[235] The immune system can be divided into two functional subsystems: the innate and adaptive immune systems. The innate immune system is the first line of defense against infections, and most potential pathogens are quickly neutralized by this system before they can cause a noticeable infection, for example. The adaptive immune system responds to molecular structures of the invading organism called antigens. Unlike the innate immune system, the adaptive immune system is highly pathogen specific. Adaptive immunity may also provide long-term protection; for example, someone who recovers from measles is now protected from measles for life. There are two types of adaptive immune responses which include humoral immune response and cell mediated immune response. In a humoral immune response, antibodies released by B cells into body fluids bind to antigens derived from pathogens, resulting in elimination of the pathogen by various mechanisms, such as complement-mediated lysis. In a cell-mediated immune response, T cells are activated that can destroy other cells. For example, if disease-associated proteins are present in a cell, they are proteolytically fragmented into peptides within the cell. Then, specific cellular proteins themselves attach to the antigen or peptide thus formed and transport them to the cell surface, where they are presented to the molecular defense mechanisms in the body's T cells. Cytotoxic T cells recognize these antigens and kill cells that contain the antigens.

[236] The term "major histocompatibility complex (MHC)", "MHC molecules" or "MHC proteins" refers to proteins capable of binding peptides resulting from proteolytic cleavage of protein antigens and presenting potential T-cell epitopes, transporting them to the surface cells and presenting them there to specific cells, for example, in cytotoxic T-lymphocytes or T-helper cells. The human MHC is also referred to as the HLA complex. Thus, the term "human leukocyte antigen (HLA) system", "HLA molecules", or "HLA proteins" refers to the gene complex encoding MHC proteins in humans. The term MHC is referred to as the "H-2" complex in mice. It should be understood by those of ordinary skill in the art that the terms "major histocompatibility complex (MHC)", "MHC molecules", "MHC proteins" and "human leukocyte antigen (HLA) system", "HLA molecules", "HLA proteins" are used used interchangeably in this document.

[237] HLA proteins are divided into two types, called HLA class I and HLA class II. The structures of the HLA proteins of the two classes are very similar; however, they have very different functions. HLA class I proteins are present on the surface of almost all body cells, including most tumor cells. HLA class I proteins load antigens, which are usually derived from endogenous proteins or from pathogens present inside cells, and then presented to naive or cytotoxic T lymphocytes (CTLs). HLA class II proteins are presented on antigen presenting cells (APCs), including but not limited to dendritic cells, B cells, and macrophages. Basically, they present peptides that process from external sources of antigens, that is, outside the cells, to T-helpers. Most of the peptides bound by HLA class I proteins are derived from cytoplasmic proteins produced in healthy host cells of the body itself and do not normally stimulate an immune response.

[238] Class I HLA molecules are composed of a heavy chain and a light chain and are capable of binding a peptide of approximately 7-13 amino acids (e.g., approximately 8-11 amino acids or 9 or 10 amino acids) if the peptide has suitable binding motifs, and present it cytotoxic T-lymphocytes. Peptides bound by class I HLA molecules originate from an endogenous protein antigen. The heavy chain of an HLA class I molecule can be an HLA-A, HLA-B or HLA-C monomer and the light chain is β-2-microglobulin. HLA class I occurs as an α-chain consisting of three domains - α1, α2 and α3. This chain is often referred to as the class I heavy chain and is referred to herein as the class I alpha chain. α1 is based on the non-HLA microglobulin β2 molecule unit (encoded on human chromosome 15). The α3 domain is transmembrane, anchoring the HLA class I molecule to the cell membrane. The presented peptide is held by the bottom of the peptide-binding groove in the central region of the α1/α2 heterodimer (a molecule consisting of two non-identical subunits). HLA-A, HLA-B or HLA-C class I are highly polymorphic. HLA class Ib exhibits limited polymorphism, expression profiles, and presented antigens. This group is subdivided into the group encoded at the HLA loci, eg HLA-E, HLA-F, HLA-G, as well as non-stress ligand groups such as ULBPs, Rae1 and H60. The antigen/ligand for many of these molecules remains unknown, but they can interact with each of CD8+ T cells, NKT cells, and NK cells.

[239] In some embodiments of the present disclosure, a non-classical allele of HLA-E class I is used. HLA-E is one of the non-classical class I molecules recognized by natural killer (NK) cells and CD8+ T cells. HLA-E is expressed in almost all tissues, including lung, liver, skin, and placental cells. HLA-E expression is also found in solid tumors (eg, osteosarcoma and melanoma). HLA-E binds to the TCR expressed on CD8+ T cells resulting in T cell activation. HLA-E is also known to bind the CD94/NKG2 receptor expressed on NK cells and CD8+ T cells. CD94 can bind to several different isoforms of NKG2 to form receptors with the potential to either inhibit (NKG2A, NKG2B) or promote (NKG2C) cellular activation. HLA-E can bind to a peptide derived from amino acid residues 3-11 of the leader sequences of most HLA-A, -B, -C and -G molecules, but cannot bind its own leader peptide. HLA-E has also been shown to present peptides derived from endogenous proteins similar to the HLA-A, -B and -C alleles. Under physiological conditions, interaction of CD94/NKG2A with HLA-E loaded with peptides from HLA class I leader sequences typically induces inhibitory signals. Cytomegalovirus (CMV) uses a mechanism to evade NK cell immune surveillance through the expression of the UL40 glycoprotein, which mimics the HLA-A leader sequence. However, CD8+ T cells are also reported to be able to recognize HLA-E loaded with the UL40 peptide derived from CMV strain Toledo and participate in CMV protection. A number of studies have identified several important functions of HLA-E in infectious diseases and cancer.

[240] Peptide antigens themselves attach to HLA class I molecules by competitive affinity binding within the endoplasmic reticulum, prior to their presentation on the cell surface. In this case, the affinity of an individual peptide antigen is directly related to its amino acid sequence and the presence of specific binding motifs at certain positions within the amino acid sequence. If the sequence of such a peptide is known, then the immune system can be manipulated against the affected cells using, for example, peptide vaccines.

[241] HLA class II molecules have two chains, α and β, each with two domains - α1 and α2 and β1 and β2 - each chain has a transmembrane domain, α2 and β2, respectively, anchoring the HLA class II molecule to the cell membrane . The peptide-binding groove is formed from an α1 and β1 heterodimer. The peptide associated with HLA class II molecules usually originates from the extracellular region of the exogenous protein antigen. The α chain and β chain are found in the HLA-DR, HLA-DQ and HLA-DP monomers (FIG. 1B). HLA class II molecules have six isotypes. Classical molecules present peptides to CD4+ lymphocytes. Non-classical molecules, additional, with intracellular functions, are located not on cell membranes, but on internal membranes in lysosomes, usually loading antigenic peptides onto classical HLA class II molecules.

[242] In HLA class II, phagocytes such as macrophages and immature dendritic cells take up objects by phagocytosis into phagosomes—although B cells exhibit more general endocytosis into endosomes—which fuse with lysosomes, whose acidic enzymes break down the ingested protein into many different peptides. Autophagy is another source of HLA class II peptides. Due to physicochemical dynamics in molecular interaction with HLA class II variants carried by the host, encoded in the host genome, a certain peptide exhibits immunodominance and is loaded into HLA class II molecules. They move and come to the surface of the cell. The most studied HLA subclass II genes are: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA and HLA-DRB1.

[243] Presentation of peptides by HLA class II molecules to CD4+ T helpers is required for immune response to foreign antigens (Roche and Furuta, 2015). Once activated, CD4+ T cells stimulate B cell differentiation and antibody production, as well as CD8+ T cell (CTL) responses. CD4+ T cells also secrete cytokines and chemokines that activate and induce the differentiation of other immune cells. Class II HLA molecules are heterodimers of α and β chains that interact to form a peptide-binding groove that is more open than class I peptide-binding grooves (Unanue et al., 2016). Peptides associated with HLA class II molecules are thought to have a 9 amino acid binding core with flanking residues on the N- or C-terminal side protruding from the groove (Jardetzky et al., 1996; Stern et al., 1994). These peptides are typically 12–16 amino acids in length and often contain 3–4 anchor residues at positions P1, P4, P6/7, and P9 of the binding register (Rossjohn et al., 2015).

[244] HLA alleles are expressed in a codominant form, which means that alleles (variants) inherited from both parents are expressed in the same way. For example, each individual carries 2 alleles of each of the 3 class I genes (HLA-A, HLA-B, and HLA-C) and therefore can express six different types of HLA class II. At the HLA class II locus, each individual inherits a pair of HLA-DP genes (DPA1 and DPB1, which encode α and β chains), a pair of HLA-DQ genes (DQA1 and DQB1, for α and β chains), one HLA-DRα gene (DRA1 ) and one or more HLA-DRβ genes (DRB1 and DRB3, -4 or -5). This means that one heterozygous individual can inherit six or eight functional HLA class II alleles, three or more from each parent. Thus, the HLA genes are highly polymorphic; different people within a population have many different alleles. The genes that code for HLA proteins have many possible variations that allow each person's immune system to respond to a wide range of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each of which is assigned a specific number. In some embodiments, the HLA class I alleles are HLA-A*02:01, HLA-B*14:02, HLA-A*23:01, HLA-E*01:01 (non-classic). In some embodiments, the HLA class II alleles are HLA-DRB*01:01, HLA-DRB*01:02, HLA-DRB 11:01, HLA-DRB*15:01, and HLA-DRB*07:01.

[245] The subject-specific HLA alleles or HLA genotype of a subject can be determined by any method known in the art. In exemplary embodiments, HLA genotypes are determined by any of the methods described in International Patent Application No. PCT/US2014/068746 published June 11, 2015 as W02015085147. Briefly, the methods include determining polymorphic gene types, which may include creating an alignment of reads extracted from a sequencing data set into a reference gene set containing polymorphic gene allele variants, determining a first posterior probability, or an estimate derived from the posterior probability, for each variant. allele in the alignment, identifying the allele variant with the highest first posterior or posterior as the first allele, identifying one or more overlapping reads that are aligned with the first allele and one or more other alleles, determining the second posterior probability or a posterior estimate for one or more other allele variants using a weighting factor, identifying a second allele variant by selecting the allele variant with the highest second posterior probability ew or an estimate obtained from the posterior probability, wherein the first and second allele variants determine the gene type for the polymorphic gene, and providing the output of the first and second allele variants.

[246] As described herein, there is a large body of evidence in both animals and humans that mutant epitopes are effective in inducing an immune response and that spontaneous tumor regression or long-term survival correlates with CD8+ T cell responses to mutant epitopes. (Buckwalter and Srivastava PK. “It is the antigen(s), stupid” and other lessons from over a decade of vaccitherapy of human cancer. Seminars in immunology 20:296-300 (2008); Karanikas et al., High frequency of cytolytic T-lymphocytes directed against a tumor-specific mutant antigen detected by HLA tetramers in the blood of a patient with long-survival lung carcinoma Cancer Res 61: 3718-3724 (2001) Lennerz et al In autologous T cell response to human melanoma mutant neoantigens predominate (Proc Natl Acad Sci US A.102: 16013 (2005)), and this "immune editing" can be traced to changes in the expression of dominant mutant antigens in mice and humans (Matsushita et al., Cancer exome analysis reveals T-cell- dependent mechanism of cancer immunoediting Nature 482: 400 (2012); DuPage et al. Cancer immunoediting is based on the expression of tumor-specific antigens. Nature 482: 405 (2012); and Sampson et al. Long-term progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed J Clin Oncol glioblastoma. 28: 4722-4729 (2010)).

[247] Sequencing technology has shown that each tumor contains many patient-specific mutations that alter the content of the protein encoding the gene. Such mutations create altered proteins ranging from single amino acid changes (caused by erroneous mutations) to the addition of long regions of a new amino acid sequence due to frameshifts, reading of stop codons, or translation of intron regions (new open reading frame mutations); neoORF). These mutant proteins are significant targets for the host's immune response to the tumor, since, unlike native proteins, they are not subject to the immunosuppressive effect of self-tolerance. Therefore, the mutant proteins are more likely to be immunogenic as well as more specific for tumor cells compared to normal patient cells.

[248] The term "T cell" includes CD4+ T cells and CD8+ T cells. The term T cell also includes both T helper type 1 T cells and T helper type 2 T cells. T cells in the context of the present invention are generally classified according to their function and cell surface antigens (cluster of differentiation or CD antigens), which also facilitate binding of the T cell receptor to the antigen, into two broad classes: T helper (T _H ) cells and cytotoxic T cells. -lymphocytes (CTL).

[249] Mature helper T cells express the CD4 surface protein and are referred to as CD4+ T cells. After T cell development, mature naive T cells leave the thymus and begin to spread throughout the body, including the lymph nodes. Naive T cells are those T cells that have never been exposed to the antigen they are programmed to respond to. Like all T cells, they express the T-cell receptor-CD3 complex. The T cell receptor (TCR) consists of constant and variable regions. The variable region determines which antigen the T cell can respond to. CD4+ T cells have a TCR with class II MHC affinity, and CD4 is involved in determining MHC affinity during maturation in the thymus. Class II MHC proteins are usually found only on the surface of specialized antigen-presenting cells (APCs). Specialized antigen presenting cells (APCs) are primarily dendritic cells, macrophages, and B cells, although dendritic cells are the only group of cells that express MHC class II consistently (all the time). Some APCs also bind native (or full-length) antigens to their surface, such as follicular dendritic cells, but full-length antigens do not interact with T cells and are not involved in their activation. Peptide antigens that bind to MHC class I proteins are generally shorter than peptide antigens that bind to MHC class II proteins.

[250] Cytotoxic T lymphocytes (CTL), also known as cytotoxic T cells, cytolytic T cells, CD+T cells, or killer T cells, refer to lymphocytes that induce apoptosis in target cells. CTLs form antigen-specific conjugates with target cells through the interaction of the TCR with processed antigen (Ag) on the surface of the target cells, which leads to apoptosis of the target cell. Apoptotic bodies are eliminated by macrophages. The term "CTL response" is used to refer to the primary immune response mediated by CTL cells. Cytotoxic T lymphocytes have both T cell receptors (TCR) and CD8 molecules on their surface. T-cell receptors are able to recognize and bind peptides formed in complex with HLA class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that is capable of binding specific MHC/peptide complexes. Most cytotoxic T cells express T cell receptors (TCRs) that can recognize a specific antigen. In order for a TCR to bind to a class I MHC molecule, the first must be accompanied by a glycoprotein called CD8, which binds to the constant portion of the class I MHC molecule. Therefore, these T cells are called CD8+ T cells. The affinity between CD8 and the MHC molecule keeps the T cell and target cell in close contact during antigen-specific activation. CD8+ T cells are recognized as T cells upon activation and are generally classified as having a predetermined cytotoxic role in the immune system. However, CD8+ T cells also have the ability to produce some cytokines.

[251] "T cell receptors (TCR)" are cell surface receptors that are involved in the activation of T cells in response to antigen presentation. The TCR typically consists of two chains, alpha and beta, which when assembled form a heterodimer and bind to CD3 transducing subunits to form a receptor-T cell complex present on the cell surface. Each TCR alpha and beta chain consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region. With respect to immunoglobulin molecules, alpha and beta chain variable regions are generated by V(D)J recombination, creating a wide variety of antigenic specificities in the T cell population. However, unlike immunoglobulins, which recognize intact antigen, T cells are activated by processed peptide fragments in association with the MHC molecule, introducing an additional dimension to T cell antigen recognition known as MHC restriction. Recognition of MHC differences between donor and recipient through the T cell receptor leads to T cell proliferation and the potential development of GVHD. Normal surface expression of the TCR has been shown to depend on the coordinated synthesis and assembly of all seven components of the complex (Ashwell and Klusner 1990). Inactivation of TCRα or TCRβ can lead to the elimination of TCR from the surface of T cells, preventing alloantigen recognition and hence GVHD. However, disruption of the TCR usually results in the elimination of the CD3 signaling component and alters the means for further expansion of T cells.

[252] The term "HLA peptide" refers to a pool of peptides that interact specifically with a particular class of HLA and may include thousands of different sequences. HLA peptidomes include a variety of peptides derived from both normal and abnormal proteins expressed in cells. Thus, HLA peptidomes can be studied to identify cancer-specific peptides, to develop an antitumor immunotherapeutic agent, and as a source of information on protein synthesis and degradation patterns in cancer cells. In some embodiments, the HLA peptide is a pool of soluble HLA molecules (sHLA). In some embodiments, the implementation of the HLA peptide is a pool of membrane HLA (mHLA).

[253] The term "antigen-presenting cell" or "APC" includes professional antigen-presenting cells (e.g., B-lymphocytes, macrophages, monocytes, dendritic cells, Langerhans cells), as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts , oligodendrocytes, thymic epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic beta cells, and vascular endothelial cells). An "antigen presenting cell" or "APC" is a cell that expresses major histocompatibility complex (MHC) molecules and can display a foreign antigen complexed with MHC on its surface.

Pipeline for Universal IP: Platform for Universal Identification of Complexes Monoallelic HLA Peptides

[254] Adaptive immune responses depend in part on the ability of cytotoxic CD8 ⁺ T cells to identify and kill cells that display disease-associated antigens associated with class I human leukocyte antigen (HLA) molecules. HLA class I proteins (HLA-A, B and C) are expressed on the surface of almost all nucleated cells in the human body and are required for the presentation of short peptides for detection by CD8+ T cell receptors. HLA-associated peptides arise from endogenous or foreign proteins that are cleaved by the proteasome and ER peptidases prior to loading and display by HLA class I proteins. HLA genes are the most polymorphic genes in the human population, and more than 10,000 HLA class I allele variants have been identified to date (Robinson et al., 2015). Each HLA allele is expected to bind and present ~1000-10000 unique peptides to T cells; ≤0.1% of ~10 million potential 9-mer peptides from genes encoding human proteins (Bassani-Sternberg et al., 2015; Hunt et al., 1992; Rammensee et al., 1995, 1999; Rock et al. ; Vita et al. ., 2015; Walz et al., 2015).

[255] In contrast to class I, HLA class II proteins (HLA-DR, DQ and DP) in response to inflammatory signals are expressed only on the surface of antigen-presenting cells (APC) and cells of epithelial, vascular and connective tissues. The presentation of class II peptides by HLA molecules, most often derived from exogenous proteins, to CD4+ T cells is necessary for the immune response to foreign antigens (Roche and Furuta, 2015). Once activated, CD4+ T cells stimulate B cell differentiation and antibody production, as well as CD8+ T cell responses. CD4+ T cells also secrete cytokines and chemokines that activate and induce the differentiation of other immune cells. HLA class II molecules are heterodimers of α and β chains that interact to form a peptide-binding groove that is more open than class I peptide-binding grooves (Unanue et al., 2016). Peptides associated with HLA class II molecules are thought to have a 9 amino acid binding core with flanking residues on the N- or C-terminal side protruding from the groove (Jardetzky et al., 1996; Stern et al., 1994). These peptides are typically 12–16 amino acids in length and often contain 3–4 anchor residues at positions P1, P4, P6/7, and P9 of the binding register (Rossjohn et al., 2015). Less is known about the allele-specific peptide-binding characteristics of HLA class II molecules due to heterogeneity in α- and β-strand pairing, data complexity limiting the ability to confidently assign nuclear-binding epitopes, and the lack of a degree of immunoprecipitation, allele-specific antibodies required for biochemical analysis with high resolution.

[256] Peptide binding rules have been extensively studied for a subset of HLA alleles (Vita et al., 2015) and encoded in modern neural network-based algorithms that predict binding (Hoof et al., 2009; Lundegaard et al., 2008). However, several factors limit the ability to predict peptides presented on HLA alleles. First, the origin of the peptide data on which these algorithms are trained is diverse, from peptide library screening to Edman degradation and mass spectrometry-based sequencing of endogenously processed and presented peptides (Boen et al., 2000; Rammensee et al., 1995 , 1999; Vita et al., 2015). Mass spectrometric identification of peptides accounts for about 30% of the total identification in the IEDB. Mass spectrometry has become a desirable method for sequencing HLA-related peptides due to the pioneering work of Donald F. Hunt and colleagues (Cobbold et al., 2013; Hunt et al., 1992; Meadows et al., 1997; Mohammed et al. . 2008; Zarling et al., 2000, 2006), as well as tool improvements demonstrated by many groups over the past two decades (Bassani-Sternberg et al., 2015; Caron et al., 2015; Mommen et al., 2014). Second, many existing prediction algorithms have focused on predicting binding but may not fully account for the endogenous processes that generate and transport peptides for binding (Larsen et al., 2007). Third, the number of binding peptides for many HLA alleles is too small to develop a reliable predictor. However, to date, the creation of high-quality resource datasets has been hampered by inefficient protocols that require an excessive amount of input cellular material, as well as a lack of database search tools for HLA peptide sequencing (Caron et al., 2015; Hoof et al. al., 2009 ; Lundegaard et al., 2008; Vita et al., 2015).

[257] This document discloses a unique strategy for the biochemical enrichment of peptide-HLA class I and II complexes from living cells and cell lysate. HLA molecules containing an N-terminal or C-terminal label sequence (eg BAP or HA) can be labeled on the cell surface or in a cell lysate. For example, HLA molecules containing an N-terminal or C-terminal biotin acceptor peptide (BAP) sequence can be enzymatically labeled with biotin on the cell surface or in a cell lysate. For example, HLA molecules containing an N-terminal or C-terminal HA sequence can be enriched in mixtures of cell complexes using an HA-specific antibody. In an exemplary embodiment, biotin-labelled HLA-peptide complexes are enriched from cell mixtures of complexes using streptavidin/NeutrAvidin beads, and the enriched HLA-peptide complexes are analyzed or characterized. In an exemplary embodiment, HA-labeled HLA peptide complexes are enriched from cell mixtures of the complexes using an HA-specific antibody, and the enriched HLA-peptide complexes are analyzed or characterized. For example, bound peptides can be eluted and sequenced using LC-MS/MS. Importantly, the currently disclosed methods provide a platform for versatile analysis and characterization of HLA-peptide complexes. For example, the currently disclosed methods provide a platform for the universal identification of endogenously presented peptides from a cell line expressing all possible class I or II constructs.

[258] Disclosed herein are separate cell lines expressing HLA class I and class II alleles that allow unambiguous peptide:allele assignment (Shimizu and DeMars, 1989; Shimizu et al., 1986). This is an improvement on current methods for detecting HLA-bound peptides, as most MS-based studies involve the elution and sequencing of a messy mixture of ligands bound to multiple HLA-A, B, and C molecules, which requires affinity prediction and sometimes deconvolution to assign alleles (Bassani- Sternberg and Gfeller, 2016). Studies with soluble HLA-transfected cell lines have yielded peptide-binding epitopes for a single HLA allele, but the most comprehensive experiments to date have identified only <200 unique peptides and required several orders of magnitude more starting cell material (Hawkins et al. 2008). By removing uncertainty about peptide:HLA assignment, the currently disclosed methods facilitate deeper and more accurate evaluation of HLA peptide ligandomes and the rules associated with peptide antigen processing and presentation using less cellular material than previous attempts.

[259] The methods and compositions described herein include, for example, chemically labeled β-variable chain (biotinylation) to differentiate between HLA class II heterodimers presented by cells, which can improve epitope mapping. The methods described herein can use class I and class II HLA constructs that contain a tag, such as a biotin acceptor peptide (BAP) sequence, at the N- or C-terminus. N- and C-terminal affinity labeling allows selective immunoaffinity purification of HLA alleles from cells expressing endogenous HLA. N-terminal affinity labeling allows selective immunoaffinity purification of alleles of HLA complexes presented on the cell surface. For example, after transfection or transduction, N-terminal biotinylation allows differentiation of HLA complexes presented on the cell surface from all HLA-peptide complexes in cell lysates. For example, biotinylation of HLA-peptide complexes on intact cell surfaces (without lysis) allows the use of methods for objective mass spectrometric (MS) sequencing of endogenously processed and presented peptides. The enrichment methods disclosed herein, such as immunoprecipitation enrichment methods, allow for high-throughput analysis of cell samples.

[260] This document provides a method for characterizing HLA-peptide complexes, comprising: providing a population of cells, wherein one or more cells of the cell population contain a polynucleic acid containing a sequence encoding an allele of an affinity-tagged HLA class I or class II acceptor, wherein the sequence , encoding HLA labeled with an affinity acceptor, contains a sequence encoding an allele of recombinant HLA class I or class II, functionally linked to a sequence encoding an affinity acceptor peptide; expressing the affinity acceptor-labeled HLA in at least one cell of one or more cells of the cell population, thereby complexing the affinity acceptor-labeled HLA peptides in at least one cell; enrichment of complexes labeled with an affinity acceptor HLA-peptides; and characterization of HLA-peptide complexes.

[261] In some embodiments, the characterization includes characterization of the peptide associated with the HLA affinity acceptor-tagged peptide complex as a result of enrichment.

[262] In some embodiments, the implementation of the method includes performing the steps of the method for two or more alleles of HLA class I and/or class II. In some embodiments, the two or more HLA class I and/or class II alleles include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 HLA class I and/or class II alleles.

[263] In some embodiments, the affinity acceptor-tagged HLA peptide complexes comprise a transmembrane domain. In some embodiments, the affinity acceptor-tagged HLA peptide complexes comprise an intracellular domain. In some embodiments, the affinity acceptor-labeled HLA peptide complexes are not excreted. In some embodiments, the affinity acceptor-tagged HLA peptide complexes are incorporated into the cell membrane upon expression. In some embodiments, the HLA acceptor affinity tagged peptide complexes are insoluble complexes of the HLA acceptor affinity tagged peptides.

[264] In some embodiments, the method further comprises creating a database of HLA allele-specific peptides.

[265] In some embodiments, the recombinant HLA Class I or Class II allele is a single recombinant HLA Class I or Class II allele.

[266] In some embodiments, the implementation of the method includes: providing a population of cells, each of which contains one or more cells containing affinity-acceptor-tagged HLA, and affinity-acceptor-tagged HLA contains another recombinant polypeptide encoded by a different HLA allele operably linked to the affinity peptide - acceptor; enrichment of complexes labeled with an affinity acceptor HLA-peptides; and characterizing the peptide, or part thereof, associated with the HLA affinity acceptor-labeled peptide complex as a result of enrichment.

[267] In some embodiments, the implementation of the method includes the introduction of one or more peptides in a population of cells.

[268] In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more peptides or the expression of one or more peptides in a population of cells. In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more nucleic acids encoding one or more peptides. In some embodiments, one or more nucleic acids encoding one or more peptides is DNA. In some embodiments, one or more nucleic acids encoding one or more peptides is RNA, optionally the RNA is mRNA.

[269] In some embodiments, enrichment does not include the use of a tetrameric reagent.

[270] In some embodiments, characterization includes determining the sequence of a peptide or portion thereof associated with an enriched HLA affinity acceptor-labeled peptide, optionally determining whether the peptide or portion thereof is modified. In some embodiments, the determination includes biochemical analysis, mass spectrometric analysis, MS analysis, MS/MS analysis, LC-MS/MS analysis, or a combination thereof. In some embodiments, the characterization includes evaluating the binding affinity or stability of the peptide, or portion thereof, associated with the enriched HLA affinity acceptor-labeled peptide complex. In some embodiments, characterization includes determining whether a peptide, or portion thereof, associated with an enriched HLA affinity acceptor-tagged peptide contains one or more mutations. In some embodiments, the characterization includes evaluating the binding of peptides to HLA molecules in complexes with affinity acceptor-labeled HLA peptides.

[271] In some embodiments, the implementation of the method includes the expression of the library of peptides in a population of cells, thereby forming a library of complexes labeled with an affinity acceptor HLA peptides. In some embodiments, the method includes contacting the cell population with a peptide library or a library of peptide coding sequences, thereby forming a library of HLA affinity acceptor tagged peptide complexes. In some embodiments, the library is a library of peptides associated with a disease or condition. In some embodiments, the disease or condition is cancer, infection with an infectious agent, or an autoimmune reaction. In some embodiments, the method includes introducing the infectious agent or parts thereof into one or more cells of the cell population. In some embodiments, the method includes characterizing one or more peptides from HLA-peptide complexes, optionally the peptides are derived from one or more target proteins of the infectious agent. In some embodiments, the method includes characterizing one or more peptide regions from one or more target proteins of the infectious agent. In some embodiments, the method includes identifying peptides from HLA-peptide complexes derived from the infectious agent.

[272] In some embodiments, the cell population is derived from a biological sample in a subject with a disease or condition. In some embodiments, the cell population is a cell line. In some embodiments, the cell population is a primary cell population. In some embodiments, the recombinant HLA Class I or Class II allele corresponds to a subject with a disease or condition.

[273] In some embodiments, the implementation of the method includes screening for hypersensitivity to drugs (eg, biologics). In some embodiments, the method includes evaluating the presentation to T cells of an administered biologic (eg, a protein, peptide, or antibody-containing drug), a fragment of the administered biologic, or a fragment of a processed biologic. These epitopes can cause an adverse effect on the subject, and thus the extent to which the administered biologics are processed in the subject should be monitored. For example, an anti-HIV drug (eg, abacavir) can bind to HLA molecules and alter peptide-binding motifs for certain HLA alleles (eg, HLA-B5701).

[274] In some embodiments, a peptide from the HLA affinity acceptor-tagged peptide complex is capable of activating a T cell in a subject when presented by an antigen presenting cell. In some embodiments, characterization includes comparing HLA-peptide complexes from cancer cells with HLA-peptide complexes from non-cancerous cells.

[275] In some embodiments, the cell population includes a plurality of cell populations, with each cell population expressing a different recombinant class I or class II HLA allele. In some embodiments, each cell population of the plurality is in the same or separate container.

[276] In some embodiments, the method further comprises isolating peptides from complexes of HLA affinity acceptor-labeled peptides prior to characterization. In some embodiments, the method further comprises removing one or more amino acids at the end of the peptide associated with the HLA affinity acceptor-tagged peptide complex.

[277] In some embodiments, the implementation of the cell population is a population of cells with low expression of class I or class II cell surface HLA. In some embodiments, a population of cells expresses one or more endogenous HLA alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles and endogenous HLA class II alleles. In some embodiments, the cell population has a knockout of one or more HLA class I alleles.

[278] In some embodiments, a population of cells has a knockout of one or more HLA class II alleles. In some embodiments, a population of cells has a knockout of all HLA class I alleles. In some embodiments, a population of cells has a knockout of all HLA class II alleles. In some embodiments, the cell population has a knockout of all HLA class I alleles and a knockout of all HLA class II alleles. In some embodiments, a sequence encoding an allele of a recombinant HLA class I or class II encodes a class I HLA. In some embodiments, Class I HLA is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the sequence encoding the allele of the recombinant HLA class I or class II encodes HLA class II. In some embodiments, the HLA class II is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP. In some embodiments, the HLA class II comprises an HLA class II α chain, an HLA class II β chain, or a combination thereof.

[279] In some embodiments, each sequence encodes at least two different HLA class I and/or class II alleles. In some embodiments, at least two different HLA class I and/or class II alleles are each operably linked to a sequence encoding a different acceptor affinity peptide. In some embodiments, at least two different HLA class I and/or class II alleles are each operably linked to a sequence encoding an affinity acceptor peptide.

[280] In some embodiments, the method includes introducing at least a second polynucleic acid containing a sequence encoding a different recombinant HLA allele operably linked to the same or a different acceptor affinity peptide.

[281] In some embodiments, the sequence encoding the acceptor affinity peptide is operably linked to a sequence that encodes the extracellular portion of the recombinant HLA class I or class II allele.

[282] In some embodiments, the encoded acceptor affinity peptide is expressed extracellularly. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to the N-terminus of a sequence encoding a recombinant HLA Class I or Class II allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence that encodes the intracellular portion of the recombinant HLA class I or class II allele. In some embodiments, the encoded acceptor affinity peptide is expressed intracellularly. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to the C-terminus of a sequence encoding a recombinant HLA Class I or Class II allele. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to a sequence encoding a recombinant HLA Class I or Class II allele via a linker.

[283] In some embodiments, the implementation of the enrichment includes the enrichment of intact cells expressing complexes labeled with an affinity acceptor HLA peptides. In some embodiments, the method does not include lysing the cells prior to enrichment. In some embodiments, the method further comprises lysing one or more cells prior to enrichment. In some embodiments, enrichment comprises contacting an acceptor affinity peptide binding molecule with the acceptor affinity-tagged HLA peptide complexes, wherein the acceptor affinity peptide binding molecule specifically binds to the acceptor affinity peptide.

[284] In some embodiments, the affinity acceptor peptide comprises a tag sequence including a biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine-glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 peptide tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep tag II, tag albumin-binding protein (ABP), alkaline phosphatase (AP) tag, bluetongue (B-tag), calmodulin-binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline binding domain (CBD) tag , chitin binding domain (CBD) tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose binding protein (GBP) tag, maltose binding protein (MBP), gluta thione S-transferase (GST), Glu-Glu tag (EE), human influenza virus hemagglutinin tag (HA), horseradish peroxidase (HA) tag, NE tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag , LacZ tag, Luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin tag, E tag, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, Xpress tag, Isopeptag, SpyTag , SnoopTag, Profinity eXact tag, protein C tag, S1 tag, S tag, carboxybiotin carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification tag (TAP), HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or a combination thereof; optionally, the affinity acceptor peptide contains two or more repeats of the tag sequence. In some embodiments, the affinity acceptor peptide binding molecule is biotin or an antibody specific for the acceptor affinity peptide. In some embodiments, enrichment includes contacting an affinity molecule with complexes of affinity acceptor-labeled HLA peptides, wherein the affinity molecule specifically binds to the affinity acceptor peptide binding molecule. In some embodiments, the affinity molecule is streptavidin, NeutrAvidin, or a derivative thereof. In some embodiments, enrichment includes immunoprecipitation of complexes labeled with an affinity acceptor HLA peptides. In some embodiments, an acceptor affinity peptide binding molecule is attached to a solid surface. In some embodiments, the affinity molecule is attached to a solid surface. In some embodiments, the hard surface is a bead. In some embodiments, enrichment comprises immunoprecipitating complexes of the affinity acceptor-labeled HLA peptides with an acceptor affinity peptide binding molecule that specifically binds to the acceptor affinity peptide. In some embodiments, the acceptor affinity peptide binding molecule does not specifically interact with the amino acid sequence of the encoded recombinant HLA class I or class II. In some embodiments, enrichment includes contacting an affinity molecule specific for the extracellular portion of a recombinant HLA Class I or Class II allele. In some embodiments, enrichment includes contacting an affinity molecule specific for the N-terminal portion of a recombinant HLA Class I or Class II allele.

[285] In some embodiments, providing includes contacting a population of cells with a polynucleic acid. In some embodiments, the introduction into contact includes transfection or transduction. In some embodiments, providing includes contacting a population of cells with a vector containing a polynucleic acid. In some embodiments, the vector is a viral vector. In some embodiments, the polynucleic acid is stably inserted into the genome of a population of cells.

[286] In some embodiments, the implementation of the sequence encoding recombinant HLA class I or class II, contains a sequence encoding the α-chain of HLA class I. In some embodiments, the implementation of the method further includes the expression of a sequence encoding β2 microglobulin in one or more cells. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding an HLA class I α chain. In some embodiments, the β2 microglobulin coding sequence is linked to the HLA class I α chain coding sequence via a linker. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding a second acceptor affinity peptide. In some embodiments, a sequence encoding a recombinant HLA class I or class II contains a sequence encoding an HLA class II α chain. In some embodiments, the method further comprises expressing a sequence encoding an HLA class II β chain in one or more cells. In some embodiments, a sequence encoding an HLA class II β chain is linked to a sequence encoding an HLA class II α chain. In some embodiments, a sequence encoding an HLA class II β chain is linked to a sequence encoding an HLA class II α chain via a linker.

[287] In some embodiments, a sequence encoding the β chain of HLA class II is linked to a sequence encoding a second acceptor affinity peptide. In some embodiments, the second acceptor affinity peptide is different from the first acceptor affinity peptide and is selected from the group consisting of a biotin acceptor peptide (BAP), a polyhistidine tag, a polyhistidine-glycine tag, a polyarginine tag, a polyaspartate tag, a polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD), FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 protein 10 peptide tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag , Strep II tags, albumin-binding protein (ABP) tags, alkaline phosphatase (AP) tags, bluetongue virus tags (B-tags), calmodulin-binding peptide (CBP) tags, chloramphenicol acetyltransferase (CAT) tags, tags choline-binding domain (CBD), chitin-binding domain (CBD) tag, cellulose-binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein (GBP) tag ), maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tags, human influenza virus hemagglutinin (HA) tags, horseradish peroxidase (HA) tags, NE-tags, HSV tags, ketosteroid tags -isomerases (KSI), KT3 tags, LacZ tags, luciferase tags, NusA tags, PDZ domain tags, AviTag, calmodulin tags, E-tags, S-tags, SBP-tags, Softag 1, Softag 3, TC tags, VSV- tags, Xpress tags, Isopeptag, SpyTag, SnoopTag, Profinity eXact tags, protein C tags, S1 tags, S tags, carboxybiotin carrier protein (BCCP) tags, green fluorescent protein (GFP) tags, small ubiquitin-like modifier tags (SUMO), tandem affinity purification (TAP) tags, HaloTag, Nus tags, Thioredoxin tags, Fc tags, CYD tags, HPC tags, TrpE tags, ubiquitin tag, VSV-G epitope tag, V5 tag, and combinations thereof; optionally, the first or second acceptor affinity peptide contains two or more repeats of the tag sequence.

[288] In some embodiments, the linker contains a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome escape site or internal ribosome entry site (IRES). In some embodiments, the ribosome escape site or IRES is cleaved when expressed in cells. In some embodiments, the ribosome exit site is selected from the group consisting of F2A, T2A, P2A, and E2A. In some embodiments, the IRES element is selected from common cellular or viral IRES sequences.

[289] In some embodiments, the determination includes performing a biochemical analysis or mass spectrometry, such as tandem mass spectrometry. In some embodiments, the determination includes obtaining a peptide sequence that corresponds to MS/MS spectra of one or more peptides isolated from enriched complexes of HLA affinity acceptor-labeled peptides from a peptide database; wherein one or more of the resulting sequences identifies the sequence of one or more peptides.

[290] In some embodiments, the cell population is a cell line selected from HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, Hep G2, SH-SY5Y, CACO-2, U937, U-2 OS , ExpiCHO, CHO and THP1. In some embodiments, the cell line is treated with one or more cytokines, checkpoint inhibitors, epigenetically active drugs, IFN-γ, agents that alter antigen processing (such as peptidase inhibitors, proteasome inhibitors, and TAP inhibitors), or a combination thereof.

[291] In some embodiments, the peptide database is a peptide database with no enzyme specificity, such as an unmodified database or a modified database. In some embodiments, the method further comprises searching the peptide database using a flipped database search strategy.

[292] In some embodiments, the cell population includes at least 10 ⁵ cells, at least 10 ⁶ cells, or at least 10 ⁷ cells. In some embodiments, the cell population is a population of dendritic cells, macrophages, cancer cells, or B cells. In some embodiments, the cell population includes tumor cells. In some embodiments, a population of cells is contacted with an agent prior to isolating said HLA-peptide complexes from one or more cells. In some embodiments, said agent is an inflammatory cytokine, a chemical agent, an adjuvant, a therapeutic agent, or radiation.

[293] In some embodiments, the HLA allele is a mutant HLA allele.

[294] In some embodiments, the sequence encoding the HLA allele is a barcoded sequence. In some embodiments, the method further comprises analyzing the expression of an allele tagged with an HLA class I or class II affinity acceptor. In some embodiments, the assay includes sequencing an allele tagged with an HLA class I or class II affinity acceptor, detecting an RNA allele tagged with an HLA class I or class II affinity acceptor, detecting a protein of the allele tagged with an HLA class I or class II affinity acceptor, or a combination thereof.

[295] In some embodiments, the implementation of the method includes performing the steps of the method for different HLA alleles. In some embodiments, each HLA allele contains a unique barcoded sequence. In some embodiments, each polynucleic acid encoding a different HLA allele contains a unique barcoded sequence.

[296] This document provides a database of sequences specifically binding alleles of HLA peptides obtained by performing the method described herein. Provided herein is a combination of two or more HLA allele-specific binding peptide sequence databases obtained by repeatedly performing the method described herein, each time using a different HLA allele. Provided herein is a method for generating a prediction algorithm for identifying HLA-specific allele-binding peptides, comprising training a machine with a sequence database of the peptides described herein or the combination described herein. In some embodiments, the machine combines one or more linear models, support vector machines, decision trees, and neural networks. In some embodiments, the variable used to train the machine includes one or more variables selected from the group consisting of peptide sequence, physical properties of amino acids, physical properties of peptides, expression level of the original peptide protein in the cell, protein stability, protein translation rate , ubiquitination sites, rate of protein degradation, translation efficiency from ribosomal profiling, protein cleavage, protein localization, host protein motifs that facilitate TAP transport, host protein undergoes autophagy, motifs that promote ribosome arrest, and protein features that promote NMD . In some embodiments, motifs that promote ribosome arrest contain polyproline or polylysine regions. In some embodiments, protein features that promote NMD are selected from the group consisting of a long 3' UTR, a stop codon greater than 50 nucleotides before the last exon:exon junction, and cleavage of the peptide. Provided herein is a method for identifying HLA allele-specific binding peptides, comprising performing a peptide sequence analysis with a machine that has been trained with a peptide sequence database obtained by performing the method described herein for HLA alleles. In some embodiments, the implementation of the method includes determining the level of expression of the original protein of the peptide in the cell; and while the expression of the original protein is the prognostic variable used by the machine. In some embodiments, the level of expression is determined by measuring the amount of the parent protein or the amount of RNA encoding said parent protein.

[297] Provided herein is a composition comprising a recombinant polynucleic acid comprising two or more sequences each encoding an affinity acceptor-tagged HLA, wherein the sequences encoding the affinity-acceptor-tagged HLA contain (a) a sequence encoding a different recombinant α allele class I HLA chains, (b) a sequence encoding an affinity acceptor peptide, and optionally, (c) a sequence encoding β2 microglobulin; wherein sequences (a) and (b), and optionally (c), are operably linked.

[298] Provided herein is a composition comprising a recombinant polynucleic acid comprising two or more sequences each containing a sequence encoding an affinity acceptor-tagged HLA, wherein the sequences encoding the affinity-acceptor-tagged HLA contain (a) a sequence encoding a recombinant an HLA class II α-chain allele, (b) a sequence encoding an affinity acceptor peptide, and optionally, (c) a sequence encoding an HLA class II β-chain; wherein sequences (a) and (b), and optionally (c), are operably linked. In some embodiments, a recombinant polynucleic acid is isolated. In some embodiments, Class I HLA is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the HLA class II is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP.

[299] In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence that encodes the extracellular portion of the recombinant HLA allele. In some embodiments, the sequence encoding the affinity acceptor molecule is operably linked to the N-terminus of the sequence encoding the recombinant HLA allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the sequence encoding the intracellular portion of the recombinant HLA allele. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to the C-terminus of a sequence encoding a recombinant HLA allele.

[300] In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to a sequence encoding a recombinant HLA allele via a linker.

[301] In some embodiments, two or more affinity acceptor-tagged HLA encoding sequences are expressed from the same polynucleotide. In some embodiments, two or more affinity acceptor-tagged HLA encoding sequences are expressed from different polynucleotides.

[302] In some embodiments, the encoded acceptor affinity peptide specifically binds to an acceptor affinity peptide binding molecule.

[303] In some embodiments, two or more affinity acceptor-tagged HLA encoding sequences contain two or more affinity acceptor peptides. In some embodiments, two or more sequences encoding an affinity-acceptor-tagged HLA comprise three or more sequences encoding an affinity-acceptor-tagged HLA, wherein at least two of the three or more sequences encoding an affinity-acceptor-tagged HLA contain the same affinity acceptor peptide. In some embodiments, the two or more affinity acceptor peptides are unique for each of the two or more sequences encoding the affinity acceptor-tagged HLA. In some embodiments, the encoded acceptor affinity peptide is selected from the group consisting of biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine-glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, glycoprotein D( gD) Herpes simplex virus tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep tag II, albumin-binding tag protein (ABP), alkaline phosphatase (AP) tags, bluetongue virus (B-tags), calmodulin-binding peptide (CBP) tags, chloramphenicol acetyltransferase (CAT) tags, choline-binding domain (CBD) tags, chitin tags binding domain (CBD), cellulose-binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein (GBP) tag, maltose-binding protein (MBP), glutathione-S-tran sferase (GST), Glu-Glu (EE) tags, human influenza virus hemagglutinin (HA) tags, horseradish peroxidase (HA) tags, NE tags, HSV tags, ketosteroid isomerase (KSI) tags, KT3 tags, LacZ tags, luciferase tag, NusA tags, PDZ domain tags, AviTag, calmodulin tag, E-tags, S-tags, SBP-tags, Softag 1, Softag 3, TC tags, VSV tags, Xpress tags, Isopepeptag, SpyTag, SnoopTag, tags Profinity eXact, protein C tags, S1 tags, S tags, carboxybiotin carrier protein (BCCP) tags, green fluorescent protein (GFP) tags, small ubiquitin-like modifier (SUMO) tags, tandem affinity purification (TAP) tags, HaloTag, Nus tags, Thioredoxin tags, Fc tags, CYD tags, HPC tags, TrpE tags, ubiquitin tag, VSV-G epitope tag, V5 tag, and combinations thereof; optionally, the first or second acceptor affinity peptide contains two or more repeats of the tag sequence. In some embodiments, the affinity acceptor peptide binding molecule is biotin or an antibody specific for the acceptor affinity peptide. In some embodiments, the affinity acceptor peptide binding molecule specifically binds to the affinity molecule. In some embodiments, the affinity molecule is streptavidin, NeutrAvidin, or a derivative thereof. In some embodiments, the acceptor affinity peptide binding molecule does not specifically interact with the recombinant HLA class I or class II amino acid sequence.

[304] In some embodiments, for two or more recombinant polynucleic acids: a sequence encoding an HLA-tagged affinity acceptor is stably inserted into the cell's genome.

[305] In some embodiments, a sequence encoding a .beta.2 microglobulin, or a sequence encoding the .beta. chain of an HLA class II, is linked to a sequence encoding a second acceptor affinity peptide. In some embodiments, the second acceptor affinity peptide contains an HA tag. In some embodiments, a sequence encoding a .beta.2 microglobulin or a sequence encoding the .beta. chain of an HLA class II is linked to a sequence encoding a recombinant HLA and an acceptor affinity peptide via a linker. In some embodiments, the linker contains a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome escape site or internal ribosome entry site (IRES). In some embodiments, the ribosome escape site or IRES is cleaved when expressed in cells. In some embodiments, the ribosome exit site is selected from the group consisting of F2A, T2A, P2A, and E2A. In some embodiments, the IRES element is selected from common cellular or viral IRES sequences.

[306] Provided herein is a composition comprising two or more isolated polypeptide molecules encoded by a polynucleic acid of the composition described herein. Provided herein is a composition comprising a population of cells containing two or more polypeptide molecules encoded by a polynucleic acid of the composition described herein. This document provides a composition containing a population of cells containing the composition described in this document. This document provides a composition containing a population of cells containing one or more cells containing the composition described herein.

[307] In some embodiments, a population of cells expresses one or more endogenous HLA class I or class II alleles. In some embodiments, a population of cells is constructed that lacks one or more endogenous HLA class I alleles. In some embodiments, a population of cells is constructed that lack alleles for endogenous HLA class I. In some embodiments, a population of cells is constructed that lacks one or more endogenous HLA class II alleles. In some embodiments, a population of cells is constructed that lack alleles for endogenous HLA class II. In some embodiments, a population of cells is constructed that lacks one or more endogenous HLA class I alleles and one or more endogenous HLA class II alleles.

In some embodiments, the cell population is a cell population with low cell surface HLA class I or class II expression. In some embodiments, a composition is formulated using peptides or polynucleic acids encoding peptides specific for the patient's HLA type.

[308] Provided herein is a method for producing a cell, comprising transducing or transfecting two or more cells with two or more polynucleic acids of the composition described herein. Provided herein is a peptide identified according to the method described herein.

[309] Provided herein is a method for eliciting an antitumor response in a mammal, comprising administering to the mammal an effective amount of a polynucleic acid containing the peptide sequence described herein. Provided herein is a method for inducing an antitumor response in a mammal, comprising administering to the mammal an effective amount of a peptide comprising the peptide sequence described herein. Provided herein is a method for eliciting an antitumor response in a mammal, comprising administering to the mammal a cell containing a peptide comprising the peptide sequence described herein. Provided herein is a method for eliciting an antitumor response in a mammal, comprising administering to the mammal a cell containing an effective amount of a polynucleic acid containing a peptide coding sequence comprising the peptide sequence described herein. In some embodiments, the cell presents the peptide as an HLA-peptide complex. Provided herein is a method of eliciting an immune response in a mammal, comprising administering to the mammal an effective amount of a polynucleic acid containing a sequence encoding a peptide described herein. Provided herein is a method of eliciting an immune response in a mammal, comprising administering to the mammal an effective amount of a peptide comprising the peptide sequence described herein. Provided herein is a method of eliciting an immune response in a mammal, comprising administering to the mammal an effective amount of cells containing a peptide comprising the peptide sequence described herein. Provided herein is a method for eliciting an immune response in a mammal comprising administering to the mammal an effective amount of cells containing a polynucleic acid containing a peptide coding sequence comprising the peptide sequence described herein.

[310] In some embodiments, the implementation of the immune response is a T-cell immune response. In some embodiments, the immune response is a CD8 T cell response. In some embodiments, the immune response is a CD4 T cell response. In some embodiments, the immune response is a humoral immune response.

[311] Provided herein is a method for treating a mammal having a disease, comprising administering to the mammal an effective amount of a polynucleic acid containing a sequence encoding a peptide described herein. Provided herein is a method of treating a mammal having a disease comprising administering to the mammal an effective amount of a peptide comprising a peptide sequence as described herein. Provided herein is a method for treating a mammal having a disease, comprising administering to the mammal an effective amount of cells containing a peptide comprising the peptide sequence described herein. Provided herein is a method for treating a mammal having a disease comprising administering to the mammal an effective amount of cells containing a polynucleic acid containing a peptide coding sequence comprising the peptide sequence described herein.

[312] In some embodiments, the disease is cancer. In some embodiments, the disease is infection by an infectious agent. In some embodiments, the infectious agent is a pathogenic microorganism, optionally a virus or bacterium or parasite. In some embodiments, the virus is selected from the group consisting of: BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4, DENV-5), Cytomegalovirus (CMV), Hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), adenovirus, human immunodeficiency virus (HIV), human T-cell lymphotrophic virus (HTLV-1), influenza virus, RSV, HPV, rabies, virus mumps, rubella, poliovirus, yellow fever, hepatitis A, hepatitis B, rotavirus, varicella-zoster virus, human papillomavirus (HPV), smallpox, shingles, and any combination thereof. In some embodiments, the bacterium is selected from the group consisting of: Klebsiella spp., Tropheryma whipplei, Mycobacterium leprae, Mycobacterium lepromatosis, and Mycobacterium tuberculosis, typhoid, pneumococcus, meningococcus, haemophilus B, anthrax, tetanus toxin, group B meningococcus, bcg, cholera, and any combination of them. In some embodiments, the parasite is a helminth or a protozoan. In some embodiments, the parasitic organism is selected from the group consisting of: Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Schistosoma spp., and any combination thereof.

[313] Provided herein is a method for enriching immunogenic peptides, comprising: providing a cell population comprising one or more cells expressing an affinity acceptor-tagged HLA, wherein the affinity-acceptor-tagged HLA contains an affinity acceptor peptide operably linked to a recombinant HLA encoded by an allele of the recombinant HLA; and enrichment of HLA-peptide complexes containing labeled HLA affinity acceptor. In some embodiments, the method further comprises determining the sequence of immunogenic peptides isolated from the HLA-peptide complexes. In some embodiments, the implementation of the definition includes the use of LC-MS/MS.

[314] Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a polynucleic acid containing a sequence encoding a peptide described herein. Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a peptide comprising the peptide sequence described herein. Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of cells containing a peptide comprising the peptide sequence described herein. Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject a cell containing an effective amount of a polynucleic acid containing a peptide coding sequence comprising the peptide sequence described herein.

Enrichment of HLA-peptide complexes

[315] The genes encoding HLA class I and class II glycoproteins are among the most polymorphic coding sequences in the human genome. However, for each of the HLA class I heavy chains and HLA class II α and β chains, there are relatively constant or unchanged regions that antibodies can target to selectively capture any HLA class I heavy chain or HLA α or β chain. II class. However, since α- or β-chains are usually associated with each other in vivo, immunoaffinity purification of the α-chain of intact soluble HLA can coprecipitate the β-chain and vice versa. Anti-HLA class II antibodies to enrich for HLA-associated polypeptides can recognize conserved epitopes present in the α or β chain.

[316] The method of enrichment using antibodies specific for HLA alleles or using non-HLA specific reagents is well known in the art. For example, HLA-C polypeptides are typically expressed by individuals at lower levels than HLA-A and HLA-B. Accordingly, in order to improve the detection of HLA-C using antibodies, it may be preferable to provide, in addition to other purification methods, specific immunoaffinity purification of HLA-C using HLA-C specific antibodies. Numerous examples of monoclonal or polyclonal antibodies are commercially available that specifically bind to individual HLA chains.

[317] Provided herein is a universal immunoaffinity purification (IP) pipeline for enrichment of one or more single-allelic HLA polypeptide complexes. An illustration of such a method for enriching an HLA-bound polypeptide is a method that includes an immunoaffinity purification step. The universal IP pipeline contains the universal IP constructs consisting of a DNA construct encoding class I or class II HLA affinity tagged alleles that are expressed from an expression vector by cell transfection or transduction. A non-limiting example of an expression vector is a lentiviral vector.

[318] Cells transfected or transduced with the universal IP constructs were either expanded or selected and then expanded prior to LC-MS/MS sequence analysis. Suitable cell populations for transfection or transduction include, for example, class I deficient cell lines expressing one HLA class I allele, class II deficient cell lines expressing one pair of HLA class II alleles, or cell lines with deficiency in class I and class II, in which one HLA class I and / or one pair of class II alleles are expressed. As an exemplary embodiment, the class I deficient B cell line is B721.221. In some embodiments, the cells are A375 or HEK293T, HeLa, or expi293. However, one skilled in the art will appreciate that other cell populations that are class I and/or class II deficient can be obtained. Methods for producing class I and/or class II deficient cells, as well as class I and/or class II deficient cell lines, are known in the art, and an illustrative method for removing/inactivating endogenous class I or class II genes includes indirectly CRISPR-Cas9 genome editing, for example, in THP-1 cells. In some embodiments, the cell populations are professional antigen presenting cells such as macrophages, B cells, and dendritic cells. The cells may be B cells or dendritic cells. In some embodiments, the cells are tumor cells or cells from a tumor cell line. In some embodiments, the cells are cells isolated from a patient. In some embodiments, the cells contain the infectious agent, or a portion thereof.

[319] In some embodiments, the universal IP constructs comprise a class I or class II HLA construct containing an affinity acceptor tag and an affinity molecule. In some embodiments, the universal IP constructs comprise at least one specific binding affinity acceptor tag and an affinity molecule. In some embodiments, the affinity acceptor tag is a polyhistidine tag, a polyhistidine-glycine tag, a polyarginine tag, a polyaspartate tag, a polycysteine tag, polyphenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, T7 protein 10 peptide tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep II tag, albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag, catarrhal virus tag fever (B-tag), calmodulin-binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline-binding domain (CBD) tag, chitin-binding domain (CBD) tag, cellulose-binding domain (CBP) tag, tag dihydrofolate reductase (DHFR), galactose-binding protein (GBP) tag, maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tag, influenza hemagglutinin tag na human (HA), horseradish peroxidase (HA) tag, NE tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, Luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin tag, E -tag, S-tag, SBP-tag, Softag 1, Softag 3, TC-tag, VSV-tag, Xpress-tag, Isopepeptag, SpyTag, SnoopTag, Profinity eXact-tag, Protein-C-tag, S1-tag, S-tag, tag carboxybiotin carrier protein (BCCP), green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, an HPC tag, a TrpE tag, a ubiquitin tag, a VSV-G epitope tag derived from the vesicular stomatitis viral glycoprotein, or a V5 tag derived from a small epitope (Pk) found on the P and V proteins of paramyxovirus simian virus 5 (SV5). In some embodiments, the affinity acceptor tag may contain multiple repeats of the tag sequence (eg, 3x polyhistidine tags, 3x FLAG tags). In some embodiments, the affinity acceptor tag may contain multiple repeats of the tag sequence (eg, 3x polyhistidine tags, 3x FLAG tags). An affinity acceptor tag is an "epitope tag", which is a type of peptide tag that adds a recognizable epitope (antibody binding site) to an HLA protein to allow the corresponding antibody to bind, allowing identification or affinity purification of the tagged protein. A non-limiting example of an epitope tag is protein A or protein G that binds to IgG. In some embodiments, the affinity acceptor tags comprise a biotin acceptor peptide (BAP) or influenza hemagglutinin (HA) peptide sequence. One of ordinary skill in the art is aware of and may consider numerous other portions of the labels that are provided herein. Any peptide tag may be used, provided that it is capable of being expressed as an element of the HLA affinity acceptor-labeled peptide complex.

[320] Affinity tags can be placed either at the N-terminus or at the C-terminus of the HLA allele. A cleavage sequence such as F2A or an internal ribosome entry site (IRES) can be placed between the α-chain and β2-microglobulin (class I) or between the α-chain and β-chain (class II). In some embodiments, the only HLA class I allele is HLA-A*02:01, HLA-A*23:01 and HLA-B*14:02, or HLA-E*01:01, and the HLA class II allele is HLA -DRB*01:01, HLA-DRB*01:02 and HLA-DRB*11:01, HLA-DRB*15:01 or HLA-DRB*07:01. In some embodiments, the cleavage sequence is a T2A, P2A, E2A, or F2A sequence. For example, the cleavage sequence could be E G R G S L L T C G D V E E N P G P (T2A), T N F S L L K Q A G D V E E N P G P (P2A), Q C T N Y A L L K L A G D V E S N P G P (E2A), or V K Q T L N F D L L K L A G D V E S N P G P (F2A).

[321] In some embodiments, the immunoaffinity purification of HLA-peptide complexes is based on biotin. In some embodiments, the immunoaffinity purification of HLA-peptide complexes is based on streptavidin or NeutrAvidin. In some embodiments, HLA-peptide complexes can also be enriched from a biological sample using chromatographic techniques such as HPLC. In some embodiments, depletion of excess whey proteins can be used to enrich HLA-peptide complexes. In some embodiments, methods for removing excess serum proteins include dye ligands (for albumin), protein A and G (for γ-globulins), or specific antibodies that bind with high affinity and selectively deplete these species from the sample (Govorukhina, Reijmers et al. . 2006). Such strategies will increase the number of HLA derived peptide sequences identified in a single mass spectrometry analysis.

[322] The degree of enrichment required to optimize the analysis of specific HLA sequences from a biological sample will depend on the initial concentration of the HLA sequence in the biological sample, as well as the concentration and nature of other non-HLA proteins in the sample.

[323] Classical protein purification methods can be used to enrich HLA-peptide complexes in a biological sample, alone or in combination with the universal IP pipeline methods provided herein. Classical protein separation (purification) methods are based on: differences in size (ultrafiltration, gel filtration or size exclusion chromatography); charge differences (pi) (anion-cation exchange chromatography or hydrophobic interaction chromatography); and combinations of differences in size and charge (1D or 2D electrophoresis). Immunoaffinity purification options include the use of monoclonal or polyclonal antibodies that specifically bind HLA proteins. Other protein affinity purification options include the use of proteins known to bind HLA, these include; CD8, which binds to the α3 domain of all HLA class I proteins; CD4, which binds to all HLA class II proteins; autologous T-cell receptors; and antigenic peptides that bind HLA with high affinity (computer simulation algorithms can be used to predict peptide/HLA binding characteristics). Any of these HLA high affinity protein variants can be immobilized on an insoluble solid support to produce an affinity matrix that can be used to capture HLA from a liquid biological sample. Suitable elution conditions will result in the concentration and purification (isolation) of the HLA content in the sample.

[324] In some embodiments, the implementation of the enrichment includes the enrichment of intact cells expressing complexes labeled with an affinity acceptor HLA peptides. In some embodiments, the method does not include lysing the cells prior to enrichment. In some embodiments, the method further comprises lysing one or more cells prior to enrichment. In some embodiments, enrichment comprises contacting an acceptor affinity peptide binding molecule with the acceptor affinity-tagged HLA peptide complexes, wherein the acceptor affinity peptide binding molecule specifically binds to the acceptor affinity peptide. In some cases, enrichment does not include the use of a tetrameric reagent.

Disease Specific Antigens

[325] In some embodiments, the size of at least one antigenic peptide molecule may include, but is not limited to, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, approximately 18, approximately 19, approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25, approximately 26, approximately 27, approximately 28, approximately 29, approximately 30, approximately 31, approximately 32, approximately 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50 , approximately 60, approximately 70, approximately but 80, about 90, about 100, about 110, about 120 or more amino acid residues, and any derived range.

[326] In some embodiments, antigenic peptide molecules are equal to or less than 50 amino acids. In some embodiments, antigenic peptide molecules are about 20 to about 30 amino acids in size. A longer peptide can be designed in several ways. For example, when HLA binding regions are predicted or known, the longer peptide may consist of either: individual binding peptides of 0-10 amino acids in length towards the N- and C-terminus of each respective gene product. The longer peptide may also consist of the concatenation of some or all of the binding peptides with extended sequences for each. Alternatively, when sequencing detects a long (>10 residue) epitope sequence present in diseased tissue (e.g., due to frameshift, readout, or intron inclusion resulting in a new peptide sequence), the longer peptide may consist of a range of new disease-specific amino acids. In both cases, the use of a longer peptide requires endogenous processing by professional antigen-presenting cells, such as dendritic cells, and may result in more efficient antigen presentation and induction of T cell responses. In some embodiments, the extended sequence is altered to improve the biochemical properties of the polypeptide (properties such as solubility or stability) or to increase the likelihood of efficient proteasome processing of the peptide.

[327] Antigenic peptides and polypeptides can bind the HLA protein. In some embodiments, antigenic peptides can bind an HLA protein with greater affinity than the corresponding native/wild type peptide. The antigenic peptide may have an IC50 of less than about 1000 nm, less than about 500 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, or less than about 50 nm. In some embodiments, the antigenic peptides do not induce an autoimmune response and/or induce immunological tolerance when administered to a subject.

[328] The present disclosure also provides compositions containing a plurality of antigenic peptides. Reference to antigenic peptides includes any suitable delivery method that may result in the introduction of a peptide into a cell of a subject (eg, a nucleic acid). In some embodiments, the composition contains at least 3 or more antigenic peptides. In some embodiments, the composition contains at least about 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, 35, 40, 45 or 50 different peptides. In some embodiments, the implementation of the composition contains at least 20 different peptides. In some embodiments, the implementation of the composition contains no more than 20 different peptides. According to the present disclosure, 2 or more different peptides can be obtained from the same polypeptide. For example, if an antigenic mutation encodes a polypeptide, two or more antigenic peptides can be obtained from the polypeptide. In one embodiment, two or more antigenic peptides derived from a polypeptide may comprise a block matrix that spans the polypeptide (eg, antigenic peptides may comprise a series of overlapping antigenic peptides that spans part or all of the polypeptide). Antigenic peptides can be obtained from any protein encoding a gene. Antigenic peptides can be obtained from human cancer mutations or from an infectious agent or autoimmune disease.

[329] Antigenic peptides, polypeptides and analogs can be further modified to contain additional chemical moieties that are not normally part of a protein. These derived fragments can improve solubility, biological half-life, protein absorption, or binding affinity. fragments can also reduce or eliminate any necessary side effects of proteins and the like. A review of these fragments can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000). For example, antigenic peptides and polypeptides having the desired activity can be modified, if necessary, to provide certain desired properties, such as improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate corresponding T cell. For example, the antigenic peptide and polypeptides can be subject to various changes, such as substitutions, either conservative or non-conservative, and such changes can provide certain advantages in their use, such as improved binding of the MHC. Such conservative substitutions may include replacing an amino acid residue with another amino acid residue that is biologically and/or chemically similar, such as one hydrophobic residue to another or one polar residue to another. The result of single amino acid substitutions can also be examined using D-amino acids. Such modifications can be made using well known peptide synthesis procedures as described in, for example, Merrifield, Science 232: 341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2d Ed. (1984).

[330] The antigenic peptide can also be modified by increasing or decreasing the amino acid sequence of the compound, for example by adding or removing amino acids. Antigenic peptides, polypeptides or analogs can also be modified by changing the order or composition of certain residues. One skilled in the art will appreciate that certain amino acid residues required for biological activity, such as those at critical contact sites or conserved residues, generally cannot be altered without adversely affecting biological activity. Non-critical amino acids need not be limited to those naturally occurring in proteins such as L-a-amino acids or their D-isomers, but may also include non-natural amino acids such as β-γ-δ-amino acids as well as many derivatives of L-a-amino acids.

[331] An antigenic peptide can be optimized using a range of peptides with single amino acid substitutions to determine the effect of electrostatic charge, hydrophobicity, etc. upon MHC binding. For example, a series of positively charged (eg, Lys or Arg) or negatively charged (eg, Glu) amino acid substitutions can be made along the length of the peptide, revealing different sensitivity profiles for different MHC molecules and T cell receptors. In addition, multiple substitutions can be used using small, relatively neutral moieties such as Ala, Gly, Pro, or similar residues. The substitutions may be homooligomers or heterooligomers. The amount and types of residues that are substituted or added depend on the necessary spacing between the major contact points and the particular functional characteristics required (eg, hydrophobicity versus hydrophilicity). Such substitutions can also increase the binding affinity for the MHC molecule or T-cell receptor relative to the affinity of the parent peptide. In any case, such substitutions should use amino acid residues or other molecular moieties chosen to avoid, for example, steric and charge interference that could interfere with binding. Amino acid substitutions usually consist of single residues. substitutions, deletions, insertions, or any combination thereof can be combined to produce the final peptide.

[332] The antigenic peptide can be modified to provide the desired properties. For example, the ability of peptides to induce CTL activity can be improved by binding to a sequence that contains at least one epitope capable of inducing a T helper cell response. In some embodiments, the immunogenic peptide/T-helper conjugates are linked by a spacer molecule. In some embodiments, the spacer contains relatively small, neutral molecules, such as amino acids or amino acid mimetics, that are substantially uncharged under physiological conditions. The spacers can be selected, for example, from Ala, Gly or other neutral non-polar amino acid spacers or neutral polar amino acids. It is understood that an optionally present spacer does not have to consist of the same residues and thus may be a hetero- or homo-oligomer. The antigenic peptide can be linked to the T helper peptide either directly or via a spacer at the amino or carboxy terminus of the peptide. The amino terminus of either the antigenic peptide or the T-helper peptide may be acylated. Exemplary T-helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria, colorosporozoitis 382-398 and 378-389.

Monoallelic HLA Cell Lines

[333] A monoallelic cell line expressing either one HLA class I allele, one pair of HLA class II alleles, or one HLA class I allele and one pair of HLA class II alleles can be obtained by transduction or transfection of an appropriate cell population with a polynucleic acid , such as a vector encoding one HLA allele. Suitable cell populations include, for example, class I deficient cell lines that express one HLA class I allele, class II deficient cell lines that express one pair of HLA class II alleles, or class I and class II deficient cell lines. in which one HLA class I and/or one pair of class II alleles are expressed. As an exemplary embodiment, the class I deficient B cell line is B721.221. However, one skilled in the art will appreciate that other populations of cells that are deficient in class I and/or class II can be obtained. An exemplary method for deleting/inactivating endogenous class I or class II genes involves CRISPR-Cas9 mediated genome editing, for example, in THP-1 cells. In some embodiments, the cell populations are professional antigen presenting cells such as macrophages, B cells, and dendritic cells. The cells may be B cells or dendritic cells. In some embodiments, the cells are tumor cells or cells of a tumor cell line. In some embodiments, the cells are cells isolated from a patient. In some embodiments, the cells contain the infectious agent, or a portion thereof. In some embodiments, the cell population contains at least 10 ⁷ cells. In some embodiments, the cell population is further modified, for example, by increasing or decreasing the expression and/or activity of at least one gene. In some embodiments, the gene encodes an element of the immunoproteasome. It is known that the immunoproteasome is involved in the processing of class I HLA-binding peptides and contains the LMP2 (β1i), MECL-1 (β2i), and LMP7 (β5i) subunits. The immunoproteasome can also be induced with interferon-gamma. Accordingly, in some embodiments, a population of cells may be contacted with one or more cytokines, growth factors, or other proteins. Cells can be stimulated with inflammatory cytokines such as interferon-gamma, IL-10, IL-6 and/or TNF-α. The cell population can also be exposed to various environmental conditions such as stress (heat stress, lack of oxygen, lack of glucose, DNA damaging agents, etc.). In some embodiments, cells are contacted with one or more chemotherapy drugs, radiation, targeted therapy, immunotherapy. Therefore, the methods disclosed herein can be used to study the effect of various genes or conditions on HLA peptide processing and presentation. In some embodiments, the conditions used are selected to match the condition of the patient for which the population of HLA peptides is to be identified.

[334] One HLA allele of the present invention can be encoded and expressed using a viral system (eg, adenovirus system, adeno-associated virus (AAV) vector, poxvirus, or lentivirus). Plasmids that can be used to deliver adeno-associated viruses, adenoviruses, and lentiviruses have been previously described (see, for example, US Pat. Among the vectors that can be used in the practice of the present disclosure, integration into the host cell genome is possible using retrovirus gene transfer techniques, which often result in long-term expression of the inserted transgene. In an exemplary embodiment, the retrovirus is a lentivirus. In addition, high transduction efficiency has been observed in many different cell types and target tissues. Retrovirus tropism can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. A retrovirus can also be engineered to conditionally express the inserted transgene so that the lentivirus only infects certain cell types. Cell type-specific promoters can be used to target expression in specific cell types. Lentiviral vectors are retroviral vectors (and therefore both lentiviral and retroviral vectors can be used in the practice of the present invention). In addition, lentiviral vectors are capable of transducing or infecting non-proliferating cells and typically produce high viral titers. An exemplary lentiviral vector that can be used to generate stable cell lines transduced to express HLA class I and class II is shown in FIG. 3.

[335] The choice of retroviral gene transfer system may depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with a packing capacity of up to 6-10 kb. foreign sequence. Minimal cis-acting LTRs are sufficient to replicate and package the vectors, which are then used to integrate the desired nucleic acid into the target cell to ensure consistent expression. Commonly used retroviral vectors that may be used in the practice of the present disclosure include those based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof. These (see e.g. Buchscher et al., (1992) J. Virol. 66:2731-2739; Johann et al., (1992) J. Virol. 66:1635-1640; Sommnerfelt et al., (1990 ) Virol 176:58-59; Wilson et al., (1998) J. Virol. 63:2374-2378; Miller et al., (1991) J. Virol. 65:2220-2224; PCT/US94/05700 ). In addition, a minimal non-primate lentiviral vector, such as the equine infectious anemia virus (EIAV) lentiviral vector, is useful in the practice of the present invention (see, for example, Balagaan, (2006) J Gene Med; 8:275-285, published online November 21, 2005 at Wiley InterScience DOI:10.1002/jgm.845). The vectors may have a cytomegalovirus (CMV) promoter that directs expression of the target gene. Accordingly, the present disclosure contemplates among the vector(s) used in the practice of the present disclosure: viral vectors, including retroviral vectors and lentiviral vectors.

[336] Any HLA allele can be expressed in a population of cells. In an exemplary embodiment, the HLA allele is an HLA class I allele. In some embodiments, the HLA class I allele is an HLA-A allele or an HLA-B allele. In some embodiments, the HLA allele is an HLA class II allele. HLA class I and class II allele sequences can be found in the IPD-IMGT/HLA database. Exemplary HLA alleles include, but are not limited to, HLA-A*02:01, HLA-B*14:02, HLA-A*23:01, HLA-E*01:01, HLA-DRB*01:01, HLA -DRB*01:02, HLA-DRB*11:01, HLA-DRB*15:01, and HLA-DRB*07:01.

[337] In some embodiments, the HLA allele is selected to match the genotype of interest. In some embodiments, the HLA allele is a mutant HLA allele, which may be a non-naturally occurring allele or a naturally occurring allele of the affected patient. The methods disclosed herein have the added benefit of identifying HLA binding peptides for HLA alleles associated with various disorders as well as alleles that are present at low frequency. Accordingly, in some embodiments, the HLA method allele is present at a frequency of less than 1% in a population, eg, in a Caucasoid population.

[338] In some embodiments, the nucleic acid sequence encoding the HLA allele further comprises an affinity acceptor tag that can be used for immunoaffinity purification of the HLA protein. Suitable labels are well known in the art. In some embodiments, the affinity acceptor tag is a polyhistidine tag, a polyhistidine-glycine tag, a polyarginine tag, a polyaspartate tag, a polycysteine tag, polyphenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, T7 protein 10 peptide tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep II tag, albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag, catarrhal virus tag fever (B-tag), calmodulin-binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline-binding domain (CBD) tag, chitin-binding domain (CBD) tag, cellulose-binding domain (CBP) tag, tag dihydrofolate reductase (DHFR), galactose-binding protein (GBP) tag, maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tag, influenza hemagglutinin tag na human (HA), horseradish peroxidase (HA) tag, NE tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, Luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin tag, E -tag, S-tag, SBP-tag, Softag 1, Softag 3, TC-tag, VSV-tag, Xpress-tag, Isopepeptag, SpyTag, SnoopTag, Profinity eXact-tag, Protein-C-tag, S1-tag, S-tag, tag carboxybiotin carrier protein (BCCP), green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, an HPC tag, a TrpE tag, a ubiquitin tag, a VSV-G epitope tag derived from the vesicular stomatitis viral glycoprotein, or a V5 tag derived from a small epitope (Pk) found on the P and V proteins of paramyxovirus simian virus 5 (SV5). In some embodiments, an affinity acceptor tag is an "epitope tag", which refers to a type of peptide tag that adds a recognizable epitope (antibody binding site) to an HLA protein to allow the corresponding antibody to bind, thus allowing identification or affinity purification of the labeled protein. A non-limiting example of an epitope tag is protein A or protein G that binds to IgG. In some embodiments, the affinity acceptor tags comprise a biotin acceptor peptide (BAP) or influenza hemagglutinin (HA) peptide sequence. One of ordinary skill in the art is aware of and may consider numerous other portions of the labels that are provided herein. Any peptide tag may be used, provided that it is capable of being expressed as an element of the HLA affinity acceptor-labeled peptide complex.

[339] The methods provided herein include isolating HLA-peptide complexes from cells transfected or transduced with universal IP HLA constructs. In some embodiments, complexes can be isolated using standard immunoprecipitation techniques known in the art with commercially available antibodies. Cells can be lysed first. HLA class I-peptide complexes can be isolated using HLA class I specific antibodies, such as the W6/32 antibody, while HLA class II-peptide complexes can be isolated using HLA class II specific antibodies, such as the M5/114 monoclonal antibody. ,15.2. In some embodiments, one (or pair) of HLA alleles is expressed as a peptide-tagged fusion protein and HLA-peptide complexes are isolated using binding molecules that recognize the peptide tags.

[340] The methods further include isolating peptides from said HLA-peptide complexes and peptide sequencing. The peptides are isolated from the complex by any method known to the person skilled in the art, such as acid elution. While any sequencing method can be used, in some embodiments methods using mass spectrometry, such as liquid chromatography-mass spectrometry (LC-MS or LC-MS/MS, or alternatively HPLC-MS or HPLC- MS/MS). These sequencing methods are well known to those skilled in the art and are discussed in Medzihradszky KF and Chalkley RJ. Weight Spectrom Rev. 2015 Jan-Feb;34(1):43-63.

[341] In some embodiments, the cell population expresses one or more endogenous HLA alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class II alleles, or an engineered cell population lacking both endogenous HLA class I alleles and endogenous HLA class II alleles. In some embodiments, the cell population includes cells that have been enriched or sorted, such as by fluorescent activated cell sorting (FACS). In some embodiments, fluorescent activated cell sorting (FACS) is used to sort the cell population. In some embodiments, the cell population is pre-sorted by FACS to express either class I or class II HLA, or both class I and class II HLA on the cell surface. For example, FACS can be used to sort a population of cells for cell surface expression of an HLA class I allele, an HLA class II allele, or a combination thereof.

Libraries of Constructs of Affinity-Acceptor-Labeled HLA

[342] The term "library" in the context of the present invention refers to a collection of nucleic acid molecules (circular or linear). In one embodiment, the library may contain multiple (ie, two or more) nucleic acid molecules, which may be from a common parent organism, organ, tissue, or cell. In some embodiments, a library represents all or a portion or a significant portion of the content of nucleic acids in an organism (a "genomic" library) or a set of nucleic acid molecules representing all or a portion or a significant portion of the expressed nucleic acid molecules (a cDNA library or segments derived from it) in cell, tissue, organ or organism. The library may also contain random sequences obtained by de novo synthesis, mutagenesis of one or more sequences, and the like. Such libraries may be contained in one or more vectors. The library of constructs labeled with an HLA acceptor acceptor in accordance with the present invention contains a DNA sequence encoding HLA allele elements, an acceptor affinity peptide, or a linker. Suitable molecular biology techniques can be found in Sambrook et al. (Molecular Cloning; A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989). Several methods have been described to facilitate the cloning of nucleic acid segments, for example, in the following references: Ferguson, J., et al., Gene 16: 191 (1981) and Hashimoto-Gotoh, T., et al., Gene 41: 125 (1986 ). Other terms within the scope of the present invention, used in the fields of recombinant nucleic acid technology and molecular and cellular biology, will be generally understood by a person skilled in the art.

[343] The various elements or domains of the recombinant HLA allele can be arranged in any order between the N-terminal and C-terminal ends of the recombinant HLA allele. An element or domain that is closer to the N-terminus of the recombinant polypeptide encoded by the recombinant HLA allele than to another element or domain is called the "N-terminus" of the other element or domain. Similarly, an element or domain that is closer to the C-terminus of a recombinant polypeptide encoded by a recombinant HLA allele than another element or domain is referred to as the "C-terminus" of the other element or domain. Unless expressly stated otherwise, the different elements or domains of a recombinant polypeptide encoded by a recombinant HLA allele need not be contiguous (ie without one or more intermediate elements or domains). In some embodiments, different elements or domains of a recombinant polypeptide encoded by a recombinant HLA allele may be contiguous.

[344] The recombinant polypeptide encoded by the recombinant HLA allele may contain one or more optional elements, for example, one or more linkers, peptide tags (eg, epitope tags), or protease recognition site(s). In some embodiments, the peptide tag is an affinity acceptor peptide. the linker has a relatively short set of amino acids that separates other elements or domains of the recombinant protein. In some embodiments, the implementation of the linker is from 1 to 100 amino acids in length; for example, length 5-75, 10-60, 15-50, 15-40, or 1-50 amino acids.

[345] Methods for expressing proteins in heterologous expression systems are well known in the art. Typically, a nucleic acid molecule encoding all or part of a protein of interest (such as an affinity acceptor-tagged recombinant HLA Class I or Class II peptide) is prepared using methods such as those described herein. The protein-coding nucleic acid sequence is cloned into an expression vector that is suitable for the particular host cell of interest using standard recombinant DNA procedures. Expression vectors include (among other elements) regulatory sequences (eg, promoters) that can be operably linked to a desired nucleic acid molecule encoding a protein to cause expression of that nucleic acid molecule in a host cell. Together, the regulatory sequences and the nucleic acid sequence encoding the protein constitute an "expression cassette". Expression vectors may also include an origin of replication, marker genes that provide phenotypic selection in transformed cells, one or more other promoters, and a polylinker region containing multiple restriction sites for insertion of heterologous nucleic acid sequences.

[346] Expression vectors used to express heterologous protein(s) in a variety of host cells are well known in the art, and some specific examples are provided herein. The host cell is transfected with the expression vector (or infected with a virus containing) the expression vector using any method suitable for the particular host cell. Such transfection methods are also well known in the art, and non-limiting examples of the methods are described herein. The transfected or transduced host cell is capable of expressing the protein encoded by the corresponding nucleic acid sequence in the expression cassette.

[347] In some embodiments, class I or class II HLA constructs contain an affinity acceptor tag and an affinity molecule at the N-terminus or C-terminus. In some embodiments, the class I or class II HLA constructs comprise at least one specific binding affinity acceptor tag and an affinity molecule. In some embodiments, the affinity acceptor tag is a polyhistidine tag, a polyhistidine-glycine tag, a polyarginine tag, a polyaspartate tag, a polycysteine tag, polyphenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, T7 protein 10 peptide tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep II tag, albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag, catarrhal virus tag fever (B-tag), calmodulin-binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline-binding domain (CBD) tag, chitin-binding domain (CBD) tag, cellulose-binding domain (CBP) tag, tag dihydrofolate reductase (DHFR), galactose-binding protein (GBP) tag, maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tag, influenza hemagglutinin tag na human (HA), horseradish peroxidase (HA) tag, NE tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, Luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin tag, E -tag, S-tag, SBP-tag, Softag 1, Softag 3, TC-tag, VSV-tag, Xpress-tag, Isopepeptag, SpyTag, SnoopTag, Profinity eXact-tag, Protein-C-tag, S1-tag, S-tag, tag carboxybiotin carrier protein (BCCP), green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, an HPC tag, a TrpE tag, a ubiquitin tag, a VSV-G epitope tag derived from the vesicular stomatitis viral glycoprotein, or a V5 tag derived from a small epitope (Pk) found on the P and V proteins of paramyxovirus simian virus 5 (SV5). In some embodiments, the affinity acceptor tag may contain multiple repeats of the tag sequence (eg, 3x polyhistidine tags, 3x FLAG tags). In some embodiments, the affinity acceptor tag may contain multiple repeats of the tag sequence (eg, 3x polyhistidine tags, 3x FLAG tags). In some embodiments, an affinity acceptor tag is an "epitope tag", which refers to a type of peptide tag that adds a recognizable epitope (antibody binding site) to an HLA protein to allow the corresponding antibody to bind, thus allowing identification or affinity purification of the labeled protein. A non-limiting example of an epitope tag is protein A or protein G that binds to IgG.

[348] In some embodiments, the affinity acceptor tags comprise a biotin acceptor peptide (BAP) or influenza hemagglutinin (HA) sequence. One of ordinary skill in the art is aware of and may consider numerous other portions of the labels that are provided herein. Any peptide tag may be used, provided that it is capable of being expressed as an element of the HLA affinity acceptor-labeled peptide complex.

[349] Affinity tags can be placed either at the N-terminus or at the C-terminus of the HLA allele. In some embodiments, an affinity tag is placed at the C-terminus of the HLA allele to allow localization of the HLA peptide to the cell surface of the vs. er. In some embodiments, an affinity tag is placed at the N-terminus of an HLA allele to allow isolation of a single HLA from cell lines expressing multiple endogenous HLA alleles. In yet another embodiment, an affinity tag is added to various β chains for immunoaffinity purification of specific HLA class II heterodimers.

[350] In some embodiments, a cleavage sequence, such as F2A or an internal ribosome entry site (IRES), can be placed between the α-chain and β2-microglobulin (class I) or between the α-chain and β-chain (class II). In some embodiments, the only HLA class I allele is HLA-A*02:01, HLA-A*23:01 and HLA-B*14:02 or HLA-E*01:01 and the HLA class II allele is HLA- DRB*01:01, HLA-DRB*01:02 and HLA-DRB*11:01, HLA-DRB*15:01 or HLA-DRB*07:01.

[351] Non-limiting illustrative constructs of affinity acceptor-tagged HLA are depicted in FIG. 2 , FIG. 6C and FIG. 7C .

Therapeutic Ways

[352] described personalized immunotherapy using tumor-specific peptides (Ott et al. Hematol. Oncol. Clin. N. Am. 28 (2014) 559-569). Efficient selection of which specific peptides to use as an immunogen requires the ability to predict which tumor-specific peptides will effectively bind to the HLA alleles present in a patient. One of the critical barriers to the development of therapeutic 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 (eg, inversions, translocations, deletions, missense mutations, splice site mutations, etc.) in malignant cells, represent the most tumor-specific class of antigens. Neoantigens have rarely been used in cancer vaccines or immunogenic compositions due to technical difficulties in identifying them, selecting optimized antigens, and generating neoantigens for use in vaccines or immunogenic compositions. These problems can be addressed by: identifying mutations in neoplasms/tumors that are present at the DNA level in the tumor but absent from the corresponding germline samples in a high proportion of subjects with cancer; analyzing the identified mutations with one or more peptide-MHC binding prediction algorithms to generate multiple T-cell neoantigen epitopes that are expressed in the neoplasm/tumor and that bind to a high proportion of the patient's HLA alleles; and synthesizing a plurality of neoantigenic peptides selected from the sets of all neoantigenic peptides and putative binding peptides for use in a cancer vaccine or immunogenic composition suitable for treating a high proportion of subjects with cancer (FIG. 18A and FIG. 18B).

[353] For example, translating peptide sequencing information into a therapeutic vaccine may include predicting mutant peptides that can bind to HLA molecules in a large proportion of individuals. Efficient selection of which particular mutations to use as an immunogen requires the ability to predict which mutant peptides will effectively bind to HLA alleles in a large proportion of patients. Recently, neural network-based learning approaches with validated binding and non-binding peptides have increased the accuracy of prediction algorithms for major HLA-A and -B alleles. However, even with the use of advanced neural network-based algorithms to encode peptide and HLA binding rules, several factors limit the ability to predict peptides presented on HLA alleles.

[354] For example, translating peptide sequencing information into a therapeutic vaccine may include formulating a long peptide multi-epitope vaccine drug. Targeting as many mutant epitopes as practicable exploits the tremendous power of the immune system, prevents evasion of immunological surveillance by down-modulating the immune targeted gene product, and compensates for the known inaccuracy of epitope prediction approaches. Synthetic peptides provide a useful means for efficiently generating a variety of immunogens and rapidly converting mutant epitope identification into an effective vaccine. Peptides can be easily synthesized chemically and easily purified using reagents free of bacterial or animal contaminants. The small size allows a clear focus on the mutant region of the protein, and also reduces irrelevant antigenic competition from other components (non-mutant protein or viral vector antigens).

[355] For example, translation of peptide sequencing information into a therapeutic vaccine may include combination with a strong vaccine adjunct. Effective vaccines may require a strong adjuvant to initiate an immune response. For example, poly-ICLC, an agonist of TLR3 and the RNA helicase domains of MDA5 and RIG3, has shown several useful properties as a vaccine adjunct. These properties include induction of local and systemic activation of immune cells in vivo, production of stimulatory chemokines and cytokines, and stimulation of DC antigen presentation. In addition, poly-ICLC can induce long-lasting CD4+ and CD8+ responses in humans. Importantly, striking similarities in the upregulation of transcriptional and signal transduction pathways were found in subjects vaccinated with poly-ICLC and in volunteers who received a highly potent, replication-competent yellow fever vaccine. In addition, >90% of ovarian carcinoma patients immunized with poly-ICLC in combination with the NYESO-1 peptide vaccine (in addition to Montanide) showed induction of CD4+ and CD8+ T cells, as well as antibody responses to the peptide in a recent phase 1 study. . At the same time, to date, poly-ICLC has been extensively tested in more than 25 clinical trials and has demonstrated a relatively favorable toxicity profile.

[356] In some embodiments, immunogenic peptides can be identified from cells of a subject with a disease or condition. In some embodiments, the implementation of immunogenic peptides may be specific for a subject with a disease or condition. In some embodiments, the immunogenic peptides can bind to an HLA that matches the HLA haplotype of the subject with the disease or condition.

[357] In some embodiments, the peptide library can be expressed in cells. In some embodiments, the cells contain peptides that need to be identified or characterized. In some embodiments, the peptides to be identified or characterized are endogenous peptides. In some embodiments, the peptides are exogenous peptides. For example, peptides to be identified or characterized can be expressed from multiple sequences encoding a peptide library.

[358] Prior to the disclosure of the present disclosure, most LC-MS/MS studies of the HLA peptidome used cells expressing multiple HLA molecules, requiring assignment of peptides of 1 of 6 class I alleles using pre-existing bioinformatic prediction tools or "deconvolution" (Bassani-Sternberg and Gfeller, 2016). Thus, peptides that do not closely match known motifs cannot be reported with certainty as linkers for a given HLA allele.

[359] Provided herein are methods for predicting peptides, such as mutant peptides, that can bind to HLA molecules of individuals. In some embodiments, the application provides methods for identifying, from a given set of antigen-containing peptides, the most appropriate peptides for preparing an immunogenic composition for a subject, said method comprising selecting, from a given set of peptides, a plurality of peptides capable of binding the HLA protein of the subject, wherein said ability to bind the HLA protein is determined by analyzing the sequence of the peptides using a machine that has been trained on databases of peptide sequences corresponding to specific HLA binding peptides for each of the HLA alleles of the specified subject. This document provides methods for identifying from a given set of antigen-containing peptides the most suitable peptides for obtaining an immunogenic composition for a subject, and this method includes the selection from a given set of peptides of a plurality of peptides determined to be capable of binding the HLA protein of the subject, while the ability to bind to the HLA protein determined by analyzing the sequence of the peptides on a machine that has been trained using a database of peptide sequences obtained using the methods described herein above. Thus, in some embodiments, the present disclosure provides methods for identifying a plurality of subject-specific peptides to produce a subject-specific immunogenic composition when the subject has a tumor and the subject-specific peptides are specific for the subject and the subject's tumor, said method comprising: sequencing a tumor sample of the subject and a non-tumor sample of the subject; determination based on nucleic acid sequencing: non-silent mutations present in the subject's cancer cell genome but not in the subject's normal tissue, and the subject's HLA genotype; and selecting, from the identified non-silent mutations, a plurality of subject-specific peptides, each having a different tumor epitope, which is an epitope specific to the subject's tumor, and each identified as capable of binding the subject's HLA protein, as determined by sequence analysis of the peptides obtained from non-silent mutations in the HLA binding prediction methods described herein.

[360] In some embodiments, provided herein is a method for characterizing HLA-peptide complexes specific to an individual.

[361] In some embodiments, a method for characterizing HLA-peptide complexes specific to an individual is used to develop an immunotherapeutic agent for an individual in need, such as a subject with a condition or disease.

[362] Provided herein is a method for providing anti-tumor immunity in a mammal, comprising administering to the mammal a polynucleic acid containing a sequence encoding a peptide identified according to the described method. Provided herein is a method for conferring anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a peptide having a peptide sequence identified according to the method described herein. Provided herein is a method for providing anti-tumor immunity in a mammal, comprising administering to the mammal a cell containing a peptide comprising a peptide sequence identified according to the method described herein. Provided herein is a method for providing antitumor immunity in a mammal, comprising administering to the mammal a cell containing a polynucleic acid containing a peptide coding sequence comprising a peptide sequence identified according to the method described herein. In some embodiments, the cell presents the peptide as an HLA-peptide complex.

[363] Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject a polynucleic acid containing a sequence encoding a peptide identified according to the method described herein. Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a peptide comprising a peptide sequence identified in accordance with the method described herein. Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject a cell containing a peptide comprising a peptide sequence identified according to the method described herein. Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject a cell containing a polynucleic acid containing a peptide coding sequence comprising a peptide sequence identified according to the method described herein. In some embodiments, the disease or disorder is cancer. In some embodiments, the method further comprises administering to the subject an immune checkpoint inhibitor.

[364] In some embodiments, disclosed herein are methods for developing an immunotherapeutic agent for an individual in need by characterizing HLA-peptide complexes, comprising: a) providing a population of cells derived from an individual in need, wherein one or more cells of the cell population contain a polynucleic acid containing a sequence encoding an allele of an affinity-acceptor-tagged HLA class I or class II, wherein the sequence encoding an affinity-acceptor-labelled HLA contains: i) a sequence encoding a recombinant HLA class I or class II allele operably linked to ii) a sequence encoding an affinity acceptor peptide; b) expressing the affinity acceptor-labeled HLA in at least one cell of one or more cells of the cell population, thereby forming complexes of the affinity acceptor-labeled HLA peptides in at least one cell; c) enrichment of complexes labeled with an affinity acceptor HLA peptides; characterization of HLA-peptide complexes specific to an individual in need thereof; and d) developing an immunotherapeutic agent based on the HLA-peptide complex, specific for the individual in need thereof; wherein the individual has a disease or condition.

[365] In some embodiments, the immunotherapeutic agent is a polynucleotide or peptide therapeutic agent.

[366] In some embodiments, the implementation of the method includes the introduction of one or more peptides in a population of cells. In some embodiments, the method includes contacting a population of cells with one or more peptides, or expressing one or more peptides in a population of cells. In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more nucleic acids encoding one or more peptides.

[367] In some embodiments, the implementation of the method includes the introduction of one or more specific for the patient HLA. In some embodiments, the implementation of the method includes the introduction of all patient-specific HLA. In some embodiments, patient-specific HLAs can be administered as a single allele. In some embodiments, a plurality of patient-specific HLAs may be administered. In some embodiments, the method includes developing an immunotherapeutic agent based on peptides identified in association with patient-specific HLA. In some embodiments, the cell population is obtained from an individual in need thereof.

[368] In some embodiments, the implementation of the method includes the expression of the library of peptides in a population of cells, thereby forming a library of complexes labeled with an affinity acceptor HLA peptides. In some embodiments, the method includes contacting the cell population with a peptide library or a library of peptide coding sequences, thereby forming a library of HLA affinity acceptor tagged peptide complexes. In some embodiments, the library is a library of peptides associated with a disease or condition. In some embodiments, the disease or condition is cancer or infection with an infectious agent or an autoimmune disease. In some embodiments, the method includes introducing the infectious agent or parts thereof into one or more cells of the cell population. In some embodiments, the method includes characterizing one or more peptides from HLA-peptide complexes specific to an individual in need thereof, optionally the peptides are derived from one or more target proteins of an infectious or autoimmune disease. In some embodiments, the method includes characterizing one or more peptide regions from one or more target proteins of an infectious agent or autoimmune disease. In some embodiments, the method includes identifying peptides from HLA-peptide complexes derived from an infectious agent or autoimmune disease.

[369] In some embodiments, the infectious agent is a pathogen. In some embodiments, the pathogen is a virus, bacterium, or parasite.

[370] In some embodiments, the virus is selected from the group consisting of: BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4, DENV-5), cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), adenovirus, human immunodeficiency virus (HIV), human T-cell lymphotrophic virus (HTLV-1), influenza virus, RSV, HPV, rabies, mumps, rubella, poliovirus, yellow fever, hepatitis A, hepatitis B, rotavirus, varicella-zoster virus, human papillomavirus (HPV), smallpox, shingles, and combinations thereof.

[371] In some embodiments, the bacterium is selected from the group consisting of: Klebsiella species, Tropheryma whipplei, Mycobacterium leprae, Mycobacterium lepromatosis, and Mycobacterium tuberculosis. In some embodiments, the bacterium is selected from the group consisting of: typhoid, pneumococcus, meningococcus, haemophilus B, anthrax, tetanus toxin, group B meningococcus, bcg, cholera, and combinations thereof.

[372] In some embodiments, the parasite is a helminth or a protozoan. In some embodiments, the parasitic organism is selected from the group consisting of: Leishmania species (e.g., L. major, L. infantum, L. braziliensis, L. donovani, L. chagasi, L. mexicana), Plasmodium species (e.g., P. falciparum, P. vivax, P. ovale, P. malariae), Trypanosoma cruzi, Ascaris lumbricoides, Trichuris trichiura, Necator americanus and Schistosoma species (S. mansoni, S. haematobium, S. japonicum).

[373] In some embodiments, the immunotherapeutic agent is an engineered receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), T cell receptor (TCR), or B cell receptor (BCR), adaptive T cell therapy (ACT), or a derivative thereof. In other aspects, the engineered receptor is a chimeric antigen receptor (CAR). In some aspects, the CAR is a first generation CAR. In other aspects, the CAR is a second generation CAR. In other aspects, the CAR is a third generation CAR.

[374] In some aspects, a CAR contains an extracellular portion, a transmembrane portion, and an intracellular portion. In some aspects, the intracellular portion contains at least one T-cell costimulatory domain. In some aspects, the T cell costimulatory domain is selected from the group consisting of CD27, CD28, TNFRS9 (4-1BB), TNFRSF4 (OX40), TNFRSF8 (CD30), CD40LG (CD40L), ICOS, ITGB2 (LFA-1), CD2 , CD7, KLRC2 (NKG2C), TNFRS18 (GITR), TNFRSF14 (HVEM), or any combination thereof.

[375] In some aspects, the engineered receptor binds the target. In some aspects, the binding is specific to the peptide identified in the method for characterizing HLA-peptide complexes specific to an individual suffering from a disease or condition.

[376] In some aspects, the immunotherapeutic agent is a cell, which is described in detail herein. In some aspects, the immunotherapeutic agent is a cell containing a receptor that specifically binds the peptide identified in the method for characterizing HLA-peptide complexes specific to an individual suffering from a disease or condition. In some aspects, the immunotherapeutic agent is a cell used in combination with the peptides/nucleic acids of the present invention. In some embodiments, the implementation of the cell is a cell of the patient. In some embodiments, the cell is a T cell. In some embodiments, the cell is a tumor-infiltrating lymphocyte.

[377] In some aspects, a subject with a condition or disease is treated based on the subject's T-cell receptor array. In some embodiments, the implementation of the antigen vaccine is selected based on the set of T-cell receptors of the subject. In some embodiments, the subject is treated with T cells expressing TCRs specific for an antigen or peptide identified using the methods described herein. In some embodiments, the subject is treated with an antigen or peptide identified using the methods described herein specific for a TCR, eg, specific for the subject's TCR. In some embodiments, the subject is treated with an antigen or peptide identified using the methods described herein that is specific for T-cells expressing a TCR, eg, specific for the subject's TCR. In some embodiments, the subject is treated with an antigen or peptide identified using the methods described herein that is specific for a subject-specific TCR.

[378] In some embodiments, an immunogenic antigenic composition or vaccine is selected based on the TCRs identified in the subject. In one embodiment, the identification of a set of T cells and testing in functional assays is used to determine an immunogenic composition or vaccine to administer to a subject with a condition or disease. In some embodiments, the immunogenic composition is an antigenic vaccine. In some embodiments, the implementation of the antigenic vaccine contains antigenic peptides specific for the subject. In some embodiments, antigenic peptides for inclusion in an antigen vaccine are selected based on the quantification of subject-specific TCRs that bind to antigens. In some embodiments, antigenic peptides are selected based on the binding affinity of the peptide to the TCR. In some embodiments, the implementation is selected based on a combination of both magnitude and binding affinity. For example, a TCR that binds strongly to an antigen in a functional assay, but that is underrepresented in a TCR array, may be a good candidate for an antigen vaccine because TCR-expressing T cells will be preferentially amplified.

[379] In some embodiments, antigens are selected for administration to a subject based on TCR binding. In some embodiments, T cells can be expanded, such as T cells in a subject with a disease or condition. Propagated T cells that express TCRs specific for an immunogenic antigenic peptide identified using the method described herein can be administered back to the subject. In some embodiments, suitable cells, such as PBMCs, are transduced or transfected with TCR expression polynucleotides specific for an immunogenic antigenic peptide identified using the method described herein and administered to a subject. T cells expressing TCRs specific for an immunogenic antigenic peptide identified using the method described herein can be expanded and administered back to the subject. In some embodiments, T cells can be expanded and administered to the subject that express TCRs specific for an immunogenic antigenic peptide identified using the method described herein that result in cytolytic activity when incubated with autologous diseased tissue. In some embodiments, T cells used in functional assays can be expanded and administered to a subject, resulting in binding to an immunogenic antigenic peptide identified using the method described herein. In some embodiments, TCRs that have been determined to bind to subject-specific immunogenic antigenic peptides identified using the method described herein can be expressed in T cells and administered to a subject.

[380] The methods described herein may include the adoptive transfer of cells of the immune system, such as T cells, specific for selected antigens, such as antigens associated with a tumor or pathogen. Various strategies can be used to genetically modify T cells by changing the specificity of the T cell receptor (TCR), for example, by introducing new TCR α and β chains with specificity for an immunogenic antigenic peptide identified using the method described herein (see ., например, патент США № 8697854; публикации патентов PCT: WO2003020763, WO2004033685, WO2004044004, WO2005114215, WO2006000830, WO2008038002, WO2008039818, WO2004074322, WO2005113595, WO2006125962, WO2013166321, WO2013039889, WO2014018863, WO2014083173; патент США № 8088379).

[381] Chimeric antigen receptors (CARs) can be used to generate immunoresponsive cells, such as T cells, specific for selected targets, such as immunogenic antigenic peptides identified using the method described herein, with a large variety of chimeric receptor constructs ( see, for example, US Pat. Alternate CAR designs can be characterized as related to later generations. First generation CARs typically consist of a single chain variable fragment of an antibody specific for an antigen, e.g., containing a VL linked to a VH of a particular antibody linked by a flexible linker, e.g., a CD8a hinge domain and a CD8a transmembrane domain, with transmembrane and intracellular signaling domains of either CD3ζ or FcRy or scFv-FcRy (see, for example, US Pat. No. 7,741,465; US Pat. No. 5,912,172; US Pat. No. 5,906,936). Second generation CARs contain intracellular domains of one or more co-stimulatory molecules such as CD28, OX40 (CD134) or 4-1BB (CD137) within an endodomain, e.g. scFv-CD28/OX40/4-lBB-CD3 (see e.g. patents US Nos. 8911993; 8916381; 8975071; 9101584; 9102760; 9102761). Third-generation CARs contain a combination of co-stimulatory endodomains such as the CD3C chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28 signaling domains, such as scFv-CD28-4- lBB-CD3C or scFv-CD28-OX40-CD3Q (see, for example, US Patent No. 8906682; US Patent No. 8399645; US Patent No. 5686281; PCT Publication No. WO2014134165; PCT Publication No. WO2012079000). In some embodiments, costimulation can be coordinated by expression of CARs in antigen-specific T cells chosen to activate and proliferate, for example, after antigen interaction on professional antigen-presenting cells upon costimulation. Additional engineered receptors can be provided on immunoresponsive cells, for example, to improve targeting of T cell attack and/or minimize side effects.

[382] Alternative methods such as protoplast fusion, lipofection, transfection, or electroporation can be used to transform immunoresponsive target cells. A wide variety of vectors can be used, such as retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated viral vectors, plasmids, or transposons such as the sleeping beauty transposon (see US Pat. Nos. 6,489,458; 7,148,203; 7,160,682; for the introduction of CARs, for example, using second generation antigen-specific CARs signaling through CD3ζ or CD28 or CD137. Viral vectors, for example, may include HIV, SV40, EBV, HSV or BPV vectors.

[383] Cells that are targeted for transformation may, for example, include T cells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, human embryonic stem cells, tumor infiltrating lymphocytes ( TIL) or a pluripotent stem cell from which lymphoid cells can be differentiated. T cells expressing the desired CAR, for example, can be selected by co-culture with γ-irradiated activating and proliferating cells (APCs) that co-express cancer antigen and costimulatory molecules. Engineered CAR T cells can be propagated, for example, by co-culturing on APC in the presence of soluble factors such as IL-2 and IL-21. This propagation, for example, can be carried out so as to provide CAR memory T cells (which, for example, are analyzed using a non-enzymatic digital matrix and/or multi-panel flow cytometry). Thus, it is possible to provide CAR T cells that have specific cytotoxic activity against antigen-bearing tumors (optionally in combination with the production of desired chemokines such as interferon-γ). CAR T cells of this type can be used, for example, in animal models, for example, to threaten tumor xenografts.

[384] Such approaches as set forth above can be adapted to provide methods of treating and/or increasing the survival of a subject having a disease, such as neoplasm or pathogen infection, for example, by administering an effective amount of immunoresponsive cells containing an antigen recognition receptor that binds a selected antigen, wherein the binding activates an immunoresponsive cell, thereby treating or preventing a disease (such as a neoplasm, pathogen infection, autoimmune disorder, or allogeneic graft reaction). The use of CAR T cell therapy may, for example, involve the administration of 10 ⁶ to 10 ⁹ cells/kg, with or without a course of lymphodepletion, for example, cyclophosphamide.

[385] To protect against possible adverse reactions, engineered immunoresponsive cells can be provided with a transgenic safety switch in the form of a transgene that makes the cells vulnerable to a specific signal. For example, the herpes simplex viral thymidine kinase (TK) gene can be used in this way, for example by introducing into allogeneic T lymphocytes used as donor lymphocyte infusions after stem cell transplantation. In such cells, administration of a nucleoside prodrug such as ganciclovir or acyclovir causes cell death. Alternative safety switch designs include inducible caspase 9, for example triggered by the introduction of a small molecular weight dimerizer that fuses two non-functional icasp9 molecules to form an active enzyme. A wide range of alternative approaches to control cell proliferation have been described (see, for example, US Patent Publication No. 20130071414; PCT Patent Publication WO2011146862; PCT Patent Publication WO2014011987; PCT Patent Application Publication WO2013040371). To further refine adoptive therapy, genome editing can be used to tailor immunoresponsive cells to alternative performance, such as providing CAR-edited T cells.

[386] Methods of cell therapy can also include the activation and expansion of T cells ex-vivo. In some embodiments, T cells can be activated prior to administration to a subject in need thereof. Examples of this type of treatment include the use of tumor infiltrating lymphocyte (TIL) cells (see US Pat. No. 5,126,132), cytotoxic T cells (see US Pat. No. 6,255,573 and US Pat. US No. 6251385) and various other preparations of lymphocytes (see US Patent No. 6194207; US Patent No. 5443983; US Patent No. 6040177; US Patent No. 5766920).

[387] An ex vivo activated T cell population may be in a state that maximally regulates the immune response to cancer, infectious diseases, or other disease states, such as an autoimmune disease. At least two signals can be delivered to T cells for activation. The first signal is usually delivered through the T cell receptor (TCR) on the surface of the T cell. The first TCR signal is usually triggered by the interaction of the TCR with peptide antigens expressed in combination with the MHC complex on the surface of an antigen presenting cell (APC). The second signal is usually delivered via costimulatory receptors on the surface of T cells. Costimulatory receptors are typically triggered by appropriate ligands or cytokines expressed on the surface of the APC.

[388] It is contemplated that T cells specific for the immunogenic antigenic peptides identified using the method described herein can be obtained and used in methods of treating or preventing a disease. In this regard, the present invention provides a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a cell population containing cells specific for immunogenic antigenic peptides identified using the method described herein in an amount effective to treat or prevent disease in the subject. In some embodiments, a method for treating or preventing a disease in a subject comprises administering to the subject a cell population enriched in disease-responsive T cells in an amount effective to treat or prevent cancer in a mammal. The cells may be cells that are allogeneic or autologous to the subject.

[389] The present disclosure further provides a method of inducing a disease-specific immune response in a subject, vaccinating against a disease, treating and/or alleviating a symptom of a disease in a subject by administering an antigenic peptide or vaccine to the subject.

[390] The peptide or composition of the invention may be administered in an amount sufficient to elicit a CTL response. The antigenic peptide or vaccine composition may be administered alone or in combination with other therapeutic agents. Illustrative therapeutic agents include, but are not limited to, a chemotherapeutic or biotherapeutic agent, radiation, or immunotherapy. Any suitable therapeutic treatment for a particular disease may be prescribed. Examples of chemotherapeutic and biotherapeutic agents include, but are not limited to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alfa, irinotecan, lansoprazole, levamisole, leucorovine, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, ondanseprazole, paclitaxel (Taxol®), pilocarpine, prochloroperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate. In addition, an anti-immunosuppressive or immunostimulatory agent may be additionally administered to the subject. For example, an anti-CTLA or anti-PD-1 or anti-PD-L1 antibody can be further administered to the subject.

[391] One skilled in the art can determine the amount of each peptide included in the vaccine composition and the administration schedule. For example, the peptide or variant thereof can be prepared for intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, intramuscular (i.m.) injection. Exemplary peptide injection methods include s.c, i.d. i.p. i.m. And i.v. Exemplary DNA injection methods include i.d. i.m. s.c, i.p. And i.v. Other methods of administering the vaccine composition are known to those skilled in the art.

[392] A pharmaceutical composition can be formulated such that the selection, number and/or amount of peptides present in the composition is/are disease and/or patient specific. For example, precise selection of peptides can be guided by expression profiles of parental proteins in a given tissue to avoid side effects. The choice may depend on the particular type of disease, the condition of the disease, previous treatment regimens, the patient's immune status, and the patient's HLA haplotype. In addition, the vaccine according to the present disclosure may contain individualized components in accordance with the personal needs of a particular patient. Examples include varying the amount of peptides according to the expression of a related antigen in a particular patient, unwanted side effects caused by personal allergies, or other treatments, and adjustments for secondary treatment after the first round or treatment regimen.

Obtaining Disease-Specific Antigens

[393] The present disclosure is based at least in part on the ability to present one or more disease-specific antigens to the patient's immune system. From this disclosure and from the knowledge of the art, one of skill in the art will appreciate that there are many ways to obtain such disease-specific antigens. Typically, such disease-specific antigens can be obtained either in vitro or in vivo. Disease-specific antigens can be produced in vitro as peptides or polypeptides, which can then be formulated into a vaccine or immunogenic composition and administered to a subject. As described herein in more detail, such in vitro production can occur by various means known to one of skill in the art, such as, for example, synthesis of a peptide or expression of a peptide/polypeptide from a DNA or RNA molecule in any of the various bacterial, eukaryotic, or viral recombinant expression systems followed by purification of the expressed peptide/polypeptide. Alternatively, disease-specific antigens can be generated in vivo by introducing molecules (eg, DNA, RNA, viral expression systems, and the like) that encode disease-specific antigens into a subject, followed by expression of the encoded disease-specific antigens. Also, this document further describes methods for producing antigens in vitro and in vivo, as they relate to pharmaceutical compositions and methods of delivery in therapy.

[394] In some embodiments, the present disclosure includes modified antigenic peptides. The modification may include a covalent chemical modification that does not change the primary amino acid sequence of the antigenic peptide itself. Modifications can create peptides with desired properties, such as prolonging in vivo half-life, increasing stability, decreasing clearance, altering immunogenicity or allergenicity, providing specific antibodies, targeting cells, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation. . Modifications to the antigenic peptide that can be made include, but are not limited to, carrier protein conjugation, ligand conjugation, antibody conjugation, PEGylation, polysialylation, HES attachment, recombinant PEG mimetics, Fc fusion, albumin fusion, nanoparticle attachment, encapsulation nanoparticles, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, addition of a surfactant, addition of amino acid mimetics, or addition of unnatural amino acids.

[395] Problems associated with short plasma half-life or susceptibility to protease degradation can be overcome by various modifications, including conjugation or linking of the polypeptide sequence to any of a variety of non-protein polymers, such as polyethylene glycol (PEG), polypropylene, glycol, or polyoxyalkylenes. (See, for example, usually via a binding moiety covalently linked to both a protein and a non-protein polymer, such as PEG). Such PEG-conjugated biomolecules have been shown to have clinically beneficial properties, including better physical and thermal stability, protection against enzymatic degradation, increased solubility, longer in vivo circulation half-life and reduced clearance, reduced immunogenicity and antigenicity, and reduced toxicity. .

[396] PEGs suitable for conjugation to a polypeptide sequence are generally water-soluble at room temperature and have the general formula R(O-CH ₂ -CH ₂ )nO-R, where R is hydrogen or a protecting group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a protecting group, it usually has 1 to 8 carbon atoms. The PEG conjugated to the polypeptide sequence may be linear or branched. The present disclosure discusses branched PEG derivatives, "star PEGs", and PEG multiarms.

[397] The present disclosure also contemplates conjugate compositions in which the PEGs have different n values and thus many different PEGs are present in specific ratios. For example, some compositions contain a mixture of conjugates where n=1, 2, 3 and 4. In some compositions, the percentage of conjugates where n=l is 18-25%, the percentage of conjugates where n=2 is 50-66%, the percentage of conjugates, where n=3, is 12-16%, and the percentage of conjugates, where n=4, is up to 5%. Such compositions can be prepared using reaction conditions and purification methods known in the art. For example, cation exchange chromatography can be used to separate conjugates and then identify a fraction that contains a conjugate having, for example, the required amount of PEG attached, clear of unmodified protein sequences, and conjugates having a different amount of PEG attached.

[398] PEG can be linked to a polypeptide of the present invention via a terminal reactive group ("spacer"). A spacer is, for example, a terminal reactive group that mediates a bond between free amino or carboxyl groups of one or more polypeptide sequences and polyethylene glycol. PEG having a spacer that can be coupled to a free amino group contains N-hydroxysuccinylimide-polyethylene glycol, which can be obtained by activating a succinic acid ester of polyethylene glycol with N-hydroxysuccinylimide. Another activated polyethylene glycol that can be coupled to a free amino group is 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine, which can be prepared by reacting polyethylene glycol monomethyl ether with cyanuric acid chloride. Activated polyethylene glycol, which is associated with a free carboxyl group, contains polyoxyethylenediamine.

[399] Conjugation of one or more polypeptide sequences of the present invention to a PEG having a spacer can be carried out in a variety of conventional ways. For example, the conjugation reaction can be carried out in solution at a pH of 5 to 10, at a temperature of 4° C. to room temperature, for 30 minutes to 20 hours using a reactant to protein molar ratio of 4:1 to 30:1. The reaction conditions can be chosen to direct the reaction to predominantly obtain the desired degree of substitution. In general, low temperature, low pH (eg pH=5) and short reaction time tend to reduce the amount of PEG attached, while high temperature, neutral to high pH (eg pH>7) and longer reaction time tend to increase the number of attached PEGs. Various methods known in the art can be used to terminate the reaction. In some embodiments, the implementation of the reaction is stopped by acidifying the reaction mixture and freezing, for example, at -20°C.

[400] The present disclosure also contemplates the use of PEG mimetics. Recombinant PEG mimetics have been developed that retain the properties of PEG (eg, increased serum half-life) while retaining some additional beneficial properties. For example, simple polypeptide chains (comprising, for example, Ala, Glu, Gly, Pro, Ser, and Thr) capable of forming an extended PEG-like conformation can be made recombinantly already fused to a peptide or protein drug of interest (e.g., Amunix's XTEN technology). Mountain View, CA). This eliminates the need for an additional conjugation step in the production process. In addition, established molecular biology techniques allow for the control of the side chain composition of polypeptide chains, thus optimizing immunogenicity and resulting properties.

[401] Glycosylation can affect the physical properties of proteins, and can also play an important role in the stability, secretion and intracellular localization of proteins. Proper glycosylation may be important for biological activity. In fact, some genes from eukaryotic organisms, when expressed in bacteria (eg E. coli), which lack the cellular processes to glycosylate proteins, produce proteins that are recovered with little or no activity due to lack of glycosylation. The addition of glycosylation sites can be done by changing the amino acid sequence. Altering the polypeptide can be accomplished, for example, by adding or replacing one or more serine or threonine residues (for O-linked glycosylation sites) or asparagine residues (for N-linked glycosylation sites). The structure of N-linked and O-linked oligosaccharides and sugar residues found in each type can be different. One type of sugar that is commonly found in both is N-acetylneuraminic acid (hereinafter referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, due to its negative charge, can impart acidic properties to the glycoprotein. Embodiments of the present invention include the creation and use of N-glycosylation variants.

[402] The polypeptide sequences of the present invention may optionally be altered by alterations at the DNA level, in particular by mutating the DNA encoding the polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids. Another way to increase the number of carbohydrate moieties in a polypeptide is to chemically or enzymatically link glycosides to the polypeptide. Removal of carbohydrates can be performed chemically or enzymatically, or by changing the codons encoding the amino acid residues that are glycosylated. Methods for chemical deglycosylation are known, and enzymatic cleavage of carbohydrate residues on polypeptides can be obtained by using various endo- and exoglycosidases.

[403] Additional suitable components and molecules for conjugation include, for example, molecules for targeting the lymphatic system thyroglobulin; albumins such as human serum albumin (HAS); tetanus toxoid; diphtheria toxoid; polyamino acids such as poly (D-lysine: D-glutamic acid); rotavirus VP6 polypeptides; influenza virus hemagglutinin, influenza virus nucleoprotein; keyhole limpet hemocyanin (KLH); and basic protein and surface antigen of hepatitis B virus; or any combination of the above.

[404] Fusion of albumin with one or more polypeptides according to the present invention, for example, can be achieved by genetic manipulation, so as to connect the DNA encoding HSA or a fragment thereof, with DNA encoding one or more polypeptide sequences. Thereafter, a suitable host can be transformed or transfected with the fused nucleotide sequences in the form of, for example, a suitable plasmid, to express the fusion polypeptide. Expression can be done in vitro, eg from prokaryotic or eukaryotic cells, or in vivo, eg from a transgenic organism. In some embodiments of the present invention, expression of the fusion protein is carried out in mammalian cell lines, such as CHO cell lines. Transformation is widely used herein to refer to the genetic change of a cell by direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its environment and uptake through the cell membrane (membranes). Transformation occurs naturally in some types of bacteria, but it can also be done artificially in other cells. In addition, albumin itself can be modified to extend its half-life from the blood. Fusion of the modified albumin with one or more polypeptides can be obtained by the genetic manipulation methods described above, or by chemical conjugation; the resulting fusion molecule has a half-life that is longer than the time of fusion with unmodified albumin. (See WO2011/051489). Several albumin binding strategies have been developed as an alternative to direct fusion, including binding of albumin via a conjugated fatty acid chain (acylation). Because serum albumin is a fatty acid transport protein, these natural ligands with albumin-binding activity have been used to extend the half-life of small protein therapeutics. For example, insulin detemir (LEVEMIR), an approved drug for the treatment of diabetes, contains a myristine chain conjugated to genetically modified insulin, resulting in a long-acting insulin analog.

[405] Another type of modification is the conjugation (e.g., linking) of one or more additional components or molecules at the N- and/or C-terminus of the polypeptide sequence, such as another protein (e.g., a protein having an amino acid sequence that is heterologous to the protein subject) or a carrier molecule. Thus, an exemplary polypeptide sequence may be provided as a conjugate to another component or molecule.

[406] Modification of the conjugate can result in a polypeptide sequence that retains activity with an additional or complementary function or the activity of a second molecule. For example, a polypeptide sequence can be conjugated to a molecule, e.g. to facilitate solubility, storage or shelf life in vivo or stability, reduced immunogenicity, sustained or controlled release in vivo, etc. Other functions or actions include a conjugate that reduces toxicity to to an unconjugated polypeptide sequence, a conjugate that targets a cell or organ type more effectively than an unconjugated polypeptide sequence, or a drug to further counteract the causes or consequences associated with a disorder or disease as set forth herein (e.g., diabetes).

[407] The polypeptide can also be conjugated to large, slowly metabolized macromolecules such as proteins; polysaccharides such as sepharose, agarose, cellulose, cellulose beads; polymeric amino acids such as polyglutamic acid, polylysine; amino acid copolymers; inactivated viral particles; inactivated bacterial toxins such as toxoid from diphtheria, tetanus, cholera, leukotoxin molecules; inactivated bacteria; and dendritic cells.

[408] Additional components and molecules candidates for conjugation include those suitable for isolation or purification. Specific non-limiting examples include binding molecules such as biotin (biotin-avidin specific binding pair), antibody, receptor, ligand, lectin, or molecules that contain a solid support, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips and membranes. To separate conjugates by charge difference, purification methods such as cation exchange chromatography can be used, which effectively separate conjugates of different molecular weights. The content of fractions obtained by cation exchange chromatography can be identified by molecular weight using conventional methods, such as mass spectroscopy, SDS-PAGE, or other known methods for separating molecular entities by molecular weight.

[409] In some embodiments, the amino or carboxyl terminus of a polypeptide sequence of the present invention can be fused to an immunoglobulin (eg, human Fc) Fc region to form a fusion conjugate (or fusion molecule). Fc fusions have been shown to increase the systemic half-life of biopharmaceuticals and thus the biopharmaceutical may require less frequent administration.

[410] Fc binds to the neonatal Fc receptor (FcRn) in the endothelial cells that line blood vessels, and once bound, the Fc fusion molecule is protected from degradation and re-released into the bloodstream, keeping the molecule in the bloodstream longer. This Fc binding is believed to be the mechanism by which endogenous IgG maintains its long plasma half-life. More recent Fc fusion technology couples a single copy of a biopharmaceutical to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical relative to traditional Fc fusions.

[411] The present disclosure provides for the use of other currently known or future-developed modifications of polypeptides to improve one or more properties. One such method for extending the circulating half-life, increasing stability, decreasing clearance, or altering the immunogenicity or allergenicity of a polypeptide of the present invention involves modifying polypeptide sequences by hesylation, which uses hydroxyethyl starch derivatives bound to other molecules in order to change the characteristics of the molecule. Various aspects of hesylation are described, for example, in US Patent Applications Nos. 2007/0134197 and 2006/0258607.

Synthesis of Peptides/Polypeptides In Vitro

[412] Proteins or peptides can be produced by any method known to those skilled in the art, including expression of proteins, polypeptides, or peptides using standard molecular biology techniques, isolation of proteins or peptides from natural sources, in vitro translation, or chemical synthesis of proteins or peptides. .

[413] Peptides can be easily synthesized chemically using reagents that do not contain bacterial or animal contaminants (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc.85:2149-54 , 1963). In some embodiments, antigenic peptides are prepared by (1) parallel solid phase synthesis on multichannel instruments using uniform synthesis and digestion conditions; (2) purification on an RP-HPLC column with distillation from the column; and rewashing, but not replacing, between peptides; followed by (3) analysis with a limited set of the most informative analyses. With regard to a set of peptides for an individual patient, a Good Manufacturing Practice (GMP) area can be defined, which requires that set-changing procedures be carried out only between peptide syntheses for different patients.

[414] Alternatively, a nucleic acid (eg, polynucleotide) encoding an antigenic peptide of the present invention can be used to produce an antigenic peptide in vitro. A polynucleotide may be, for example, DNA, cDNA, PNA, CNA, RNA, single- and/or double-stranded or native or stabilized forms of polynucleotides, such as, for example, polynucleotides with a phosphorothiate backbone, or combinations thereof, and may contain introns if they encode a peptide. In one embodiment, in vitro translation is used to produce the peptide. There are many exemplary systems that can be used by one of skill in the art (eg, Retic Lysate IVT kit, Life Technologies, Waltham, MA). It is also possible to obtain an expression vector capable of expressing the polypeptide. Expression vectors for various cell types are well known in the art and can be selected without undue experimentation. Typically, the DNA is inserted into an expression vector, such as a plasmid, in the correct orientation and in the correct reading frame for expression. If desired, the DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (eg, bacteria), although such controls are usually available in the expression vector. The vector is then introduced into host bacteria for cloning using standard techniques (see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

[415] Expression vectors containing isolated polynucleotides as well as host cells containing expression vectors are also contemplated. The antigenic peptides may be provided in the form of RNA or cDNA molecules encoding the antigenic peptides of interest. One or more antigenic peptides according to the invention may encode one expression vector.

[416] In some embodiments, the polynucleotides may comprise a coding sequence for a disease-specific antigenic peptide fused in the same reading frame to a polynucleotide that facilitates, for example, expression and/or secretion of the polypeptide from a host cell (e.g., a leader sequence , which functions as a secretory sequence to control the transport of the polypeptide out of the cell). A polypeptide having a leader sequence is a preprotein and may have a leader sequence that is cleaved by the host cell to form the mature form of the polypeptide.

[417] In some embodiments, the polynucleotides may contain a coding sequence for a disease-specific antigenic peptide fused in the same reading frame with a marker sequence, which allows, for example, to purify the encoded polypeptide, which can then be included in a personalized disease vaccine or an immunogenic composition. For example, the marker sequence may be a hexahistidine tag supplied by the pQE-9 vector to allow purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence may be a haemagglutinin (HA) tag derived from influenza hemagglutinin protein when a mammalian host is used (eg, COS-7 cells). Additional tags include, but are not limited to, calmodulin tags, FLAG tags, Myc tags, S tags, SBP tags, Softag 1 tags, Softag 3 tags, V5 tags, Xpress tags, Isopeptag tags, SpyTag, carboxybiotin carrier protein (BCCP) tags, GST tags, fluorescent protein tags (eg, green fluorescent protein tags), maltose binding protein tags, Nus tags, Strep tag, Thioredoxin tag, TC tag, Ty tag, and the like.

[418] In some embodiments, the polynucleotides may comprise a coding sequence for one or more disease-specific antigenic peptides fused in the same reading frame to create a single concatamerized antigenic peptide construct capable of generating multiple antigenic peptides.

[419] In some embodiments, isolated nucleic acid molecules can be provided having a nucleotide sequence that is 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 the polynucleotide encoding a disease-specific antigenic peptide according to the present invention.

[420] The isolated disease-specific antigenic peptides described herein can be prepared in vitro (eg, in a laboratory) by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding isolated polypeptide sequences and expressing these sequences in a suitable transformed host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized using site-directed mutagenesis to obtain its functional analogues. See, for example, Zoeller et al., Proc. Nat'l. Acad. sci. USA 81:5662-5066 (1984) and US patent. No. 4588585.

[421] In some embodiments, a DNA sequence encoding a polypeptide of interest can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and the selection of those codons that are preferred in the host cell in which the recombinant polypeptide of interest is being produced. Standard methods can be used to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, the complete amino acid sequence can be used to construct a back translated gene. In addition, a DNA oligomer containing a nucleotide sequence encoding a particular isolated polypeptide can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' ends for complementary assembly.

[422] Once assembled (e.g., by synthesis, site-directed mutagenesis, or other method), the polynucleotide sequences encoding the particular isolated polypeptide of interest are inserted into an expression vector and optionally operably linked to an expression control sequence suitable for expression of the protein in the desired host. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of the biologically active polypeptide in a suitable host. As is well known in the art, to obtain high levels of expression of the transfected gene in the host, the gene can be operably linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.

[423] Recombinant expression vectors can be used to amplify and express DNA encoding disease-specific antigenic peptides. Recombinant expression vectors are replicable DNA constructs that have synthetic or cDNA-derived DNA fragments encoding a disease-specific antigenic peptide or bioequivalent analogue operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. . A transcription unit typically contains a complex of (1) a genetic element or elements that play a regulatory role in gene expression, such as transcriptional promoters or enhancers, (2) a structural or coding sequence that is transcribed into an mRNA and translated into a protein, and (3) the corresponding sequences initiation and termination of transcription and translation, as detailed herein. Such regulatory elements may contain an operator sequence to direct transcription. Additionally, the ability to replicate in the host, usually conferred by the source of replication, and a selection gene can be included to facilitate recognition of transformants. Regions of DNA are functionally linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it directs the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is located to allow translation. In general, operably linked means adjacent, and in the case of secretory leaders, means adjacent and in reading frame. Structural elements for use in yeast expression systems include a leader sequence that allows extracellular secretion of the translated protein by the host cell. Alternatively, when the recombinant protein is expressed without a leader or transport sequence, it may contain an N-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to obtain the final product.

[424] Suitable expression vectors for eukaryotic hosts, especially mammals or humans, include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Suitable expression vectors for bacterial hosts include known bacterial plasmids such as plasmids from Escherichia coli including pCR 1, pBR322, pMB9 and their derivatives, broader host spectrum plasmids such as M13 and single stranded DNA filamentous phages.

[425] Suitable host cells for expression of the polypeptide include prokaryotes, yeast, insect cells or higher eukaryotes under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive organisms such as E. coli or bacilli. Higher eukaryotic cells include established mammalian cell lines. Cell-free translation systems can also be used. Suitable cloning and expression vectors for use with bacterial, fungal, yeast and mammalian host cells are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).

[426] Various mammalian or insect cell culture systems are also advantageously used to express the recombinant protein. Expression of recombinant proteins can be performed in mammalian cells because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include the COS-7 monkey kidney cell lines 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) cell lines, 293, HeLa and BHK. Mammalian expression vectors may contain non-transcribed elements such as an origin of replication, a suitable promoter and enhancer associated with the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences and 5' or 3' non-translated sequences such as essential ribosome binding sites, site polyadenylation, donor and acceptor splicing sites, and transcription termination sequences. Baculovirus systems for producing heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

[427] Proteins produced by the transformed host can be purified by any suitable method. Such standard methods include chromatography (eg, ion exchange, affinity and calibration column chromatography, and the like), centrifugation, differential solubility, or any other standard protein purification technique. Affinity tags such as hexahistidine, maltose-binding domain, influenza coat sequence, glutathione-S-transferase and the like can be attached to the protein to allow for easy purification by passing through an appropriate affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography. For example, supernatants from systems that secrete recombinant protein into the culture medium can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin, such as a matrix or substrate, having pendent diethylaminoethyl (DEAE) groups can be used. The matrices may be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers contain various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reverse phase high performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, such as silica gel having pendent methyl or other aliphatic groups, can be used to further purify the cancer stem cell Fc protein. The writing. Some or all of the above purification steps in various combinations can also be used to obtain a homogeneous recombinant protein.

[428] A recombinant protein produced in a bacterial culture can be isolated, for example, by initial extraction from cell pellets followed by one or more steps of concentration, salting out, water ion exchange, or size exclusion chromatography. High Performance Liquid Chromatography (HPLC) can be used for final purification steps. The microbial cells used to express the recombinant protein may be disrupted by any convenient means, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysing agents.

Synthesis of Peptides/Polypeptides In Vivo

[429] The present disclosure also contemplates the use of nucleic acid molecules as carriers for delivering antigenic peptides/polypeptides to a subject in need in vivo, in the form of, for example, DNA/RNA vaccines (see, for example, WO2012/159643 and WO2012/ 159754, incorporated herein by reference in their entirety).

[430] In some embodiments, antigens can be administered to a patient in need using a plasmid. These are plasmids that typically consist of a strong viral promoter to drive in vivo transcription and translation of the gene (or complementary DNA) of interest (Mor, et al., (1995). The Journal of Immunology 155 (4): 2039-2046). Intron A can sometimes be included to improve mRNA stability and hence increase protein expression (Leitner, et al. (1997). The Journal of Immunology 159 (12): 6112-6119). Plasmids also include a strong polyadenylation/transcriptional termination signal such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410; Robinson et al. , (2000) Adv. Virus Res. Advances in Virus Research 55: 1-74 Böhmet al., (1996) Journal of Immunological Methods 193 (1): 29-40. For the expression of more than one immunogen, or for the expression of an immunogen and an immunostimulatory protein, multicistronic vectors are sometimes constructed (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88).

[431] Plasmids can be introduced into animal tissues in various ways. The two most popular approaches are injection of DNA in saline using a standard hypodermic needle and gene gun delivery. A schematic description of the construction of the DNA vaccine plasmid and its subsequent delivery by these two methods to the host is illustrated in Scientific American (Weiner et al., (1999) Scientific American 281 (1): 34-41). Injection in saline is usually given intramuscularly (IM) in skeletal muscle or intradermally (ID), with delivery of DNA to extracellular spaces. This can be aided by electroporation by temporarily damaging muscle fibers with myotoxins such as bupivacaine; or with hypertonic saline or sucrose solutions (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410). Many factors can influence immune responses with this delivery method, including needle type, needle alignment, injection speed, injection volume, muscle type, and age, sex, and physiology of the injected animal (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410).

[432] Gene gun delivery, another widely used delivery method, ballistically accelerates plasmid DNA (pDNA), which is adsorbed on gold or tungsten microparticles, into target cells using pressurized helium as an accelerator (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410; Lewis et al., (1999) Advances in Virus Research (Academic Press) 54: 129-88).

[433] Alternative methods of delivery may include aerosol instillation of naked DNA on mucosal surfaces, such as those of the nose and lungs, (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88) and local introduction of pDNA into the mucous membrane of the eye and vagina (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88). Surface mucosal delivery is also achieved using cationic DNA liposome preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors. DNA or RNA can also be delivered to cells after slight mechanical disruption of the cell membrane, making the cells temporarily permeable. This slight mechanical disruption of the membrane can be achieved by gently pushing the 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.0118803 April 13, 2015).

[434] In some embodiments, a disease-specific vaccine or immunogenic composition may comprise separate DNA plasmids encoding, for example, one or more antigenic peptides/polypeptides as defined in accordance with the present invention. As discussed herein, the exact choice of expression vectors may depend on the peptide/polypeptides to be expressed and is within the skill of the ordinary person. The expected persistence of DNA constructs (eg, in episomal, non-replicating, non-integrated form in muscle cells) is expected to provide increased duration of protection.

[435] One or more antigenic peptides of the present invention can be encoded and expressed in vivo using a virus-based system (eg, adenovirus system, adeno-associated virus (AAV) vector, poxvirus, or lentivirus). In one embodiment, a vaccine against a disease or immunogenic composition for use in a human patient in need thereof may comprise a vector based on a virus, such as, for example, an adenovirus (see, for example, 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 15;207(2):240-7, incorporated herein by reference in its entirety). Plasmids that can be used to deliver adeno-associated viruses, adenoviruses, and lentiviruses have been previously described (see, for example, US Pat.

[436] The peptides and polypeptides of the invention may also be expressed by a vector, such as a nucleic acid molecule as discussed herein, such as an RNA or DNA plasmid, a viral vector such as a poxvirus, such as orthopoxvirus, avipoxvirus, or adenovirus, AAV, or lentivirus. . This approach involves using a vector to express the nucleotide sequences that encode the peptide of the present invention. When administered to an acutely or chronically infected host or an uninfected host, the vector expresses an immunogenic peptide and thereby elicits a host CTL response.

[437] Among the vectors that can be used in the practice of disclosure, integration into the genome of the host cell is possible using retrovirus gene transfer techniques, which often results in long-term expression of the inserted transgene. In some embodiments, the retrovirus is a lentivirus. In addition, high transduction efficiency has been observed in many different cell types and target tissues. Retrovirus tropism can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. A retrovirus can also be engineered to conditionally express the inserted transgene so that the lentivirus only infects certain cell types. Cell type-specific promoters can be used to target expression in specific cell types. Lentiviral vectors are retroviral vectors (and therefore both lentiviral and retroviral vectors can be used in the practice of disclosure). In addition, lentiviral vectors are capable of transducing or infecting non-dividing cells and typically produce high viral titers. Therefore, the choice of retroviral gene transfer system may depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with a packing capacity of up to 6-10 kb. foreign sequence. The minimal cis-acting LTRs are sufficient to replicate and package the vectors, which are then used to integrate the desired nucleic acid into the target cell to ensure consistent expression. Commonly used retroviral vectors that can be used in the practice of disclosure include those based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see for example Buchscher et al. .176:58-59; Wilson et al., (1998) J. Virol.63: 2374-2378; Miller et al. (1991) J. Virol.65: 2220-2224; PCT/US94/05700).

[438] Also useful in the practice of the disclosure is a minimal non-primate lentiviral vector, such as an equine infectious anemia virus (EIAV) lentiviral vector (see, for example, Balagaan, (2006) J Gene Med; 8: 275-285, published online November 21, 2005 at Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jgm.845). The vectors may have a cytomegalovirus (CMV) promoter that directs expression of the target gene. Accordingly, this disclosure relates to vector(s) useful in the practice of disclosure: viral vectors, including retroviral vectors and lentiviral vectors.

[439] Lentiviral vectors have been described as a treatment for Parkinson's disease, see, for example, US Patent Publications No. 20120295960 and US Patent Nos. 7,303,910 and 7,351,585. Lentiviral vectors have also been disclosed for delivery to the brain, see, for example, Patent Publications USA No. US20110293571; US20040013648, US20070025970, US20090111106 and US Patent No. US7259015. In another embodiment, lentiviral vectors are used to deliver the vectors to the brain of those being treated for a disease. With respect to lentiviral vector systems useful in the practice of disclosure, reference is made to US Pat. In one embodiment of the present invention, delivery is via a lentivirus. Zou et al. About 10 μl of recombinant lentivirus having a titer of 1×10 ⁹ transducing units (TU)/ml was injected using an intrathecal catheter. These types of dosages can be adapted or extrapolated for the use of a retroviral or lentiviral vector in the present disclosure. For transduction in tissues such as the brain, very small volumes must be used, so the viral preparation is concentrated by ultracentrifugation. You can use other methods of concentration, such as ultrafiltration or binding and elution from the matrix. In other embodiments, the amount of lentivirus administered may be ^1x105 or about ^1x105 plaque forming units (PFU), 5x105 or about 5x105 PFU, 1x106 or about 1x106 PFU, 5x106 or about 5x106 PFU, 1x107 or about 1x107 PFU, 5x107 or about 5x107 or about 5x107 or about 5x107 PFU, 1x108 or about 1x108 PFU, 5x108 or about 5x108 PFU, 1x109 or about 1x109 PFU, 5x109 or about 5x109 PFU, 1x1010 or about 1x1010 PFU or 5x1010 or about 5x1010 PFU as a single total dose for an average person weighing 75 kg, or it can be adjusted for weight and size and types of subject. A person skilled in the art can determine the appropriate dosage. Suitable doses for the virus can be determined empirically.

[440] Also useful in the practice of disclosure is an adenoviral vector. One advantage is the ability of recombinant adenoviruses to efficiently transfer and express recombinant genes in various mammalian cells and tissues in vitro and in vivo, resulting in high expression of transferred nucleic acids. In addition, the ability to productively infect resting cells expands the possibilities of using recombinant adenoviral vectors. In addition, high levels of expression ensure that nucleic acid products are expressed to levels sufficient to generate an immune response (see, for example, US Pat. No. 7,029,848, incorporated herein by reference). With regard to adenoviral vectors useful in the practice of the disclosure, mention should be made of US Pat. No. 6,955,808. The adenovirus vector used may be selected from the group consisting of Ad5, Ad35, Ad11, C6 and C7 vectors. The genome sequence of adenovirus 5 ("Ad5") 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; contents of which are included in present description as a reference). Ad35 vectors are described in US Pat. Nos. 6,974,695, 6,913,922 and 6,869,794. Ad11 vectors are described in US Pat. 6537594; 6309647; 6265189; 6156567; 6090393; 5,942,235 and 5,833,975. C7 vectors are described in US Pat. No. 6,277,558. Adenovirus vectors with E1 defective or deletion, E3 defective or deletion, or E4 defective or deletion can also be used. Some adenoviruses with mutations in the E1 region have an improved margin of safety because E1-defective adenovirus mutants are replication-defective in non-permissive cells, or at least severely attenuated. Adenoviruses having mutations in the E3 region may have enhanced immunogenicity by disrupting the mechanism by which adenovirus suppresses MHC class I molecules. Adenoviruses having E4 mutations may have reduced adenoviral vector immunogenicity due to suppression of late gene expression. Such vectors can be especially useful when re-vaccination using the same vector is needed. Adenovirus vectors with deleted or mutated E1, E3, E4, E1 and E3, as well as E1 and E4, can be used in accordance with the present disclosure. In addition, in accordance with the present disclosure, it is also possible to use "gutted" adenoviral vectors in which all viral genes have been removed. Such vectors require a helper virus for replication and require a special human cell line 293 expressing both E1a and Cre, a condition that does not exist in the natural environment. Such "gutted" vectors are non-immunogenic and therefore the vectors can be inoculated several times for re-vaccination. Gutted adenoviral vectors can be used to introduce heterologous inserts/genes, such as the transgenes of the present invention, and can even be used to co-deliver a large number of heterologous inserts/genes. In some embodiments, delivery is via adenovirus, which may be in a single booster dose. In some embodiments, the adenovirus is delivered in multiple doses. In terms of in vivo delivery, AAV has an advantage over other viral vectors due to low toxicity and low insertional mutagenesis because it does not integrate into the host genome. AAV has a packaging limit of 4.5 or 4.75 KB. Constructs larger than 4.5 or 4.75 kb result in a significant reduction in virus production. There are many promoters that can be used to direct the expression of nucleic acid molecules. The AAV ITR can serve as a promoter and is preferred to eliminate the need for an additional promoter element. The following promoters can be used for ubiquitous expression: CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chains, etc. For expression in the brain, the following promoters can be used: SynapsinI for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. Promoters used to direct RNA synthesis may include: Pol III promoters such as U6 or H1. The use of the Pol II promoter and intron cassettes can be used to express guide RNA (gRNA). With respect to AAV vectors useful in the practice of disclosure, mention should be made of US Pat. As for AAV, AAV can be AAV1, AAV2, AAV5, or any combination thereof. You can select AAV in relation to the target cells; for example, AAV 1, 2, 5 serotypes or AAV1, AAV2, AAV5 hybrid capsid or any combination thereof can be selected to target brain cells or neurons; and for targeting heart tissue, AAV4 can be selected. AAV8 is useful for liver delivery. In some embodiments, delivery is via AAV. Dosage may be adjusted to balance therapeutic benefit against side effects.

[441] In some embodiments, effective activation of a cellular immune response for a disease vaccine or immunogenic composition can be achieved by expressing appropriate antigens in the vaccine or immunogenic composition in a non-pathogenic microorganism. Well-known examples of such microorganisms are Mycobacterium bovis BCG, Salmonella and Pseudomona (see US Pat. No. 6,991,797, incorporated herein by reference in its entirety).

[442] In some embodiments, the variola virus is used in a disease vaccine or immunogenic composition. It includes orthopoxvirus, avipoxvirus, vaccinia, MVA, NYVAC, canaripox, ALVAC, canarypox, TROVAC, etc. (See, for example, Verardi et al., Hum Vaccin Immunother. 2012 Jul; 8 (7): 961 -70 and Moss, Vaccine 2013; 31 (39): 4220-4222). Poxvirus expression vectors were described in 1982 and quickly became widely used for vaccine development, as well as for research in many areas. The advantages of vectors include simple construction, the ability to accommodate large amounts of foreign DNA, and high levels of expression. Information regarding poxviruses that can be used in disclosure practice, such as poxviruses of the subfamily Chordopoxvirinae (vertebrate poxviruses), e.g. orthopoxviruses and avipoxviruses, e.g. vaccinia virus (e.g. Wyeth strain, WR strain (e.g. ATCC® VR-1354) , strain Copenhagen, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypox virus (e.g. strain Wheatley C93, ALVAC), avian pox virus (e.g. strain FP9, strain Webster, TROVAC), pox turtledoves, pigeon pox, quail pox and raccoon pox, among others, their synthetic or non-naturally occurring recombinants, their use and methods for preparing and using such recombinants can be found in the scientific and patent literature.

[443] In some embodiments, a vaccinia virus is used in the disease vaccine or immunogenic composition to express the antigen. (Rolph et al., Recombinant viruses as vaccines and immunological tools. Curr Opin Immunol 9:517-524, 1997). The recombinant vaccinia virus is able to replicate in the cytoplasm of an infected host cell and therefore the polypeptide of interest can induce an immune response. In addition, poxviruses are widely used as vaccine vectors or immunogenic compositions due to their ability to target encoded antigens to process the class I major histocompatibility complex pathway by directly infecting immune cells, in particular antigen presenting cells, but also due to their ability to have adjuvant properties. .

[444] In some embodiments, ALVAC is used as a vector in a disease vaccine or immunogenic composition. ALVAC is a canarypox virus that can be modified to express foreign transgenes and has been used as a vaccination method against prokaryotic and eukaryotic antigens (Horig H, Lee DS, Conkright W, et al. Phase I clinical trial of a recombinant canarypoxvirus (ALVAC ) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule Cancer Immunol Immunother 2000;49:504-14; von Mehren M, Arlen P, Tsang KY, et al. both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas Clin Cancer Res 2000;6:2219-28;Musey L, Ding Y, Elizaga M, et al.Vaccination against HIV-1 administered intramuscularly can induce both systemic and mucosal T-cell immunity in people not infected with HIV-1.J Immunol 2003;171:1094-101;Paoletti E. Applications of pox virus vectors to vaccination: a n update. Proc Natl Acad Sci U S A 1996; 93:11349-53; US patent No. 7255862). In a phase I clinical trial, ALVAC virus expressing the CEA tumor antigen demonstrated an excellent safety profile and resulted in increased CEA-specific T cell responses in selected patients; objective clinical responses, however, were not observed (Marshall JL, Hawkins MJ, Tsang KY, et al. Phase I study in cancer patients of a replication-defective avipox recombinant vaccine that expresses human carcinoembryonic antigen. J Clin Oncol 1999; 17:332- 7).

[445] In some embodiments, a modified Vaccinia Ankara (MVA) virus can be used as a viral vector for an antigenic vaccine or immunogenic composition. MVA is a member of the orthopoxvirus family and about 570 serial passages have been made on chick embryo fibroblasts of the Ankara strain of vaccinia virus (CVA) (for a review, see Mayr, A., et al., Infection 3, 6-14, 1975). As a result of these passages, the resulting MVA virus contains 31 kb less genomic information compared to CVA and is highly restricted by host cells (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038, 1991). MVA is characterized by extreme attenuation, namely reduced virulence or infectivity, but still has excellent immunogenicity. When tested in various animal models, MVA has been shown to be avirulent, even in immunosuppressed individuals. In addition, MVA-BN®-HER2 is a potential immunotherapy developed for the treatment of HER-2 positive breast cancer and is currently in clinical trials. (Mandl et al., Cancer Immunol Immunother. Jan 2012; 61(1): 19-29). Methods for the preparation and use of recombinant MVA are described (eg, see US Pat. Nos. 8,309,098 and 5,185,146 incorporated herein in their entirety).

[446] In some embodiments, the recombinant viral particles of the vaccine or immunogenic composition are administered to patients in need thereof.

[447] Provided herein is a method for developing a therapeutic agent for a subject with a disease or condition, comprising providing a population of cells derived from a subject with the disease or condition, expressing in one or more cells of the cell population an allele labeled with an HLA class I or class II affinity acceptor by introduction into one or more cells of a polynucleic acid encoding a sequence containing: a sequence encoding an allele of recombinant HLA class I or class II, operably linked to a sequence encoding an affinity acceptor peptide, thereby forming complexes of affinity acceptor-labeled HLA peptides in one or more cells; enrichment and characterization of complexes labeled with an affinity acceptor HLA-peptides; and, optionally, designing a therapeutic agent based on the characteristic obtained.

[448] Provided herein is a method for identifying at least one subject-specific immunogenic antigen and preparing a subject-specific immunogenic composition that contains at least one subject-specific immunogenic antigen, wherein the subject has a disease and at least one subject-specific subject, the immunogenic antigen is specific for the subject and the disease of the subject, and the method includes: providing a population of cells obtained from a subject with a disease or condition, expression in one or more cells of the cell population in the subject of an allele labeled with an HLA class I or class II affinity acceptor by introducing into one or more cells of a polynucleic acid encoding a sequence containing: a sequence encoding an allele of recombinant HLA class I or class II, operably linked to a sequence encoding an affinity acceptor peptide, thereby forming sword complexes affinity acceptor-enhanced HLA peptides in one or more cells; enrichment of complexes labeled with an affinity acceptor HLA peptides from one or more cells; identifying an immunogenic peptide from the enriched complexes labeled with an affinity acceptor HLA peptides that is specific for the subject and the subject's disease; and formulating a subject-specific immunogenic composition based on one or more subject-specific immunogenic peptides identified.

[449] In some embodiments, the therapeutic or subject-specific immunogenic composition comprises a peptide from enriched complexes of affinity acceptor labeled HLA peptides or a polynucleotide encoding a polypeptide from enriched complexes of affinity acceptor labeled HLA peptides.

[450] In some embodiments, the therapeutic or subject-specific immunogenic composition comprises a T cell expressing a T cell receptor (TCR) that specifically binds to a polypeptide from enriched complexes of HLA affinity acceptor tagged peptides. In some embodiments, the subject-specific immunogenic composition comprises a chimeric antigen receptor (CAR), a T cell expressing a receptor that specifically binds to a polypeptide from enriched complexes of HLA affinity acceptor tagged peptides. In some embodiments, the method further comprises administering to the subject another therapeutic agent, optionally an immune checkpoint inhibitor. In some embodiments, the method further comprises administering to the subject an adjuvant, optionally a poly-ICLC.

[451] In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, the disease or disorder is an infection. In some embodiments, the infection is infection with an infectious agent. In some embodiments, the infectious agent is a pathogen, virus, bacterium, or parasite. In some embodiments, the virus is selected from the group consisting of: BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4, DENV-5), Cytomegalovirus (CMV), Hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), adenovirus, human immunodeficiency virus (HIV), human T-cell lymphotrophic virus (HTLV-1), influenza virus, RSV, HPV, rabies, virus mumps, rubella, poliovirus, yellow fever, hepatitis A, hepatitis B, rotavirus, varicella-zoster virus, human papillomavirus (HPV), smallpox, shingles, and any combination thereof. In some embodiments, the bacterium is selected from the group consisting of: Klebsiella spp., Tropheryma whipplei, Mycobacterium leprae, Mycobacterium lepromatosis, and Mycobacterium tuberculosis, typhoid, pneumococcus, meningococcus, haemophilus B, anthrax, tetanus toxin, group B meningococcus, bcg, cholera, and their combinations. In some embodiments, the parasite is a helminth or a protozoan. In some embodiments, the parasitic organism is selected from the group consisting of: Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Schistosoma spp., and any combination thereof.

[452] Provided herein is a method for developing a therapeutic agent for a subject with a disease or condition, comprising: providing a cell population, wherein one or more cells of the cell population comprise a polynucleic acid containing a sequence encoding at least two alleles of an affine acceptor-tagged HLA class I or class II, wherein the sequence encoding at least two class I or class II HLA labeled with an affinity acceptor contains a first recombinant sequence containing a sequence encoding the first class I or class II HLA allele operably linked to the sequence encoding the first affinity peptide - acceptor; and a second recombinant sequence containing a sequence encoding a second HLA class I or class II allele operably linked to a sequence encoding a second acceptor affinity peptide; expressing at least two affinity acceptor-labeled HLA peptides in at least one cell of one or more cells of the cell population, thereby forming complexes of the affinity acceptor-labeled HLA peptides in at least one cell; enrichment of complexes labeled with an affinity acceptor HLA-peptides; and identification of the peptide from the enriched complexes labeled with an affinity acceptor HLA peptides; and formulating an immunogenic composition based on one or more of the identified peptides, wherein the alleles of the first and second recombinant HLA class I or class II match the HLA haplotype of the subject.

[453] In some embodiments, the subject has a disease or condition. In some embodiments, the first allele of the recombinant HLA class I or class II is different from the allele of the second recombinant HLA class I or class II. In some embodiments, the first acceptor affinity peptide is the same as the second acceptor affinity peptide. In some embodiments, the method includes characterizing the peptide associated with the first and/or second complexes of affinity acceptor-labeled HLA peptides by enrichment. In some embodiments, the at least two alleles of class I or class II affinity-acceptor-tagged HLA include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 HLA class I and/or class II alleles. In some embodiments, the implementation of the first and/or second complexes labeled with an affinity acceptor HLA peptides contain a transmembrane domain. In some embodiments, the implementation of the first and/or second complexes labeled with an affinity acceptor HLA peptides contain an intracellular domain. In some embodiments, the implementation of the first and/or second complexes labeled with an affinity acceptor HLA peptides are not excreted. In some embodiments, the first and/or second complexes of the affinity acceptor-tagged HLA peptides are incorporated into the cell membrane upon expression. In some embodiments, the first and/or second complexes of the HLA acceptor affinity labeled peptides are insoluble complexes of the HLA acceptor affinity labeled peptides. In some embodiments, the method further comprises creating a database of HLA allele-specific peptides. In some embodiments, the implementation of the method includes the introduction of one or more exogenous peptides in a population of cells. In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more exogenous peptides or the expression of one or more exogenous peptides in a population of cells. In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more nucleic acids encoding one or more exogenous peptides. In some embodiments, one or more nucleic acids encoding one or more peptides is DNA. In some embodiments, one or more nucleic acids encoding one or more peptides is RNA, optionally the RNA is mRNA. In some embodiments, enrichment does not include the use of a tetrameric reagent. In some embodiments, the method includes determining the sequence of the peptide or portion thereof associated with the first and/or second complex of the enriched HLA affinity acceptor-labeled peptide. In some embodiments, the determination includes biochemical analysis, mass spectrometric analysis, MS analysis, MS/MS analysis, LC-MS/MS analysis, or a combination thereof. In some embodiments, the method includes evaluating the binding affinity or stability of the peptide or portion thereof associated with the first and/or second complex of the affinity acceptor-tagged HLA peptide as a result of enrichment.

[454] In some embodiments, the implementation of the method includes determining whether the peptide or part thereof associated with the first and/or second complex affinity-labeled HLA acceptor peptide as a result of enrichment, one or more mutations. In some embodiments, the method includes evaluating the binding of peptides to HLA molecules in the first and/or second complex of an HLA affinity acceptor-labeled peptide.

[455] In some embodiments, the implementation of the method includes the expression of the library of peptides in a population of cells, thereby forming a library of complexes labeled with an affinity acceptor HLA peptides. In some embodiments, the method includes contacting the cell population with a peptide library or a library of peptide coding sequences, thereby forming a library of HLA affinity acceptor-labeled peptide complexes. In some embodiments, the library is a library of peptides associated with a disease or condition. In some embodiments, the disease or condition is cancer or infection with an infectious agent.

[456] In some embodiments, the implementation of the method includes the introduction of the infectious agent or parts thereof in one or more cells of the population of cells. In some embodiments, the method includes characterizing one or more peptides from the first and/or second HLA-peptide complexes, optionally the peptides are derived from one or more target proteins of the infectious agent. In some embodiments, the method includes characterizing one or more peptide regions from one or more target proteins of the infectious agent. In some embodiments, the method includes identifying peptides from the first and/or second HLA-peptide complexes derived from the infectious agent.

[457] In some embodiments, the cell population is derived from a biological sample in a subject with a disease or condition. In some embodiments, the cell population is a cell line. In some embodiments, the cell population is a primary cell population.

[458] In some embodiments, the peptide from the first and/or second complex, the affinity acceptor-tagged HLA peptide, is capable of activating a T cell in a subject when presented by an antigen presenting cell. In some embodiments, the method includes comparing HLA-peptide complexes from diseased cells with HLA-peptide complexes from an unaffected cell.

[459] In some embodiments, the method further comprises isolating the peptides from the first and/or second complexes of the affinity acceptor-labeled HLA peptides prior to identification.

[460] In some embodiments, the implementation of the cell population is a population of cells with low expression of class I or class II cell surface HLA. In some embodiments, a population of cells expresses one or more endogenous HLA alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles and endogenous HLA class II alleles. In some embodiments, the cell population has a knockout of one or more HLA class I alleles. In some embodiments, the cell population has a knockout of one or more HLA class II alleles. In some embodiments, a population of cells has a knockout of all HLA class I alleles.

[461] In some embodiments, a population of cells has a knockout of all HLA class II alleles. In some embodiments, a population of cells has a knockout of all HLA class I alleles and a knockout of all HLA class II alleles.

[462] In some embodiments, a sequence encoding at least two alleles of an affinity-acceptor-tagged class I or class II HLA encodes a class I HLA. In some embodiments, the Class I HLA is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the first allele of the recombinant HLA class I or class II is the first allele of HLA class I, and the second allele of the recombinant HLA class I or class II is the second allele of HLA class I. In some embodiments, a sequence encoding at least two alleles of an affinity-acceptor-tagged class I or class II HLA encodes a class II HLA. In some embodiments, the HLA class II is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP. In some embodiments, the HLA class II comprises an HLA class II α chain, an HLA class II β chain, or a combination thereof. In some embodiments, the first allele of the recombinant HLA class I or class II is the first allele of HLA class II, and the second allele of the recombinant HLA class I or class II is the second allele of HLA class II.

[463] In some embodiments, each of the first sequence and the second sequence is operably linked. In some embodiments, the first sequence and the second sequence are in different polynucleotide molecules.

[464] In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to a sequence that encodes the extracellular portion of the first and/or second HLA class I or class II allele. In some embodiments, the first and/or second encoded acceptor affinity peptide is extracellularly expressed. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to the N-terminus of the sequence encoding the first and/or second HLA class I or class II allele.

[465] In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to a sequence that encodes the intracellular portion of the first and/or second HLA class I or class II allele. In some embodiments, the encoded first and/or second acceptor affinity peptide is expressed intracellularly. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to the C-terminus of the sequence encoding the first and/or second HLA class I or class II allele. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to the sequence encoding the first and/or second HLA class I or class II allele via a linker.

[466] In some embodiments, enrichment includes enrichment of intact cells expressing the first and/or second complexes of affinity acceptor-tagged HLA peptides. In some embodiments, the method does not include lysing the cells prior to enrichment. In some embodiments, the method further comprises lysing one or more cells prior to enrichment.

[467] In some embodiments, enrichment comprises contacting an acceptor affinity peptide binding molecule with a first and/or second complexes of affinity acceptor-labeled HLA peptides, wherein the acceptor affinity peptide binding molecule specifically binds to the first and/or second affinity acceptor peptide. In some embodiments, the first and/or second affinity acceptor peptide comprises a tag sequence comprising a biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine-glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine, c- tag myc, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep tag II , albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus (B-tag), calmodulin-binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline-binding domain tag ( CBD), chitin-binding domain (CBD) tag, cellulose-binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein (GBP) tag, maltose-binding protein to (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza virus hemagglutinin (HA) tag, horseradish peroxidase (HA) tag, NE-tag, HSV tag, ketosteroid isomerase ( KSI), KT3 tag, LacZ tag, Luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin tag, E tag, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, tag Xpress, Isopepeptag, SpyTag, SnoopTag, Profinity eXact tag, protein C tag, S1 tag, S tag, carboxybiotin carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag , tandem affinity purification (TAP) tag, HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or a combination thereof; optionally, the first and/or second affinity acceptor peptide contains two or more repeats of the tag sequence. In some embodiments, the affinity acceptor peptide binding molecule is biotin or an antibody specific for the first and/or second acceptor affinity peptide.

[468] In some embodiments, enrichment includes contacting the affinity molecule with the first and/or second complexes of affinity acceptor-labeled HLA peptides, wherein the affinity molecule specifically binds to the affinity acceptor peptide binding molecule. In some embodiments, the affinity molecule is streptavidin, NeutrAvidin, or a derivative thereof. In some embodiments, enrichment includes immunoprecipitation of the first and/or second complexes of affinity acceptor-tagged HLA peptides. In some embodiments, an acceptor affinity peptide binding molecule is attached to a solid surface. In some embodiments, the affinity molecule is attached to a solid surface. In some embodiments, the hard surface is a bead.

[469] In some embodiments, enrichment includes immunoprecipitating the first and/or second complexes of the affinity acceptor-labeled HLA peptides with an acceptor affinity peptide binding molecule that specifically binds to the first and/or second acceptor affinity peptide. In some embodiments, the affinity acceptor peptide binding molecule does not specifically interact with the amino acid sequence of the encoded first and/or second class I or class II HLA. In some embodiments, enrichment includes contacting an affinity molecule specific for the extracellular portion of the first and/or second HLA class I or class II allele. In some embodiments, enrichment includes contacting an affinity molecule specific for the N-terminal portion of the first and/or second HLA class I or class II allele.

[470] In some embodiments, providing includes contacting a population of cells with a polynucleic acid. In some embodiments, the introduction into contact includes transfection or transduction. In some embodiments, providing includes contacting a population of cells with a vector containing a polynucleic acid. In some embodiments, the vector is a viral vector. In some embodiments, the polynucleic acid is stably inserted into the genome of a population of cells.

[471] In some embodiments, the sequence encoding the first and/or second HLA class I or class II contains a sequence encoding the α-chain of HLA class I. In some embodiments, the first allele of the recombinant HLA class I or class II is the α chain of the first HLA class I, and the second allele of the recombinant HLA class I or class II is the α chain of the second HLA class I. In some embodiments, the implementation of the method further includes the expression of a sequence encoding β2 microglobulin in one or more cells. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding a first and/or second class I or class II HLA. In some embodiments, the β2 microglobulin coding sequence is linked to the first and/or second class I or class II HLA coding sequence via a linker. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding a third acceptor affinity peptide. In some embodiments, the third acceptor affinity peptide is different from the first and/or second acceptor affinity peptide.

[472] In some embodiments, the sequence encoding the first and/or second HLA class I or class II comprises a sequence encoding an HLA class II α chain and/or an HLA class II β chain. In some embodiments, the sequence encoding the first and/or second HLA class I or class II contains a sequence encoding the α chain of the first HLA class II and the α chain of the second HLA class II. In some embodiments, the method further comprises expressing an HLA class II β chain coding sequence in one or more cells. In some embodiments, a sequence encoding a first HLA class II α chain and a second HLA class II α chain are linked to a sequence encoding an HLA class II β chain. In some embodiments, the sequence encoding the first and/or second HLA class I or class II contains a sequence encoding the β chain of the first HLA class II and the β chain of the second HLA class II. In some embodiments, the method further comprises expressing a sequence encoding an HLA class II α chain in one or more cells. In some embodiments, a sequence encoding a first HLA class II β chain and a second HLA class II β chain are linked to a sequence encoding an HLA class II α chain via a linker. In some embodiments, a sequence encoding an HLA class II β chain or an HLA class II α chain is linked to a sequence encoding a third acceptor affinity peptide. In some embodiments, the third acceptor affinity peptide is different from the first and/or second acceptor affinity peptide.

[473] In some embodiments, the third acceptor affinity peptide is different from the first acceptor affinity peptide and is selected from the group consisting of a biotin acceptor peptide (BAP), a polyhistidine tag, a polyhistidine glycine tag, a polyarginine tag, a polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tags, Strep II tags, albumin binding protein (ABP) tags, alkaline phosphatase (AP) tags, bluetongue virus tags (B tags), calmodulin binding peptide (CBP) tags, chloramphenicol acetyltransferase (CAT) tags ), choline-binding domain (CBD) tags, chitin-binding domain (CBD) tags, cellulose-binding domain (CBP) tags, dihydrofolate reductase (DHFR) tags, galactose-binding protein tags a (GBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tags, human influenza virus hemagglutinin (HA) tags, horseradish peroxidase (HA) tags, NE-tags, HSV tags , ketosteroid isomerase (KSI) tags, KT3 tags, LacZ tags, luciferase tags, NusA tags, PDZ domain tags, AviTag, calmodulin tags, E-tags, S-tags, SBP-tags, Softag 1, Softag 3, TC tags , VSV tags, Xpress tags, Isopeptag, SpyTag, SnoopTag, Profinity eXact tags, protein C tags, S1 tags, S tags, carboxybiotin carrier protein (BCCP) tags, green fluorescent protein (GFP) tags, small ubiquitin tags -like modifier (SUMO), tandem affinity purification (TAP) tags, HaloTag, Nus tags, Thioredoxin tags, Fc tags, CYD tags, HPC tags, TrpE tags, ubiquitin tags, VSV-G epitope tags, V5 tags and their combinations; optionally, the first or second acceptor affinity peptide contains two or more repeats of the tag sequence.

[474] In some embodiments, the linker contains a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome escape site element or an internal ribosome entry site (IRES). In some embodiments, the ribosome escape site or IRES is cleaved when expressed in cells. In some embodiments, the ribosome exit site is selected from the group consisting of F2A, T2A, P2A, and E2A. In some embodiments, the IRES element is selected from common cellular or viral IRES sequences.

[475] In some embodiments, the implementation of the method includes conducting biochemical analysis or mass spectrometry, such as tandem mass spectrometry.

[476] In some embodiments, the method includes obtaining a peptide sequence that corresponds to MS/MS spectra of one or more peptides isolated from enriched complexes of HLA affinity acceptor-labeled peptides from a peptide database; wherein one or more of the resulting sequences identifies the sequence of one or more peptides.

[477] In some embodiments, the cell population is a cell line selected from HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, Hep G2, SH-SY5Y, CACO-2, U937, U-2 OS , ExpiCHO, CHO and THP1.

[478] In some embodiments, the cell line is treated with one or more cytokines, checkpoint inhibitors, epigenetically active drugs, IFN-γ, or a combination thereof.

[479] In some embodiments, the cell population includes at least 10 ⁵ cells, at least 10 ⁶ cells, or at least 10 ⁷ cells. In some embodiments, the cell population is a population of dendritic cells, macrophages, cancer cells, or B cells. In some embodiments, the cell population includes tumor cells.

[480] In some embodiments, a population of cells is contacted with an agent prior to isolation of the first and/or second HLA-peptide complexes from one or more cells. In some embodiments, the agent is an inflammatory cytokine, a chemical agent, an adjuvant, a therapeutic agent, or radiation.

[481] In some embodiments, the first and/or second HLA allele is a mutant HLA allele. In some embodiments, the sequence encoding the first and/or second HLA allele contains a barcoded sequence. In some embodiments, the method further comprises analyzing the expression of the first and/or second allele labeled with an HLA class I or class II affinity acceptor. In some embodiments, the assay includes sequencing a first and/or second allele tagged with an HLA class I or class II affinity acceptor, detecting RNA encoding the RNA of the first and/or second allele tagged with an HLA class I or class II affinity acceptor, detecting a protein of the first and /or a second allele labeled with an HLA class I or class II affinity acceptor, or a combination thereof. In some embodiments, the implementation of the first and second allele labeled with an affinity acceptor HLA class I or class II contains a unique barcoded sequence. In some embodiments, the first sequence and the second sequence comprise a unique barcoded sequence.

EXAMPLES

[482] The examples provided below are for illustrative purposes only, and not to limit the scope of the claims provided herein.

EXAMPLE 1 Universal IP Pipeline: Universal Identification Platform for Uniallelic HLA-Peptide Complexes

[483] The universal immunoaffinity purification (IP) constructs disclosed herein consist of a DNA construct encoding class I or class II HLA affinity tagged alleles that are expressed from a mammalian expression vector via cell transfection or transduction (FIG. 1A and FIG . .1B ). Non-limiting illustrative HLA Class I and Class II constructs are shown in FIG. 2 . Non-limiting exemplary affinity tags comprise a biotin acceptor peptide (BAP) or influenza hemagglutinin (HA) peptide sequence. Affinity tags can be placed either at the N-terminus or at the C-terminus of the HLA allele. A cleavage sequence such as F2A shown in FIG. 2 , or an internal ribosome entry site (IRES) can be placed between the α-chain and β2-microglobulin (class I) or between the α-chain and β-chain (class II). Non-limiting illustrative vectors comprise a lentiviral vector as shown in FIG. 3. Antibody resistance genes, such as puromycin resistance (Puro), are included in the constructs to allow selection after transfection or transduction. Cells transfected or transduced with universal IP constructs are either expanded (FIG. 4A) or selected and then expanded (FIG. 4B) prior to LC-MS/MS analysis. Platform schematics for universal immunopurification of class I and class I HLA are shown in FIG. 5.

EXAMPLE 2 Cell Culture and Immunoaffinity Purification and HLA Peptide Sequencing

[484] Monoallelic HLA cells were obtained by transducing B721.221, A375, JEG-3, K562, Jurkat, or HEK293T, HeLa, or expi293 cells with a retroviral vector encoding a single HLA class I allele (e.g., HLA-A*02:01, HLA -A*23:01 and HLA-B*14:02 or HLA-E*01:01) or an HLA class II allele (e.g. HLA-DRB*01:01, HLA-DRB*01:02 and HLA-DRB *11:01 or HLA-DRB*15:01 or HLA-DRB*07:01) as previously described (Reche et al., 2006). HLA type I or class II cell lines were confirmed using standard molecular typing. Cells were cultured and immunoaffinity purification of the HLA peptide was performed.

[485] Proof of concept for transduction of HLA class I alleles (FIG. 6C) into HEK293T cells is shown in FIG. 6A-6C. Mimic, GFP, and empty plasmid transductions were performed using the HLA-A*02:01 constructs for biotinylation-based universal immunopurification, and the biotinylation was confirmed by Western blotting (FIG. 6A). Ponceau stained gel was used as a loading control for Western blot (FIG. 6B). Optimization of transfection and biotinylation of HLA-BAP class I and class II alleles (FIG. 7C) expressed by HEK293T cells is shown in FIG. 7A-7C. The biotinylation over time experiment showed that biotinylation of C- and N-terminally labeled HLA-BAP was completed after 10 minutes for cells expressing class I and class II HLA-BAP (FIG. 7A and FIG. 7B).

[486] The generic IP pipeline disclosed herein was tested in several cell types (FIGS. 8A-8D). Generic IP constructs for HLA class I and class II were transfected into HEK293T (human fetal kidney) (Figure 8A), HeLa (human cervical cancer) (Figure 8B), A375 (human malignant melanoma) (Figure 8C) and Expi293 cells (primary human kidney genetically engineered for high density culture and protein expression) (FIG. 8D). Western blot was performed using anti-streptavidin for BAP label and anti-HA for HA label, and Ponceau stained gel was used as loading control for Western blot. Western blotting confirmed the expression of class I and class II constructs in all cell types tested (FIGS. 8A-8D).

[487] The following describes the materials and methods used in this Example.

Universal IP of HLA class I and class II alleles (Biotin)

[488] Cells were transfected or transduced to express the universal IP constructs following standard methods. After transduction, cells were resuspended in medium and transferred to 50 ml falcon tubes. The tubes were rotated at 1500 rpm for 5 minutes and the medium was removed. The cells were then resuspended in 1.5 ml cold PBS and transferred to a 1.5 ml Eppendorf tube. Then the tubes were centrifuged (550×g at 4°C) for 5 minutes. The PBS was removed and the cells were then resuspended in 1.2 ml lysis buffer. Cells were resuspended in buffer followed by the addition of benzonase. The tubes were incubated on ice with occasional stirring. After 15 minutes of incubation on ice, the tubes were centrifuged (15000×g at 4°C) for 20 minutes. Supernatants (500 µl) were transferred to another 1.5 ml tube (pre-washed) for biotinylation. Biotinylation of cell lysates was obtained by adding biotin, ATP and BirA to each sample. The sample was then incubated at room temperature for 10 minutes and then placed on ice until immunoprecipitation.

[489] Immunoprecipitation with NeutrAvidin or streptavidin beads was performed by adding a pre-washed suspension of streptavidin or NeutrAvidin agarose resin to the biotinylated lysate. The sample was then placed in a test tube and incubated for 30 minutes at 4°C. After a 30 minute incubation, the beads were pelleted by centrifugation (1500×g, 1 min, 4°C) and the supernatant removed and discarded. The beads were then resuspended in 1 ml wash buffer. The beads were then pelleted by centrifugation (1500×g, 1 min, 4°C) and the wash buffer removed and discarded. This step was repeated to give a total of four washes in wash buffer. The precipitated beads were resuspended in 1 ml Tris buffer, pelleted by centrifugation (1500×g, 1 min, 4°C) and the Tris buffer was removed. This step was repeated to obtain a total of four washes in Tris buffer. A final wash was performed in MS water by resuspending the beads in 1 ml Mass Spec water and centrifuging (1500×g, 1 min, 4°C) to pellet the beads. The supernatant was removed, and the beads were either stored at -80°C or immediately subjected to elution and desalting of the HLA peptides.

Serial Generic IP HLA class II alleles (labeled with HA and Biotin)

[490] Cells were transfected or transduced to express the universal IP constructs according to standard protocols. After transduction, cells were resuspended in medium and transferred to 50 ml falcon tubes. The tubes were centrifuged at 1500 rpm for 5 minutes and the medium was removed. The cells were then resuspended in 1.5 ml cold PBS and transferred to a 1.5 ml Eppendorf tube. Then the tubes were centrifuged (550×g at 4°C) for 5 minutes. The PBS was removed and the cells were then resuspended in 1.2 ml lysis buffer. Cells were resuspended in buffer followed by the addition of benzonase. The tubes were incubated on ice with occasional stirring. After 15 minutes of incubation on ice, the tubes were centrifuged (15000×g at 4°C) for 20 minutes. The supernatants were transferred to another 1.5 ml tube (pre-washed) for biotinylation. Biotinylation of cell lysates was achieved by adding biotin, ATP and BirA to each sample. The sample was then incubated at room temperature for 10 minutes and then placed on ice until immunoprecipitation.

[491] Immunoprecipitation of class II HA labeled alleles was performed by adding pre-washed Protein G agarose resin that had been pre-coupled to the anti-HA antibody. The sample was then incubated for 60 minutes at 4° C. on a test tube turner. After a 60 minute incubation, the beads were pelleted by centrifugation (1500×g, 1 min, 4°C) and the supernatant removed and discarded. The beads were washed twice with lysis buffer and resuspended in lysis buffer containing the free HA peptide and incubated for 15 minutes at 4° C. in a tube spinner. The beads were then pelleted by centrifugation (1500×g, 1 min, 4°C) and the supernatant was transferred to a 1.5 ml Eppendorf tube containing 200 μl of pre-washed NeutrAvidin or streptavidin agarose beads. The sample was then placed in a test tube and incubated for 30 minutes at 4°C. After a 30 minute incubation, the beads were pelleted by centrifugation (1500×g, 1 min, 4°C) and the supernatant removed and discarded. The beads were then resuspended in 1 ml wash buffer. The beads were then pelleted by centrifugation (1500×g, 1 min, 4°C) and the wash buffer removed and discarded. This step was repeated to give a total of four washes in wash buffer. The precipitated beads were resuspended in 1 ml Tris buffer, pelleted by centrifugation (1500×g, 1 min, 4°C) and the wash buffer was removed. This step was repeated to obtain a total of four washes in Tris buffer. A final wash was performed in MS water by resuspending the beads in 1 ml Mass Spec water and centrifuging (1500×g, 1 min, 4°C) to pellet the beads. The supernatant was removed and the beads were either stored at -80° C. or immediately eluted and desalted from the HLA peptides.

Elution and Desalting of HLA Peptides

[492] Peptides were eluted from the HLA complexes and desalted on Empore C18 StageTips (3M, 2315) built in (Rappsilber et al., 2007). Samples were loaded, washed, and eluted on a benchtop centrifuge at a maximum speed of 1500-3000 x g. The StageTips were balanced with two methanol washes, two acetonitrile/formic acid washes, and two formic acid washes. In vitro, the dried IP HLA-associated peptide pellets were thawed at 4° C., reconstituted in ACN/formic acid, and loaded onto StageTips. The beads were washed with formic acid and the peptides were further eluted using two cycles of 5 minute incubations in 10% acetic acid. The combined wash and elution volumes were pooled and loaded onto the StageTips. The tubes containing the IP beads were washed again with formic acid and this volume was also loaded into the StageTips. The peptides were washed twice on StageTips or formic acid desalting cartridges. The peptides were eluted using a stepwise gradient of mixtures of ACN and formic acid. To complete the elution, the stages were combined and dried.

EXAMPLE 3 Sequencing of HLA Class I and Class II Related Peptides by LC-MS/MS

[493] All nano-LC-ESI-MS/MS analyzes used the same LC separation conditions, which are described below. Samples were separated chromatographically using a Proxeon Easy Nano LC 1000 (Thermo Scientific, San Jose, CA) equipped with a 75 µm ID PicoFrit capillary with a 10 µm emitter and packed under pressure to ~20 cm with C18 Reprosil beads (particle size 1, 9 μm, pore size 200 Å Dr. Maisch GmBH) and heated to 50°C during separation.

[494] Samples were loaded into CAN and a mixture of formic acid and peptides were eluted with a linear gradient of 7-30% buffer B (0.1% FA or 0.5% AcOH and 80% or 90% ACN) over 82 minutes, 30 -90% buffer B for 6 minutes and then incubated in 90% buffer B for 15 minutes at 200 nL/min (buffer A, 0.1% FA and 3% ACN) to give a peak width of ~13 (FA ) seconds. At the time of collection, depending on the data, the eluted peptides were injected into an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific) equipped with a 2.2 kV nanoelectrospray source. A full MS scan was obtained with a resolution of 30,000 from 300 to 1800 m/s. Each full scan was followed by the top 10 MS2 scans depending on the data at 15000 resolution with an isolation width of 0.7 m/z.

[495] FIG. 9A shows the number of total unique HLA-associated peptides identified from a variety of cell types expressing affinity-tagged class I and class II HLA constructs used in the universal IP pipeline. In FIG. 9B shows the number of unique peptides from HLA class I monoallelic peptide profiling. In FIG. 9C shows the number of unique peptides from HLA class II monoallelic peptide profiling. LC-MS/MS analysis of HLA-associated peptides revealed characteristics of HLA-associated class I and class II peptides (FIGS. 10A and 10B). In FIG. 10A shows sequence ligature representations of isolated and sequenced HLA-A*02:01 class I associated peptides and HLA-DRβ*11:01 class II associated peptides. Comparison of the length distribution of peptides associated with HLA-A*02:01 class I (red) and HLA-DRβ*11:01 associated with class II peptides (blue) showed that HLA-associated class I and class II peptides followed expected trends (Fig. 10B).

[496] 70 HLA class I alleles and 47 HLA class II alleles were evaluated for the monoallelic approach described herein. 70 unique HLA class I alleles with affinity tags were generated (Table 1A) and 47 unique HLA class II affinity tagged alleles (Table 2A). Table 1B shows the details of 96 unique experiments using 70 unique HLA class I alleles (in some cases the same allele was placed in multiple cell lines). Table 2B shows the details of 54 unique experiments performed using 47 unique HLA class II alleles (in some cases the same allele was placed in multiple cell lines).

Table 1A. 70 Unique alleles of HLA class I

# Unique Class I Alleles # Unique Class I Alleles one A*0201 36 B*3802 2 A*0202 37 B*3901 3 A*0203 38 B*3906 four A*0206 39 B*4001 5 A*0207 40 B*4002 6 A*1101 41 B*4006 7 A*2301 42 B*4101 eight A*2501 43 B*4201 9 A*2601 44 B*4501 ten A*3001 45 B*4601 eleven A*3002 46 B*4801 12 A*3101 47 B*4901 13 A*3201 48 B*5001 fourteen A*3301 49 B*5201 fifteen A*3303 fifty B*5301 16 A*3402 51 B*5401 17 A*3601 52 B*5501 eighteen A*6801 53 B*5703 19 A*7401 54 B*5801 twenty B*0702 55 B*5802 21 B*0801 56 B*8101 22 B*1302 57 C*0102 23 B*1401 58 C*0202 24 B*1402 59 C*0303 25 B*1501 60 C*0401 26 B*1502 61 C*0602 27 B*1503 62 C*0701 28 B*1509 63 C*0702 29 B*1510 64 C*0704 thirty B*1801 65 C*1203 31 B*270502 66 C*1701 32 B*3502 67 C*1801 33 B*3503 68 F*0101 34 B*3701 69 G*0101 35 B*3801 70 E*0101

Table 1B. Libraries and Cell Lines for HLA Class I Alleles

# Unique Experiments # library wave Class I allele cell line # library wave Class I allele cell line one W1 A0201 Hek293 49 W2 B4101 expi293 2 W1 A0201 HeLa fifty W2 B4201 expi293 3 W1 A0201 A375 51 W2 B4501 expi293 four W1 A0201 expi293 52 W2 B4601 expi293 5 W1 A1101 expi293 53 W2 B4801 expi293 6 W1 A1101 HeLa 54 W2 B4901 expi293 7 W1 A2301 Hek293 55 W2 B5001 expi293 eight W1 A2301 A375 56 W2 B5201 expi293 9 W1 A2301 expi293 57 W2 B5301 expi293 ten W1 A2601 A375 58 W2 B5501 expi293 eleven W1 A2601 HeLa 59 W2 B5501 A375 12 W1 A2601 expi293 60 W2 B5703 expi293 13 W1 A3201 A375 61 W2 B5801 expi293 fourteen W1 A3201 HeLa 62 W2 B5801 A375 fifteen W1 A3201 expi293 63 W2 B5802 expi293 16 W1 A3601 expi293 64 W2 B8101 expi293 17 W1 B0702 expi293 65 W2 C0401 expi293 eighteen W1 B0702 HeLa 66 W2 C0401 A375 19 W1 B0801 expi293 67 W2 C0602 expi293 twenty W1 B0801 HeLa 68 W2 C0602 A375 21 W1 B0801 A375 69 W2 C0701 expi293 22 W1 B1402 Hek293 70 W2 C0701 A375 23 W1 B1402 A375 71 W2 C0702 expi293 24 W1 B1402 expi293 72 W2 C0702 A375 25 W1 B1402 HeLa 73 W3 A3301 expi293 26 W1 B1501 expi293 74 W3 A0206 expi293 27 W1 B1509 HeLa 75 W3 A0202 expi293 28 W1 B1509 expi293 76 W3 A3402 expi293 29 W1 B1801 HeLa 77 W3 A0207 expi293 thirty W1 B1801 expi293 78 W3 A0203 expi293 31 W1 B270502 HeLa 79 W3 B3502 expi293 32 W1 B270502 expi293 80 W3 B1401 expi293 33 W1 B4001 HeLa 81 W3 B3906 expi293 34 W1 B4001 expi293 82 W3 B1510 expi293 35 W2 A2501 expi293 83 W3 B4006 expi293 36 W2 A3001 expi293 84 W3 B1502 expi293 37 W2 A3002 expi293 85 W3 B3802 expi293 38 W2 A3101 expi293 86 W3 B5401 expi293 39 W2 A3303 expi293 87 W3 C0303 expi293 40 W2 A6801 expi293 88 W3 C0202 expi293 41 W2 A7401 expi293 89 W3 C1203 expi293 42 W2 B1302 expi293 90 W3 C0102 expi293 43 W2 B1503 expi293 91 W3 C1701 expi293 44 W2 B3503 expi293 92 W3 C0704 expi293 45 W2 B3701 expi293 93 W3 C1801 expi293 46 W2 B3801 expi293 94 W2 E0101 expi293 47 W2 B3901 expi293 95 W3 F0101 expi293 48 W2 B4002 expi293 96 W3 G0101 expi293

Table 2A. 47 Unique HLA Class II Alleles

# Unique Class II Alleles # Unique Class II Alleles one DPB1*0101 25 DRB1*0901 2 DPB1*0101 DPA*01:03 26 DRB1*1001 3 DPB1*0201 DPA*01:03 27 DRB1*1101 four DPB1*0401 DPA*01:03 28 DRB1*1102 5 DPB1*0402 DPA*01:03 29 DRB1*1104 6 DQ2*B0201*A0501 thirty DRB1*1201 7 DQ2*B0202*A0201 31 DRB1*1202 eight DQ6*B0602*A0102 32 DRB1*1301 9 DQ6*B1*0602 33 DRB1*1302 ten DRB1*0101 34 DRB1*1303 eleven DRB1*0102 35 DRB1*1401 12 DRB1*0301 36 DRB1*1501 13 DRB1*0302 37 DRB1*1502 fourteen DRB1*0401 38 DRB1*1503 fifteen DRB1*0402 39 DRB1*1601 16 DRB1*0403 40 DRB3*0101 17 DRB1*0404 41 DRB3*0202 eighteen DRB1*0405 42 DRB3*0301 19 DRB1*0407 43 DRB4*0103 twenty DRB1*0701 44 DRB5*0101 21 DRB1*0801 45 HLA DM no affinity label 22 DRB1*0802 46 HLA-DM with affinity label 23 DRB1*0803 47 HLA-DO 24 DRB1*0804

Table 2B. Libraries and Cell Lines for HLA Class II Alleles

# Unique Experiments # library wave class II allele cell line # library wave class II allele cell line one W1 DRB1_0101 Hek293 28 W2 DRB1_1303 expi293 2 W1 DRB1_0101 expi293 29 W2 DRB1_1401 expi293 3 W1 DRB1_0102 Hek293 thirty W2 DRB1_1502 expi293 four W1 DRB1_0102 expi293 31 W2 DRB1_1503 expi293 5 W1 DRB1_0701 Hek293 32 W2 DRB1_1601 expi293 6 W1 DRB1_0701 expi293 33 W2 DRB4_0103 expi293 7 W1 DRB1_1101 Hek293 34 W2 DPB1_0101 expi293 eight W1 DRB1_1101 HeLa 35 W2 DPB1_0201 expi293 9 W1 DRB1_1101 A375 36 W2 DPB1_0401 expi293 ten W1 DRB1_1101 expi293 37 W2 DPB1_0402 expi293 eleven W1 DRB1_1501 Hek293 38 W2 DQ2_B0201_A0501 expi293 12 W1 DRB1_1501 expi293 39 W2 DQ2_B0202_A0201 expi293 13 W2 DRB1_0301 expi293 40 W2 DQ6_B0602_A0102 expi293 fourteen W2 DRB1_0302 expi293 41 W2 HLA DM no tag expi293 fifteen W2 DRB1_0401 expi293 42 W3 DRB1*0402 expi293 16 W2 DRB1_0404 expi293 43 W3 DRB1*0403 expi293 17 W2 DRB1_0405 expi293 44 W3 DRB1*1102 expi293 eighteen W2 DRB1_0407 expi293 45 W3 DRB1*1202 expi293 19 W2 DRB1_0801 expi293 46 W3 DRB1*0803 expi293 twenty W2 DRB1_0802 expi293 47 W3 DRB3*0101 expi293 21 W2 DRB1_0804 expi293 48 W3 DRB3*0202 expi293 22 W2 DRB1_0901 expi293 49 W3 DRB5*0101 expi293 23 W2 DRB1_1001 expi293 fifty W3 DRB3*0301 expi293 24 W2 DRB1_1104 expi293 51 W3 HLA-DO expi293 25 W2 DRB1_1201 expi293 52 W3 DPB10101 expi293 26 W2 DRB1_1301 expi293 53 W3 DQ6 B1 0602 expi293 27 W2 DRB1_1302 expi293 54 W3 HLA-DM labeled expi293

EXAMPLE 4. Tandem Universal IP of Class II HLA Complexes with Multiple Affinity Labels

[497] Class II HLA complexes were formed by pairing an α-strand and a β-strand, each of which can be labeled with a different affinity tag. Serial IP using both affinity tags provides deconvolution of α-strand and β-strand pairing and unambiguous peptide binding assignments to HLA class II complexes. In FIG. 11A shows a schematic representation of HLA class II constructs designed for expression by different cell types for the universal IP pipeline. In FIG. 11B shows schematic representations of possible HLA class II complexes that may be formed upon expression of the FIG. 11A in cell lines expressing the α-chain and β-chain subunits of endogenous HLA class II.

[498] FIG. 12A depicts schematics of a sequential universal IP strategy that can be used to deconvolve α-strand-β-strand pairings and unambiguous peptide binding assignments to specific HLA class II complexes. Cells expressing dual affinity tagged HLA class II constructs were lysed, biotinylated, and incubated with anti-HA antibody-coupled beads. HLA class II complexes with HA labeled subunits were isolated, washed and eluted using the HA peptide (YPYDVPDYA). The eluate was then incubated with beads coupled to either NeutrAvidin or streptavidin to isolate HA labeled and biotin labeled HLA class II complexes. The peptides associated with double-labeled HLA class II complexes were then eluted and sequenced by LC-MS/MS. Western blotting and transfer quality control (Ponceau S stained gel) showed the specificity of the pipeline for serial universal IP. Western blotting confirmed the serial universal IP strategy in HEK293T expressing double-labeled HLA-DRB*11:01 constructs (FIG. 12B ). Anti-HA antibody was used for the serial enrichment process. A stained Ponceau S gel was used as a Western blot transfer quality control. In FIG. 12C shows a Western blot of a negative control experiment where cells expressing the HLA-DRB*11:01 dual affinity class II construct HLA-DRB*11:01 were lysed and incubated with anti-HA antibody bound beads without biotinylation. As shown in FIG. 12C , no enrichment was observed when the biotinylation step was omitted from the serial universal IP protocol.

EXAMPLE 5 Monoallelic HLA peptidome profiling approach using a Biotin affinity tag.

[499] FIG . 14A and FIG. 14B shows a schematic representation of an HLA monoallelic peptidome profiling approach using a biotin affinity tag. In an exemplary embodiment of the present disclosure, a biotin acceptor peptide (BAP) is used that is biotinylated at a lysine (K) residue by the enzyme BirA. The BAP peptide sequence contains a lysine residue that is biotinylated with the addition of the BirA enzyme, biotin and ATP. The biotinylated product shows high affinity for streptavidin/NeutrAvidin. Streptavidin/NeutrAvidin beads can be used to enrich the biotinylated BAP peptide sequence.

EXAMPLE 6 Target Epitope Detection Platform

[500] Into a cell line of interest (e.g., 2HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, Hep G2, SH-SY5Y, CACO-2, U937, U-2 OS, ExpiCHO, CHO, or THP1) or primary cells (e.g., cells in a subject with a disease or condition) can be transfected/transduced with an HLA class I or II construct containing a tag (e.g., a BAP sequence) at the N- or C-terminus, with selection for enrichment of HLA-expressing cells, or without it (Fig. 15 ). The cells can then be transfected or transduced with a second plasmid that contains an epitope fragment or chain of epitopes that can be expressed and presented on the HLA-tagged molecule. Alternatively, both the HLA allele plasmid and the epitope plasmid can be co-delivered into cells, followed by expansion and/or selection. These engineered cells are then lysed, biotinylated, and the HLA molecule enriched from the lysate (eg using streptavidin beads). The peptides are eluted from the HLA molecule and analyzed, for example, by LC-MS/MS. This method allows one to analyze how different alleles process and present epitopes. This method can also be used to improve the delivery and development of epitopes.

EXAMPLE 7 Allele Multiplexing

[501] A DNA construct can be engineered to express multiple class I heavy chains or multiple < or ® class II chains that contain one or more labels (FIG. 16 ). Each HLA construct can be expressed from the same gene construct that contains a ribosome escape sequence (F2A, T2A, P2A, etc.) or an IRES element. This plasmid can be transduced or transfected into the desired cell line to cause the expression of multiple HLA alleles that are labeled and subsequently enriched. Alternatively, a plurality of plasmids each containing a single HLA allele can be transduced or transfected into a cell line. The peptides associated with the HLA alleles can then be analyzed, for example, by LC-MS/MS. This platform enables the generation of cell lines with multiple alleles. It can be used, for example, to match the patient's HLA type. This makes it possible to obtain profiles of peptide epitopes for different combinations of alleles.

EXAMPLE 8 Improved Prediction of Processing and Specific Allele Binding

[502] NetMHC is an allele-specific method that trains a separate predictor on a dataset for each allele, while NetMHCpan is a panel method whose inputs are vector encodings for both the peptide and the subsequence of a specific MHC molecule. The conventional wisdom is that NetMHC performs better on alleles with multiple ligands analyzed, while NetMHCpan performs better on less well-characterized alleles. However, it has been shown that NetMHCpan is not accurate when appropriate data was not included in the training sets.

[503] The monoallelic approach that is described herein (FIG. 21) identified HLA binding peptides that were poorly evaluated by NetMHCpan but biochemically confirmed as strong binders. In FIG. 20A shows exemplary HLA binding peptides for alleles A*01:01, B*51:01, A*29:02, and B*54:01, disclosed using the currently described monoallelic approach. In FIG. 20B shows the misassignment rate in 100 simulated deconvolutions. A random HLA genotype of the patient was generated with six alleles (2 alleles each of HLA-A, HLA-B, and HLA-C taken at US allele frequency). For each allele, 500 peptides from the respective mono-allelic experiment were selected and pooled to create a simulated 3000 peptide multi-allelic dataset. Each peptide was assigned to the allele that gave the best NetMHCpan% rank score to determine the percentage of peptides misassigned by NetMHCpan. This process was repeated 100 times. As shown in FIG. 22, the prediction of both processing and allele-specific binding was significantly improved.

Disclosure paragraphs

[504] Provided herein is a method for characterizing HLA-peptide complexes, comprising: providing a cell population, wherein one or more cells of the cell population contain a polynucleic acid containing a sequence encoding an allele of an affinity-acceptor-tagged HLA class I or class II, wherein the sequence , encoding HLA labeled with an affinity acceptor, contains a sequence encoding an allele of recombinant HLA class I or class II, functionally linked to a sequence encoding an affinity acceptor peptide; expressing the affinity acceptor-labeled HLA in at least one cell of one or more cells of the cell population, thereby complexing the affinity acceptor-labeled HLA peptides in at least one cell; enrichment of complexes labeled with an affinity acceptor HLA-peptides; and characterization of HLA-peptide complexes. In some embodiments, the encoded allele of class I or class II affinity-acceptor-labelled HLA is a soluble allele of class I or class II affinity-acceptor-labelled HLA.

[505] In some embodiments, characterization includes characterization of the peptide associated with the HLA affinity acceptor-tagged peptide complex as a result of enrichment. In some embodiments, the implementation of the method includes performing the steps of the method for two or more alleles of HLA class I and/or class II. In some embodiments, the two or more HLA class I and/or class II alleles include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 HLA class I and/or class II alleles. In some embodiments, the affinity acceptor-tagged HLA peptide complexes comprise a transmembrane domain. In some embodiments, the affinity acceptor-tagged HLA peptide complexes comprise an intracellular domain. In some embodiments, the affinity acceptor-tagged HLA peptide complexes are not secreted. In some embodiments, the affinity acceptor-tagged HLA peptide complexes are incorporated into the cell membrane upon expression. In some embodiments, the affinity acceptor-tagged HLA peptide complexes are soluble complexes of the affinity-acceptor-tagged HLA peptides. In some embodiments, the HLA acceptor affinity tagged peptide complexes are insoluble complexes of the HLA acceptor affinity tagged peptides. In some embodiments, the method further comprises creating a database of HLA allele-specific peptides. In some embodiments, the recombinant HLA Class I or Class II allele is a single recombinant HLA Class I or Class II allele.

[506] In some embodiments, the implementation of the method includes: providing a population of cells, each of which contains one or more cells containing affinity-acceptor-tagged HLA, and affinity-acceptor-tagged HLA contains another recombinant polypeptide encoded by a different HLA allele operably linked to the affinity peptide - acceptor; enrichment of complexes labeled with an affinity acceptor HLA-peptides; and characterizing the peptide, or part thereof, associated with the HLA affinity acceptor-labeled peptide complex as a result of enrichment.

[507] In some embodiments, the implementation of the method includes the introduction of one or more peptides in a population of cells. In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more peptides or the expression of one or more peptides in a population of cells. In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more nucleic acids encoding one or more peptides. In some embodiments, one or more nucleic acids encoding one or more peptides is DNA. In some embodiments, one or more nucleic acids encoding one or more peptides is RNA, optionally the RNA is mRNA. In some embodiments, enrichment does not include the use of a tetrameric reagent.

[508] In some embodiments, characterization includes determining the sequence of the peptide or portion thereof associated with the enriched HLA affinity acceptor-tagged peptide complex, optionally determining whether the peptide or portion thereof is modified. In some embodiments, the determination includes biochemical analysis, mass spectrometric analysis, MS analysis, MS/MS analysis, LC-MS/MS analysis, or a combination thereof. In some embodiments, the characterization includes evaluating the binding affinity or stability of the peptide, or portion thereof, associated with the enriched HLA affinity acceptor-labeled peptide complex. In some embodiments, characterization includes determining whether a peptide, or portion thereof, associated with an enriched HLA affinity acceptor-tagged peptide contains one or more mutations. In some embodiments, the characterization includes evaluating the binding of peptides to HLA molecules in complexes with affinity acceptor-labeled HLA peptides.

[509] In some embodiments, the implementation of the method includes the expression of the library of peptides in a population of cells, thereby forming a library of complexes labeled with an affinity acceptor HLA peptides. In some embodiments, the method includes contacting the cell population with a peptide library or a library of peptide coding sequences, thereby forming a library of HLA affinity acceptor-labeled peptide complexes. In some embodiments, the library is a library of peptides associated with a disease or condition. In some embodiments, the library is a library of peptides derived from a polypeptide drug, such as a biological drug (eg, an antibody containing drug).

[510] In some embodiments, the disease or condition is cancer, infection with an infectious agent, or an autoimmune reaction. In some embodiments, the method includes introducing the infectious agent or parts thereof into one or more cells of the cell population. In some embodiments, the method includes administering a polypeptide drug, such as a biological drug (eg, an antibody-containing drug), or portions thereof, to one or more cells of a cell population. In some embodiments, the method includes characterizing one or more peptides from HLA-peptide complexes, optionally the peptides are derived from one or more target proteins of the infectious agent or polypeptide drug. In some embodiments, the method includes characterizing one or more regions of peptides from one or more target proteins of the infectious agent or drug polypeptide.

[511] In some embodiments, the method includes identifying peptides from HLA-peptide complexes derived from the infectious agent. In some embodiments, the cell population is derived from a biological sample in a subject with a disease or condition. In some embodiments, the cell population is a cell line. In some embodiments, the cell population is a primary cell population. In some embodiments, the recombinant HLA class I or class II allele corresponds to a subject with a disease or condition.

[512] In some embodiments, a peptide from the HLA affinity acceptor-tagged peptide complex is capable of activating a T cell in a subject when presented by an antigen presenting cell. In some embodiments, characterization includes comparing HLA-peptide complexes from cancer cells with HLA-peptide complexes from non-cancerous cells. In some embodiments, the cell population includes a plurality of cell populations, with each cell population expressing a different recombinant HLA class I or class II allele. In some embodiments, each cell population of the plurality is in the same or separate container.

[513] In some embodiments, the method further comprises isolating peptides from complexes of affinity acceptor-labeled HLA peptides prior to characterization. In some embodiments, the HLA-peptide complex is isolated using anti-HLA antibodies. In some cases, an HLA-peptide complex with or without an affinity tag is isolated using anti-HLA antibodies. In some cases, soluble HLA (sHLA) with or without an affinity tag is isolated from the cell culture medium. In some cases, soluble HLA (sHLA) with or without an affinity tag is isolated using anti-HLA antibodies. For example, HLA, such as soluble HLA (sHLA) with or without an affinity tag, can be isolated using a bead or column containing an anti-HLA antibody. In some embodiments, the peptides are isolated using anti-HLA antibodies. In some cases, soluble HLA (sHLA) with or without an affinity tag is isolated using anti-HLA antibodies. In some cases, soluble HLA (sHLA) with or without an affinity tag is isolated using a column containing an anti-HLA antibody. In some embodiments, the method further comprises removing one or more amino acids at the end of the peptide associated with the HLA affinity acceptor-tagged peptide complex.

[514] In some embodiments, the implementation of the cell population is a population of cells with low expression of class I or class II cell surface HLA. In some embodiments, a population of cells expresses one or more endogenous HLA alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles and endogenous HLA class II alleles. In some embodiments, the cell population has a knockout of one or more HLA class I alleles. In some embodiments, the cell population has a knockout of one or more HLA class II alleles. In some embodiments, a population of cells has a knockout of all HLA class I alleles. In some embodiments, a population of cells has a knockout of all HLA class II alleles. In some embodiments, the cell population has a knockout of all HLA class I alleles and a knockout of all HLA class II alleles. In some embodiments, a sequence encoding an allele of a recombinant HLA class I or class II encodes a class I HLA. In some embodiments, Class I HLA is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the sequence encoding the allele of the recombinant HLA class I or class II encodes HLA class II. In some embodiments, the HLA class II is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP. In some embodiments, the HLA class II comprises an HLA class II α chain, an HLA class II β chain, or a combination thereof. In some embodiments, each sequence encodes for at least two different HLA class I and/or class II alleles.

[515] In some embodiments, at least two different HLA class I and/or class II alleles are each operably linked to a sequence encoding an affinity acceptor peptide. In some embodiments, at least two different HLA class I and/or class II alleles are each operably linked to a sequence encoding a different acceptor affinity peptide. In some embodiments, at least two different HLA class I and/or class II alleles are each operably linked to a sequence encoding an affinity acceptor peptide. In some embodiments, one or more of at least two different HLA class I and/or class II alleles is operably linked to a sequence encoding a first acceptor affinity peptide and one or more of at least two different HLA class I and/or alleles. or class II is operably linked to a sequence encoding a second acceptor affinity peptide. In some embodiments, at least two different HLA class I and/or class II alleles are each operably linked to a sequence encoding a different acceptor affinity peptide. In some embodiments, each of at least two different HLA class I and/or class II alleles is operably linked to a sequence encoding a different acceptor affinity peptide. In some embodiments, at least two different HLA class I and/or class II alleles are each operably linked to an affinity tag encoding sequence. In some embodiments, the method includes administering at least a second polynucleic acid containing a sequence encoding a different recombinant HLA allele operably linked to the same or a different acceptor affinity peptide.

[516] In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence that encodes the extracellular portion of the recombinant HLA class I or class II allele. In some embodiments, the encoded acceptor affinity peptide is expressed extracellularly. In some embodiments, the encoded acceptor affinity peptide is located on the extracellular portion of the recombinant HLA class I or class II allele. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to the N-terminus of a sequence encoding a recombinant HLA Class I or Class II allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence that encodes the intracellular portion of the recombinant HLA class I or class II allele. In some embodiments, the encoded acceptor affinity peptide is expressed intracellularly. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to the C-terminus of a sequence encoding a recombinant HLA Class I or Class II allele. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to an internal sequence of a sequence encoding a recombinant HLA Class I or Class II allele, such as a flexible loop sequence. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to a sequence encoding a recombinant HLA Class I or Class II allele via a linker. In some embodiments, enrichment includes enrichment of intact cells expressing complexes of affinity acceptor-tagged HLA peptides. In some embodiments, the method does not include lysing the cells prior to enrichment. In some embodiments, the method further comprises lysing one or more cells prior to enrichment. In some embodiments, enrichment comprises contacting an acceptor affinity peptide binding molecule with the acceptor affinity-tagged HLA peptide complexes, wherein the acceptor affinity peptide binding molecule specifically binds to the acceptor affinity peptide.

[517] In some embodiments, the affinity acceptor peptide comprises a tag sequence including a biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine-glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 peptide tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep tag II, tag albumin-binding protein (ABP), alkaline phosphatase (AP) tag, bluetongue (B-tag), calmodulin-binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline binding domain (CBD) tag , chitin binding domain (CBD) tag, cellulose binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose binding protein (GBP) tag, maltose binding protein (MBP), gluta thione S-transferase (GST), Glu-Glu tag (EE), human influenza virus hemagglutinin tag (HA), horseradish peroxidase (HA) tag, NE tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag , LacZ tag, Luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin tag, E tag, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, Xpress tag, Isopeptag, SpyTag , SnoopTag, Profinity eXact tag, protein C tag, S1 tag, S tag, carboxybiotin carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification tag (TAP), HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, sortase tag, covalent peptide bond tag, or a combination of them; optionally, the affinity acceptor peptide contains two or more repeats of the tag sequence.

[518] In some embodiments, the affinity acceptor peptide binding molecule is biotin or an antibody specific for the acceptor affinity peptide. In some embodiments, enrichment includes contacting an affinity molecule with complexes of affinity acceptor-labeled HLA peptides, wherein the affinity molecule specifically binds to the affinity acceptor peptide binding molecule.

[519] In some embodiments, the affinity molecule is a molecule that binds to biotin. For example, the affinity molecule may contain streptavidin, NeutrAvidin, including protein homologues from other organisms and derivatives thereof.

[520] In some embodiments, enrichment includes immunoprecipitation of complexes labeled with an affinity acceptor HLA peptides. In some embodiments, an acceptor affinity peptide binding molecule is attached to a solid surface. In some embodiments, the affinity molecule is attached to a solid surface. In some embodiments, the hard surface is a bead. In some embodiments, enrichment comprises immunoprecipitating complexes of the affinity acceptor-labeled HLA peptides with an acceptor affinity peptide binding molecule that specifically binds to the acceptor affinity peptide.

[521] In some embodiments, the acceptor affinity peptide binding molecule does not specifically interact with the amino acid sequence of the encoded recombinant HLA Class I or Class II. In some embodiments, enrichment includes contacting an affinity molecule specific for the extracellular portion of a recombinant HLA Class I or Class II allele. In some embodiments, enrichment includes contacting an affinity molecule specific for the N-terminal portion of a recombinant HLA Class I or Class II allele.

[522] In some embodiments, providing includes contacting a population of cells with a polynucleic acid. In some embodiments, the introduction into contact includes transfection or transduction. In some embodiments, providing includes contacting a population of cells with a vector containing a polynucleic acid. In some embodiments, the vector is a viral vector. In some embodiments, the polynucleic acid is stably inserted into the genome of a population of cells.

[523] In some embodiments, the implementation of the sequence encoding recombinant HLA class I or class II, contains a sequence encoding the α-chain of HLA class I. In some embodiments, the implementation of the method further includes the expression of a sequence encoding β2 microglobulin in one or more cells. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding an HLA class I α chain. In some embodiments, the β2 microglobulin coding sequence is linked to the HLA class I α chain coding sequence via a linker. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding a second acceptor affinity peptide. In some embodiments, a sequence encoding a recombinant HLA class I or class II contains a sequence encoding an HLA class II α chain. In some embodiments, the method further comprises expressing an HLA class II β chain coding sequence in one or more cells. In some embodiments, a sequence encoding an HLA class II β chain is linked to a sequence encoding an HLA class II α chain. In some embodiments, a sequence encoding an HLA class II β chain is linked to a sequence encoding an HLA class II α chain via a linker. In some embodiments, a sequence encoding an HLA class II β chain is linked to a sequence encoding a second acceptor affinity peptide.

[524] In some embodiments, the second acceptor affinity peptide is different from the first acceptor affinity peptide and is selected from the group consisting of a biotin acceptor peptide (BAP), a polyhistidine tag, a polyhistidine glycine tag, a polyarginine tag, a polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tags, Strep II tags, albumin-binding protein (ABP) tags, alkaline phosphatase (AP) tags, bluetongue virus tags (B-tags), calmodulin-binding peptide (CBP) tags, chloramphenicol acetyltransferase (CAT) tags ), choline-binding domain (CBD) tags, chitin-binding domain (CBD) tags, cellulose-binding domain (CBP) tags, dihydrofolate reductase (DHFR) tags, galactose-binding protein tags a (GBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tags, human influenza virus hemagglutinin (HA) tags, horseradish peroxidase (HA) tags, NE-tags, HSV tags , ketosteroid isomerase (KSI) tags, KT3 tags, LacZ tags, luciferase tags, NusA tags, PDZ domain tags, AviTag, calmodulin tags, E-tags, S-tags, SBP-tags, Softag 1, Softag 3, TC tags , VSV tags, Xpress tags, Isopeptag, SpyTag, SnoopTag, Profinity eXact tags, protein C tags, S1 tags, S tags, carboxybiotin carrier protein (BCCP) tags, green fluorescent protein (GFP) tags, small ubiquitin tags -like modifier (SUMO), tandem affinity purification (TAP) tags, HaloTag, Nus tags, Thioredoxin tags, Fc tags, CYD tags, HPC tags, TrpE tags, ubiquitin tags, VSV-G epitope tags, V5 tags and their combinations; optionally, the first or second acceptor affinity peptide contains two or more repeats of the tag sequence.

[525] In some embodiments, the linker contains a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome escape site or internal ribosome entry site (IRES). In some embodiments, the ribosome escape site or IRES is cleaved when expressed in cells. In some embodiments, the ribosome exit site is selected from the group consisting of F2A, T2A, P2A, and E2A. In some embodiments, the IRES element is selected from common cellular or viral IRES sequences.

[526] In some embodiments, the determination includes performing a biochemical analysis or mass spectrometry, such as tandem mass spectrometry. In some embodiments, the determination includes obtaining a peptide sequence that corresponds to MS/MS spectra of one or more peptides isolated from enriched complexes of HLA affinity acceptor-labeled peptides from a peptide database; wherein one or more of the resulting sequences identifies the sequence of one or more peptides. In some embodiments, the peptide database is a peptide database with no enzyme specificity, such as an unmodified database or a modified database. In some embodiments, the method further comprises searching the peptide database using a flipped database search strategy.

[527] In some embodiments, the cell population is a cell line. In some embodiments, the cell population is a human cell line. In some embodiments, the cell population is a mouse cell line. In some embodiments, the cell population is a CHO cell line. In some embodiments, the cell population is a cell line selected from HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, and THP1. In some embodiments, the cell population is treated with one or more cytokines, checkpoint inhibitors, epigenetically active drugs, IFN-γ, agents that alter antigen processing (such as peptidase inhibitors, proteasome inhibitors, and TAP inhibitors), or a combination thereof. In some embodiments, a population of cells is treated with one or more reagents that modulate the metabolic pathway or metabolic state of the cells. In some embodiments, a population of cells is treated with one or more reagents that modulate the cellular proteome of the cells. In some embodiments, the cell population is treated with one or more reagents that modulate or regulate cellular expression or transcription (eg, AIRE or CREB binding protein or modulators thereof) of the cells. In some embodiments, a population of cells is treated with one or more reagents that modulate or regulate a cell's transcription factor. In some embodiments, a population of cells is treated with one or more reagents that modulate or regulate cellular expression or transcription of HLA cells. In some embodiments, a population of cells is treated with one or more reagents that modulate or regulate cellular expression or transcription of a cell proteome.

[528] In some embodiments, the cell population includes at least 10 ⁵ cells, at least 10 ⁶ cells, or at least 10 ⁷ cells. In some embodiments, the cell population is a population of dendritic cells, macrophages, cancer cells, or B cells. In some embodiments, the cell population includes tumor cells. In some embodiments, a population of cells is contacted with an agent prior to isolating said HLA-peptide complexes from one or more cells. In some embodiments, said agent is an inflammatory cytokine, a chemical agent, an adjuvant, a therapeutic agent, or radiation.

[529] In some embodiments, the HLA allele is a mutant HLA allele. In some embodiments, the implementation of the sequence encoding the HLA allele is a barcoded sequence. In some embodiments, the method further comprises analyzing the expression of an allele tagged with an HLA class I or class II affinity acceptor. In some embodiments, the assay includes sequencing an allele tagged with an HLA class I or class II affinity acceptor, detecting an RNA allele tagged with an HLA class I or class II affinity acceptor, detecting a protein of the allele tagged with an HLA class I or class II affinity acceptor, or a combination thereof. In some embodiments, expression analysis may include Western blotting, fluorescence activated cell sorting (FACS), mass spectrometry (MS), microarray hybridization analysis, RNA sequencing analysis, polymerase chain reaction analysis, LAMP analysis, ligation analysis. chain reaction, southern blot, northern blot, or enzyme immunoassay (ELISA).

[530] In some embodiments, the method includes performing the steps of the method for different HLA alleles. In some embodiments, each HLA allele contains a unique barcoded sequence. In some embodiments, each polynucleic acid encoding a different HLA allele contains a unique barcoded sequence.

[531] This document provides a database of sequences specifically binding alleles of HLA peptides obtained by performing the method described herein. Provided herein is a combination of two or more HLA allele-specific binding peptide sequence databases obtained by repeatedly performing the method described herein, each time using a different HLA allele. Provided herein is a method for generating a prediction algorithm for identifying HLA-specific allele-binding peptides, comprising training a machine with a sequence database of the peptides described herein or the combination described herein.

[532] In some embodiments, the machine combines one or more linear models, support vector machines, decision trees, and neural networks. In some embodiments, the variable used to train the machine includes one or more variables selected from the group consisting of peptide sequence, physical properties of amino acids, physical properties of peptides, expression level of the original peptide protein in the cell, protein stability, protein translation rate , ubiquitination sites, rate of protein degradation, translation efficiency from ribosomal profiling, protein cleavage, protein localization, host protein motifs that facilitate TAP transport, host protein undergoes autophagy, motifs that promote ribosome arrest, and protein features that promote NMD .

[533] In some embodiments, motifs that promote ribosome arrest contain polyproline or polylysine regions. In some embodiments, protein features that promote NMD are selected from the group consisting of a long 3' UTR, a stop codon greater than 50 nucleotides before the last exon:exon junction, and cleavage of the peptide.

[534] Provided herein is a method for identifying HLA allele-specific binding peptides, comprising performing a peptide sequence analysis with a machine that has been trained with a peptide sequence database obtained by performing the method described herein for HLA alleles. In some embodiments, the implementation of the method includes determining the level of expression of the original protein of the peptide in the cell; and while the expression of the original protein is the prognostic variable used by the machine. In some embodiments, the level of expression is determined by measuring the amount of the parent protein or the amount of RNA encoding said parent protein.

[535] Provided herein is a composition comprising a recombinant polynucleic acid comprising two or more sequences each encoding an affinity-acceptor-tagged HLA, wherein the sequences encoding the affinity-acceptor-tagged HLA contain a sequence encoding a different recombinant HLA α-chain allele class I, a sequence encoding an affinity acceptor peptide, and optionally, a sequence encoding β2 microglobulin; wherein sequences (a) and (b), and optionally (c), are operably linked.

[536] Provided herein is a composition comprising a recombinant polynucleic acid comprising two or more sequences each containing a sequence encoding an affinity-acceptor-tagged HLA, wherein the sequences encoding the affinity-acceptor-tagged HLA contain a sequence encoding a recombinant α- HLA class II chains, a sequence encoding an affinity acceptor peptide, and optionally, a sequence encoding a class II HLA β chain; wherein sequences (a) and (b), and optionally (c), are operably linked. In some embodiments, a recombinant polynucleic acid is isolated. In some embodiments, Class I HLA is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the HLA class II is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP.

[537] In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence that encodes the extracellular portion of the recombinant HLA allele. In some embodiments, the sequence encoding the affinity acceptor molecule is operably linked to the N-terminus of the sequence encoding the recombinant HLA allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the sequence encoding the intracellular portion of the recombinant HLA allele. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to the C-terminus of a sequence encoding a recombinant HLA allele. In some embodiments, a sequence encoding an affinity acceptor peptide is operably linked to a sequence encoding a recombinant HLA allele via a linker.

[538] In some embodiments, two or more affinity acceptor-tagged HLA encoding sequences are expressed from the same polynucleotide. In some embodiments, two or more affinity acceptor-tagged HLA encoding sequences are expressed from different polynucleotides. In some embodiments, the encoded acceptor affinity peptide specifically binds to an acceptor affinity peptide binding molecule. In some embodiments, two or more sequences encoding an affinity acceptor-tagged HLA contain two or more affinity acceptor peptides. In some embodiments, two or more sequences encoding an affinity-acceptor-tagged HLA comprise three or more sequences encoding an affinity-acceptor-tagged HLA, wherein at least two of the three or more sequences encoding an affinity-acceptor-tagged HLA contain the same affinity acceptor peptide. In some embodiments, the two or more affinity acceptor peptides are unique for each of the two or more sequences encoding the affinity acceptor-tagged HLA.

[539] In some embodiments, the encoded acceptor affinity peptide is selected from the group consisting of a biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 peptide tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tags, Strep tags II, tags albumin-binding protein (ABP), alkaline phosphatase (AP) tags, bluetongue (B-tags), calmodulin-binding peptide (CBP) tags, chloramphenicol acetyltransferase (CAT) tags, choline binding domain (CBD) tags , chitin-binding domain (CBD) tags, cellulose-binding domain (CBP) tags, dihydrofolate reductase (DHFR) tags, galactose-binding protein (GBP), maltose-binding protein (MBP), glutathione- S-transferases (GST), Glu-Glu tags (EE), human influenza virus hemagglutinin (HA) tags, horseradish peroxidase (HA) tags, NE tags, HSV tags, ketosteroid isomerase (KSI) tags, KT3 tags, tags LacZ, luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, TC-tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag , Profinity eXact tags, protein C tags, S1 tags, S tags, carboxybiotin carrier protein (BCCP) tags, green fluorescent protein (GFP) tags, small ubiquitin-like modifier (SUMO) tags, tandem affinity purification (TAP) tags ), HaloTag, Nus tags, Thioredoxin tags, Fc tags, CYD tags, HPC tags, TrpE tags, ubiquitin tag, VSV-G epitope tag, V5 tag, and combinations thereof; optionally, the first or second acceptor affinity peptide contains two or more repeats of the tag sequence.

[540] In some embodiments, the affinity acceptor peptide binding molecule is biotin or an antibody specific for the acceptor affinity peptide. In some embodiments, the affinity acceptor peptide binding molecule specifically binds to the affinity molecule. In some embodiments, the affinity molecule is streptavidin, NeutrAvidin, or a derivative thereof. In some embodiments, the acceptor affinity peptide binding molecule does not specifically interact with the recombinant HLA class I or class II amino acid sequence. In some embodiments, for two or more recombinant polynucleic acids: a sequence encoding an affinity acceptor-tagged HLA is stably inserted into the cell's genome. In some embodiments, a sequence encoding a .beta.2 microglobulin or a sequence encoding the .beta. chain of an HLA class II is linked to a sequence encoding a second acceptor affinity peptide. In some embodiments, the second acceptor affinity peptide contains an HA tag. In some embodiments, a sequence encoding a .beta.2 microglobulin or a sequence encoding the .beta. chain of an HLA class II is linked to a sequence encoding a recombinant HLA and an acceptor affinity peptide via a linker.

[541] In some embodiments, the linker contains a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome escape site or internal ribosome entry site (IRES). In some embodiments, the ribosome escape site or IRES is cleaved when expressed in cells. In some embodiments, the ribosome exit site is selected from the group consisting of F2A, T2A, P2A, and E2A. In some embodiments, the IRES element is selected from common cellular or viral IRES sequences.

[542] Provided herein is a composition comprising two or more isolated polypeptide molecules encoded by a polynucleic acid of the composition described herein. Provided herein is a composition comprising a population of cells containing two or more polypeptide molecules encoded by a polynucleic acid of the composition described herein. This document provides a composition containing a population of cells containing the composition described in this document. This document provides a composition containing a population of cells containing one or more cells containing the composition described herein.

[543] In some embodiments, a population of cells expresses one or more endogenous HLA class I or class II alleles. In some embodiments, a population of cells is constructed that lacks one or more endogenous HLA class I alleles. In some embodiments, a population of cells is constructed that lack alleles for endogenous HLA class I. In some embodiments, a population of cells is constructed that lacks one or more endogenous HLA class II alleles. In some embodiments, a population of cells is constructed that lack alleles for endogenous HLA class II. In some embodiments, a population of cells is constructed that lacks one or more endogenous HLA class I alleles and one or more endogenous HLA class II alleles. In some embodiments, the cell population is a cell population with low cell surface HLA class I or class II expression. In some embodiments, a composition is formulated using peptides or polynucleic acids encoding peptides specific for the patient's HLA type. Provided herein is a method for producing a cell, comprising transduction or transfection of two or more cells with two or more polynucleic acids of the composition described herein.

[544] Provided herein is a peptide identified according to the method described herein. Provided herein is a method for eliciting an antitumor response in a mammal, comprising administering to the mammal an effective amount of a polynucleic acid containing the peptide sequence described herein. Provided herein is a method for inducing an antitumor response in a mammal, comprising administering to the mammal an effective amount of a peptide comprising the peptide sequence described herein. Provided herein is a method for eliciting an antitumor response in a mammal, comprising administering to the mammal a cell containing a peptide comprising the peptide sequence described herein. Provided herein is a method for eliciting an antitumor response in a mammal, comprising administering to the mammal a cell containing an effective amount of a polynucleic acid containing a peptide coding sequence comprising the peptide sequence described herein. In some embodiments, the cell presents the peptide as an HLA-peptide complex. Provided herein is a method of eliciting an immune response in a mammal, comprising administering to the mammal an effective amount of a polynucleic acid containing a sequence encoding a peptide described herein. Provided herein is a method of eliciting an immune response in a mammal, comprising administering to the mammal an effective amount of a peptide comprising the peptide sequence described herein. Provided herein is a method of eliciting an immune response in a mammal, comprising administering to the mammal an effective amount of cells containing a peptide comprising the peptide sequence described herein. Provided herein is a method for eliciting an immune response in a mammal comprising administering to the mammal an effective amount of cells containing a polynucleic acid containing a peptide coding sequence comprising the peptide sequence described herein.

[545] In some embodiments, the implementation of the immune response is a T-cell immune response. In some embodiments, the immune response is a CD8 T cell response. In some embodiments, the immune response is a CD4 T cell response. In some embodiments, the immune response is a humoral immune response.

[546] Provided herein is a method for treating a mammal having a disease, comprising administering to the mammal an effective amount of a polynucleic acid containing a sequence encoding a peptide described herein. Provided herein is a method of treating a mammal having a disease comprising administering to the mammal an effective amount of a peptide comprising a peptide sequence as described herein. Provided herein is a method for treating a mammal having a disease, comprising administering to the mammal an effective amount of cells containing a peptide comprising the peptide sequence described herein. Provided herein is a method for treating a mammal having a disease comprising administering to the mammal an effective amount of cells containing a polynucleic acid containing a peptide coding sequence comprising the peptide sequence described herein. In some embodiments, the disease is cancer. In some embodiments, the disease is infection by an infectious agent. In some embodiments, the infectious agent is a pathogenic microorganism, optionally a virus or bacterium or parasite.

[547] In some embodiments, the virus is selected from the group consisting of: BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4, DENV-5), cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), adenovirus, human immunodeficiency virus (HIV), human T-cell lymphotrophic virus (HTLV-1), influenza virus, RSV, HPV, rabies, mumps, rubella, poliovirus, yellow fever, hepatitis A, hepatitis B, rotavirus, varicella-zoster virus, human papillomavirus (HPV), smallpox, shingles, and any combination thereof.

[548] In some embodiments, the bacterium is selected from the group consisting of: Klebsiella species, Tropheryma whipplei, Mycobacterium leprae, Mycobacterium lepromatosis, and Mycobacterium tuberculosis, typhoid, pneumococcus, meningococcus, haemophilus B, anthrax, tetanus toxin, group B meningococcus, bcg , cholera, and any combination thereof.

[549] In some embodiments, the parasite is a helminth or a protozoan. In some embodiments, the parasitic organism is selected from the group consisting of: Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Schistosoma spp., and any combination thereof.

[550] Provided herein is a method for enriching immunogenic peptides, comprising: providing a cell population comprising one or more cells expressing an affinity acceptor-tagged HLA, wherein the affinity-acceptor-tagged HLA contains an affinity acceptor peptide operably linked to a recombinant HLA encoded by an allele of the recombinant HLA; and enrichment of HLA-peptide complexes containing labeled HLA affinity acceptor. In some embodiments, the method further comprises determining the sequence of immunogenic peptides isolated from the HLA-peptide complexes. In some embodiments, the implementation of the definition includes the use of LC-MS/MS.

[551] Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a polynucleic acid containing a sequence encoding a peptide described herein. Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a peptide comprising the peptide sequence described herein. Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of cells containing a peptide comprising the peptide sequence described herein. Provided herein is a method for treating a disease or disorder in a subject, the method comprising administering to the subject a cell containing an effective amount of a polynucleic acid containing a peptide coding sequence comprising the peptide sequence described herein.

[552] Provided herein is a method for developing a therapeutic agent for a subject with a disease or condition, comprising providing a population of cells derived from a subject with the disease or condition, expressing in one or more cells of the cell population an allele labeled with an HLA class I or class II affinity acceptor by introduction into one or more cells of a polynucleic acid encoding a sequence containing: a sequence encoding an allele of recombinant HLA class I or class II, operably linked to a sequence encoding an affinity acceptor peptide, thereby forming complexes of affinity acceptor-labeled HLA peptides in one or more cells; enrichment and characterization of complexes labeled with an affinity acceptor HLA-peptides; and, optionally, developing a therapeutic agent based on the resulting characteristic.

[553] Provided herein is a method for identifying at least one subject specific immunogenic antigen and preparing a subject specific immunogenic composition that contains at least one subject specific immunogenic antigen wherein the subject has a disease and at least one specific for a subject, the immunogenic antigen is specific for the subject and the subject's disease, said method comprising: providing a population of cells obtained from a subject with a disease or condition, expression in one or more cells of the cell population in the subject of an allele labeled with an HLA class I or class II affinity acceptor by introduction into one or more cells of a polynucleic acid encoding a sequence containing: a sequence encoding an allele of recombinant HLA class I or class II, functionally linked to a sequence encoding an affinity acceptor peptide, thereby forming complexes affinity acceptor labeled HLA peptides in one or more cells; enrichment of complexes labeled with an affinity acceptor HLA peptides from one or more cells; identifying an immunogenic peptide from the enriched complexes labeled with an affinity acceptor HLA peptides that is specific for the subject and the subject's disease; and formulating a subject-specific immunogenic composition based on one or more subject-specific immunogenic peptides identified.

[554] In some embodiments, the therapeutic or subject-specific immunogenic composition comprises a peptide from enriched complexes of HLA affinity acceptor tagged peptides or a polynucleotide encoding a polypeptide from enriched complexes of HLA affinity acceptor tagged peptides. In some embodiments, the therapeutic or subject-specific immunogenic composition comprises a T cell expressing a T cell receptor (TCR) that specifically binds to a polypeptide from enriched complexes of HLA affinity acceptor tagged peptides. In some embodiments, the subject-specific immunogenic composition comprises a chimeric antigen receptor (CAR), a T cell expressing a receptor that specifically binds to a polypeptide from enriched complexes of HLA affinity acceptor tagged peptides.

[555] In some embodiments, the method further comprises administering to the subject another therapeutic agent, optionally an immune checkpoint inhibitor. In some embodiments, the method further comprises administering to the subject an adjuvant, optionally a poly-ICLC.

[556] In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, the disease or disorder is an infection. In some embodiments, the infection is infection with an infectious agent. In some embodiments, the infectious agent is a pathogen, virus, bacterium, or parasite.

[557] In some embodiments, the virus is selected from the group consisting of: BK virus (BKV), Dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4, DENV-5), cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), adenovirus, human immunodeficiency virus (HIV), human T-cell lymphotrophic virus (HTLV-1), influenza virus, RSV, HPV, rabies, mumps, rubella, poliovirus, yellow fever, hepatitis A, hepatitis B, rotavirus, varicella-zoster virus, human papillomavirus (HPV), smallpox, shingles, and any combination thereof.

[558] In some embodiments, the bacterium is selected from the group consisting of: Klebsiella species, Tropheryma whipplei, Mycobacterium leprae, Mycobacterium lepromatosis, and Mycobacterium tuberculosis, typhoid, pneumococcus, meningococcus, haemophilus B, anthrax, tetanus toxin, group B meningococcus, bcg , cholera, and combinations thereof.

[559] In some embodiments, the parasite is a helminth or a protozoan. In some embodiments, the parasitic organism is selected from the group consisting of: Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Schistosoma spp., and any combination thereof.

[560] Provided herein is a method for developing a therapeutic agent for a subject with a disease or condition, comprising: providing a cell population, wherein one or more cells of the cell population comprise a polynucleic acid containing a sequence encoding at least two alleles of an affinity acceptor-tagged HLA class I or class II, wherein the sequence encoding at least two class I or class II HLA labeled with an affinity acceptor contains a first recombinant sequence containing a sequence encoding the first class I or class II HLA allele operably linked to the sequence encoding the first affinity peptide - acceptor; and a second recombinant sequence containing a sequence encoding a second HLA class I or class II allele operably linked to a sequence encoding a second acceptor affinity peptide; expressing at least two affinity acceptor-labeled HLA peptides in at least one cell of one or more cells of the cell population, thereby forming complexes of the affinity acceptor-labeled HLA peptides in at least one cell; enrichment of complexes labeled with an affinity acceptor HLA-peptides; and identification of the peptide from the enriched complexes labeled with an affinity acceptor HLA peptides; and formulating an immunogenic composition based on one or more of the identified peptides, wherein the alleles of the first and second recombinant HLA class I or class II match the HLA haplotype of the subject. In some embodiments, the subject has a disease or condition.

[561] In some embodiments, the first allele of the recombinant HLA class I or class II is different from the allele of the second recombinant HLA class I or class II. In some embodiments, the first acceptor affinity peptide is the same as the second acceptor affinity peptide. In some embodiments, the method includes characterizing the peptide associated with the first and/or second complexes of affinity acceptor-labeled HLA peptides by enrichment. In some embodiments, the at least two alleles of class I or class II affinity-acceptor-tagged HLA include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 HLA class I and/or class II alleles. In some embodiments, the implementation of the first and/or second complexes labeled with an affinity acceptor HLA peptides contain a transmembrane domain. In some embodiments, the implementation of the first and/or second complexes labeled with an affinity acceptor HLA peptides contain an intracellular domain. In some embodiments, the implementation of the first and/or second complexes labeled with an affinity acceptor HLA peptides are not excreted. In some embodiments, the first and/or second complexes of the affinity acceptor-tagged HLA peptides are incorporated into the cell membrane upon expression. In some embodiments, the first and/or second complexes of the HLA acceptor affinity labeled peptides are insoluble complexes of the HLA acceptor affinity labeled peptides.

[562] In some embodiments, the method further comprises creating a database of HLA allele-specific peptides. In some embodiments, the implementation of the method includes the introduction of one or more exogenous peptides in a population of cells. In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more exogenous peptides or the expression of one or more exogenous peptides in a population of cells. In some embodiments, the implementation includes the introduction of a population of cells in contact with one or more nucleic acids encoding one or more exogenous peptides.

[563] In some embodiments, one or more nucleic acids encoding one or more peptides is DNA. In some embodiments, one or more nucleic acids encoding one or more peptides is RNA, optionally the RNA is mRNA.

[564] In some embodiments, enrichment does not include the use of a tetrameric reagent. In some embodiments, the method includes determining the sequence of the peptide or portion thereof associated with the first and/or second complex of the enriched HLA affinity acceptor-labeled peptide. In some embodiments, the determination includes biochemical analysis, mass spectrometric analysis, MS analysis, MS/MS analysis, LC-MS/MS analysis, or a combination thereof.

[565] In some embodiments, the implementation of the method includes assessing the binding affinity or stability of the peptide or part thereof associated with the first and/or second complex affinity-acceptor labeled HLA peptide as a result of enrichment. In some embodiments, the method includes determining whether the peptide or portion thereof associated with the first and/or second complex enriched with an HLA acceptor affinity peptide has one or more mutations. In some embodiments, the method includes evaluating the binding of peptides to HLA molecules in the first and/or second complex of an HLA affinity acceptor-labeled peptide.

[566] In some embodiments, the implementation of the method includes the expression of the library of peptides in a population of cells, thereby forming a library of complexes labeled with an affinity acceptor HLA peptides. In some embodiments, the method includes contacting the cell population with a peptide library or a library of peptide coding sequences, thereby forming a library of HLA affinity acceptor-labeled peptide complexes. In some embodiments, the library is a library of peptides associated with a disease or condition.

[567] In some embodiments, the disease or condition is cancer or infection with an infectious agent. In some embodiments, the method includes introducing the infectious agent or parts thereof into one or more cells of the cell population. In some embodiments, the method includes characterizing one or more peptides from the first and/or second HLA-peptide complexes, optionally the peptides are derived from one or more target proteins of the infectious agent. In some embodiments, the method includes characterizing one or more peptide regions from one or more target proteins of the infectious agent. In some embodiments, the method includes identifying peptides from the first and/or second HLA-peptide complexes derived from the infectious agent.

[568] In some embodiments, the cell population is derived from a biological sample in a subject with a disease or condition. In some embodiments, the cell population is a cell line. In some embodiments, the cell population is a primary cell population. In some embodiments, a peptide from the first and/or second complex, the affinity acceptor-tagged HLA peptide, is capable of activating a T cell in a subject when presented by an antigen presenting cell. In some embodiments, the method includes comparing HLA-peptide complexes from diseased cells with HLA-peptide complexes from unaffected cells. In some embodiments, the method further comprises isolating peptides from the first and/or second complexes of HLA affinity acceptor-labeled peptides prior to identification. In some embodiments, the cell population is a cell population with low cell surface HLA class I or class II expression.

[569] In some embodiments, the cell population expresses one or more endogenous HLA alleles. In some embodiments, the cell population expresses endogenous HLA alleles normally expressed by the cell population. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles. In some embodiments, the cell population is an engineered cell population lacking one or more endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class II alleles. In some embodiments, the cell population is an engineered cell population lacking endogenous HLA class I alleles and endogenous HLA class II alleles. In some embodiments, the cell population has a knockout of one or more HLA class I alleles. In some embodiments, the cell population has a knockout of one or more HLA class II alleles. In some embodiments, a population of cells has a knockout of all HLA class I alleles. In some embodiments, a population of cells has a knockout of all HLA class II alleles. In some embodiments, the cell population has a knockout of all HLA class I alleles and a knockout of all HLA class II alleles. In some embodiments, a sequence encoding at least two alleles of an affinity-acceptor-tagged class I or class II HLA encodes a class I HLA. In some embodiments, Class I HLA is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the first allele of the recombinant HLA class I or class II is the first allele of HLA class I, and the second allele of the recombinant HLA class I or class II is the second allele of HLA class I. In some embodiments, a sequence encoding at least two alleles of an affinity-acceptor-tagged class I or class II HLA encodes a class II HLA. In some embodiments, the HLA class II is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP. In some embodiments, the HLA class II comprises an HLA class II α chain, an HLA class II β chain, or a combination thereof. In some embodiments, the first allele of the recombinant HLA class I or class II is the first allele of HLA class II, and the second allele of the recombinant HLA class I or class II is the second allele of HLA class II.

[570] In some embodiments, each of the first sequence and the second sequence is operably linked. In some embodiments, the first sequence and the second sequence are in different polynucleotide molecules. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to a sequence that encodes the extracellular portion of the first and/or second HLA class I or class II allele. In some embodiments, the first and/or second encoded acceptor affinity peptide is extracellularly expressed. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to the N-terminus of the sequence encoding the first and/or second HLA class I or class II allele. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to a sequence that encodes the intracellular portion of the first and/or second HLA class I or class II allele. In some embodiments, the encoded first and/or second acceptor affinity peptide is expressed intracellularly. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to the C-terminus of the sequence encoding the first and/or second HLA class I or class II allele. In some embodiments, the sequence encoding the first and/or second acceptor affinity peptide is operably linked to the sequence encoding the first and/or second HLA class I or class II allele via a linker.

[571] In some embodiments, enrichment includes enrichment of intact cells expressing the first and/or second complexes of affinity acceptor-tagged HLA peptides. In some embodiments, the method does not include lysing the cells prior to enrichment. In some embodiments, the method further comprises lysing one or more cells prior to enrichment. In some embodiments, enrichment comprises contacting an acceptor affinity peptide binding molecule with a first and/or second complexes of affinity acceptor-labeled HLA peptides, wherein the acceptor affinity peptide binding molecule specifically binds to the first and/or second acceptor affinity peptide. .

[572] In some embodiments, the first and/or second affinity acceptor peptide comprises a tag sequence including a biotin acceptor peptide (BAP), polyhistidine tag, polyhistidine-glycine tag, polyarginine tag, polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tag, Strep -tag II, albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag, bluetongue virus tag (B-tag), calmodulin-binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline- binding domain (CBD), chitin-binding domain (CBD) tag, cellulose-binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein (GBP) tag, maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu tag (EE), human influenza virus hemagglutinin (HA) tag, horseradish peroxidase (HA) tag, NE-tag, HSV tag, ketosteroid isomerase tag (KSI), KT3 tag, LacZ tag, Luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin tag, E tag, S tag, SBP tag, Softag 1, Softag 3, TC tag, VSV tag, Xpress tag, Isopepeptag, SpyTag, SnoopTag, Profinity eXact tag, protein C tag, S1 tag, S tag, carboxybiotin carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag ), tandem affinity purification (TAP) tag, HaloTag, Nus tag, Thioredoxin tag, Fc tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or a combination thereof; optionally, the first and/or second affinity acceptor peptide contains two or more repeats of the tag sequence.

[573] In some embodiments, the affinity acceptor peptide binding molecule is biotin or an antibody specific for the first and/or second acceptor affinity peptide. In some embodiments, enrichment includes contacting the affinity molecule with the first and/or second complexes of affinity acceptor-labeled HLA peptides, wherein the affinity molecule specifically binds to the affinity acceptor peptide binding molecule. In some embodiments, the affinity molecule is streptavidin, NeutrAvidin, or a derivative thereof. In some embodiments, enrichment includes immunoprecipitation of the first and/or second complexes of affinity acceptor-tagged HLA peptides.

[574] In some embodiments, an acceptor affinity peptide binding molecule is attached to a solid surface. In some embodiments, the affinity molecule is attached to a solid surface. In some embodiments, the hard surface is a bead.

[575] In some embodiments, enrichment comprises immunoprecipitating the first and/or second complexes of the affinity acceptor-labeled HLA peptides with an acceptor affinity peptide binding molecule that specifically binds to the first and/or second acceptor affinity peptide. In some embodiments, the affinity acceptor peptide binding molecule does not specifically interact with the amino acid sequence of the encoded first and/or second class I or class II HLA. In some embodiments, enrichment includes contacting an affinity molecule specific for the extracellular portion of the first and/or second HLA class I or class II allele. In some embodiments, enrichment includes contacting an affinity molecule specific for the N-terminal portion of the first and/or second HLA class I or class II allele.

[576] In some embodiments, providing includes contacting a population of cells with a polynucleic acid. In some embodiments, the introduction into contact includes transfection or transduction. In some embodiments, providing includes contacting a population of cells with a vector containing a polynucleic acid. In some embodiments, the vector is a viral vector. In some embodiments, the polynucleic acid is stably inserted into the genome of a population of cells.

[577] In some embodiments, the sequence encoding the first and/or second HLA class I or class II contains a sequence encoding the α-chain of HLA class I. In some embodiments, the first allele of the recombinant HLA class I or class II is the α chain of the first HLA class I, and the second allele of the recombinant HLA class I or II is the α chain of the second HLA class I.

[578] In some embodiments, the implementation of the method further includes the expression of a sequence encoding β2 microglobulin in one or more cells. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding a first and/or second class I or class II HLA. In some embodiments, the β2 microglobulin coding sequence is linked to the first and/or second class I or class II HLA coding sequence via a linker. In some embodiments, a sequence encoding a β2 microglobulin is linked to a sequence encoding a third acceptor affinity peptide.

[579] In some embodiments, the third acceptor affinity peptide is different from the first and/or second acceptor affinity peptide. In some embodiments, the sequence encoding the first and/or second HLA class I or class II comprises a sequence encoding an HLA class II α chain and/or an HLA class II β chain. In some embodiments, the sequence encoding the first and/or second HLA class I or class II contains a sequence encoding the α chain of the first HLA class II and the α chain of the second HLA class II. In some embodiments, the method further comprises expressing an HLA class II β chain coding sequence in one or more cells. In some embodiments, a sequence encoding a first HLA class II α chain and a second HLA class II α chain are linked to a sequence encoding an HLA class II β chain. In some embodiments, the sequence encoding the first and/or second HLA class I or class II contains a sequence encoding the β chain of the first HLA class II and the β chain of the second HLA class II.

[580] In some embodiments, the implementation of the method further includes the expression of the sequence encoding the α-chain of HLA class II, in one or more cells. In some embodiments, a sequence encoding a first HLA class II β chain and a second HLA class II β chain are linked to a sequence encoding an HLA class II α chain via a linker. In some embodiments, a sequence encoding an HLA class II β chain or an HLA class II α chain is linked to a sequence encoding a third acceptor affinity peptide. In some embodiments, the third acceptor affinity peptide is different from the first and/or second acceptor affinity peptide.

[581] In some embodiments, the third acceptor affinity peptide is different from the first acceptor affinity peptide and is selected from the group consisting of a biotin acceptor peptide (BAP), a polyhistidine tag, a polyhistidine glycine tag, a polyarginine tag, a polyaspartate tag, polycysteine tag, polyphenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene protein 10 tag, streptavidin tag, streptavidin-binding peptide (SPB) tag, Strep tags, Strep II tags, albumin binding protein (ABP) tags, alkaline phosphatase (AP) tags, bluetongue virus tags (B tags), calmodulin binding peptide (CBP) tags, chloramphenicol acetyltransferase (CAT) tags ), choline-binding domain (CBD) tags, chitin-binding domain (CBD) tags, cellulose-binding domain (CBP) tags, dihydrofolate reductase (DHFR) tags, galactose-binding protein tags a (GBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tags, human influenza virus hemagglutinin (HA) tags, horseradish peroxidase (HA) tags, NE-tags, HSV tags , ketosteroid isomerase (KSI) tags, KT3 tags, LacZ tags, luciferase tags, NusA tags, PDZ domain tags, AviTag, calmodulin tags, E-tags, S-tags, SBP-tags, Softag 1, Softag 3, TC tags , VSV tags, Xpress tags, Isopeptag, SpyTag, SnoopTag, Profinity eXact tags, protein C tags, S1 tags, S tags, carboxybiotin carrier protein (BCCP) tags, green fluorescent protein (GFP) tags, small ubiquitin tags -like modifier (SUMO), tandem affinity purification (TAP) tags, HaloTag, Nus tags, Thioredoxin tags, Fc tags, CYD tags, HPC tags, TrpE tags, ubiquitin tags, VSV-G epitope tags, V5 tags and their combinations; optionally, the first or second acceptor affinity peptide contains two or more repeats of the tag sequence.

[582] In some embodiments, the linker contains a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome escape site or internal ribosome entry site (IRES). In some embodiments, the ribosome escape site or IRES is cleaved when expressed in cells. In some embodiments, the ribosome exit site is selected from the group consisting of F2A, T2A, P2A, and E2A. In some embodiments, the IRES element is selected from common cellular or viral IRES sequences.

[583] In some embodiments, the implementation of the method includes conducting biochemical analysis or mass spectrometry, such as tandem mass spectrometry. In some embodiments, the method includes obtaining a peptide sequence that corresponds to MS/MS spectra of one or more peptides isolated from enriched complexes of HLA affinity acceptor-labeled peptides from a peptide database; wherein one or more of the resulting sequences identifies the sequence of one or more peptides.

[584] In some embodiments, the cell population is a cell line selected from HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, and THP1. In some embodiments, the cell line is treated with one or more cytokines, checkpoint inhibitors, epigenetically active drugs, IFN-γ, or a combination thereof. In some embodiments, the cell population includes at least 10 ⁵ cells, at least 10 ⁶ cells, or at least 10 ⁷ cells. In some embodiments, the cell population is a population of dendritic cells, macrophages, cancer cells, or B cells. In some embodiments, the cell population includes tumor cells.

[585] In some embodiments, a population of cells is contacted with an agent prior to isolation of the first and/or second HLA-peptide complexes from one or more cells. In some embodiments, the agent is an inflammatory cytokine, a chemical agent, an adjuvant, a therapeutic agent, or radiation.

[586] In some embodiments, the first and/or second HLA allele is a mutant HLA allele. In some embodiments, the sequence encoding the first and/or second HLA allele contains a barcoded sequence. In some embodiments, the method further comprises analyzing the expression of a first and/or second allele labeled with an HLA class I or class II affinity acceptor.

[587] In some embodiments, the analysis includes sequencing a first and/or second allele labeled with an HLA class I or class II affinity acceptor, detecting RNA encoding the RNA of the first and/or second allele labeled with an HLA class I or class II affinity acceptor, detecting a protein of the first and/or second allele labeled with an HLA class I or class II affinity acceptor, or a combination thereof. In some embodiments, the first and second allele of an affinity-acceptor-tagged class I or class II HLA contains a unique barcoded sequence. In some embodiments, the first sequence and the second sequence comprise a unique barcoded sequence.

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List of Sequences

<110> NEON THERAPEUTICS, INC.

<120> HLA-BASED METHODS AND COMPOSITIONS AND THEIR USE

<130> 50401-704.601

<140> PCT/US2018/017849

<141> 2018-02-12

<150> 62/461.162

<151> 2017-02-20

<150> 62/457.978

<151> 2017-02-12

<160> 37

<170>PatentIn version 3.5

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<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

Synthetic peptide"

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Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

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<210> 2

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<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

Synthetic His label"

<220>

<221> MISC_FEATURE

<222> (1)..(15)

<223> /note="this sequence can span 4-15 residues"

<220>

<221> source

<223> /note="See the description filed for a detailed description

substitutions and preferred embodiments"

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His His His His His His His His His His His His His His His His

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<210> 3

<211> 18

<212> PRT

<213> Thosea asigna virus

<400> 3

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

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GlyPro

<210> 4

<211> 19

<212> PRT

<213> Porcine teschovirus

<400> 4

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

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Pro Gly Pro

<210> 5

<211> 20

<212> PRT

<213> Equine rhinitis virus A

<400> 5

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

1 5 10 15

Asn Pro Gly Pro

twenty

<210> 6

<211> 22

<212> PRT

<213> foot-and-mouth disease virus

<400> 6

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

1 5 10 15

Glu Ser Asn Pro Gly Pro

twenty

<210> 7

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

Synthetic 6xHis label"

<400> 7

His His His His His His His

fifteen

<210> 8

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

Synthetic peptide"

<400> 8

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

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<210> 9

<211> 9

<212> PRT

<213> Homo sapiens

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<210> 10

<211> 9

<212> PRT

<213> Homo sapiens

<400> 10

Tyr Val Pro Glu His Trp Glu Tyr Tyr

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<210> 11

<211> 9

<212> PRT

<213> Homo sapiens

<400> 11

Lys Leu Asp Leu Leu Glu Gln Glu Tyr

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<210> 12

<211> 9

<212> PRT

<213> Homo sapiens

<400> 12

Thr Thr Asp Gly Tyr Leu Leu Arg Leu

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<210> 13

<211> 9

<212> PRT

<213> Homo sapiens

<400> 13

Asp Thr Glu Phe Pro Asn Phe Lys Tyr

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<210> 14

<211> 9

<212> PRT

<213> Homo sapiens

<400> 14

Ala Thr Asp Gly Lys Val Leu Leu Trp

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<211> 9

<212> PRT

<213> Homo sapiens

<400> 15

Asp Gly Leu Leu Arg Val Leu Thr Val

fifteen

<210> 16

<211> 9

<212> PRT

<213> Homo sapiens

<400> 16

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<210> 17

<211> 9

<212> PRT

<213> Homo sapiens

<400> 17

Asp Gly Leu Arg Asp Leu Pro Ser Ile

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<210> 18

<211> 9

<212> PRT

<213> Homo sapiens

<400> 18

Asp Gly Tyr Val Val Lys Glu Thr Ile

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<210> 19

<211> 9

<212> PRT

<213> Homo sapiens

<400> 19

Asp Gly Arg Leu Val Ile Asn Arg Val

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<210> 20

<211> 9

<212> PRT

<213> Homo sapiens

<400> 20

Asp Ala Tyr Pro Gln Arg Ile Lys Phe

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<210> 21

<211> 9

<212> PRT

<213> Homo sapiens

<400> 21

Ile Ile Tyr Pro Thr Pro Lys Val Val

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<210> 22

<211> 9

<212> PRT

<213> Homo sapiens

<400> 22

Val Pro Pro Leu Val Pro Lys Val Leu

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<210> 23

<211> 9

<212> PRT

<213> Homo sapiens

<400> 23

Thr Pro Glu Ser Lys Ile Arg Phe Val

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<210> 24

<211> 9

<212> PRT

<213> Homo sapiens

<400> 24

Val Ala Leu Leu Val Gly Glu Lys Val

fifteen

<210> 25

<211> 9

<212> PRT

<213> Homo sapiens

<400> 25

Tyr Ile Ile Glu Arg Glu Pro Leu Ile

fifteen

<210> 26

<211> 9

<212> PRT

<213> Homo sapiens

<400> 26

Phe Tyr Pro Glu Leu Lys Leu Ala Tyr

fifteen

<210> 27

<211> 9

<212> PRT

<213> Homo sapiens

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His Phe Leu Asp Arg His Leu Val Phe

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<210> 28

<211> 9

<212> PRT

<213> Homo sapiens

<400> 28

Tyr Leu Pro Ala Leu Lys Val Glu Tyr

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<210> 29

<211> 9

<212> PRT

<213> Homo sapiens

<400> 29

Leu Pro Leu Asp Ile Gln Ile Phe Tyr

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<210> 30

<211> 9

<212> PRT

<213> Homo sapiens

<400> 30

Ala Leu Ser Asp Leu Ala Leu His Phe

fifteen

<210> 31

<211> 9

<212> PRT

<213> Homo sapiens

<400> 31

Gly Leu Asp Asp Lys Leu Leu His Tyr

fifteen

<210> 32

<211> 9

<212> PRT

<213> Homo sapiens

<400> 32

Gly Leu Pro Asp Leu Val Ala Lys Tyr

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<211> 9

<212> PRT

<213> Homo sapiens

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Tyr Pro Val Tyr Gln Pro Val Gly Pro

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<211> 9

<212> PRT

<213> Homo sapiens

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Tyr Gly Phe Val Asn Tyr Val Thr Ala

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<212> PRT

<213> Homo sapiens

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Phe Val Glu Ile Leu Ile Pro Val Ala

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<212> PRT

<213> Homo sapiens

<400> 36

Phe Pro Lys Glu Thr Phe Glu Gly Pro

fifteen

<210> 37

<211> 9

<212> PRT

<213> Homo sapiens

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Ser Ala Pro Val Asn Phe Ile Ser Ala

fifteen

<---

Claims (34)

1. A method for selecting peptide sequences for preparing a vaccine composition, including: (a) processing amino acid sequence information of a plurality of candidate peptide sequences expressed by cancer cells of a single human subject using a machine learning HLA peptide presentation prediction model to generate a plurality of presentation predictions, where each presentation prediction of said set of presentation predictions indicates the probability that the peptide sequence of the plurality of candidate peptide sequences binds to the MHC protein of said single human subject; where a model for predicting the presentation of an HLA peptide using machine learning is trained using training data containing sequence information of training peptide sequences identified by mass spectrometry to be represented by a recombinant MHC protein expressed in training cells, where the recombinant MHC protein is incorporated into the cell membrane of learning cells upon expression, and where: (i) learning cells do not express endogenous MHC, or (ii) the recombinant MHC proteins contain an affinity acceptor peptide sequence; and (b) selecting, based on at least a plurality of presentation predictions, a peptide sequence from a plurality of peptide sequences that is predicted to be presented by the MHC protein of said one individual, wherein the one or more selected peptide sequences are for preparing a vaccine composition comprising a polypeptide with one or more selected peptide sequences, or a polynucleotide encoding said polypeptide. 2. The method of claim 1, wherein the affinity acceptor peptide sequence is operably linked to the extracellular portion of the recombinant MHC protein. 3. The method of claim 1 wherein the affinity acceptor peptide is a Biotin Acceptor Protein (BAP). 4. The method of claim 1, wherein the MHC protein of one human subject is a class II MHC protein. 5. The method of claim 1 wherein the learning cells express one recombinant MHC protein. 6. The method according to p. 1, where the learning cells express: (a) a first recombinant MHC protein comprising a first MHC molecule and a first acceptor affinity peptide; (b) a second recombinant MHC protein comprising a second MHC molecule and a second acceptor affinity peptide, where the first MHC molecule and the second MHC molecule are not identical; and wherein the first acceptor affinity peptide and the second acceptor affinity peptide are not identical. 7. The method of claim 1 wherein the learning cell is an antigen presenting cell line. 8. The method of claim 1, wherein the machine learning HLA peptide presentation prediction model is trained by a method comprising the step of immunoprecipitating training cells expressing recombinant MHCs and determining sequences of training peptides associated with recombinant MHC proteins using mass spectrometry. 9. The method of claim 8, wherein the identification further comprises performing sequence analysis, biochemical analysis, or a combination thereof. 10. The method of claim 1, wherein the machine learning HLA peptide presentation prediction model is trained by a method comprising the step of introducing as a variable information about a plurality of training peptides identified by mass spectrometry and information about the MHC protein from said recombinant MHC proteins in a presentation prediction algorithm. 11. The method of claim 10 wherein the variable includes one or more variables selected from the group consisting of peptide sequence, physical properties of amino acids, physical properties of peptides, expression level of the parent peptide protein in the cell, protein stability, protein translation rate, ubiquitination sites , protein degradation rate, translation efficiency from ribosomal profiling, protein cleavage, protein localization, host protein motifs that facilitate TAP transport, the host protein undergoes autophagy, motifs that promote ribosome arrest, and protein features that promote non-sense mediated degradation ( NMD). 12. The method of claim 1, further comprising preparing a vaccine composition comprising a polypeptide with one or more selected peptide sequences or a polynucleotide encoding said polypeptide. 13. The method of claim 1 wherein the learning peptides comprise endogenous learning cell peptides. 14. The method of claim 8, wherein the method further comprises the step of generating a peptide library containing disease-associated peptides prior to the step of immunoprecipitation. 15. The method of claim 8, wherein the recombinant MHC protein contains at least 10 different recombinant MHC proteins. 16. The method of claim 10, wherein the method further comprises the step of generating a database of HLA allele-specific peptide sequences for a plurality of HLA alleles. 17. The method of claim 1, further comprising administering to the subject a vaccine composition comprising a polypeptide with one or more selected peptide sequences or a polynucleotide encoding said polypeptide. 18. The method of claim 1, further comprising administering to the subject a composition comprising: (a) one or more APCs containing one or more selected peptide sequences, (b) a cell containing a T cell receptor (TCR) specific for one or more selected peptide sequences in complex with the MHC of one human subject, or (c) a cell containing a TCR or chimeric T cell receptor (CAR) specific for one or more selected peptide sequences in complex with the MHC of one human subject. 19. The method of claim 1, wherein the recombinant MHC proteins contain an acceptor affinity peptide sequence, wherein the sequence of each training peptide is associated with an MHC protein identity, where the MHC protein identity is based on the MHC protein identity of the recombinant MHC protein immunoprecipitated from training cells using a reagent against an affinity acceptor peptide that binds to an affinity acceptor peptide sequence.

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