HLA-based methods and compositions and uses thereof

Abstract

To provide HLA-based methods and compositions, and to provide uses thereof.SOLUTION: The present disclosure provides compositions and methods for isolating HLA-peptides from cells. The present disclosure provides a universal platform and methods for profiling the HLA-peptidome, enabling identification of endogenously presented HLA-peptides from cell lines expressing any possible class I or II construct. The methods and compositions described herein find uses in a wide range of applications. For example, the methods and compositions described herein can be used to identify immunogenic antigen peptides and can be used to develop drugs, such as personalized medicine drugs.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application claims priority to 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 is incorporated by reference herein in its entirety. [Background technology]

[0002] The major histocompatibility complex (MHC) is a gene complex that encodes human leukocyte antigen (HLA) genes. HLA genes are expressed as protein heterodimers that are displayed on the surface of human cells to circulating T cells. HLA genes are highly polymorphic, allowing for fine-tuning of the adaptive immune system. The adaptive immune response relies, in part, on the ability of T cells to identify and eliminate cells that display disease-associated peptide antigens bound to human leukocyte antigen (HLA) heterodimers.

[0003] In humans, endogenous and exogenous proteins are processed into peptides by the proteasome and by cytoplasmic and endosomal / lysosomal proteases and peptidases, which can then be presented by two classes of cell surface proteins encoded by the MHC. These cell surface proteins are called human leukocyte antigens (HLA class I and class II), and the peptides that bind to them and elicit an immune response are called HLA epitopes. HLA epitopes are important components that enable the immune system to detect danger signals, such as pathogen infection and self-transformation. Circulating CD8+ T cells recognize class I MHC (HLA-A, HLA-B, and HLA-C) epitopes derived from the endogenous processing pathway and displayed on nearly all nucleated cells. CD4+ T cells recognize class II MHC (HLA-DR, HLA-DQ, and HLA-DP) epitopes displayed on antigen-presenting cells (APCs), such as dendritic cells and macrophages. HLA class II peptide presentation activates helper T cells, which subsequently promote 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.

[0004] Genes encoding HLA heterodimers are highly polymorphic, with over 12,000 class I and 4,000 class II allelic variants identified across the human population. Individuals can inherit different alleles at each of the class I and class II HLA loci from maternal and paternal HLA haplotypes. Class I HLA molecules are heterodimers composed of an α heavy chain encoded by a class I HLA gene and β-2-microglobulin (B2M). Class II HLA molecules are heterodimers of α and β chains, both encoded by class II HLA genes. Due to the combination of α and β chain pairing, the population of HLA heterodimers is highly complex. Furthermore, it is estimated that each HLA heterodimer binds thousands of peptides with allele-specific binding selectivity. In fact, it is estimated that each HLA allele binds and presents approximately 1,000–10,000 unique peptides to T cells, representing less than 0.1% of the approximately 10 million potential 9-mer peptides derived from human protein-coding genes. Given such diversity in HLA binding, accurate prediction of whether a peptide will bind to a particular HLA allele is extremely challenging. Due to heterogeneity in α-chain and β-chain pairing, data complexity that limits the ability to unambiguously assign core binding epitopes, and the lack of immunoprecipitation-grade allele-specific antibodies required for high-resolution biochemical analysis, little is known about the allele-specific peptide-binding characteristics of HLA class II molecules. Furthermore, analysis of peptide epitopes derived from a given HLA allele can be ambiguous when multiple HLA alleles are presented on the cell surface.

[0005] Understanding the binding selectivity of each HLA heterodimer is key to successfully predicting which neoantigens are likely to elicit tumor-specific T cell responses. Clearly, methods are needed to identify and isolate specific class I and class II HLA-associated peptides (e.g., neoantigen peptides). Such methods and isolated molecules are useful, for example, for the study of HLA-associated peptides and for the development of therapeutics, including, but not limited to, immune-based therapeutics.

[0006] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention [Means for solving the problem]

[0007] The methods and compositions described herein find use in a variety 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 medicine drugs.

[0008] Provided herein are methods for characterizing HLA-peptide complexes, the methods comprising: providing a cell population, wherein one or more cells of the cell population comprise a polynucleic acid comprising a sequence encoding an affinity acceptor-tagged class I or class II HLA allele, the sequence encoding the affinity acceptor-tagged HLA comprising a sequence encoding a recombinant class I or class II HLA allele operably linked to a sequence encoding an affinity acceptor peptide; expressing the affinity acceptor-tagged HLA in at least one cell of the one or more cells of the cell population, thereby forming an affinity acceptor-tagged HLA-peptide complex in the at least one cell; enriching the affinity acceptor-tagged HLA-peptide complex; and characterizing the HLA-peptide complex. In some embodiments, the encoded affinity acceptor-tagged class I or class II HLA allele is a soluble affinity acceptor-tagged class I or class II HLA allele.

[0009] In some embodiments, the characterizing step comprises characterizing peptides bound to the affinity acceptor-tagged HLA-peptide complexes from the enriching step. In some embodiments, the method comprises performing the method steps for two or more class I and / or class II HLA alleles. In some embodiments, the two or more class I and / or class II HLA alleles comprise 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 class I and / or class II HLA 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 cell membranes when expressed. In some embodiments, the affinity acceptor-tagged HLA-peptide complexes are soluble affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the affinity acceptor-tagged HLA-peptide complexes are not soluble affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the method further comprises generating an HLA-allele-specific peptide database. In some embodiments, the recombinant class I or class II HLA allele is a single recombinant class I or class II HLA allele.

[0010] In some embodiments, the method includes providing a cell population comprising one or more cells each comprising affinity acceptor-tagged HLA, the cells comprising different recombinant polypeptides encoded by different HLA alleles operably linked to affinity acceptor peptides; enriching the affinity acceptor-tagged HLA-peptide complexes; and characterizing peptides or portions thereof bound to the affinity acceptor-tagged HLA-peptide complexes resulting from the enriching step.

[0011] In some embodiments, the method includes introducing one or more peptides into a cell population. In some embodiments, the introducing step includes contacting the cell population with one or more peptides or expressing one or more peptides in the cell population. In some embodiments, the introducing step includes contacting the cell population with one or more nucleic acids encoding one or more peptides. In some embodiments, the one or more nucleic acids encoding the one or more peptides are DNA. In some embodiments, the one or more nucleic acids encoding the one or more peptides are RNA, and optionally, the RNA is mRNA. In some embodiments, the enriching step does not include the use of a tetramer reagent.

[0012] In some embodiments, the characterizing step comprises determining the sequence of the peptide or portion thereof bound to the affinity acceptor-tagged HLA-peptide complexes from the enriching step, and optionally determining whether the peptide or portion thereof is modified. In some embodiments, the determining step comprises biochemical analysis, mass spectrometry, MS analysis, MS / MS analysis, LC-MS / MS analysis, or a combination thereof. In some embodiments, the characterizing step comprises evaluating the binding affinity or stability of the peptide or portion thereof bound to the affinity acceptor-tagged HLA-peptide complexes from the enriching step. In some embodiments, the characterizing step comprises determining whether the peptide or portion thereof bound to the affinity acceptor-tagged HLA-peptide complexes from the enriching step contains one or more mutations. In some embodiments, the characterizing step comprises evaluating the association of the peptide with an HLA molecule in the affinity acceptor-tagged HLA-peptide complex.

[0013] In some embodiments, the method comprises expressing a library of peptides in the cell population, thereby forming a library of affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the method comprises contacting the cell population with a library of peptides or a library of sequences encoding peptides, thereby forming a library of affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the library comprises a library of peptides associated with a disease or condition. In some embodiments, the library comprises a library of peptides derived from a polypeptide drug, such as a biopharmaceutical (e.g., an antibody drug).

[0014] In some embodiments, the disease or condition is cancer, infection by an infectious agent, or an autoimmune response. In some embodiments, the method includes introducing an infectious agent, or a portion thereof, into one or more cells of the cell population. In some embodiments, the method includes introducing a polypeptide drug, such as a biopharmaceutical (e.g., an antibody drug), or a portion thereof, into one or more cells of the cell population. In some embodiments, the method includes characterizing one or more peptides derived from HLA-peptide complexes, optionally wherein 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 the peptides derived from one or more target proteins of the infectious agent or polypeptide drug.

[0015] In some embodiments, the method includes identifying peptides derived from HLA-peptide complexes derived from an infectious agent. In some embodiments, the cell population is derived from a biological sample from 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 population of primary cells. In some embodiments, recombinant class I or class II HLA alleles are matched to a subject with a disease or condition.

[0016] In some embodiments, peptides derived from affinity acceptor-tagged HLA-peptide complexes can activate T cells from the subject when presented by antigen-presenting cells. In some embodiments, the characterizing step includes comparing HLA-peptide complexes derived from cancer cells with HLA-peptide complexes derived from non-cancer cells. In some embodiments, the cell population comprises a plurality of cell populations, 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 containers.

[0017] In some embodiments, the method further comprises isolating peptides derived from the affinity acceptor-tagged HLA-peptide complexes prior to the characterizing step. In some embodiments, the HLA-peptide complexes are isolated using an anti-HLA antibody. In some cases, HLA-peptide complexes with or without an affinity tag are 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 beads or a column containing an anti-HLA antibody. In some embodiments, the peptides are isolated using an anti-HLA antibody. In some cases, soluble HLA (sHLA) 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 using a column containing an anti-HLA antibody. In some embodiments, the method further comprises removing one or more amino acids from the termini of the peptides bound to the affinity acceptor-tagged HLA-peptide complexes.

[0018] In some embodiments, the cell population is a population of low cell surface HLA class I or class II expressing cells. 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 I alleles and endogenous HLA class II alleles. In some embodiments, the cell population is a knockout of one or more HLA class I alleles. In some embodiments, the cell population is a knockout of one or more HLA class II alleles. In some embodiments, the cell population is a knockout of all HLA class I alleles. In some embodiments, the cell population is knockout of all HLA class II alleles. In some embodiments, the cell population is knockout of all HLA class I alleles and knockout of all HLA class II alleles. In some embodiments, the sequences encoding the recombinant class I or class II HLA alleles encode 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 sequences encoding the recombinant class I or class II HLA alleles encode class II HLA. In some embodiments, the class II HLA is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP. In some embodiments, the class II HLA comprises an HLA class II α chain, an HLA class II β chain, or a combination thereof. In some embodiments, each sequence encodes at least two different class I and / or class II HLA alleles.

[0019] In some embodiments, at least two different class I and / or class II HLA alleles are each operably linked to a sequence encoding an affinity acceptor peptide. In some embodiments, at least two different class I and / or class II HLA alleles are each operably linked to a sequence encoding a different affinity acceptor peptide. In some embodiments, at least two different class I and / or class II HLA alleles are each operably linked to a sequence encoding an affinity acceptor peptide. In some embodiments, one or more of the at least two different class I and / or class II HLA alleles are operably linked to a sequence encoding a first affinity acceptor peptide, and one or more of the at least two different class I and / or class II HLA alleles are operably linked to a sequence encoding a second affinity acceptor peptide. In some embodiments, at least two different class I and / or class II HLA alleles are each operably linked to a sequence encoding a different affinity acceptor peptide. In some embodiments, each of the at least two different class I and / or class II HLA alleles is operably linked to a sequence encoding a different affinity acceptor peptide. In some embodiments, each of the at least two different class I and / or class II HLA alleles is operably linked to a sequence encoding an affinity tag. In some embodiments, the method includes administering at least a second polynucleic acid comprising a sequence encoding a different recombinant HLA allele operably linked to the same or a different affinity acceptor peptide.

[0020] In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence encoding the extracellular portion of a recombinant class I or class II HLA allele. In some embodiments, the encoded affinity acceptor peptide is expressed extracellularly. In some embodiments, the encoded affinity acceptor peptide is located on an extracellular portion of a recombinant class I or class II HLA allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the N-terminus of a sequence encoding a recombinant class I or class II HLA allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence encoding an intracellular portion of a recombinant class I or class II HLA allele. In some embodiments, the encoded affinity acceptor peptide is expressed intracellularly. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the C-terminus of a sequence encoding a recombinant class I or class II HLA allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to an internal sequence of a sequence encoding a recombinant class I or class II HLA allele, such as a flexible loop sequence. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the sequence encoding the recombinant class I or class II HLA allele by a linker. In some embodiments, the enriching step comprises enriching intact cells expressing the affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the method does not comprise lysing the cells prior to the enriching step. In some embodiments, the method further comprises lysing one or more cells prior to the enriching step. In some embodiments, the enriching step comprises contacting an affinity acceptor peptide-binding molecule with the affinity acceptor-tagged HLA-peptide complexes, wherein the affinity acceptor peptide-binding molecule specifically binds to the affinity acceptor peptide.

[0021] In some embodiments, the affinity acceptor peptide is a biotin acceptor peptide (BAP), a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin-binding peptide (SPB) tag, a Strep tag, a Strep tag II, an albumin-binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag (B-tag), a calmodulin-binding peptide (CBP) tag, a chloramphenicol acetyltransferase (CAT) tag, a 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), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, ProfinityThe tag sequence comprises an eXact tag, protein C tag, S1-tag, S-tag, biotin-carboxy 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, sortase tag, a tag that forms a covalent peptide bond to the bead, or a combination thereof, and optionally the affinity acceptor peptide comprises two or more repeats of the tag sequence.

[0022] In some embodiments, the affinity acceptor peptide-binding molecule is biotin or an antibody specific for the affinity acceptor peptide. In some embodiments, the enriching step comprises contacting an affinity molecule with the affinity acceptor-tagged HLA-peptide complex, wherein the affinity molecule specifically binds to the affinity acceptor peptide-binding molecule.

[0023] In some embodiments, the affinity molecule comprises a molecule that binds to biotin. For example, the affinity molecule may include streptavidin, NeutrAvidin, including protein homologs and derivatives thereof from other organisms.

[0024] In some embodiments, the enriching step comprises immunoprecipitating the affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the 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 solid surface is a bead. In some embodiments, the enriching step comprises immunoprecipitating the affinity acceptor-tagged HLA-peptide complexes using an affinity acceptor peptide binding molecule that specifically binds to the affinity acceptor peptide.

[0025] In some embodiments, the affinity acceptor peptide binding molecule does not specifically interact with the amino acid sequence of the encoded recombinant class I HLA or class II HLA. In some embodiments, the enriching step comprises contacting an affinity molecule specific for the extracellular portion of the recombinant class I or class II HLA allele. In some embodiments, the enriching step comprises contacting an affinity molecule specific for the N-terminal portion of the recombinant class I or class II HLA allele.

[0026] In some embodiments, the providing step comprises contacting the cell population with the polynucleic acid. In some embodiments, the contacting step comprises transfecting or transducing. In some embodiments, the providing step comprises contacting the cell population with a vector comprising the polynucleic acid. In some embodiments, the vector is a viral vector. In some embodiments, the polynucleic acid is stably integrated into the genome of the cell population.

[0027] In some embodiments, the sequence encoding the recombinant class I or class II HLA comprises a sequence encoding an HLA class I α chain. In some embodiments, the method further comprises expressing a sequence encoding β2 microglobulin in one or more cells. In some embodiments, the sequence encoding β2 microglobulin is connected to a sequence encoding an HLA class I α chain. In some embodiments, the sequence encoding β2 microglobulin is connected to a sequence encoding an HLA class I α chain by a linker. In some embodiments, the sequence encoding β2 microglobulin is connected to a sequence encoding a second affinity acceptor peptide. In some embodiments, the sequence encoding the recombinant class I or class II HLA comprises 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, the sequence encoding an HLA class II β chain is connected to a sequence encoding an HLA class II α chain. In some embodiments, the sequence encoding an HLA class II β chain is connected to a sequence encoding an HLA class II α chain by a linker. In some embodiments, the sequence encoding an HLA class II β chain is connected to a sequence encoding a second affinity acceptor peptide.

[0028] In some embodiments, the second affinity acceptor peptide is different from the first affinity acceptor peptide and is a biotin acceptor peptide (BAP), poly-histidine tag, poly-histidine-glycine tag, poly-arginine tag, poly-aspartic acid tag, poly-cysteine ​​tag, poly-phenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene 10 protein peptide 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) tag, 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) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinityand optionally the first or second affinity acceptor peptide comprises two or more repeats of the tag sequence.

[0029] In some embodiments, the linker comprises a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome skipping site or an internal ribosome entry site (IRES) element. In some embodiments, the ribosome skipping site or IRES is cleaved when expressed in a cell. In some embodiments, the ribosome skipping 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.

[0030] In some embodiments, the determining step includes performing biochemical analysis or mass spectrometry, such as tandem mass spectrometry. In some embodiments, the determining step includes obtaining peptide sequences corresponding to MS / MS spectra of one or more peptides isolated from the enriched affinity acceptor-tagged HLA-peptide complexes from a peptide database, wherein the obtained one or more sequences identify the sequences of the one or more peptides. In some embodiments, the peptide database is a non-enzyme-specific peptide database, such as one that does not include or includes a modified database. In some embodiments, the method further includes searching the peptide database using a reverse 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, HepG2, SH-SY5Y, CACO-2, U937, U-2 OS, ExpiCHO, CHO, and THP1.

[0031] 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 combinations thereof. In some embodiments, the cell population is treated with one or more reagents that modulate the metabolic pathway or metabolic state of the cell. In some embodiments, the cell population is treated with one or more reagents that modulate the cellular proteome of the cell. In some embodiments, the cell population is treated with one or more reagents that modulate or regulate the cellular expression or transcription of the cell (e.g., AIRE or CREB binding protein or modulators thereof). In some embodiments, the cell population is treated with one or more reagents that modulate or regulate the cellular expression or transcription of the cell. In some embodiments, the cell population is treated with one or more reagents that modulate or regulate the cellular expression or transcription of an HLA of the cell. In some embodiments, the cell population is treated with one or more reagents that modulate or regulate the cellular expression or transcription of the proteome of the cell.

[0032] In some embodiments, the cell population comprises at least 10 5 cells, at least 10 6 cells or at least 10 7 In some embodiments, the cell population comprises a population of dendritic cells, macrophages, cancer cells, or B cells. In some embodiments, the cell population comprises tumor cells. In some embodiments, the cell population is contacted with an agent prior to isolating the 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.

[0033] In some embodiments, the HLA allele is a mutated HLA allele. In some embodiments, the sequence encoding the HLA allele comprises a barcode sequence. In some embodiments, the method further comprises assaying for expression of the affinity acceptor-tagged class I or class II HLA allele. In some embodiments, the assaying comprises sequencing the affinity acceptor-tagged class I or class II HLA allele, detecting affinity acceptor-tagged class I or class II HLA allele RNA, detecting affinity acceptor-tagged class I or class II HLA allele protein, or a combination thereof. In some embodiments, the assaying for expression may comprise a Western blot assay, fluorescence-activated cell sorting (FACS), mass spectrometry (MS), a microarray hybridization assay, an RNA-seq assay, a polymerase chain reaction assay, a LAMP assay, a ligase chain reaction assay, a Southern blot assay, a Northern blot assay, or an enzyme-linked immunosorbent assay (ELISA).

[0034] In some embodiments, the method comprises performing the steps of the method for different HLA alleles. In some embodiments, each different HLA allele comprises a unique barcode sequence. In some embodiments, each polynucleic acid encoding a different HLA allele comprises a unique barcode sequence.

[0035] Provided herein is an HLA-allele-specific binding peptide sequence database 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 predictive algorithm for identifying HLA-allele-specific binding peptides, comprising training a machine using the peptide sequence database described herein or the combination described herein.

[0036] In some embodiments, the machine combines one or more linear models, support vector machines, decision trees, and neural networks. In some embodiments, the variables used to train the machine include one or more variables selected from the group consisting of peptide sequence, amino acid physical properties, peptide physical properties, expression level of the peptide's source protein in cells, protein stability, protein translation rate, ubiquitination site, protein degradation rate, translation efficiency from ribosome profiling, protein cleavability, protein localization, host protein motifs that facilitate TAP transport, host proteins that undergo autophagy, motifs that favor ribosome stalling, and protein properties that favor NMD.

[0037] In some embodiments, the motif that favors ribosome stalling comprises a polyproline or polylysine stretch. In some embodiments, the protein trait that favors NMD is selected from the group consisting of a long 3'UTR, a stop codon more than 50 nucleic acids upstream of the last exon:exon junction, and peptide cleavability.

[0038] Provided herein are methods for identifying HLA-allele-specific binding peptides, comprising analyzing peptide sequences using a machine trained with a peptide sequence database obtained by performing the method described herein on HLA-alleles. In some embodiments, the method comprises determining the expression level of a source protein of the peptide in a cell, wherein the source protein expression is a predictor variable used by the machine. In some embodiments, the expression level is determined by measuring the amount of source protein or the amount of RNA encoding the source protein.

[0039] 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 comprise sequences encoding different recombinant HLA class I α chain alleles, sequences encoding an affinity acceptor peptide, and optionally, sequences encoding β2 microglobulin, and wherein sequences (a) and (b), and optionally (c), are operably linked.

[0040] Provided herein are compositions comprising recombinant polynucleic acids comprising two or more sequences, each comprising a sequence encoding an affinity acceptor-tagged HLA, wherein the affinity acceptor-tagged HLA-encoding sequences comprise a sequence encoding a recombinant HLA class II α chain allele, a sequence encoding an affinity acceptor peptide, and optionally a sequence encoding an HLA class II β chain, wherein the sequences of (a) and (b), and optionally (c), are operably linked. In some embodiments, the recombinant polynucleic acid is isolated. 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 class II HLA is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP.

[0041] In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the sequence encoding 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, the sequence encoding the affinity acceptor peptide is operably linked to the C-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 recombinant HLA allele by a linker.

[0042] In some embodiments, two or more sequences encoding affinity acceptor-tagged HLA are expressed from the same polynucleotide. In some embodiments, two or more sequences encoding affinity acceptor-tagged HLA are expressed from different polynucleotides. In some embodiments, the encoded affinity acceptor peptide specifically binds to an affinity acceptor peptide-binding molecule. In some embodiments, two or more sequences encoding affinity acceptor-tagged HLA comprise two or more affinity acceptor peptides. In some embodiments, two or more sequences encoding affinity acceptor-tagged HLA comprise three or more sequences encoding affinity acceptor-tagged HLA, wherein at least two of the three or more sequences encoding affinity acceptor-tagged HLA comprise 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 affinity acceptor-tagged HLA.

[0043] In some embodiments, the encoded affinity acceptor peptide is a biotin acceptor peptide (BAP), a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin binding peptide (SPB) tag, a Strep tag, a Strep tag II, an albumin binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag (B-tag), a calmodulin binding peptide (CBP) tag, a chloramphenicol acetyltransferase (CAT) tag, a coli acetyltransferase (CTA) tag, a guanine acetyltransferase (GAT ... Cellulose-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), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinityand optionally the first or second affinity acceptor peptide comprises two or more repeats of the tag sequence.

[0044] In some embodiments, the affinity acceptor peptide-binding molecule is biotin or an antibody specific for the affinity acceptor 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 affinity acceptor peptide-binding molecule does not specifically interact with the amino acid sequence of the recombinant class I HLA or the amino acid sequence of the class II HLA. In some embodiments, for two or more of the recombinant polynucleic acids, the sequence encoding the affinity acceptor-tagged HLA is stably integrated into the genome of the cell. In some embodiments, the sequence encoding β2 microglobulin or the sequence encoding the HLA class II β chain is connected to the sequence encoding the second affinity acceptor peptide. In some embodiments, the second affinity acceptor peptide comprises an HA tag. In some embodiments, the sequence encoding β2 microglobulin or the sequence encoding the HLA class II β chain is connected to the sequence encoding the recombinant HLA and the affinity acceptor peptide by a linker.

[0045] In some embodiments, the linker comprises a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome skipping site or an internal ribosome entry site (IRES) element. In some embodiments, the ribosome skipping site or IRES is cleaved when expressed in a cell. In some embodiments, the ribosome skipping 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.

[0046] Provided herein are compositions comprising two or more isolated polypeptide molecules encoded by the polynucleic acids of the compositions described herein. Provided herein are compositions comprising cell populations comprising two or more polypeptide molecules encoded by the polynucleic acids of the compositions described herein. Provided herein are compositions comprising cell populations comprising the compositions described herein. Provided herein are compositions comprising cell populations comprising one or more cells comprising the compositions described herein.

[0047] In some embodiments, the cell population comprises one or more endogenous class I or class II The cell population expresses HLA alleles. In some embodiments, the cell population is engineered to lack one or more endogenous HLA class I alleles. In some embodiments, the cell population is engineered to lack endogenous HLA class I alleles. In some embodiments, the cell population is engineered to lack one or more endogenous HLA class II alleles. In some embodiments, the cell population is engineered to lack endogenous HLA class II alleles. In some embodiments, the cell population is engineered to lack 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 population of low cell surface HLA class I or class II expressing cells. In some embodiments, the composition is formulated with a peptide or a polynucleic acid encoding a peptide specific to the patient's HLA type. Provided herein is a method of producing cells, comprising transducing or transfecting two or more cells with two or more polynucleic acids of the compositions described herein.

[0048] Provided herein are peptides identified according to the methods described herein. Provided herein are methods of inducing an anti-tumor response in a mammal, comprising administering to the mammal an effective amount of a polynucleic acid comprising the sequence of a peptide described herein. Provided herein are methods of inducing an anti-tumor response in a mammal, comprising administering to the mammal an effective amount of a peptide comprising the sequence of a peptide described herein. Provided herein are methods of inducing an anti-tumor response in a mammal, comprising administering to the mammal cells comprising a peptide comprising the sequence of a peptide described herein. Provided herein are methods of inducing an anti-tumor response in a mammal, comprising administering to the mammal cells comprising an effective amount of a polynucleic acid comprising a sequence encoding a peptide comprising the sequence of a peptide described herein. In some embodiments, the cells present the peptide as an HLA-peptide complex. Provided herein are methods for inducing an immune response in a mammal, comprising administering to the mammal an effective amount of a polynucleic acid comprising a sequence encoding a peptide described herein. Provided herein are methods for inducing an immune response in a mammal, comprising administering to the mammal an effective amount of a peptide comprising the sequence of a peptide described herein. Provided herein is a method for inducing an immune response in a mammal, the method comprising administering to the mammal an effective amount of cells comprising a peptide comprising the sequence of a peptide described herein. Provided herein is a method for inducing an immune response in a mammal, the method comprising administering to the mammal an effective amount of cells comprising a polynucleic acid comprising a sequence encoding a peptide comprising the sequence of a peptide described herein.

[0049] In some embodiments, 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.

[0050] Provided herein is a method for treating a mammal having a disease, the method comprising administering to the mammal an effective amount of a polynucleic acid comprising a sequence encoding a peptide described herein. Provided herein is a method for treating a mammal having a disease, the method comprising administering to the mammal an effective amount of a peptide comprising a sequence of a peptide described herein. Provided herein is a method for treating a mammal having a disease, the method comprising administering to the mammal an effective amount of cells comprising a peptide comprising a sequence of a peptide described herein. Provided herein is a method for treating a mammal having a disease, the method comprising administering to the mammal an effective amount of cells comprising a polynucleic acid comprising a sequence encoding a peptide comprising a sequence of a peptide described herein. In some embodiments, the disease is cancer. In some embodiments, the disease is an infection by an infectious agent. In some embodiments, the infectious agent is a pathogen, optionally a virus or bacterium, or a parasite.

[0051] In some embodiments, the virus is selected from the group consisting of BK virus (BKV), dengue virus (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 lymphotropic virus (HTLV-1), influenza virus, RSV, HPV, rabies, mumps, rubella virus, poliovirus, yellow fever, hepatitis A, hepatitis B, rotavirus, varicella virus, human papillomavirus (HPV), smallpox, shingles, and any combination thereof.

[0052] In some embodiments, the bacteria is selected from the group consisting of Klebsiella spp., Tropheryma whipplei, Mycobacterium leprae, Mycobacterium lepromatosis, and Mycobacterium tuberculosis, typhoid, pneumococcus, meningococcus, Haemophilus B, Bacillus anthracis, tetanus toxoid, meningococcus B, bcg, cholera, and any combination thereof.

[0053] In some embodiments, the parasite is a parasitic helminth or protozoan. In some embodiments, the parasite 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.

[0054] Provided herein are methods for enriching immunogenic peptides, the methods comprising: providing a cell population comprising one or more cells expressing affinity acceptor-tagged HLA, the affinity acceptor peptide operably linked to a recombinant HLA encoded by a recombinant HLA allele; and enriching HLA-peptide complexes comprising the affinity acceptor-tagged HLA. In some embodiments, the method further comprises determining the sequence of the immunogenic peptide isolated from the HLA-peptide complex. In some embodiments, the determining step comprises using LC-MS / MS.

[0055] Provided herein are methods of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a polynucleic acid comprising a sequence encoding a peptide described herein. Provided herein are methods of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a peptide comprising the sequence of a peptide described herein. Provided herein are methods of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of cells comprising a peptide comprising the sequence of a peptide described herein. Provided herein are methods of treating a disease or disorder in a subject, the method comprising administering to the subject cells comprising an effective amount of a polynucleic acid comprising a sequence encoding a peptide comprising the sequence of a peptide described herein.

[0056] Provided herein are methods for developing a therapeutic agent for a subject having a disease or condition, the methods comprising: providing a cell population derived from a subject having the disease or condition; expressing affinity acceptor-tagged class I or class II HLA alleles in one or more cells of the cell population by introducing into the one or more cells a polynucleic acid encoding a sequence comprising a sequence encoding a recombinant class I or class II HLA allele operably linked to a sequence encoding an affinity acceptor peptide, thereby forming affinity acceptor-tagged HLA-peptide complexes in the one or more cells; enriching and characterizing the affinity acceptor-tagged HLA-peptide complexes; and, if desired, developing a therapeutic agent based on the characterization.

[0057] Provided herein are methods for identifying at least one subject-specific immunogenic antigen and preparing a subject-specific immunogenic composition comprising the at least one subject-specific immunogenic antigen, wherein the subject has a disease and the at least one subject-specific immunogenic antigen is specific to the subject and the subject's disease, the method comprising the steps of: providing a cell population derived from a subject having the disease or condition; expressing affinity acceptor-tagged class I or class II HLA alleles in one or more cells of the cell population derived from the subject by introducing into the one or more cells a polynucleic acid encoding a sequence comprising a sequence encoding a recombinant class I or class II HLA allele operably linked to a sequence encoding an affinity acceptor peptide, thereby forming affinity acceptor-tagged HLA-peptide complexes in the one or more cells; enriching the affinity acceptor-tagged HLA-peptide complexes from the one or more cells; identifying immunogenic peptides from the enriched affinity acceptor-tagged HLA-peptide complexes that are specific to the subject and the subject's disease; and formulating the subject-specific immunogenic composition based on one or more of the identified subject-specific immunogenic peptides.

[0058] In some embodiments, a therapeutic agent or subject-specific immunogenic composition comprises a polynucleotide encoding a peptide derived from enriched affinity acceptor-tagged HLA-peptide complexes or a polypeptide derived from enriched affinity acceptor-tagged HLA-peptide complexes. In some embodiments, a therapeutic agent or subject-specific immunogenic composition comprises a T cell expressing a T cell receptor (TCR) that specifically binds to a polypeptide derived from enriched affinity acceptor-tagged HLA-peptide complexes. In some embodiments, a subject-specific immunogenic composition comprises a chimeric antigen receptor (CAR) T cell expressing a receptor that specifically binds to a polypeptide derived from enriched affinity acceptor-tagged HLA-peptide complexes.

[0059] In some embodiments, the method further comprises administering to the subject another therapeutic agent, optionally an immune checkpoint inhibitor, hi some embodiments, the method further comprises administering to the subject an adjuvant, optionally poly-ICLC.

[0060] 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 an infection by an infectious agent. In some embodiments, the infectious agent is a pathogen, a virus, a bacterium, or a parasite.

[0061] In some embodiments, the virus is selected from the group consisting of BK virus (BKV), dengue virus (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 lymphotropic virus (HTLV-1), influenza virus, RSV, HPV, rabies, mumps, rubella virus, poliovirus, yellow fever, hepatitis A, hepatitis B, rotavirus, varicella virus, human papillomavirus (HPV), smallpox, shingles, and any combination thereof.

[0062] In some embodiments, the bacteria is selected from the group consisting of Klebsiella spp., Tropheryma whipplei, Mycobacterium leprae, Mycobacterium lepromatosis, and Mycobacterium tuberculosis, typhoid, pneumococcus, meningococcus, Haemophilus B, Bacillus anthracis, tetanus toxoid, meningococcus B, bcg, cholera, and combinations thereof.

[0063] In some embodiments, the parasite is a parasitic helminth or protozoan. In some embodiments, the parasite 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.

[0064] 1. A method of developing a therapeutic agent for a subject having a disease or condition, comprising the steps of: providing a cell population, wherein one or more cells of the cell population comprise a polynucleic acid comprising a sequence encoding at least two affinity acceptor-tagged class I or class II HLA alleles, wherein the sequence encoding the at least two affinity acceptor-tagged class I or class II HLA alleles comprises a first recombinant sequence comprising a sequence encoding a first class I or class II HLA allele operably linked to a sequence encoding a first affinity acceptor peptide, and a second class I or class II HLA allele operably linked to a sequence encoding a second affinity acceptor peptide. Provided herein is a method comprising the steps of: including a second recombinant sequence comprising a sequence encoding an HLA allele; expressing at least two affinity acceptor-tagged HLAs in at least one cell of one or more cells of a cell population, thereby forming an affinity acceptor-tagged HLA-peptide complex in the at least one cell; enriching the affinity acceptor-tagged HLA-peptide complexes; and identifying peptides derived from the enriched affinity acceptor-tagged HLA-peptide complexes; and formulating an immunogenic composition based on one or more of the identified peptides, wherein the first and second recombinant class I or class II HLA alleles match the subject's HLA haplotype. In some embodiments, the subject has a disease or condition.

[0065] In some embodiments, the first recombinant class I or class II HLA allele is different from the second recombinant class I or class II HLA allele. In some embodiments, the first affinity acceptor peptide is the same as the second affinity acceptor peptide. In some embodiments, the method includes characterizing peptides bound to the first and / or second affinity acceptor-tagged HLA-peptide complexes from the enriching step. In some embodiments, the at least two affinity acceptor-tagged class I or class II HLA alleles comprise 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 class I and / or class II HLA alleles. In some embodiments, the first and / or second affinity acceptor-tagged HLA-peptide complexes comprise a transmembrane domain. In some embodiments, the first and / or second affinity acceptor-tagged HLA-peptide complexes comprise an intracellular domain. In some embodiments, the first and / or second affinity acceptor-tagged HLA-peptide complexes are not excreted. In some embodiments, the first and / or second affinity acceptor-tagged HLA-peptide complexes, when expressed, are incorporated into the cell membrane. In some embodiments, the first and / or second affinity acceptor-tagged HLA-peptide complexes are not soluble affinity acceptor-tagged HLA-peptide complexes.

[0066] In some embodiments, the method further comprises generating an HLA-allele-specific peptide database. In some embodiments, the method comprises introducing one or more exogenous peptides into the cell population. In some embodiments, the introducing comprises contacting the cell population with the one or more exogenous peptides or expressing the one or more exogenous peptides in the cell population. In some embodiments, the introducing comprises contacting the cell population with one or more nucleic acids encoding the one or more exogenous peptides.

[0067] In some embodiments, the one or more nucleic acids encoding the one or more peptides are DNA. In some embodiments, the one or more nucleic acids encoding the one or more peptides are RNA, and optionally, the RNA is mRNA.

[0068] In some embodiments, the enriching step does not include the use of a tetramer reagent. In some embodiments, the method includes determining the sequence of peptides or portions thereof bound to the first and / or second affinity acceptor-tagged HLA-peptide complexes from the enriching step. In some embodiments, the determining step includes biochemical analysis, mass spectrometry, MS analysis, MS / MS analysis, LC-MS / MS analysis, or a combination thereof.

[0069] In some embodiments, the method includes assessing the binding affinity or stability of the peptides or portions thereof bound to the first and / or second affinity acceptor-tagged HLA-peptide complexes derived from the enriching step. In some embodiments, the method includes determining whether the peptides or portions thereof bound to the first and / or second affinity acceptor-tagged HLA-peptide complexes derived from the enriching step contain one or more mutations. In some embodiments, the method includes assessing the association of the peptides in the first and / or second affinity acceptor-tagged HLA-peptide complexes with HLA molecules.

[0070] In some embodiments, the method comprises expressing a library of peptides in the cell population, thereby forming a library of affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the method comprises contacting the cell population with a library of peptides or a library of sequences encoding peptides, thereby forming a library of affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the library comprises a library of peptides associated with a disease or condition.

[0071] In some embodiments, the disease or condition is cancer or infection by an infectious agent. In some embodiments, the method includes introducing an infectious agent, or a portion thereof, into one or more cells of a cell population. In some embodiments, the method includes characterizing one or more peptides derived from a first and / or second HLA-peptide complex, optionally wherein the peptides are derived from one or more target proteins of the infectious agent. In some embodiments, the method includes characterizing one or more regions of peptides derived from one or more target proteins of the infectious agent. In some embodiments, the method includes identifying peptides derived from the first and / or second HLA-peptide complexes derived from the infectious agent.

[0072] In some embodiments, the cell population is derived from a biological sample from 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 population of primary cells. In some embodiments, peptides derived from the first and / or second affinity acceptor-tagged HLA-peptide complexes can activate T cells from the subject when presented by antigen-presenting cells. In some embodiments, the method includes comparing HLA-peptide complexes derived from diseased cells with HLA-peptide complexes derived from non-diseased cells. In some embodiments, the method further includes isolating peptides derived from the first and / or second affinity acceptor-tagged HLA-peptide complexes prior to the identifying step. In some embodiments, the cell population is a population of low cell-surface HLA class I or class II expressing cells.

[0073] 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 is a knockout of one or more HLA class I alleles. In some embodiments, the cell population is a knockout of one or more HLA class II alleles. In some embodiments, the cell population is a knockout of all HLA class I alleles. In some embodiments, the cell population is knockout of all HLA class II alleles. In some embodiments, the cell population is knockout of all HLA class I alleles and knockout of all HLA class II alleles. In some embodiments, the sequence encoding at least two affinity acceptor-tagged class I or class II HLA alleles 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 recombinant class I or class II HLA allele is a first class I HLA allele and the second recombinant class I or class II HLA allele is a second class I HLA allele. In some embodiments, the sequence encoding at least two affinity acceptor-tagged class I or class II HLA alleles encodes a class II HLA. In some embodiments, the class II HLA is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP.In some embodiments, the class II HLA comprises an HLA class II α chain, an HLA class II β chain, or a combination thereof. In some embodiments, the first recombinant class I or class II HLA allele is a first class II HLA allele, and the second recombinant class I or class II HLA allele is a second class II HLA allele.

[0074] In some embodiments, the first sequence and the second sequence are operably linked to each other. In some embodiments, the first sequence and the second sequence are comprised on different polynucleotide molecules. In some embodiments, the sequence encoding the first and / or second affinity acceptor peptide is operably linked to a sequence encoding the extracellular portion of the first and / or second class I or class II HLA allele. In some embodiments, the first and / or second encoded affinity acceptor peptide is expressed extracellularly. In some embodiments, the sequence encoding the first and / or second affinity acceptor peptide is operably linked to the N-terminus of a sequence encoding the first and / or second class I or class II HLA allele. In some embodiments, the sequence encoding the first and / or second affinity acceptor peptide is operably linked to a sequence encoding the intracellular portion of the first and / or second class I or class II HLA allele. In some embodiments, the encoded first and / or second affinity acceptor peptide is expressed intracellularly. In some embodiments, the sequence encoding the first and / or second affinity acceptor peptide is operably linked to the C-terminus of the sequence encoding the first and / or second class I or class II HLA allele. In some embodiments, the sequence encoding the first and / or second affinity acceptor peptide is operably linked to the sequence encoding the first and / or second class I or class II HLA allele by a linker.

[0075] In some embodiments, the enriching step comprises enriching for intact cells expressing the first and / or second affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the method does not comprise lysing the cells prior to the enriching step. In some embodiments, the method further comprises lysing one or more cells prior to the enriching step. In some embodiments, the enriching step comprises contacting an affinity acceptor peptide-binding molecule with the first and / or second affinity acceptor-tagged HLA-peptide complexes, wherein the affinity acceptor peptide-binding molecule specifically binds to the first and / or second affinity acceptor peptides.

[0076] In some embodiments, the first and / or second affinity acceptor peptide is a biotin acceptor peptide (BAP), a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin-binding peptide (SPB) tag, a Strep tag, a Strep tag II, an albumin-binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag (B-tag), a calmodulin-binding peptide (CBP) tag, a 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), Glutathione-S-Transferase (GST), Glu-Glu (EE) Tag, Human Influenza Hemagglutinin (HA) Tag, Horseradish Peroxidase (HRP) 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, Softag1, Softag3, TC Tag, VSV-Tag, Xpress Tag, Isopeptag, SpyTag, SnoopTag, Profinityand a tag sequence comprising an eXact tag, a protein C tag, an S1-tag, an S-tag, a biotin-carboxy carrier protein (BCCP) tag, a green fluorescent protein (GFP) tag, a small ubiquitin-like modifier (SUMO) tag, a tandem affinity purification (TAP) tag, a HaloTag, a Nus-tag, a thioredoxin tag, an Fc-tag, a CYD tag, an HPC tag, a TrpE tag, a ubiquitin tag, a VSV-G epitope tag, a V5 tag, or a combination thereof, and optionally, the first and / or second affinity acceptor peptides comprise two or more repeats of the tag sequence.

[0077] In some embodiments, the affinity acceptor peptide-binding molecule is biotin or an antibody specific for the first and / or second affinity acceptor peptide. In some embodiments, the enriching step comprises contacting an affinity molecule with the first and / or second affinity acceptor-tagged HLA-peptide complexes, 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, the enriching step comprises immunoprecipitating the first and / or second affinity acceptor-tagged HLA-peptide complexes.

[0078] In some embodiments, the 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 solid surface is a bead.

[0079] In some embodiments, the enriching step comprises immunoprecipitating the first and / or second affinity acceptor-tagged HLA-peptide complexes with an affinity acceptor peptide-binding molecule that specifically binds to the first and / or second affinity acceptor 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 HLA or class II HLA. In some embodiments, the enriching step comprises contacting an affinity molecule specific for the extracellular portion of the first and / or second class I or class II HLA allele. In some embodiments, the enriching step comprises contacting an affinity molecule specific for the N-terminal portion of the first and / or second class I or class II HLA allele.

[0080] In some embodiments, the providing step comprises contacting the cell population with the polynucleic acid. In some embodiments, the contacting step comprises transfecting or transducing. In some embodiments, the providing step comprises contacting the cell population with a vector comprising the polynucleic acid. In some embodiments, the vector is a viral vector. In some embodiments, the polynucleic acid is stably integrated into the genome of the cell population.

[0081] In some embodiments, the first and / or second class I or class II HLA-encoding sequence comprises an HLA class I α chain-encoding sequence, hi some embodiments, the first recombinant class I or class II HLA allele is a first HLA class I α chain and the second recombinant class I or class II HLA allele is a second HLA class I α chain.

[0082] In some embodiments, the method further comprises expressing a sequence encoding β2 microglobulin in one or more cells. In some embodiments, the sequence encoding β2 microglobulin is connected to a sequence encoding a first and / or second class I or class II HLA. In some embodiments, the sequence encoding β2 microglobulin is connected to a sequence encoding a first and / or second class I or class II HLA by a linker. In some embodiments, the sequence encoding β2 microglobulin is connected to a sequence encoding a third affinity acceptor peptide.

[0083] In some embodiments, the third affinity acceptor peptide is different from the first and / or second affinity acceptor peptide. In some embodiments, the first and / or second class I or class II HLA-encoding sequence comprises a sequence encoding an HLA class II α chain and / or an HLA class II β chain. In some embodiments, the first and / or second class I or class II HLA-encoding sequence comprises a sequence encoding a first HLA class II α chain and a second 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, the sequence encoding the first HLA class II α chain and the second HLA class II α chain is connected to a sequence encoding an HLA class II β chain. In some embodiments, the first and / or second class I or class II HLA-encoding sequence comprises a sequence encoding a first HLA class II β chain and a second HLA class II β chain.

[0084] 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, the sequences encoding the first HLA class II β chain and the second HLA class II β chain are connected to the sequence encoding the HLA class II α chain by a linker. In some embodiments, the sequence encoding the HLA class II β chain or the HLA class II α chain is connected to a sequence encoding a third affinity acceptor peptide. In some embodiments, the third affinity acceptor peptide is different from the first and / or second affinity acceptor peptide.

[0085] In some embodiments, the third affinity acceptor peptide is different from the first affinity acceptor peptide and is a biotin acceptor peptide (BAP), poly-histidine tag, poly-histidine-glycine tag, poly-arginine tag, poly-aspartic acid tag, poly-cysteine ​​tag, poly-phenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene 10 protein peptide 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) tag, 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) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinityand optionally the first or second affinity acceptor peptide comprises two or more repeats of the tag sequence.

[0086] In some embodiments, the linker comprises a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome skipping site or an internal ribosome entry site (IRES) element. In some embodiments, the ribosome skipping site or IRES is cleaved when expressed in a cell. In some embodiments, the ribosome skipping 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.

[0087] In some embodiments, the method includes performing biochemical analysis or mass spectrometry, such as tandem mass spectrometry. In some embodiments, the method includes obtaining peptide sequences corresponding to MS / MS spectra of one or more peptides isolated from enriched affinity acceptor-tagged HLA-peptide complexes from a peptide database, wherein the obtained sequence or sequences identify the sequence of the one or more peptides.

[0088] In some embodiments, the cell population is a cell line selected from HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, HepG2, SH-SY5Y, CACO-2, U937, U-2OS, 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 is at least 10 5 cells, at least 10 6 cells or at least 10 7 In some embodiments, the cell population comprises a population of dendritic cells, macrophages, cancer cells, or B cells. In some embodiments, the cell population comprises tumor cells.

[0089] In some embodiments, the cell population is contacted with an agent prior to isolating the first and / or second HLA-peptide complex from one or more cells, hi some embodiments, the agent is an inflammatory cytokine, a chemical agent, an adjuvant, a therapeutic agent, or radiation.

[0090] In some embodiments, the first and / or second HLA alleles are mutated HLA alleles. In some embodiments, the sequences encoding the first and / or second HLA alleles comprise barcode sequences. In some embodiments, the method further comprises assaying for expression of the first and / or second affinity acceptor-tagged class I or class II HLA alleles.

[0091] In some embodiments, the assaying step comprises sequencing the first and / or second affinity acceptor-tagged class I or class II HLA alleles, The method includes detecting RNA encoding an HLA allele RNA, detecting first and / or second affinity acceptor-tagged class I or class II HLA allele proteins, or a combination thereof. In some embodiments, the first and second affinity acceptor-tagged class I or class II HLA alleles comprise unique barcode sequences. In some embodiments, the first sequence and the second sequence comprise unique barcode sequences.

[0092] The features of the present disclosure are set forth with particularity 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 that sets forth illustrative embodiments in which the principles of the disclosure are utilized and the accompanying drawings. [Brief explanation of the drawings]

[0093] [Figure 1A] Figure 1A is a schematic diagram of the universal immunopurification and data generation pipeline. Class I and / or class II HLA molecules are introduced into any cells, including cells that do not express class I or class II HLA, to express specific class I or class II HLA alleles in the cells. A population of cells expressing the engineered HLA is harvested, lysed, and the HLA-peptide complexes are tagged (e.g., biotinylated) and immunopurified (e.g., using biotin-streptavidin interactions). HLA-associated peptides specific for a single HLA can be eluted from the tagged (e.g., biotinylated) complexes and evaluated (e.g., sequenced using high-resolution LC-MS / MS).

[0094] [Figure 1B] Figure 1B is a diagram of the structure of HLA class II molecules -DP, -DQ, and -DR. HLA-DR molecules are heterodimers containing a constant α chain and a variable β chain. HLA-DQ and HLA-DP molecules are heterodimers containing a variable α chain and a variable β chain.

[0095] [Figure 2] Figure 2 is a schematic diagram of constructs designed for expression of HLA class I and II in cultured cell lines. The HLA-A*02:01 construct is an HLA class I design incorporating a biotin acceptor peptide (BAP) for biotinylation and immunopurification. The HLA-DRB1*11:01 construct is an HLA class II design incorporating a biotin acceptor peptide (BAP) for biotinylation and immunopurification.

[0096] [Figure 3] FIG. 3 is a diagram of an exemplary lentiviral vector that can be used to generate stable cell lines expressing HLA class I and class II constructs.

[0097] [Figure 4A] FIG. 4A is a schematic representation of transfection-based delivery of class I or class II HLA constructs for universal IP and HLA-associated peptide sequencing by LC-MS / MS.

[0098] [Figure 4B] Figure 4B is a schematic representation of the transfection-based introduction of class I or class II HLA constructs followed by a selection process, e.g., the inclusion of antibiotic resistance genes. Selected cells can then be submitted to universal IP and HLA-associated peptide sequencing by LC-MS / MS.

[0099] [Figure 5]Figure 5 is a diagram of universal immunopurification for class I and class II HLA. Cells, such as HEK293T (human embryonic kidney), are transfected or transduced to express a single class I or class II HLA allele with an affinity tag for immunopurification. Cells expressing the HLA tag are harvested, lysed, and the HLA-peptide complexes are biotinylated and immunopurified using biotin-streptavidin interactions. HLA-associated peptides specific for a single HLA are eluted from the biotinylated complex and analyzed (e.g., sequenced using high-resolution LC-MS / MS).

[0100] [Figure 6A] Figure 6A is a Western blot (anti-biotinylated) comparing mock, GFP, and empty plasmid transfection with the HLA-A*02:01 construct for biotinylation-based immunoprecipitation showing expression of class I HLA alleles in HEK293T cells.

[0101] [Figure 6B] Figure 6B is a Ponceau stained gel used as a loading control for Western blot analysis.

[0102] [Figure 6C] FIG. 6C is a schematic representation of the class I HLA constructs used to generate the engineered HEK293T cells imaged in FIGS. 6A and 6B.

[0103] [Figure 7A] Figure 7A shows images of Western blots (top) and loading controls (bottom) from a biotinylation time course experiment demonstrating that C- and N-terminally labeled HLA-BAP biotinylation is complete in 10 minutes for both class I and class II HLA-BAP-expressing cells. The results demonstrate the optimization of transfection and biotinylation of class I and class II HLA-BAP alleles expressed by HEK293T cells.

[0104] [Figure 7B] FIG. 7B is a Western blot against anti-BAP (top) and loading control (bottom) from cells expressing both class I and class II HLA constructs tagged with N- and C-terminal BAP.

[0105] [Figure 7C] Figure 7C is a schematic representation of both N- and C-terminal BAP-tagged class I (HLA-A*02:01) and class II (HLA-DRβ*11:01) constructs used for transfection and biotinylation optimization.

[0106] [Figure 8A] Figure 8A is a Western blot image showing the expression of biotinylated class I and class II HLA constructs used for HLA immunoprecipitation in HEK293T cells (anti-streptavidin for BAP labeling and anti-HA for HA labeling) and a loading control (Ponceau S). Lysates were analyzed before the addition of biotin (-biotin), after the addition of biotin (+biotin input), and after biotinylation and subsequent pulldown with streptavidin beads (+biotin FT). The decrease in signal in the +biotin FT lane indicates that biotinylated MHC has been removed from the lysate and binds to the streptavidin beads.

[0107] [Figure 8B]Figure 8B is a Western blot image showing the expression of biotinylated class I and class II HLA constructs used for HLA immunoprecipitation in HeLa (human cervical carcinoma) cells (anti-streptavidin for BAP labeling and anti-HA for HA labeling) and a loading control (Ponceau S). Lysates were analyzed before the addition of biotin (-biotin), after the addition of biotin (+biotin input), and after biotinylation and subsequent pull-down with streptavidin beads (+biotin FT). The decrease in signal in the +biotin FT lane indicates that biotinylated MHC has been removed from the lysate and binds to the streptavidin beads.

[0108] [Figure 8C] Figure 8C is a Western blot image showing the expression of biotinylated class I and class II HLA constructs used for HLA immunoprecipitation in A375 (human malignant melanoma) cells (anti-streptavidin for BAP labeling and anti-HA for HA labeling) and a loading control (Ponceau S). Lysates were analyzed before the addition of biotin (-biotin), after the addition of biotin (+biotin input), and after biotinylation and subsequent pulldown with streptavidin beads (+biotin FT). The decrease in signal in the +biotin FT lane indicates that biotinylated MHC has been removed from the lysate and binds to the streptavidin beads.

[0109] [Figure 8D]Figure 8D shows a Western blot image showing the expression of biotinylated class I and class II HLA constructs used for HLA immunoprecipitation in Expi293 cells (human embryonic kidneys genetically engineered for high-density culture and protein expression) (anti-streptavidin for BAP labeling and anti-HA for HA labeling) and a loading control (Ponceau S). Lysates were analyzed before the addition of biotin (-biotin), after the addition of biotin (+biotin input), and after biotinylation and subsequent pulldown with streptavidin beads (+biotin FT). The decrease in signal in the +biotin FT lane indicates that biotinylated MHC has been removed from the lysate and binds to the streptavidin beads.

[0110] [Figure 9A] Figure 9A is a bar graph of an exemplary LC-MS / MS analysis of HLA-associated peptides isolated using the universal HLA immunoprecipitation (universal IP) pipeline. A bar plot representation of the total unique HLA-associated peptides identified from multiple cell types (A375; gray, HEK293T; orange, HeLa; blue) expressing affinity-tagged class I and class II HLA constructs used in the universal IP pipeline is shown.

[0111] [Figure 9B] Figure 9B is a bar plot showing representative data from class I HLA monoallelic peptide profiling by LC-MS / MS, with each bar representing the total number of unique HLA-associated peptides identified from a class I monoallelic experiment implementing affinity-tagged HLA constructs.

[0112] [Figure 9C]Figure 9C is a bar plot showing representative data from class II HLA monoallelic peptide profiling by LC-MS / MS, with each bar representing the total number of unique HLA-associated peptides identified from a class II monoallelic experiment implementing affinity-tagged HLA constructs.

[0113] [Figure 10A] 10A is an exemplary diagram of the characteristics of class I and class II HLA-associated peptides discovered using the universal IP pipeline. Exemplary sequence logo representations 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 are shown.

[0114] [Figure 10B] Figure 10B is a bar graph showing the length distribution of HLA-associated peptides comparing class I (red; HLA-A*02:01) and class II (blue; HLA-DRβ*11:01) HLA-associated peptides identified using the universal IP pipeline. The length distribution of both class I and class II HLA-associated peptides identified using universal IP follows the expected trend.

[0115] [Figure 11A] FIG. 11A is a schematic representation of class II HLA constructs engineered for expression by different cell types for a universal IP pipeline.

[0116] [Figure 11B] Figure 11B is a schematic representation of a class II HLA complex that can form upon expression of the construct shown in Figure 11A in a cell line expressing endogenous class II HLA α and β chain subunits. The class II HLA complex is formed by pairing of an α chain and a β chain, each tagged with a different affinity handle.

[0117] [Figure 12A]Figure 12A is a schematic diagram of a series of universal IP strategies that can be used to deconvolve the class II HLA α and β chain pairings shown in Figure 11B and unambiguously assign peptide binding to specific class II HLA complexes. This figure shows validation of a series of universal IPs for class II HLA complexes containing multiple affinity tags. Cells expressing dual affinity-tagged class II HLA constructs are lysed and incubated with beads conjugated to biotinylated anti-HA antibodies. Class II HLA complexes containing HA-tagged subunits are isolated, washed, and eluted using an HA peptide (e.g., YPYDVPDYA). The eluate is then incubated with beads conjugated to NeutrAvidin or streptavidin to isolate HA-tagged and biotin-tagged class II HLA complexes. Peptides bound to the dual-tagged class II HLA complexes are then eluted and sequenced by LC-MS / MS.

[0118] [Figure 12B] Figure 12B shows Western blot validation of the sequential universal IP strategy in HEK293T expressing the dual-tagged HLA-DRB*11:01 construct. A sequential enrichment process was performed using an anti-HA antibody. A loading control (Ponceau S stained gel) is shown.

[0119] [Figure 12C] Figure 12C shows results from an exemplary negative control experiment in which cells expressing the dual affinity-tagged class II HLA construct HLA-DRB*11:01 were lysed and incubated with beads conjugated to anti-HA antibodies without biotinylation. Western blots and loading controls (Ponceau S-stained gels) are shown to demonstrate the specificity of the universal IP pipeline. When the biotinylation step was removed from the universal IP protocol, no enrichment was observed.

[0120] [Figure 13]Figure 13 is a schematic overview of an HLA class II trimming experiment that allows for the identification of core binding epitopes. HLA class II molecules bind to a nested set of peptides, typically 12-18 amino acids in length, generated from the same source protein. While longer peptides protrude from the N- and C-terminal sides of the HLA class II molecule, the core epitope interacts most strongly with the peptide-binding groove. Peptides bound to HLA class II molecules are trimmed using peptidases specific for the N- and C-termini. After trimming, the core peptide epitopes are sequenced using LC-MS / MS.

[0121] [Figure 14A] Figure 14A is a schematic diagram of a monoallelic HLA-peptidome profiling approach implementing a biotin affinity tag. An exemplary embodiment of the present disclosure uses biotin acceptor peptides (BAPs) that are biotinylated on lysine (K) residues by BirA enzyme. The BAP peptide sequence contains a lysine residue that is biotinylated upon addition of BirA enzyme, biotin, and ATP. The biotinylated product exhibits high affinity for streptavidin / NeutrAvidin. Streptavidin / NeutrAvidin beads can be used to enrich for biotinylated BAP peptide sequences.

[0122] [Figure 14B]Figure 14B is a schematic diagram of biotin-based immunopurification of engineered HLA molecules. Specific HLA alleles bearing a BAP sequence at either the N- or C-terminus of the HLA protein are introduced into cells, for example, by transfection or transduction of a plasmid. Note that the plasmid contains a DNA barcode, which allows for PCR-based methods to monitor cell lines for each allele. The barcode length can be at least 5 base pairs, at least 10 base pairs, at least 15 base pairs, at least 20 base pairs, or longer. Cells expressing the HLA-BAP protein are lysed and biotinylated. HLA-BAP-peptide complexes can be immunopurified from the complex lysate mixture and subjected to LC-MS / MS analysis for peptide identification.

[0123] [Figure 15] Figure 15 is a schematic diagram of an exemplary application of the universal IP platform for target epitope validation and discovery. A cell line of interest is engineered to express an allele-specific HLA-tagged (e.g., BAP) construct. Cells expressing the HLA-tagged (e.g., BAP) molecule are genetically engineered to express a single epitope or multiple epitopes. The epitope-expressing cells are lysed, and the HLA-BAP-peptide complexes are immunopurified. The isolated peptide antigens can be examined by any suitable means, e.g., sequenced by LC-MS / MS, and peptide fragments generated from the introduced epitopes can be used as a high-efficiency readout for the processing and presentation of HLA-allele-matched antigens.

[0124] [Figure 16]Figure 16 is a schematic diagram of HLA allele multiplexing within a universal IP pipeline. Multiple class I and class II alleles can be expressed from a single HLA construct. For example, multiple heavy chains can be included in a class I construct, and multiple beta and / or alpha chains can be included in a class II construct. By multiplexing HLA alleles within a single construct, multiple HLA molecules can be delivered and expressed in a cell line of interest. Allele multiplexing allows for matching with a patient's HLA type and application of the universal IP pipeline for personalized peptide antigen readout and subsequent complex and / or peptide analysis, e.g., LC-MS / MS readout.

[0125] [Figure 17] Figure 17 is a diagram of multi-allelic and mono-allelic approaches to HLA ligand profiling. In multi-allelic approaches, HLA ligands are co-immunoprecipitated with HLA heterodimers derived directly from patient material or cell lines (top). Because these cells naturally express multiple HLA alleles, peptides identified from such multi-allelic approaches must be deconvoluted to assign binding to specific HLA heterodimers if the HLA type is known. In mono-allelic approaches, HLA ligands are co-immunoprecipitated with HLA heterodimers derived from cell lines genetically modified to express only a single HLA allele (bottom). Therefore, peptides identified from mono-allelic approaches do not require deconvolution for HLA heterodimer binding assignment.

[0126] [Figure 18A] FIG. 18A shows mutated neo-antigenic peptides presented on MHC.

[0127] [Figure 18B] FIG. 18B is a diagram of the method for developing personalized neo-antigen targeted therapy described herein.

[0128] [Figure 19] Figure 19 shows a diagram illustrating different experimental approaches for HLA ligand profiling. Biochemical peptide:MHC (p:MHC) binding assays are slow, inefficient, and lack insight into processing. Multi-allelic mass spectrometry is highly efficient and has the ability to learn processing rules; however, it requires in silico imputation to assign peptides to alleles. Mono-allelic mass spectrometry provides a rapid, unbiased, and clean approach to defining peptide-binding motifs across diverse MHC alleles. Mono-allelic mass spectrometry allows for rapid and systematic filling of allele coverage gaps and exploitation of allele-specific peptide length selection.

[0129] [Figure 20A] Figure 20A shows a table of exemplary HLA-binding peptides for the A*01:01, B*51:01, A*29:02, and B*54:01 alleles revealed using the single-allele approach. The single-allele approach reveals HLA-binding peptides that score poorly by NetMHCpan but validate biochemically as strong binders.

[0130] [Figure 20B] Figure 20B is a bar graph showing the proportion of incorrect assignments across 100 simulated deconvolutions. Random six-allele patient HLA genotypes (two alleles each of HLA-A, HLA-B, and HLA-C, sampled at US allele frequencies) were generated. For each allele, 500 peptides from related single-allele experiments were sampled and combined to create a mock 3000-peptide multi-allelic dataset. Each peptide was assigned to the allele that yielded the best NetMHCpan % rank score, and the percentage of peptides incorrectly assigned by NetMHCpan was determined. This process was repeated 100 times.

[0131] [Figure 21] Figure 21 is an illustration of MHC presentation predictors for various individual MHC class I alleles using MS data. Model training and evaluation are performed on non-overlapping source proteins. Peptides observed by MS are assigned to training / test according to source protein. The evaluation procedure uses a 5000:1 excess of decoys over true binders.

[0132] [Figure 22] FIG. 22 is a bar graph showing significant improvement in prediction for both processing and allele-specific binding. DETAILED DESCRIPTION OF THE INVENTION

[0133] The following description and examples illustrate in detail the embodiments of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that there are numerous variations and modifications of the present disclosure that fall within its scope.

[0134] All terms are intended to be understood as they would be understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0135] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0136] 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 the present disclosure may, for clarity, be described herein in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0137] The following definitions supplement those in the art and are directed to the current application, and are not attributable to any related or unrelated cases, for example, any commonly owned patents or applications. Any methods and materials similar or equivalent to those described herein can be used in carrying out the test of the present disclosure, but exemplary materials and methods are described herein.Therefore, the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0138] definition In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "include," "includes," and "included" is not limiting.

[0139] The terms "one or more" or "at least one," such as one or more, or at least one member of a group of members, are self-explanatory by further example, and specifically encompass reference to any one of said members, or any two or more of said members, for example, any three or more, four or more, five or more, six or more, or seven or more, etc., of said members, and up to all of said members.

[0140] References herein to "some embodiments," "embodiments," "one embodiment," or "other embodiments" mean that a feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments, but not necessarily in all embodiments of the present disclosure.

[0141] As used in this specification and claims, the words "comprising" (and any form of "comprising," such as "comprise" and "comprises"), "having" (and any form of "having," such as "have" and "has"), "including" (and any form of "including," such as "includes" and "include"), or "containing" (and any form of "containing," such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0142] As used herein, the term "about" or "approximately" when referring to a measurable value, such as a parameter, amount, time period, or the like, is meant to encompass a variation of + / - 20% or less, + / - 10% or less, + / - 5% or less, or + / - 1% or less from the particular value, to the extent that such variation is appropriate for practice in this disclosure. It should be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically disclosed.

[0143] The term "immune response" includes T cell-mediated and / or B cell-mediated immune responses that are influenced by modulation of T cell costimulation. Exemplary immune responses include T cell responses, e.g., cytokine production, and cellular cytotoxicity. Additionally, the term immune response includes immune responses that are indirectly influenced by T cell activation, e.g., antibody production (humoral response), and activation of cytokine-responsive cells, e.g., macrophages.

[0144] "Receptor" should be understood to mean a biological molecule or group of molecules that can bind to a ligand. Receptors can function to transmit information in cells, cell formations, or organisms. A receptor includes at least one receptor unit and may contain two or more receptor units, each of which may be a protein molecule, e.g., a glycoprotein molecule. A receptor has a structure complementary to that of a ligand and can complex with the ligand as a binding partner. Signaling information can be transmitted by a conformational change of the receptor after binding to the ligand on the cell surface. According to the present disclosure, a receptor may refer to MHC class I and II proteins that can form a receptor / ligand complex with a ligand, e.g., a peptide or peptide fragment of a suitable length.

[0145] A "barcode" sequence may be a nucleic acid sequence that can encode an item of information about a sequence, 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.

[0146] "Ligand" means a molecule capable of forming a complex with a receptor. According to the present disclosure, a ligand is understood to mean, for example, a peptide or peptide fragment having a suitable length and suitable binding motives in its amino acid sequence, so that the peptide or peptide fragment can form a complex with a protein of MHC class I or MHC class II.

[0147] An "antigen" is a molecule capable of stimulating an immune response and may be produced by cancer cells or infectious agents or autoimmune diseases. Helper T lymphocytes (T HAntigens recognized by T cells, whether T cells (immune cells) or cytotoxic T lymphocytes (CTLs), are not recognized as intact proteins but rather as small peptides associated with class I or class II MHC proteins on the cell surface. During the course of a naturally occurring immune response, antigens recognized in association with class II MHC molecules on antigen-presenting cells (APCs) are acquired from the outside of the cell, internalized, and processed into small peptides that associate with class II MHC molecules. APCs can also cross-present peptide antigens by processing exogenous antigens and presenting the processed antigens on class I MHC molecules. Antigens that give rise to proteins recognized in association with class I MHC molecules are generally proteins produced within the cell, which are processed and associated with class I MHC molecules. It is understood that peptides associated with a given class I or class II MHC molecule are characterized by a common binding motif, and binding motifs for many different class I and II MHC molecules have been determined. Synthetic peptides can also be synthesized that correspond to the amino acid sequence of a given antigen and contain binding motifs for a given class I or II MHC molecule. These peptides can then be added to appropriate APCs, which can be used to stimulate helper T cell or CTL responses in vitro or in vivo. The binding motifs, methods for synthesizing peptides, and methods for stimulating helper T cell or CTL responses are all known and readily available to those skilled in the art.

[0148] The term "peptide" is used interchangeably herein with "mutant peptide" and "neoantigenic peptide." Similarly, the term "polypeptide" is used interchangeably herein with "mutant polypeptide" and "neoantigenic polypeptide." "Neoantigen" or "neoepitope" refers to a class of tumor antigens or tumor epitopes that arise from tumor-specific mutations in expressed proteins. The present disclosure further includes peptides containing tumor-specific mutations, peptides containing known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the methods of the present disclosure. These peptides and polypeptides are referred to herein as "neoantigenic peptides" or "neoantigenic polypeptides." The polypeptides or peptides may be of various lengths, in their neutral (uncharged) or salt forms, free of or containing glycosylation, side chain oxidation, phosphorylation, or any post-translational modifications, and may be subjected to conditions such that the modifications do not destroy the biological activity of the polypeptides described herein. In some embodiments, a neo-antigenic peptide of the present disclosure may comprise, for MHC class I, 22 residues or less in length, e.g., 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, or about 14 to about 18 residues in length. In some embodiments, the neo-antigenic peptide or neo-antigenic polypeptide comprises a neo-epitope.

[0149] The term "epitope" includes any protein determinant capable of specific binding to an antibody, antibody peptide, and / or antibody-like molecule (including, but not limited to, a T-cell receptor) as defined herein. Epitopic determinants typically consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0150] By "T cell epitope" is meant a peptide sequence that can be bound by class I or II MHC molecules in the form of a peptide-presenting MHC molecule or MHC complex, and then in this form recognized and bound by cytotoxic T lymphocytes or helper T cells, respectively.

[0151] As used herein, the term "antibody" refers to whole antibodies, including IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY, including single-chain whole antibodies, and antigen-binding (Fab) fragments thereof. Antigen-binding antibody fragments include, but are not limited to, Fab, Fab', and F(ab'), Fd (consisting of VH and CH1), single-chain variable fragments (scFv), single-chain antibodies, disulfide-linked variable fragments (dsFv), and fragments containing the VL or VH domains. Antibodies may be derived from any animal origin. Antigen-binding antibody fragments, including single-chain antibodies, may contain the variable region alone or in combination with all or part of the following: hinge region, CH1, CH2, and CH3 domains. Also included are any combinations of the variable region with the hinge region, CH1, CH2, and CH3 domains. The antibodies may be, for example, monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies that specifically bind to HLA-associated polypeptides or HLA-peptide complexes. Those skilled in the art will recognize that various immunoaffinity techniques are suitable for enriching soluble proteins, such as soluble HLA-peptide complexes, or membrane-bound HLA-associated polypeptides, for example, proteolytically cleaved from the membrane. These include (1) immobilizing one or more antibodies capable of specifically binding to soluble proteins on a fixed or mobile substrate (e.g., plastic wells or resins, latex, or paramagnetic beads), and (2) passing a solution containing soluble proteins from a biological sample over the antibody-coated substrate, allowing the soluble proteins to bind to the antibodies. The antibody-containing substrate and the bound soluble proteins are separated from the solution, and if necessary, the antibodies and soluble proteins are dissociated, for example, by changing the pH and / or ionic strength and / or ionic composition of the antibody bath solution. Alternatively, immunoprecipitation techniques can be used in which antibodies and soluble proteins are mixed and allowed to form macromolecular aggregates, which can be separated from the solution by size exclusion techniques or centrifugation.

[0152] The term "immunopurification (IP)" (or immunoaffinity purification or immunoprecipitation) is a well-known process in the art and widely used for the isolation of a desired antigen from a sample. Generally, this process involves contacting a sample containing the desired antigen with an affinity matrix containing an antibody against the antigen covalently attached to a solid phase. The antigen in the sample becomes bound to the affinity matrix via immunochemical binding. The affinity matrix is ​​then washed to remove any unbound species. The antigen is removed from the affinity matrix by changing the chemical composition of the solution contacting the affinity matrix. Immunopurification can be performed on a column containing the affinity matrix, in which case the solution is the eluent. Alternatively, immunopurification can be a batch process, in which the affinity matrix is ​​maintained as a suspension in a solution. A critical step in the process is the removal of the antigen from the matrix. This is typically achieved by increasing the ionic strength of the solution contacting the affinity matrix, for example, by adding inorganic salts. Changing the pH can also be effective in dissociating the immunochemical bond between the antigen and the affinity matrix.

[0153] By "agent" is meant any small molecule compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0154] "Alteration" or "change" means an increase or decrease. The alteration may be as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or 40%, 50%, 60%, or even up to 70%, 75%, 80%, 90%, or 100%.

[0155] "Biological sample" refers to any tissue, cell, fluid, or other material derived from an organism. As used herein, the term "sample" includes biological samples, such as any tissue, cell, fluid, or other material derived from an organism. "Specifically binds" refers to a compound (e.g., a peptide) that recognizes and binds to a molecule (e.g., a polypeptide) but does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample.

[0156] By "capture reagent" is meant a reagent that specifically binds to a molecule (eg, a nucleic acid molecule or a polypeptide) to select or isolate the molecule (eg, a nucleic acid molecule or a polypeptide).

[0157] As used herein, the terms "determining," "evaluating," "assaying," "measuring," "detecting," and grammatical equivalents refer to both quantitative and qualitative determinations; thus, the term "determining" is used interchangeably herein with "assaying," "measuring," etc. Where a quantitative determination is intended, the phrase "determining the amount" of an analyte, etc. is used. Where a qualitative and / or quantitative determination is intended, the phrase "determining the level" of an analyte or "detecting" an analyte is used.

[0158] 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, the 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.

[0159] The terms "isolated," "purified," "biologically pure," and their grammatical equivalents refer to material that is free, to varying degrees, from components that normally accompany it when found in its native state. "Isolated" indicates a degree of separation from the original source or environment. "Purified" indicates a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other materials so that impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the present disclosure is purified if it is substantially free from cellular material, viral material, or culture medium, if produced by recombinant DNA technology, or from chemical precursors or other chemicals, if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" may indicate that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For proteins that may undergo modifications, such as phosphorylation or glycosylation, different modifications may result in different isolated proteins that can be purified separately.

[0160] An "isolated" polypeptide (e.g., a peptide derived from an HLA-peptide complex) or polypeptide complex (e.g., an HLA-peptide complex) refers to a polypeptide or polypeptide complex of the present disclosure that has been separated from components that naturally accompany it. Typically, a polypeptide or polypeptide complex is isolated when it is at least 60% by weight free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preparations may be at least 75%, at least 90%, or at least 99% by weight of a polypeptide or polypeptide complex of the present disclosure. Isolated polypeptides or polypeptide complexes of the present disclosure can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding one or more components of such a polypeptide or polypeptide complex, or by chemically synthesizing one or more components of the polypeptide or polypeptide complex. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0161] The term "vector" refers to a nucleic acid molecule capable of transporting or mediating the expression of heterologous nucleic acid. Plasmids are species within the genus encompassed by the term "vector." A vector typically refers to a nucleic acid sequence containing an origin of replication and other entities necessary for replication and / or maintenance in a host cell. A vector capable of directing the expression of an operably linked gene and / or nucleic acid sequence is referred to herein as an "expression vector." Generally, useful expression vectors are not bound to a chromosome in their vector form and are typically in the form of a "plasmid," which refers to a circular double-stranded DNA molecule containing an entity or encoded DNA for stable or transient expression. Other expression vectors that can be used in the methods disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, or viral vectors; such vectors can be integrated into the host genome or replicate autonomously in the cell. The vector may be a DNA or RNA vector. Other forms of expression vectors known to those skilled in the art which serve equivalent functions can also be used, e.g., autonomously replicating extrachromosomal vectors or vectors capable of integrating into a host genome. Exemplary vectors are those which are autonomously replicating and / or capable of expressing nucleic acids to which they are linked.

[0162] By "molecular profile" is meant a characterization of the expression or expression levels of two or more markers (eg, polypeptides or polynucleotides).

[0163] The term "spacer" or "linker" when used in reference to a fusion protein refers to a peptide that joins proteins comprising the fusion protein. Generally, a spacer has no specific biological activity other than connecting or maintaining a minimum distance or other spatial relationship between protein or RNA sequences. However, in some embodiments, the constituent amino acids of a spacer can be selected to affect certain properties of the molecule, such as the folding, net charge, or hydrophobicity of the molecule. Suitable linkers for use in embodiments of the present disclosure are well known to those of skill in the art and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In some embodiments, a linker is used to separate two antigenic peptides by a distance sufficient to ensure proper folding of each antigenic peptide. Exemplary peptide linker sequences adopt a flexible, extended conformation and do not tend to develop an ordered secondary structure. Typical amino acids in flexible protein regions include Gly, Asn, and Ser. Virtually any permutation of an amino acid sequence containing Gly, Asn, and Ser is expected to meet the above criteria for a linker sequence. Other near-neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Further amino acid sequences that can be used as linkers are described in Maratea et al. (1985), Gene 40:39-46; Murphy et al. (1986), (2003), Proc. Nat'l. Acad. Sci. USA 83:8258-62; U.S. Pat. No. 4, 935,233; and U.S. Pat. No. 4,751,180.

[0164] The term "neoplasm" refers to any disease caused by or resulting from inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Glioblastoma is one non-limiting example of neoplasm or cancer. The term "cancer" or "tumor" or "hyperproliferative disorder" refers to the presence of cell processing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological characteristics. Cancer cells are often in the form of tumors, but such cells may exist alone in animals or may be non-tumorigenic cancer cells, such as leukemia cells. Cancers include, but are not limited to, B-cell cancers such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases such as alpha, gamma, and mu chain diseases, benign monoclonal gammopathy and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone-refractory prostate cancer), pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, 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 gland cancer, osteosarcoma, chondrosarcoma, cancers of hematological tissues, and the like.Other non-limiting examples of cancer types applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, seminal vesicle carcinoma, ovarian cancer ... These include follicular carcinoma, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'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, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, 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 still other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers can be characterized in various other ways, including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or anaplastic. In some embodiments, the present disclosure is used in the treatment, diagnosis, and / or prognosis of lymphoma or its subtypes, including, but not limited to, mantle cell lymphoma. Lymphoproliferative disorders are also considered to be proliferative diseases.

[0165] The term "vaccine" should be understood to mean a composition for generating immunity for the prevention and / or treatment of a disease (e.g., neoplasm / tumor / infectious agent / autoimmune disease). Thus, a vaccine is a pharmaceutical that contains an antigen and is intended for use in humans or animals to generate specific defenses and protective substances by vaccination. A "vaccine composition" may include a pharmaceutically acceptable excipient, carrier, or diluent. Aspects of the present disclosure relate to the use of the technology in the preparation of antigen-based vaccines. In these embodiments, vaccine is meant to refer to one or more disease-specific antigenic peptides (or corresponding nucleic acids encoding them). In some embodiments, an antigen-based vaccine contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or more antigenic peptides.In some embodiments, the antigen-based vaccine is selected from the group consisting of 2 to 100, 2 to 75, 2 to 50, 2 to 25, 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 100, 3 to 75, 3 to 50, 3 to 25, 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 10, 3 to 9, 3 to 8, 3 to Contains 7, 3 to 6, 3 to 5, 4 to 100, 4 to 75, 4 to 50, 4 to 25, 4 to 20, 4 to 19, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 100, 5 to 75, 5 to 50, 5 to 25, 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 10, 5 to 9, 5 to 8, or 5 to 7 antigenic peptides. In some embodiments, the antigen-based 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 neo-antigenic peptides. In some cases, the antigenic peptides comprise one or more neo-epitopes.

[0166] The term "pharmaceutically acceptable" refers to a substance that is or may be approved by a regulatory agency of the U.S. federal or state government, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia 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 together with a drug, does not destroy its pharmacological activity, and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug. The "pharmaceutically acceptable salts" of the pooled disease-specific antigens described herein may be acidic or basic salts generally considered in the art to be suitable for use in contact with human or animal tissues without undue toxicity, irritation, allergic response, or other problems or complications. Such salts include inorganic and organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, acetic acid, alkanoic acids such as HOOC-(CH)-COOH, where n is 0 to 4. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize from this disclosure and knowledge in the art additional pharmaceutically acceptable salts for the pooled disease-specific antigens provided herein, such as those listed by Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, PA, p. 1418 (1985).In general, pharmaceutically acceptable acid or base salts can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[0167] Nucleic acid molecules useful in the methods of the present disclosure include any nucleic acid molecule encoding a polypeptide of the present disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having substantial identity to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to pairing between complementary polynucleotide sequences, or portions thereof, to form a double-stranded molecule under various stringency conditions (see, e.g., Wahl, GM and SL Berger (1988)). See, for example, Kimmel, AR (1987), Methods Enzymol. 152:399; Kimmel, AR (1987), Methods Enzymol. 152:507. The salt concentration may typically be less than about 750 mM NaCl and less than 75 mM trisodium citrate, less than about 500 mM NaCl and less than 50 mM trisodium citrate, or less than about 250 mM NaCl and less than 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions may typically include a temperature 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, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In an exemplary embodiment, hybridization may be performed at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another exemplary embodiment, hybridization may be performed 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 exemplary embodiment, hybridization may be performed at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations on these conditions will be readily apparent to those of skill in the art. For most applications, the wash steps following hybridization may also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentrations for the wash steps may be less than about 30 mM NaCl and less than 3 mM trisodium citrate, or less than about 15 mM NaCl and less than 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps may include a temperature of at least about 25°C, at least about 42°C, or at least about 68°C. In an exemplary embodiment, the wash steps may be performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the wash steps may be performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the wash steps may be performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations on these conditions will be readily apparent to those of skill in the art. Hybridization techniques are well known to those skilled in the art, see, eg, 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.

[0168] "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the 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 typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, a BLAST program can be used, with a probability score between e-3 and em° indicating closely related sequences. "Reference" refers to a standard of comparison.

[0169] 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, mammals, including humans, or non-human mammals such as non-human primates, murines, bovines, equines, canines, ovines, or felines.

[0170] The terms "treat," "treated," "treating," "treatment," and the like are meant to refer to reducing, preventing, or ameliorating a disorder and / or its associated symptoms (e.g., a neoplasm or tumor or an infectious agent or an autoimmune disease). "Treating" may also refer to administering a therapy to a subject after the onset or suspected onset of a disease (e.g., cancer or infection by an infectious agent or an autoimmune disease). "Treating" includes the concept of "alleviating," which refers to reducing the frequency or severity of the occurrence or recurrence of any symptoms or other adverse effects associated with a disease and / or side effects associated with a treatment. The term "treating" also encompasses the concept of "managing," which refers to reducing the severity of a disease or disorder in a patient, for example, extending the life of a patient with a disease or extending the patient's survival, or delaying its recurrence, for example, extending the period of remission in a patient afflicted with a disease. Although not excluded, it is understood that treating a disorder or condition does not require that the disorder, condition, or its associated symptoms be completely eliminated.

[0171] As used herein, the terms "prevent," "preventing," "prevention," and grammatical equivalents mean to avert or delay the onset of symptoms associated with a disease or condition in a subject who has not developed such symptoms at the time administration of an agent or compound begins.

[0172] The term "therapeutic effect" refers to some alleviation of one or more of the symptoms of a disorder (e.g., a neoplasm, a tumor, or an infection by an infectious agent or an autoimmune disease) or its associated pathology. As used herein, a "therapeutically effective amount" refers to an amount of an agent that, upon administration to a cell or a subject in a single or multiple doses, is effective in prolonging the survival of a patient with such a disorder, reducing, preventing, or delaying one or more signs or symptoms of the disorder, etc., beyond that expected in the absence of such treatment. A "therapeutically effective amount" is intended to quantify the amount required to achieve a therapeutic effect. A physician or veterinarian of ordinary skill in the art will be able to determine the "therapeutically effective amount" (e.g., ED) of the pharmaceutical composition required. 50 ) can be readily determined and prescribed. For example, a physician or veterinarian can start a dose of a compound of the present disclosure used in a pharmaceutical composition at a level lower than that 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 herein.

[0173] In some embodiments, the nucleic acid sequence encoding the HLA allele further comprises a peptide tag, affinity tag, epitope tag, or affinity acceptor tag that can be used to immunopurify the HLA-protein. Those skilled in the art will recognize that the terms "peptide tag," "affinity tag," "epitope tag," or "affinity acceptor tag" are used interchangeably herein. As used herein, the term "affinity acceptor tag" refers to an amino acid sequence that allows the tagged protein to be easily detected or purified, for example, by affinity purification. The affinity acceptor tag is typically (but not necessarily) located at or near the N- or C-terminus of the HLA allele. A variety of peptide tags are well known in the art. Non-limiting examples include a poly-histidine tag (e.g., 4 to 15 consecutive His residues, such as 8 consecutive His residues); a poly-histidine-glycine tag; an HA tag (e.g., Field et al., Mol. Cell. Biol., 8:2159, 1988); a c-myc tag (e.g., Evans et al., Mol. Cell. Biol., 5:3610, 1985); a herpes simplex virus glycoprotein D (gD) tag (e.g., Paborsky et al., Protein Engineering, 3:547, 1990); a FLAG tag (e.g., Hopp et al., BioTechnology, 6:1204, 1988; U.S. Patent Nos. 4,703,004 and 4,851,341); KT3 epitope tag (e.g., Martine et al., Science, 255:192, 1992); tubulin epitope tag (e.g., Skinner, Biol. Chem., 266:15173, , 1991); T7 gene 10 protein peptide tag (e.g., Lutz-Freyemuth et al., Proc. Natl. Acad. Sci. USA 87:6393, 1990); streptavidin tag (StrepTag™ or StrepTagII™; see, e.g., Schmidt et al., J. Mol. Biol. 255(5):753-766, 1996 or U.S. Pat. No. 5,506,121; also commercially available from Sigma-Genosys); or VSV-G epitope tag, derived from vesicular stomatitis virus glycoprotein; or V5 tag, derived from a small epitope (Pk) found on the P and V proteins of the paramyxovirus simian virus 5 (SV5). In some embodiments, the affinity acceptor tag is an "epitope tag," a type of peptide tag that adds a recognizable epitope (antibody binding site) to an HLA-protein to provide binding of the corresponding antibody, thereby enabling identification or affinity purification of the tagged protein. Non-limiting examples of epitope tags are Protein A or Protein G, which bind to IgG. In some embodiments, a matrix of IgG Sepharose 6 Fast Flow chromatography resin is covalently attached to human IgG. This resin allows for high flow rates for rapid and convenient purification of Protein A-tagged proteins. Numerous other tag moieties are known to and can be envisioned by those skilled in the art and are contemplated herein. Any peptide tag can be used, as long as it can be expressed as an element of an affinity acceptor-tagged HLA-peptide complex.

[0174] As used herein, the term "affinity molecule" refers to a molecule or ligand that binds with chemical specificity to an affinity acceptor peptide. Chemical specificity is the ability of a protein's binding site to bind a particular ligand. The fewer ligands a protein can bind, the higher its specificity. Specificity describes the strength of binding between a given protein and a ligand. This relationship is expressed as the dissociation constant (K), which characterizes the equilibrium between the bound and unbound states for a protein-ligand system. D )

[0175] The term "affinity acceptor-tagged HLA-peptide complex" refers to a complex comprising an HLA class I or class II associated peptide or portion thereof specifically bound to a single allelic recombinant class I or class II HLA peptide containing an affinity acceptor peptide.

[0176] The terms "specific binding" or "specifically binding," when used in reference to the interaction of an affinity molecule with an affinity acceptor tag, or the interaction of an epitope with an HLA peptide, mean that the interaction is dependent on the presence of a particular structure (i.e., an antigenic determinant or epitope) on the protein; in other words, the affinity molecule recognizes and binds to a specific affinity acceptor peptide structure, rather than to proteins in general.

[0177] As used herein, the term "affinity" refers to a measure of the strength of binding between two members of a binding pair, e.g., an "affinity binding tag" and an "affinity molecule" and an HLA-binding peptide and class I or II HLA. D is the dissociation constant and has units of molar concentration. The affinity constant is the reciprocal of the dissociation constant. Affinity constant is sometimes used as a general term to describe this chemical entity. It is a direct measure of the energy of binding. Affinity can be determined experimentally, for example, by surface plasmon resonance (SPR) using a commercially available Biacore SPR instrument. Affinity is measured as the inhibitory concentration 50 (IC), which is the concentration at which 50% of the peptide is displaced.50 ) can also be expressed as ln(IC 50 ) is an IC 50 It refers to the natural logarithm of K. off refers, for example, to the off-rate constant for dissociation of an affinity molecule from an affinity acceptor-tagged HLA-epitope complex.

[0178] In some embodiments, affinity acceptor-tagged HLA-peptide complexes containing biotin acceptor peptides (BAPs) are immunopurified from complex cell mixtures using streptavidin / NeutrAvidin beads. Biotin-avidin / streptavidin binding is the strongest non-covalent interaction known in nature. This property has been exploited as a biological tool for various applications, such as immunopurification of proteins to which biotin is covalently attached. In an exemplary embodiment, nucleic acid sequences encoding HLA alleles incorporate biotin acceptor peptides (BAPs) as affinity acceptor tags for immunopurification. BAPs can be specifically biotinylated in vivo or in vitro at a single lysine residue within the tag (e.g., U.S. Pat. Nos. 5,723,584; 5,874,239; and 5,932,433; and UK Patent No. GB2370039). BAPs are typically 15 amino acids long and contain a single lysine as a biotin acceptor residue. In some embodiments, the BAP is placed at or near the N- or C-terminus of a monoallelic HLA peptide. In some embodiments, the BAP is placed between the heavy chain domain and the β2 microglobulin domain of a class I HLA peptide. In some embodiments, the BAP is placed between the β chain domain and the α chain domain of a class II HLA peptide. In some embodiments, the BAP is placed in the loop region between the α1, α2, and α3 domains of the heavy chain of a class I HLA, or between the α1 and α2 and β1 and β2 domains of the α and β chains of a class II HLA, respectively. Exemplary constructs designed for HLA class I and II expression incorporating a BAP for biotinylation and immunopurification are described in Figure 2.

[0179] As used herein, the term "biotin" refers to the compound biotin itself as well as 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 derivatives and analogs thereof, 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, sulfosuccinimidoiminoviotin, biotin bromoacetylhydrazide, p-diazobenzoylbiocytin, 3-(N-maleimidopropionyl)biocytin, desthiobiotin, etc. The term "biotin" also includes biotin variants that are capable of specifically binding to one or more of lysavidin, avidin, streptavidin, tamavidin moieties, or other avidin-like peptides.

[0180] HLA Ligand Profiling Methods Biochemical peptide-MHC binding assays for HLA epitope discovery were the basis for NetMHC, an allele-specific predictor that uses artificial neural networks; however, biochemical p:MHC binding assays are slow and inefficient (Figure 19). Endogenously processed and presented HLA-ligands profiled from cell lines and patient-derived materials are typically multiallelic, meaning that LC-MS / MS data generated from these samples contain a mixed population of ligands that can bind to one of multiple simultaneously expressed HLA alleles, as shown in Figures 17 and 19. Multiallelic datasets require deconvolution to confirm which peptides bind to different HLA heterodimers presented by an individual. Therefore, ligands derived from multiallelic datasets must be assigned to their corresponding HLA heterodimers using either (1) a binding predictor trained with the original data or (2) a deconvolution algorithm that exploits the overlap across HLA alleles represented in the large ligand dataset. It is important to note that only LC-MS / MS datasets with available HLA typing information can be unambiguously deconvoluted. In fact, nearly 40% of naturally processed ligands bound to HLA class I complexes reported from multi-allelic studies in the Immune Epitope Database (IEDB) lack HLA allele-specific assignments due to the lack of HLA typing information or their inability to be deconvoluted, making it difficult to use this subset of data for allele-specific epitope prediction. Furthermore, due to the lack of sufficient annotated data for deconvolution, it is difficult to identify peptides bound to rare class I HLA heterodimers and many class II HLA heterodimers. Multi-allelic data generation methods also limit the discovery of novel binding motifs because their deconvolution relies on existing knowledge.Although there are cautions against using multi-allelic datasets for allele-specific epitope prediction, they are extremely valuable for determining patterns of ligand presentation that require the co-expression of multiple alleles, and for validating epitope prediction algorithms.

[0181] An orthogonal approach for multi-allelic data generation and subsequent deconvolution is the creation of mono-allelic datasets, in which peptide populations presented by a single HLA allele are identified (Figures 17 and 19). One method for generating mono-allelic data uses cell lines deficient in HLA expression. These cells can be transfected or transduced with a single HLA allele, and the ligands can then be profiled by LC-MS / MS to generate allele-specific ligand libraries. Peptides bound to soluble HLA (sHLA) molecules can also be isolated from cell culture media and profiled by LC-MS / MS to produce mono-allelic data. The main advantage of mono-allelic datasets is that they do not require deconvolution and allow unambiguous peptide-HLA allele assignments without pre-existing data. Mono-allelic approaches also rapidly provide data for previously uncharacterized HLA alleles—a task that can only be performed if sufficient overlap exists in a large dataset. Furthermore, because no prior knowledge is required for unambiguous HLA-binding assignment, novel peptide-binding motifs can be easily discovered using monoallelic systems. Monoallelic data can be leveraged to assign ligands from multiallelic datasets even when deconvolution methods fail to do so.

[0182] A limiting factor of currently available single-allele approaches is that they require an HLA-deficient cell line. A key innovative feature of the present disclosure is that an HLA-deficient cell line is not required for single-allele data generation. The affinity-tagged constructs provided herein can be placed into any cell line that displays endogenous HLA-peptide complexes, and the affinity tag can be used to isolate the allele of interest. Another advantage of the present disclosure is that the same reagent can be used for any class I or class II allele in the library, provided that the reagents have the same affinity tag, making the methods disclosed herein scalable (automatable). In some embodiments, the method includes expressing a library of peptides in a cell population, thereby forming a library of affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the method includes contacting the cell population with a library of peptides or a library of peptide-encoding sequences, thereby forming a library of affinity acceptor-tagged HLA-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.

[0183] In some embodiments, the method further comprises isolating peptides derived from the affinity acceptor-tagged HLA-peptide complexes prior to the characterizing step. In some embodiments, the peptides are isolated using an anti-HLA antibody. In some cases, soluble HLA (sHLA) comprising an affinity tag is isolated using an anti-HLA antibody. In some cases, soluble HLA (sHLA) comprising an affinity tag is isolated using a column containing an anti-HLA antibody.

[0184] Methods and Compositions Provided herein is a method for characterizing an HLA-peptide complex, the method comprising the steps of: providing a cell population, wherein one or more cells of the cell population comprise a polynucleic acid comprising a sequence encoding an affinity acceptor-tagged class I or class II HLA allele, wherein the sequence encoding the affinity acceptor-tagged HLA comprises a sequence encoding a recombinant class I or class II HLA allele operably linked to a sequence encoding an affinity acceptor peptide; expressing the affinity acceptor-tagged HLA in at least one cell of the one or more cells of the cell population, thereby forming an affinity acceptor-tagged HLA-peptide complex in the at least one cell; enriching the affinity acceptor-tagged HLA-peptide complex; and characterizing the HLA-peptide complex.

[0185] In some embodiments, the characterizing step comprises characterizing peptides derived from the affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the method comprises performing the method steps for different class I and / or class II HLA alleles. In some embodiments, the method comprises using more than one class I and / or class II HLA allele. In some embodiments, the cell population is derived from a subject (e.g., a patient with a disease). In some embodiments, the cell population is a class I and / or class II negative cell line. In some embodiments, the method further comprises generating an HLA-allele-specific peptide database.

[0186] Provided herein is a method for generating an HLA-allele-specific peptide database, the method comprising the steps of: providing first and second cell populations, each comprising one or more cells comprising affinity acceptor-tagged HLA, the cells comprising different recombinant polypeptides encoded by different HLA alleles operably linked to affinity acceptor peptides; enriching the affinity acceptor-tagged HLA-peptide complexes; characterizing peptides or portions thereof bound to the affinity acceptor-tagged HLA-peptide complexes resulting from the enriching step; and generating the HLA-allele-specific peptide database.

[0187] In some embodiments, the enriching step does not include the use of a tetramer reagent.

[0188] In some embodiments, the characterizing step includes determining the sequence of the peptide or portion thereof bound to the affinity acceptor-tagged HLA-peptide complex from the enriching step. In some embodiments, the characterizing step includes determining whether the peptide or portion thereof is modified (e.g., post-translationally modified). In some embodiments, the determining step includes biochemical analysis. In some embodiments, the determining step includes mass spectrometry. 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 determining step may include determining the mass of the intact peptide (e.g., MS analysis). In some embodiments, MS / MS analysis is used to determine the mass of peptide fragments. For example, the determining step may include determining the mass of peptide fragments, which can be used to determine the amino acid sequence of the peptide or portion thereof (e.g., MS / MS analysis). In some embodiments, the mass of the peptide fragments is used to determine the sequence of amino acids within the peptide. In some embodiments, LC-MS / MS analysis is used to separate the complex peptide mixture. For example, the determining step may include separating the complex peptide mixture, such as by liquid chromatography, and determining the masses of intact peptides, peptide fragments, or a combination thereof (e.g., LC-MS / MS analysis). This data can be used, for example, for peptide sequencing.

[0189] In some embodiments, the characterizing step comprises assessing the binding affinity or stability of the peptides or portions thereof bound to the affinity acceptor-tagged HLA-peptide complexes from the enriching step. In some embodiments, the characterizing step comprises determining whether the peptides or portions thereof bound to the affinity acceptor-tagged HLA-peptide complexes from the enriching step contain one or more mutations. In some embodiments, the characterizing step comprises determining whether the peptides or portions thereof are modified (e.g., post-translationally modified). In some embodiments, the characterizing step comprises assessing the association of the peptides in the affinity acceptor-tagged HLA-peptide complexes with HLA alleles.

[0190] In some embodiments, the method comprises expressing a library of peptides in the cell population, thereby forming a library of affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the method comprises contacting the cell population with a library of peptides or a library of sequences encoding peptides, thereby forming a library of affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the library comprises 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.

[0191] In some embodiments, the cell population is a cell line. In some embodiments, the cell population is a population of primary cells.

[0192] In some embodiments, the recombinant class I or class II HLA alleles are matched to a subject with a disease or condition. In some embodiments, antigen-presenting cells containing the peptide or variant thereof bound to the affinity acceptor-tagged HLA-peptide complex are reactive to T cells expressing a T cell receptor derived from the subject. In some embodiments, the characterizing step includes comparing the HLA-peptide complex derived from the cancer cell with the HLA-peptide complex derived from a non-cancer cell.

[0193] In some embodiments, the cell population is knocked out of one or more HLA class I alleles. In some embodiments, the cell population is knocked out of one or more HLA class II alleles. In some embodiments, the cell population is knocked out of all HLA class I alleles. In some embodiments, the cell population is knocked out of all HLA class II alleles. In some embodiments, the cell population is knocked out of all HLA class I alleles and all HLA class II alleles. In some embodiments, the knockout of HLA class I or class II alleles comprises the elimination of the function of an HLA class I or class II allele. In some embodiments, the knockout of HLA class I or class II alleles is achieved by gene editing. In some embodiments, the gene editing is performed by administering to an individual in need thereof a nuclease that targets an HLA class I or class II allele to be knocked out. In some embodiments, the nuclease is a CRISPR-associated protein (e.g., a Cas protein, e.g., Cas9), a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a meganuclease. In some embodiments, gene editing is achieved by administering a CRISPR-Cas9 system to an individual in need thereof. In some embodiments, any suitable nuclease that induces a nick or double-strand break at the desired recognition site is used. In some embodiments, a naturally occurring or native nuclease is used. In some embodiments, a modified or engineered nuclease is used.

[0194] In some embodiments, the cell population has one or more HLA class I alleles knocked down. In some embodiments, the cell population has one or more HLA class II alleles knocked down. In some embodiments, the cell population has all HLA class I alleles knocked down. In some embodiments, the cell population has all HLA class II alleles knocked down. In some embodiments, the cell population has all HLA class I alleles knocked down and all HLA class II alleles knocked out. In some embodiments, the knockdown of HLA class I or class II alleles comprises reducing the expression of HLA class I or class II alleles. In some embodiments, the knockdown of HLA class I or class II alleles is achieved by administering to an individual in need thereof a therapeutically effective amount of small double-stranded interfering RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA), wherein the siRNA, miRNA, or shRNA targets the HLA class I or class II allele to be knocked down. 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 when the HLA class I or class II allele is not knocked down.

[0195] In some embodiments, the cell population comprises cells enriched or sorted for cell surface expression of HLA class I alleles, HLA class II alleles, or a combination thereof, such as by fluorescence-activated cell sorting (FACS). In some embodiments, fluorescence-activated cell sorting (FACS) is used to sort the cell population. In some embodiments, fluorescence-activated cell sorting (FACS) is used to sort the cell population for cell surface expression of HLA class I alleles, HLA class II alleles, or a combination thereof. In some embodiments, FACS is used to enrich or sort for low cell surface HLA class I or class II expressing cells.

[0196] In some embodiments, the population of cells comprises a plurality of cell populations, each expressing a different recombinant class I or class II HLA allele, hi some embodiments, each cell population of the plurality is in a separate container.

[0197] In some embodiments, the method further comprises isolating peptides from the affinity acceptor-tagged HLA-peptide complexes prior to the characterizing step, hi some embodiments, the method further comprises trimming the ends of the peptides bound to the HLA-peptide complexes (Figure 13).

[0198] 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, the sequence encoding the recombinant class I or class II HLA allele encodes a class I HLA. In some embodiments, the sequence encoding the recombinant class I or class II HLA allele encodes a class II HLA. In some embodiments, the class I HLA is selected from the group consisting of HLA-A, HLA-B, and HLA-C. In some embodiments, the class I HLA is a non-classical class Ib 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 is a non-classical class Ib group selected from the group consisting of HLA-E, HLA-F, and HLA-G. In some embodiments, the class II HLA comprises an HLA class II alpha chain, an HLA class II beta chain, or a combination thereof.

[0199] In some embodiments, each sequence encoding a different class I and / or class II HLA allele is operably linked to a sequence encoding a different affinity acceptor peptide. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence encoding a recombinant class I or class II HLA allele that encodes the extracellular portion of the recombinant class I or class II HLA allele. In some embodiments, the encoded affinity acceptor peptide is expressed extracellularly. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the N-terminus of the sequence encoding a recombinant class I or class II HLA allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the N-terminus of the sequence encoding a recombinant class I or class II HLA allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the C-terminus of the sequence encoding a recombinant class I or class II HLA allele.

[0200] In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the sequence encoding the recombinant class I or class II HLA allele by a linker.

[0201] In some embodiments, the enriching step comprises enriching for intact cells that express affinity acceptor-tagged HLA-peptide complexes.

[0202] In some embodiments, the method does not include lysing one or more cells prior to the enriching step, hi some embodiments, the method further includes lysing one or more cells prior to the enriching step.

[0203] In some embodiments, the enriching step comprises contacting the affinity acceptor-tagged HLA-peptide complexes with an affinity acceptor peptide binding molecule that specifically binds to the affinity acceptor peptide.In some embodiments, the affinity acceptor peptide is a biotin acceptor peptide (BAP), a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin-binding peptide (SPB) tag, a Strep tag, a Strep tag II, an albumin-binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag (B-tag), a calmodulin-binding peptide (CBP) tag, a chloramphenicol acetyltransferase (CAT) tag, a 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), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity The affinity acceptor peptide may comprise an eXact tag, a protein C tag, an S1-tag, an S-tag, a biotin-carboxy carrier protein (BCCP) tag, a green fluorescent protein (GFP) tag, a small ubiquitin-like modifier (SUMO) tag, a tandem affinity purification (TAP) tag, a HaloTag, a Nus-tag, a thioredoxin tag, an Fc-tag, a CYD tag, an HPC tag, a TrpE tag, a ubiquitin tag, a VSV-G epitope tag, a V5 tag, or a combination thereof; optionally, the affinity acceptor peptide comprises two or more repeats of the tag sequence.In some embodiments, the affinity acceptor peptide binding molecule is an antibody specific for biotin or the affinity acceptor peptide.

[0204] In some embodiments, the enriching step comprises contacting an affinity molecule with the affinity acceptor-tagged HLA-peptide complexes, 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, the enriching step comprises immunoprecipitating the affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the 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 solid surface is a bead.

[0205] In some embodiments, the enriching step comprises immunoprecipitating the affinity acceptor-tagged HLA-peptide complexes with an affinity acceptor peptide-binding molecule that specifically binds to the affinity acceptor peptide. In some embodiments, the affinity acceptor peptide-binding molecule does not specifically interact with the amino acid sequence of the encoded recombinant class I HLA or class II HLA. In some embodiments, the enriching step comprises contacting an affinity molecule specific for the extracellular portion of the HLA-peptide complexes. In some embodiments, the enriching step comprises contacting an affinity molecule specific for the N-terminal portion of the HLA-peptide complexes.

[0206] In some embodiments, the providing step comprises contacting the cell population with a polynucleic acid comprising a sequence encoding an affinity acceptor-tagged HLA. In some embodiments, the contacting step comprises transfecting or transducing. In some embodiments, the providing step comprises contacting the cell population with a vector or plasmid comprising a polynucleic acid comprising a sequence encoding an affinity acceptor-tagged HLA. In some embodiments, the vector is a viral vector.

[0207] Any suitable biochemical assay can be used to determine the HLA expressed in cells (e.g., engineered cell lines). Exemplary methods for determining the identity of HLA alleles expressed in cells (e.g., engineered cell lines) include, for example, Western blot analysis to determine the class (class I or class II) of the HLA allele, or sequence analysis to sequence individual alleles (e.g., using different primers to distinguish different alleles of similar sequences). In some embodiments, the polynucleic acid encoding the HLA allele comprises a barcode sequence. The barcode sequence can be used to identify the HLA allele expressed in the cell. In some embodiments, the barcode sequence is unique to a single HLA. In some embodiments, the barcode sequence is unique to a single HLA class I or class II allele.

[0208] In some embodiments, the polynucleic acid comprising a sequence encoding an affinity acceptor-tagged HLA is stably integrated into the genome of the cell population. In some embodiments, the sequence encoding the recombinant class I or class II HLA comprises a sequence encoding an HLA class I α chain. In some embodiments, the method further comprises expressing a sequence encoding β2 microglobulin in one or more cells. In some embodiments, the sequence encoding β2 microglobulin is connected to a sequence encoding an HLA class I α chain. In some embodiments, the sequence encoding β2 microglobulin is connected to a sequence encoding an HLA class I α chain by a linker. In some embodiments, the sequence encoding β2 microglobulin is connected to a sequence encoding a second affinity acceptor peptide.

[0209] In some embodiments, the recombinant class I or class II HLA encoding sequence comprises 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, the sequence encoding an HLA class II β chain is connected to a sequence encoding an HLA class II α chain. In some embodiments, the sequence encoding an HLA class II β chain is connected to a sequence encoding an HLA class II α chain by a linker. In some embodiments, the sequence encoding an HLA class II β chain is connected to a sequence encoding a second affinity acceptor peptide.

[0210] In some embodiments, the second affinity acceptor peptide is different from the first affinity acceptor peptide and is a biotin acceptor peptide (BAP), poly-histidine tag, poly-histidine-glycine tag, poly-arginine tag, poly-aspartic acid tag, poly-cysteine ​​tag, poly-phenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene 10 protein peptide 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) tag, 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) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity The tag may include an eXact tag, a protein C tag, an S1-tag, an S-tag, a biotin-carboxy carrier protein (BCCP) tag, a green fluorescent protein (GFP) tag, a small ubiquitin-like modifier (SUMO) tag, a tandem affinity purification (TAP) tag, a HaloTag, a Nus-tag, a thioredoxin tag, an Fc-tag, a CYD tag, an HPC tag, a TrpE tag, a ubiquitin tag, a VSV-G epitope tag, a V5 tag, or a combination thereof.

[0211] In some embodiments, the linker comprises a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome skipping site or an internal ribosome entry site (IRES) element. In some embodiments, the ribosome skipping site or IRES is cleaved when expressed in a cell. In some embodiments, the ribosome skipping 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.

[0212] In some embodiments, the determining step comprises performing mass spectrometry, such as tandem mass spectrometry. In some embodiments, the determining step comprises obtaining peptide sequences corresponding to MS / MS spectra of one or more peptides isolated from the enriched affinity acceptor-tagged HLA-peptide complexes from a peptide database, wherein the obtained sequence or sequences identify the sequence of the one or more peptides.

[0213] In some embodiments, the cell population is a cell line selected from HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, HepG2, SH-SY5Y, CACO-2, U937, U-2OS, 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 (e.g., peptidase inhibitors, proteasome inhibitors, and TAP inhibitors), or combinations thereof. In some embodiments, the peptide database is a non-enzyme-specific peptide database, such as one that does not include a modification database or that includes a modification (e.g., phosphorylation or cysteinylation) database. 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 reverse database search strategy. In some embodiments, the method further comprises searching a protein database using a reverse database searching strategy, hi some embodiments, a de novo search is performed, e.g., to discover new peptides that are not contained in either conventional peptide or protein databases.

[0214] In some embodiments, the cell population comprises at least 10 5 cells, at least 10 6 cells or at least 10 7 In some embodiments, the cell population comprises a population of dendritic cells, macrophages, cancer cells, or B cells. In some embodiments, the cell population comprises tumor cells or cells infected by an infectious agent, or portions thereof.

[0215] In some embodiments, the cell population is contacted with an agent prior to isolating the HLA-peptide complexes from one or more cells, hi some embodiments, the agent is an inflammatory cytokine, a chemical agent, an adjuvant, a therapeutic agent, or radiation.

[0216] In some embodiments, the HLA allele is a mutated HLA allele.

[0217] In some embodiments, the method comprises performing the steps of the method for different HLA alleles.

[0218] Provided herein is an HLA-allele-specific binding peptide sequence database 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 predictive algorithm for identifying HLA-allele-specific binding peptides, comprising training a machine using 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.

[0219] The creation of predictive algorithms by training machines is a well-known technique. The most important factor in machine training is the quality of the database used for training. Typically, machines combine one or more linear models, support vector machines, decision trees, and / or neural networks.

[0220] In some embodiments, the variables used to train the machine or algorithm comprise one or more variables selected from the group consisting of peptide sequence, amino acid physical properties, peptide physical properties, expression level of the peptide's source protein in a cell, protein stability, protein translation rate, ubiquitination sites, protein degradation rate, translation efficiency from ribosome profiling, protein cleavability, protein localization, motifs in host proteins that facilitate TAP transport, host proteins that undergo autophagy, motifs that favor ribosome stalling (e.g., polyproline or polylysine stretches), protein characteristics that favor NMD (e.g., long 3' UTR, stop codons more than 50 nt upstream of the last exon:exon junction, and peptide cleavability).

[0221] Provided herein are methods for identifying HLA-allele-specific binding peptides, comprising analyzing peptide sequences using a machine trained with a peptide sequence database obtained by performing the method described herein on HLA-alleles. In some embodiments, the method comprises determining the expression level of a source protein of the peptide in a cell, wherein the source protein expression is a predictor variable used by the machine. In some embodiments, the expression level is determined by measuring the amount of source protein or the amount of RNA encoding the source protein.

[0222] Provided herein is a composition comprising first and second recombinant polynucleic acids, each comprising a sequence encoding an affinity acceptor-tagged HLA, wherein the sequence encoding the affinity acceptor-tagged HLA comprises (a) a sequence encoding a different recombinant HLA class I α chain allele, (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.

[0223] Provided herein is a composition comprising first and second recombinant polynucleic acids, each comprising a sequence encoding an affinity acceptor-tagged HLA, wherein the sequence encoding the affinity acceptor-tagged HLA comprises (a) a sequence encoding a recombinant 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.

[0224] In some embodiments, the first and second recombinant polynucleic acids are isolated.

[0225] In some embodiments, the sequence encodes a recombinant class I or class II HLA allele. In some embodiments, the class I HLA is selected from the group consisting of HLA-A, HLA-B, and HLA-C. In some embodiments, the class I HLA is a non-classical class Ib 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 is a non-classical class Ib group selected from the group consisting of HLA-E, HLA-F, and HLA-G.

[0226] In some embodiments, for both the first and second recombinant polynucleic acids, the sequence encoding the affinity acceptor peptide is operably linked to a sequence encoding a different recombinant HLA allele that encodes the extracellular portion of the different 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 a sequence encoding a different recombinant HLA allele. In some embodiments, for both the first and second recombinant polynucleic acids, the sequence encoding the affinity acceptor peptide is operably linked to a sequence encoding a different recombinant HLA allele that encodes the intracellular portion of the different recombinant HLA allele. In some embodiments, for both the first and second recombinant polynucleic acids, the sequence encoding the affinity acceptor peptide is operably linked to the C-terminus of a sequence encoding a different recombinant HLA allele. In some embodiments, for both the first and second recombinant polynucleic acids, the sequence encoding the affinity acceptor peptide is operably linked to a sequence encoding a different recombinant HLA allele by a linker. In some embodiments, the encoded affinity acceptor peptide specifically binds to an affinity acceptor peptide binding molecule, hi some embodiments, the affinity acceptor peptides of the first and second recombinant polynucleic acids are different.

[0227] In some embodiments, the encoded affinity acceptor peptide is a biotin acceptor peptide (BAP), a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin binding peptide (SPB) tag, a Strep tag, a Strep tag II, an albumin binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag (B-tag), a calmodulin binding peptide (CBP) tag, a chloramphenicol acetyltransferase (CAT) tag, a coli acetyltransferase (CTA) tag, a guanine acetyltransferase (GAT ... Cellulose-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), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity The affinity acceptor peptide may comprise an eXact tag, a protein C tag, an S1-tag, an S-tag, a biotin-carboxy carrier protein (BCCP) tag, a green fluorescent protein (GFP) tag, a small ubiquitin-like modifier (SUMO) tag, a tandem affinity purification (TAP) tag, a HaloTag, a Nus-tag, a thioredoxin tag, an Fc-tag, a CYD tag, an HPC tag, a TrpE tag, a ubiquitin tag, a VSV-G epitope tag, a V5 tag, or a combination thereof; optionally, the affinity acceptor peptide comprises 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 affinity acceptor 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 affinity acceptor peptide-binding molecule does not specifically interact with the amino acid sequence of the encoded recombinant class I HLA or class II HLA. In some embodiments, for both the first and second recombinant polynucleic acids, the sequence encoding the affinity acceptor-tagged HLA is stably integrated into the genome of the cell. In some embodiments, the sequence encoding β2-microglobulin or the sequence encoding the HLA class II β chain is connected to the sequence encoding the second affinity acceptor peptide.

[0228] In some embodiments, the second affinity acceptor peptide comprises an HA tag.In some embodiments, the second affinity acceptor peptide is a biotin acceptor peptide (BAP), a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin-binding peptide (SPB) tag, a Strep tag, a Strep tag II, an albumin-binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag (B-tag), a calmodulin-binding peptide (CBP) tag, a chloramphenicol acetyltransferase (CAT) tag, a 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), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity The tag may comprise an eXact tag, a protein C tag, an S1-tag, an S-tag, a biotin-carboxy carrier protein (BCCP) tag, a green fluorescent protein (GFP) tag, a small ubiquitin-like modifier (SUMO) tag, a tandem affinity purification (TAP) tag, a HaloTag, a Nus-tag, a thioredoxin tag, an Fc-tag, a CYD tag, an HPC tag, a TrpE tag, a ubiquitin tag, a VSV-G epitope tag, a V5 tag, or a combination thereof; optionally, the second affinity acceptor peptide comprises two or more repeats of the tag sequence.

[0229] In some embodiments, for both the first and second recombinant polynucleic acids, the sequence encoding β2 microglobulin or the sequence encoding the HLA class II β chain is connected to the sequence encoding the different recombinant HLA and affinity acceptor peptides by a linker. In some embodiments, the linker comprises a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome skipping site or an internal ribosome entry site (IRES) element. In some embodiments, the ribosome skipping site or IRES is cleaved when expressed in a cell. In some embodiments, the ribosome skipping 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.

[0230] Provided herein are compositions comprising first and second isolated polypeptide molecules encoded by the first and second polynucleic acids, respectively, of the compositions described herein. Provided herein are compositions comprising first and second cells comprising the first and second polypeptide molecules encoded by the first and second polynucleic acids, respectively, of the compositions described herein. Provided herein are compositions comprising first and second cells comprising the first and second polynucleic acids, respectively, of the compositions described herein. Provided herein are compositions comprising first and second cell populations comprising one or more cells comprising the first and second polynucleic acids, respectively, of the compositions described herein.

[0231] In some embodiments, the first and second cell populations express one or more endogenous class I or class II HLA alleles. In some embodiments, the first and second cell populations are engineered to lack one or more endogenous HLA class I alleles. In some embodiments, the first and second cell populations are engineered to lack endogenous HLA class I alleles. In some embodiments, the first and second cell populations are engineered to lack one or more endogenous HLA class II alleles. In some embodiments, the first and second cell populations are engineered to lack endogenous HLA class II alleles. In some embodiments, the first and second cell populations are engineered to lack endogenous HLA class I alleles and endogenous HLA class II alleles.

[0232] Provided herein is a method of producing a cell, the method comprising transducing or transfecting a first and a second cell with a first and a second polynucleic acid, respectively, of a composition described herein.

[0233] Provided herein are peptides identified according to the methods described herein.

[0234] Provided herein are methods for enriching immunogenic peptides, the methods comprising: providing a cell population comprising one or more cells expressing affinity acceptor-tagged HLA, the affinity acceptor peptide operably linked to a recombinant HLA encoded by a recombinant HLA allele; and enriching HLA-peptide complexes comprising the affinity acceptor-tagged HLA. In some embodiments, the method further comprises determining the sequence of the immunogenic peptide isolated from the HLA-peptide complex. In some embodiments, the determining step comprises using LC-MS / MS.

[0235] Human leukocyte antigen (HLA) system The immune system can be divided into two functional subsystems: the innate immune system and the adaptive immune system. The innate immune system is the first line of defense against infection; most potential pathogens are quickly neutralized by this system before they can cause significant infection. The adaptive immune system responds to molecular structures, called antigens, of invading organisms. Unlike the innate immune system, the adaptive immune system is highly pathogen-specific. Adaptive immunity can also provide long-term protection; for example, a person who recovers from measles is now protected against measles for the rest of their life. There are two types of adaptive immune responses: humoral and cell-mediated. In the humoral immune response, antibodies secreted into bodily fluids by B cells bind to pathogen-derived antigens and lead to their elimination by various mechanisms, such as complement-mediated lysis. In the cellular immune response, T cells are activated, which can destroy other cells. For example, if disease-associated proteins are present in cells, they are proteolytically fragmented into peptides within the cell. Certain cellular proteins then attach themselves to the antigens or peptides formed in this manner and transport them to the cell surface, where they are presented to the body's molecular defense mechanisms in T cells. Cytotoxic T cells recognize these antigens and kill cells that bear them.

[0236] The terms "major histocompatibility complex (MHC)," "MHC molecule," or "MHC protein" refer to proteins that bind peptides and representative potential T cell epitopes resulting from proteolytic cleavage of protein antigens and transport them to the cell surface, where they can be presented to specific cells, for example, in cytotoxic T lymphocytes or helper T cells. Human MHC is also called the HLA complex. Thus, the terms "human leukocyte antigen (HLA) system," "HLA molecule," or "HLA protein" refer to the gene complex that encodes MHC proteins in humans. The term MHC is called the "H-2" complex in murine species. Those skilled in the art will recognize that the terms "major histocompatibility complex (MHC)," "MHC molecule," "MHC protein," and "human leukocyte antigen (HLA) system," "HLA molecule," and "HLA protein" are used interchangeably herein.

[0237] HLA proteins are classified into two types, HLA class I and HLA class II. Although the structures of the two HLA class proteins are very similar, they have very different functions. Class I HLA proteins are present on the surface of almost all cells in the body, including most tumor cells. Class I HLA proteins load antigens, usually originating from endogenous proteins or pathogens present inside the cell, and then present them to naive or cytotoxic T lymphocytes (CTLs). HLA class II proteins are present on antigen-presenting cells (APCs), including, but not limited to, dendritic cells, B cells, and macrophages. They primarily present peptides, which are processed by helper T cells from external antigen sources, i.e., from outside the cell. Most peptides bound by HLA class I proteins originate from cytoplasmic proteins produced in the organism's own healthy host cells and do not usually stimulate an immune response.

[0238] Class I HLA molecules consist of heavy and light chains and can bind peptides of approximately 7 to 13 amino acids (e.g., approximately 8 to 11 amino acids, or 9 or 10 amino acids). If the peptide has a suitable binding motif, it can be presented to cytotoxic T lymphocytes. The peptides bound by class I HLA molecules originate from endogenous protein antigens. The heavy chains of class I HLA molecules can be HLA-A, HLA-B, or HLA-C monomers, and the light chains are β-2 microglobulin. Class I HLA exists as an α chain composed 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 found in the unit of the non-HLA molecule β2 microglobulin (encoded on human chromosome 15). The α3 domain is transmembrane and anchors the HLA class I molecule to the cell membrane. The presented peptide is held by the floor of the peptide-binding groove in the central region of the α1 / α2 heterodimer (a molecule composed of two non-identical subunits). Class I HLA-A, HLA-B, and HLA-C are highly polymorphic. Class Ib HLA exhibits limited polymorphism, expression patterns, and antigen presentation. This group is further divided into groups encoded within the HLA locus, such as HLA-E, HLA-F, HLA-G, and non-stress ligands, such as ULBP, Rae1, and H60. The antigens / ligands for many of these molecules remain unknown, but they can interact with CD8+ T cells, NKT cells, and NK cells, respectively.

[0239] In some embodiments, the present disclosure uses non-classical class I HLA-E alleles. HLA-E is involved in the regulation of natural killer (NK) cells and CD8 + HLA-E is one of the non-classical class I molecules recognized by T cells. It is expressed in almost all tissues, including lung, liver, skin, and placental cells. HLA-E expression is also detected in solid tumors (e.g., osteosarcoma and melanoma). HLA-E is a CD8 +HLA-E binds to TCRs expressed on T cells, leading to T cell activation. HLA-E also binds to NK cells and CD8 + It is also known to bind to the CD94 / NKG2 receptor expressed on T cells. CD94 can pair with several different isoforms of NKG2 to form receptors that have the potential to either inhibit (NKG2A, NKG2B) or promote (NKG2C) cell activation. HLA-E can bind peptides 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 HLA-A, -B, and -C alleles. Under physiological conditions, engagement of CD94 / NKG2A with HLA-E loaded with peptides derived from the HLA class I leader sequence usually induces an inhibitory signal. Cytomegalovirus (CMV) uses a mechanism for evasion from NK cell immune surveillance by expression of the UL40 glycoprotein, which mimics the HLA-A leader. However, CD8 + It has also been reported that T cells can recognize HLA-E loaded with the UL40 peptide derived from the Toledo strain of CMV and play a role in defense against CMV.Several studies have demonstrated several important functions of HLA-E in infectious diseases and cancer.

[0240] Peptide antigens attach themselves to HLA class I molecules through competitive affinity binding in the endoplasmic reticulum, and then they are presented on the cell surface. Here, the affinity of individual peptide antigens is directly related to their amino acid sequence and the presence of specific binding motifs at defined positions within the amino acid sequence. If the sequences of such peptides are known, for example, peptide vaccines can be used to manipulate the immune system against diseased cells.

[0241] Class II HLA molecules have two chains, α and β, each with two domains—α1 and α2 and β1 and β2—with transmembrane domains α2 and β2, respectively, that anchor the HLA class II molecule to the cell membrane. The peptide-binding groove is formed by a heterodimer of α1 and β1. Peptides bound by class II HLA molecules usually originate extracellularly from exogenous protein antigens. The α and β chains are present in HLA-DR, HLA-DQ, and HLA-DP monomers (Figure 1B). Class II HLA molecules have six isotypes. Classical molecules present peptides to CD4+ lymphocytes. Non-classical molecules with intracellular functions, accessory molecules, are not exposed on the cell membrane but are located in the inner membrane of lysosomes and usually load antigenic peptides onto classical HLA class II molecules.

[0242] For HLA class II peptides, phagocytes such as macrophages and immature dendritic cells ingest the entity by phagocytosis into phagosomes, whereas B cells more commonly endocytose into endosomes that fuse with lysosomes, where acidic enzymes cleave the ingested protein into many different peptides. Autophagy is another source of HLA class II peptides. Due to physicochemical interactions with host-born HLA class II variants encoded in the host genome, certain peptides exhibit immunodominance and are loaded onto HLA class II molecules. These are transported to the cell surface and externalized. The most studied subclass II HLA genes are HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0243] Presentation of peptides by HLA class II molecules to CD4+ helper T cells is necessary for immune responses to foreign antigens (Roche and Furuta, 2015). Once activated, CD4+ T cells promote B cell differentiation and antibody production, as well as CD8+ T cell (CTL) responses. CD4+ T cells also secrete cytokines and chemokines that activate other immune cells and induce their differentiation. HLA class II molecules are heterodimers of α and β chains that interact to form a peptide-binding groove that is more open than the class I peptide-binding groove (Unanue et al., 2016). Peptides bound to HLA class II molecules are thought to have a nine-amino acid binding core, with adjacent 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 long and often contain three to four anchor residues at positions P1, P4, P6 / 7, and P9 of the binding register (Rossjohn et al., 2015).

[0244] HLA alleles are expressed in a codominant manner, meaning that alleles (variants) inherited from both parents are equally expressed. For example, each person carries two alleles of each of the three class I genes (HLA-A, HLA-B, and HLA-C), and can therefore express six different types of class II HLA. At the class II HLA locus, each person inherits a pair of HLA-DP genes (DPAl and DPB1, encoding the α and β chains), a pair of HLA-DQ genes (DQA1 and DQB1 for the 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 a heterozygous individual can inherit six or eight functional class II HLA alleles, three or more, from each parent. Thus, HLA genes are highly polymorphic; many different alleles exist in different individuals within a population. The genes encoding HLA proteins have many possible variations, allowing each person's immune system to respond to a variety of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each given a specific number. In some embodiments, class I HLA alleles are HLA-A * 02:01, HLA-B * 14:02, HLA-A * 23:01, HLA-E * 01:01 (non-classical). In some embodiments, Class II HLA alleles are HLA-DRB * 01:01, HLA-DRB * 01:02, HLA-DRB * 11:01, HLA-DRB * 15:01, and HLA-DRB * It is 07:01.

[0245] The subject-specific HLA allele or the subject's HLA genotype can be determined by any method known in the art. In an exemplary embodiment, the HLA genotype is determined by any method described in International Patent Application No. PCT / US2014 / 068746, published on June 11, 2015 as WO2015085147. Briefly, the method includes a step of determining a polymorphic genotype, which may include generating an alignment of reads extracted from a sequencing dataset with a genetic reference set including allelic variants of the polymorphic gene, determining a first posterior probability or posterior probability-derived score for each allelic variant in the alignment, identifying the allelic variant with the largest first posterior probability or posterior probability-derived score as the first allelic variant, identifying one or more overlapping reads aligned with the first allelic variant and one or more other allelic variants, determining a second posterior probability or posterior probability-derived score for the one or more other allelic variants using a weighting factor, identifying the second allelic variant by selecting the allelic variant with the largest second posterior probability or posterior probability-derived score, the first and second allelic variants defining a genotype for the polymorphic gene, and providing an output of the first and second allelic variants.

[0246] As described herein, mutated epitopes are effective in inducing immune responses, and cases of spontaneous tumor regression or long-term survival correlate with CD8+ T cell responses to mutated 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 mutated antigen detectable with HLA tetramers in the blood of a lung carcinoma patient with long survival, Cancer Res. 61:3718-3724 (2001); Lennerz et al., The response of autologous T cells to a human melanoma is dominated by mutated neoantigens, Proc Natl Acad Sci U S A. 102:16013 (2005)), and "immunoediting" can be traced to changes in the expression of dominant mutant antigens in mice and humans (Matsushita et al., Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting, Nature 482:400). (2012); DuPage et al., Expression of tumor-specific antigens underlies cancer immunoediting, Nature 482:405 (2012); and Sampson et al. , Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma, J Clin Oncol. 28:4722~47 There is a large body of evidence in both animals and humans (p. 29 (2010)).

[0247] Sequencing techniques have shown that each tumor contains multiple patient-specific mutations that alter the protein-coding content of genes. Such mutations create altered proteins, ranging from single amino acid changes (caused by missense mutations) to the addition of long stretches of novel amino acid sequence due to frameshifts, stop codon readthrough, or translation of intronic regions (novel open reading frame mutations; neoORFs). These mutated proteins are valuable targets for the host's immune response against tumors because, unlike native proteins, they are not susceptible to the immune response of self-tolerance. Therefore, mutated proteins are more likely to be immunogenic and more specific to tumor cells than normal cells in the patient.

[0248] The term "T cell" includes CD4+ T cells and CD8+ T cells. The term T cell also includes both type 1 helper T cells and type 2 helper T cells. As used herein, T cells are generally divided into two main classes, depending on their function and cell surface antigens (cluster differentiation antigens, or CDs), which also facilitate T cell receptor binding to antigen: helper T (T H ) cells and cytotoxic T lymphocytes (CTLs).

[0249] Mature helper T (T HCD4+ T cells express the surface protein CD4 and are called CD4+ T cells. After T cell development, mature naive T cells leave the thymus and begin to disseminate throughout the body, including lymph nodes. Naive T cells are T cells that have never been exposed to the antigen to which they are programmed to respond. Like all T cells, they express the T cell receptor-CD3 complex. The T cell receptor (TCR) consists of both a constant region and a variable region. The variable region determines the antigen to which the T cell can respond. CD4+ T cells have TCRs with affinity for class II MHC, and CD4 is involved in determining MHC affinity during maturation in the thymus. Class II MHC proteins are generally found only on the surface of specialized antigen-presenting cells (APCs). Specialized antigen-presenting cells (APCs) are primarily dendritic cells, macrophages, and B cells, but dendritic cells are the only cell population that constitutively (constantly) express MHC class II. Some APCs, such as follicular dendritic cells, also bind naive (or unprocessed) antigens on their surface, but unprocessed antigens do not interact with T cells and are not involved in their activation. Peptide antigens that bind to MHC class I proteins are typically shorter than peptide antigens that bind to MHC class II proteins.

[0250] Cytotoxic T lymphocytes (CTLs), also known as cytotoxic T cells, cytolytic T cells, CD8+ T cells, or killer T cells, are lymphocytes that induce apoptosis in targeted cells. CTLs form antigen-specific conjugates with target cells through the interaction of TCRs with processed antigens (Ag) on ​​the target cell surface, resulting in apoptosis of the targeted cells. The apoptotic bodies are removed by macrophages. The term "CTL response" refers to the primary immune response mediated by CTL cells. Cytotoxic T lymphocytes possess both T cell receptors (TCRs) and CD8 molecules on their surface. T cell receptors can recognize and bind to peptides complexed with HLA class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that can bind to a specific MHC / peptide complex. Most cytotoxic T cells express a T cell receptor (TCR) that can recognize a specific antigen. For the TCR to bind to a class I MHC molecule, it 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 of CD8 for MHC molecules keeps the T cell and target cell tightly bound together during antigen-specific activation. Once activated, CD8+ T cells are recognized as T cells and are generally classified as having a specific cytotoxic role within the immune system. However, CD8+ T cells also have the ability to produce some cytokines.

[0251] A "T cell receptor (TCR)" is a cell surface receptor involved in T cell activation in response to antigen presentation. TCRs are typically made up of two chains, alpha and beta, which assemble to form a heterodimer and associate with the CD3 transducing subunit to form the T cell receptor complex present on the cell surface. Each alpha and beta chain of a TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region. As with immunoglobulin molecules, the variable regions of the alpha and beta chains are generated by V(D)J recombination, creating a large diversity of antigen specificities within T cell populations. However, in contrast to immunoglobulins that recognize intact antigens, T cells are activated by processed peptide fragments that associate with MHC molecules, introducing an additional dimension to antigen recognition by T cells, known as MHC restriction. Differences in MHC recognition between donor and recipient T cell receptors can lead to T cell proliferation and the potential for graft-versus-host disease (GVHD). Normal surface expression of the TCR has been shown to depend on the coordinate synthesis and assembly of all seven components of the complex (Ashwell and Klusner, 1990). Inactivation of TCRα or TCRβ can result in removal of the TCR from the surface of the T cell, preventing recognition of alloantigen and therefore GVHD. However, disruption of the TCR generally results in removal of the CD3 signaling components, altering the means for further T cell expansion.

[0252] The term "HLA peptidome" refers to a pool of peptides that specifically interact with a particular HLA class and may contain thousands of different sequences. The HLA peptidome includes diverse peptides derived from both normal and abnormal proteins expressed in cells. Therefore, the HLA peptidome can be studied to identify cancer-specific peptides for the development of tumor immunotherapeutics and as a source of information on protein synthesis and degradation schemes in cancer cells. In some embodiments, the HLA peptidome is a pool of soluble HLA molecules (sHLA). In some embodiments, the HLA peptidome is a pool of membrane HLA (mHLA).

[0253] 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 foreign antigens complexed with MHC on its surface.

[0254] Universal IP Pipeline: A universal monoallelic HLA-peptide complex discrimination platform The adaptive immune response is driven, in part, by cytotoxic CD8 + It relies on the ability of T cells to identify and eliminate cells that display disease-associated antigens bound to human leukocyte antigen (HLA) class I molecules. HLA class I proteins (HLA-A, B, and C) are expressed on the surface of nearly all nucleated cells in the human body and are expressed by CD8 + HLA-binding proteins are required for the presentation of short peptides for detection by T cell receptors. HLA-bound peptides originate from endogenous or foreign proteins that are cleaved by proteasomes and ER peptidases before loading and display by HLA class I proteins. HLA genes are the most polymorphic genes across human populations, with over 10,000 HLA class I allelic variants identified to date (Robinson et al., 2015). It is estimated that each HLA allele binds and presents approximately 1,000–10,000 unique peptides to T cells—less than 0.1% of the approximately 10 million potential 9-mer peptides derived from human protein-coding genes (Bassani-Sternberg et al., 2015; Hunt et al., 1992; Rammensee et al., 1995, 1999; Rock et al., 2015; Vita et al., 2015; Walz et al., 2015).

[0255] Unlike class I, HLA class II proteins (HLA-DR, DQ, and DP) are expressed exclusively on the surface of antigen-presenting cells (APCs) and epithelial, vascular, and connective tissue cells in response to inflammatory signals. Presentation of proteins, most often derived from exogenous proteins, by HLA class II molecules to CD4+ T cells is necessary for the immune response to foreign antigens (Roche and Furuta, 2015). Once activated, CD4+ T cells promote B cell differentiation and antibody production, as well as CD8+ T cell responses. CD4+ T cells also secrete cytokines and chemokines that activate other immune cells and induce their differentiation. HLA class II molecules are heterodimers of α and β chains that interact to form a peptide-binding groove that is more open than the class I peptide-binding groove (Unanue et al., 2016). Peptides bound to HLA class II molecules are thought to have a nine-amino acid binding core with adjacent residues on the N- or C-terminus protruding from the groove (Jardetzky et al., 2016). (Stern et al., 1994; Stern et al., 1996). These peptides usually contain 12–16 amino acids. They are typically 10-amino acid long and often contain 3–4 anchor residues at positions P1, P4, P6 / 7, and P9 of the binding register (Rossjohn et al., 2015). Due to heterogeneity in α and β chain pairing, data complexity that limits the ability to unambiguously assign core binding epitopes, and the lack of immunoprecipitation-grade allele-specific antibodies required for high-resolution biochemical analysis, little is known about the allele-specific peptide-binding characteristics of HLA class II molecules.

[0256] Peptide binding rules have been extensively studied for a subset of HLA alleles (Vita et al., 2015) and have been encoded in evolved neural network-based algorithms that predict binding (Hoof et al., 2009; Lundegaard et al., 2008). However, several factors limit their power to predict peptides presented on HLA alleles. First, the origins of the peptide data on which these algorithms are trained are diverse, ranging 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 spectrometry-based peptide identifications account for approximately 30% of all identifications in the IEDB. Mass spectrometry has been used since the pioneering work of Donald F. Hunt and colleagues (Cobbold et al., 2013; Hunt et al., 2019). Due to the advances in sequencing (e.g., 1992; Meadows et al., 1997; Mohammed et al., 2008; Zarling et al., 2000, 2006), as well as instrumentation improvements demonstrated by many groups over the past two decades (Bassani-Sternberg et al., 2015; Caron et al., 2015; Mommen et al., 2014), sequencing has become a desirable method for HLA-associated peptide sequencing. Second, many existing prediction algorithms have focused on predicting binding but have not fully taken into account the endogenous processes that generate and transport peptides prior to binding (Larsen et al., 2007). Third, the number of binding peptides for many HLA alleles is too small to develop reliable predictors. However, to date, the generation of high-quality resource datasets has been hampered by inadequate protocols requiring prohibitively large amounts of input cellular material and a lack of database searching tools for HLA-peptide sequencing (Caron et al., 2014). , 2015 ; Hoof et al., 2009 ; Lundegaard et al., 2008 ; Vita et al., 2015 ).

[0257] Disclosed herein is a unique biochemical enrichment strategy for peptide-HLA class I and II complexes from live cells and cell lysates. HLA molecules containing N- or C-terminal tag sequences (e.g., BAP or HA) can be labeled on the cell surface or in cell lysates. For example, HLA molecules containing N- or C-terminal biotin acceptor peptide (BAP) sequences can be enzymatically labeled with biotin on the cell surface or in cell lysates. For example, HLA molecules containing N- or C-terminal HA sequences can be enriched from complex cell mixtures using HA-specific antibodies. In an exemplary embodiment, biotin-labeled HLA-peptide complexes are enriched from complex cell mixtures using streptavidin / NeutrAvidin beads, and the enriched HLA-peptide complexes are analyzed or characterized. In an exemplary embodiment, HA-tagged HLA-peptide complexes are enriched from complex cell mixtures using HA-specific antibodies, and the enriched HLA-peptide complexes are analyzed or characterized. For example, the associated peptides can be eluted and sequenced by LC-MS / MS. Importantly, the methods disclosed herein provide a universal platform for analyzing and characterizing HLA-peptide complexes. For example, the methods disclosed herein provide a universal platform for identifying endogenously presented peptides from cell lines expressing all possible class I or class II constructs.

[0258] Disclosed herein are cell lines expressing single HLA class I and class II alleles that allow for unambiguous peptide:allele assignment (Shimizu and DeMars, 1989; Shimizu et al., 1986). Most MS-based studies have focused on affinity prediction and This is an improvement over current methods for detecting HLA-bound peptides, as they involve eluting and sequencing a promiscuous mixture of ligands bound to multiple HLA-A, B, and C molecules, which sometimes requires deconvolution for allele assignment (Bassani-Sternberg and Gfeller, 2016). While studies using cell lines transfected with soluble HLA have been able to derive peptide-binding epitopes for single HLA alleles, most comprehensive experiments to date have identified fewer than 200 unique peptides and required several orders of magnitude more starting cell material (Hawkins et al., 2008). By removing the uncertainty of peptide:HLA assignment, the methods disclosed herein facilitate a deeper and more accurate assessment of the HLA-peptide ligandome and the rules associated with peptide antigen processing and presentation, using less cell material than previous efforts.

[0259] The methods and compositions described herein include chemically labeled variable β chains (biotinylated) to distinguish between class II HLA heterodimers presented by cells, enabling, for example, improved epitope mapping. HLA class I and class II constructs containing tags, such as biotin acceptor peptide sequences (BAPs), at the N- or C-terminus can be used in the methods described herein. N- and C-terminal affinity tagging allows for HLA-allele-selective immunopurification from cells expressing endogenous HLA. N-terminal affinity tagging allows for HLA-allele-selective immunopurification of complexes presented on the cell surface. For example, after transfection or transduction, N-terminal biotinylation allows for the 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 for unbiased mass spectrometry (MS) sequencing of endogenously processed and presented peptides. The enrichment methods disclosed herein, such as immunoprecipitation enrichment methods, allow for highly efficient analysis of cell samples.

[0260] Provided herein is a method for characterizing an HLA-peptide complex, the method comprising the steps of: providing a cell population, wherein one or more cells of the cell population comprise a polynucleic acid comprising a sequence encoding an affinity acceptor-tagged class I or class II HLA allele, wherein the sequence encoding the affinity acceptor-tagged HLA comprises a sequence encoding a recombinant class I or class II HLA allele operably linked to a sequence encoding an affinity acceptor peptide; expressing the affinity acceptor-tagged HLA in at least one cell of the one or more cells of the cell population, thereby forming an affinity acceptor-tagged HLA-peptide complex in the at least one cell; enriching the affinity acceptor-tagged HLA-peptide complex; and characterizing the HLA-peptide complex.

[0261] In some embodiments, the characterizing step comprises characterizing peptides bound to the affinity acceptor-tagged HLA-peptide complexes from the enriching step.

[0262] In some embodiments, the method comprises performing the steps of the method for two or more class I and / or class II HLA alleles, in some embodiments, the two or more class I and / or class II HLA alleles comprise 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 class I and / or class II HLA alleles.

[0263] In some embodiments, the affinity acceptor-tagged HLA-peptide complex comprises a transmembrane domain. In some embodiments, the affinity acceptor-tagged HLA-peptide complex comprises an intracellular domain. In some embodiments, the affinity acceptor-tagged HLA-peptide complex is not excreted. In some embodiments, the affinity acceptor-tagged HLA-peptide complex, when expressed, is incorporated into the cell membrane. In some embodiments, the affinity acceptor-tagged HLA-peptide complex is not a soluble affinity acceptor-tagged HLA-peptide complex.

[0264] In some embodiments, the method further comprises generating an HLA-allele specific peptide database.

[0265] In some embodiments, the recombinant class I or class II HLA allele is a single recombinant class I or class II HLA allele.

[0266] In some embodiments, the method includes providing a cell population comprising one or more cells each comprising affinity acceptor-tagged HLA, the cells comprising different recombinant polypeptides encoded by different HLA alleles operably linked to affinity acceptor peptides; enriching the affinity acceptor-tagged HLA-peptide complexes; and characterizing peptides or portions thereof bound to the affinity acceptor-tagged HLA-peptide complexes resulting from the enriching step.

[0267] In some embodiments, the method comprises introducing one or more peptides into the cell population.

[0268] In some embodiments, the introducing step comprises contacting the cell population with one or more peptides or expressing one or more peptides in the cell population. In some embodiments, the introducing step comprises contacting the cell population with one or more nucleic acids encoding one or more peptides. In some embodiments, the one or more nucleic acids encoding one or more peptides are DNA. In some embodiments, the one or more nucleic acids encoding one or more peptides are RNA, and optionally, the RNA is mRNA.

[0269] In some embodiments, the enriching step does not include the use of a tetramer reagent.

[0270] In some embodiments, the characterizing step comprises determining the sequence of the peptide or portion thereof bound to the affinity acceptor-tagged HLA-peptide complexes from the enriching step, and optionally determining whether the peptide or portion thereof is modified. In some embodiments, the determining step comprises biochemical analysis, mass spectrometry, MS analysis, MS / MS analysis, LC-MS / MS analysis, or a combination thereof. In some embodiments, the characterizing step comprises evaluating the binding affinity or stability of the peptide or portion thereof bound to the affinity acceptor-tagged HLA-peptide complexes from the enriching step. In some embodiments, the characterizing step comprises determining whether the peptide or portion thereof bound to the affinity acceptor-tagged HLA-peptide complexes from the enriching step contains one or more mutations. In some embodiments, the characterizing step comprises evaluating the association of the peptide with an HLA molecule in the affinity acceptor-tagged HLA-peptide complex.

[0271] In some embodiments, the method includes expressing a library of peptides in a cell population, thereby forming a library of affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the method includes contacting a cell population with a library of peptides or a library of sequences encoding peptides, thereby forming a library of affinity acceptor-tagged HLA-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, infection by an infectious agent, or an autoimmune response. In some embodiments, the method includes introducing an infectious agent, or a portion thereof, into one or more cells of the cell population. In some embodiments, the method includes characterizing one or more peptides derived from the HLA-peptide complexes, optionally wherein the peptides are derived from one or more target proteins of the infectious agent. In some embodiments, the method includes characterizing one or more regions of peptides derived from one or more target proteins of the infectious agent. In some embodiments, the method includes identifying peptides derived from HLA-peptide complexes derived from the infectious agent.

[0272] In some embodiments, the cell population is derived from a biological sample from 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 population of primary cells. In some embodiments, recombinant class I or class II HLA alleles are matched to subjects with the disease or condition.

[0273] In some embodiments, the method includes screening for drug (e.g., biopharmaceutical) hypersensitivity. In some embodiments, the method includes evaluating whether an administered biopharmaceutical (e.g., a protein, peptide, or antibody drug), an administered biopharmaceutical fragment, or a processed biopharmaceutical fragment is presented to T cells. These epitopes can cause adverse effects in subjects, so monitoring how the administered biopharmaceutical is processed in subjects should be performed. For example, HIV drugs (e.g., Abacavir) can bind to HLA molecules and alter the peptide-binding motif for certain HLA alleles (e.g., HLA-B5701).

[0274] In some embodiments, peptides derived from affinity acceptor-tagged HLA-peptide complexes are capable of activating T cells derived from the subject when presented by antigen-presenting cells. In some embodiments, the characterizing step comprises comparing HLA-peptide complexes derived from cancer cells with HLA-peptide complexes derived from non-cancer cells.

[0275] In some embodiments, the cell population comprises a plurality of cell populations, each cell population expressing a different recombinant class I or class II HLA allele, hi some embodiments, each cell population of the plurality is in the same or a separate container.

[0276] In some embodiments, the method further comprises isolating the peptide from the affinity acceptor-tagged HLA-peptide complex prior to the characterizing step, hi some embodiments, the method further comprises removing one or more amino acids from the termini of the peptide bound to the affinity acceptor-tagged HLA-peptide complex.

[0277] In some embodiments, the cell population is a population of cells with low cell surface HLA class I or class II expression. 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, the cell population is a knockout of one or more HLA class I alleles.

[0278] In some embodiments, the cell population is knocked out of one or more HLA class II alleles. In some embodiments, the cell population is knocked out of all HLA class I alleles. In some embodiments, the cell population is knocked out of all HLA class II alleles. In some embodiments, the cell population is knocked out of all HLA class I alleles and all HLA class II alleles. In some embodiments, the sequence encoding the recombinant class I or class II HLA alleles 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 recombinant class I or class II HLA alleles encodes a class II HLA. In some embodiments, the class II HLA is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP. In some embodiments, the class II HLA comprises an HLA class II alpha chain, an HLA class II beta chain, or a combination thereof.

[0279] In some embodiments, each sequence encodes at least two different class I and / or class II HLA alleles. In some embodiments, at least two different class I and / or class II HLA alleles are each operably linked to a sequence encoding a different affinity acceptor peptide. In some embodiments, at least two different class I and / or class II HLA alleles are each operably linked to a sequence encoding an affinity acceptor peptide.

[0280] In some embodiments, the method comprises administering at least a second polynucleic acid comprising a sequence encoding a different recombinant HLA allele operably linked to the same or a different affinity acceptor peptide.

[0281] In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence encoding the extracellular portion of a recombinant class I or class II HLA allele.

[0282] In some embodiments, the encoded affinity acceptor peptide is expressed extracellularly. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the N-terminus of a sequence encoding a recombinant class I or class II HLA allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence encoding the intracellular portion of a recombinant class I or class II HLA allele. In some embodiments, the encoded affinity acceptor peptide is expressed intracellularly. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the C-terminus of a sequence encoding a recombinant class I or class II HLA allele. In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to a sequence encoding a recombinant class I or class II HLA allele by a linker.

[0283] In some embodiments, the enriching step comprises enriching for intact cells that express the affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the method does not comprise lysing the cells prior to the enriching step. In some embodiments, the method further comprises lysing one or more cells prior to the enriching step. In some embodiments, the enriching step comprises contacting an affinity acceptor peptide-binding molecule with the affinity acceptor-tagged HLA-peptide complexes, wherein the affinity acceptor peptide-binding molecule specifically binds to the affinity acceptor peptide.

[0284] In some embodiments, the affinity acceptor peptide is a biotin acceptor peptide (BAP), a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin-binding peptide (SPB) tag, a Strep tag, a Strep tag II, an albumin-binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag (B-tag), a calmodulin-binding peptide (CBP) tag, a chloramphenicol acetyltransferase (CAT) tag, a 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), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity The affinity acceptor peptide comprises a tag sequence comprising an eXact tag, a protein C tag, an S1-tag, an S-tag, a biotin-carboxy carrier protein (BCCP) tag, a green fluorescent protein (GFP) tag, a small ubiquitin-like modifier (SUMO) tag, a tandem affinity purification (TAP) tag, a HaloTag, a Nus-tag, a thioredoxin tag, an Fc-tag, a CYD tag, an HPC tag, a TrpE tag, a ubiquitin tag, a VSV-G epitope tag, a V5 tag, or a combination thereof, and optionally, the affinity acceptor peptide comprises 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 affinity acceptor peptide. In some embodiments, the enriching step comprises contacting an affinity molecule with the affinity acceptor-tagged HLA-peptide complex, 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, the enriching step comprises immunoprecipitating the affinity acceptor-tagged HLA-peptide complex. In some embodiments, the 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 solid surface is a bead. In some embodiments, the enriching step comprises immunoprecipitating the affinity acceptor-tagged HLA-peptide complex with an affinity acceptor peptide-binding molecule that specifically binds to the affinity acceptor peptide. In some embodiments, the affinity acceptor peptide-binding molecule does not specifically interact with the amino acid sequence of the encoded recombinant class I HLA or class II HLA. In some embodiments, the enriching step comprises contacting an affinity molecule specific for the extracellular portion of the recombinant class I or class II HLA allele, hi some embodiments, the enriching step comprises contacting an affinity molecule specific for the N-terminal portion of the recombinant class I or class II HLA allele.

[0285] In some embodiments, the providing step comprises contacting the cell population with the polynucleic acid. In some embodiments, the contacting step comprises transfecting or transducing. In some embodiments, the providing step comprises contacting the cell population with a vector comprising the polynucleic acid. In some embodiments, the vector is a viral vector. In some embodiments, the polynucleic acid is stably integrated into the genome of the cell population.

[0286] In some embodiments, the sequence encoding the recombinant class I or class II HLA comprises a sequence encoding an HLA class I α chain. In some embodiments, the method further comprises expressing a sequence encoding β2 microglobulin in one or more cells. In some embodiments, the sequence encoding β2 microglobulin is connected to a sequence encoding an HLA class I α chain. In some embodiments, the sequence encoding β2 microglobulin is connected to a sequence encoding an HLA class I α chain by a linker. In some embodiments, the sequence encoding β2 microglobulin is connected to a sequence encoding a second affinity acceptor peptide. In some embodiments, the sequence encoding the recombinant class I or class II HLA comprises 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, the sequence encoding an HLA class II β chain is connected to a sequence encoding an HLA class II α chain. In some embodiments, the sequence encoding an HLA class II β chain is connected to a sequence encoding an HLA class II α chain by a linker.

[0287] In some embodiments, the sequence encoding the HLA class II β chain is connected to a sequence encoding a second affinity acceptor peptide. In some embodiments, the second affinity acceptor peptide is different from the first affinity acceptor peptide and is selected from the group consisting of a biotin acceptor peptide (BAP), a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine tag, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin-binding peptide (SPB) tag, a Strep tag, a Strep tag II, an albumin-binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag (B-tag), a calmodulin-binding peptide (CBP) tag, a chloramphenicol acetyltransferase (ACAT) tag, a phosphodiesterase (PHOS) tag, a phosphodiesterase (PAS ... 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), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinityand optionally the first or second affinity acceptor peptide comprises two or more repeats of the tag sequence.

[0288] In some embodiments, the linker comprises a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome skipping site or an internal ribosome entry site (IRES) element. In some embodiments, the ribosome skipping site or IRES is cleaved when expressed in a cell. In some embodiments, the ribosome skipping 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.

[0289] In some embodiments, the determining step comprises performing biochemical analysis or mass spectrometry, such as tandem mass spectrometry. In some embodiments, the determining step comprises obtaining peptide sequences corresponding to MS / MS spectra of one or more peptides isolated from the enriched affinity acceptor-tagged HLA-peptide complexes from a peptide database, wherein the obtained sequence or sequences identify the sequence of the one or more peptides.

[0290] In some embodiments, the cell population is a cell line selected from HEK293T, expi293, HeLa, A375, 721.221, JEG-3, K562, Jurkat, HepG2, SH-SY5Y, CACO-2, U937, U-2 OS, ExpiCHO, CHO, and THP 1. 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 combinations thereof.

[0291] In some embodiments, the peptide database is a non-enzyme-specific peptide database, such as one that does not include a modified database or one that includes a modified database. In some embodiments, the method further comprises searching the peptide database using a reverse database search strategy.

[0292] In some embodiments, the cell population comprises at least 10 5 cells, at least 10 6 cells or at least 10 7 In some embodiments, the cell population comprises a population of dendritic cells, macrophages, cancer cells, or B cells. In some embodiments, the cell population comprises tumor cells. In some embodiments, the cell population is contacted with an agent prior to isolating the 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.

[0293] In some embodiments, the HLA allele is a mutated HLA allele.

[0294] In some embodiments, the sequence encoding the HLA allele comprises a barcode sequence. In some embodiments, the method further comprises assaying for expression of affinity acceptor-tagged class I or class II HLA alleles. In some embodiments, the assaying comprises assaying for expression of affinity acceptor-tagged class I or class II HLA alleles. This includes sequencing the HLA allele, detecting affinity acceptor-tagged class I or class II HLA allele RNA, detecting affinity acceptor-tagged class I or class II HLA allele protein, or a combination thereof.

[0295] In some embodiments, the method comprises performing the steps of the method for different HLA alleles. In some embodiments, each different HLA allele comprises a unique barcode sequence. In some embodiments, each polynucleic acid encoding a different HLA allele comprises a unique barcode sequence.

[0296] Provided herein is an HLA-allele-specific binding peptide sequence database 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 predictive algorithm for identifying HLA-allele-specific binding peptides, comprising training a machine using the peptide sequence databases 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 variables used to train the machine include one or more variables selected from the group consisting of peptide sequence, amino acid physical properties, peptide physical properties, expression level of the peptide's source protein in cells, protein stability, protein translation rate, ubiquitination site, protein degradation rate, translation efficiency from ribosome profiling, protein cleavability, protein localization, host protein motifs that facilitate TAP transport, host proteins that undergo autophagy, motifs that favor ribosome stalling, and protein properties that favor NMD. In some embodiments, the motif that favors ribosome stalling comprises a polyproline or polylysine stretch. In some embodiments, the protein characteristic that favors NMD is selected from the group consisting of a long 3'UTR, a stop codon more than 50 nt upstream of the last exon:exon junction, and peptide cleavability. Provided herein is a method for identifying HLA-allele-specific binding peptides, the method comprising analyzing the sequence of the peptide using a machine trained with a peptide sequence database obtained by performing the method described herein on the HLA-allele. In some embodiments, the method comprises determining the expression level of the source protein of the peptide in a cell, wherein the source protein expression is a predictor variable used by the machine.In some embodiments, the expression level is determined by measuring the amount of source protein or the amount of RNA encoding said source protein.

[0297] 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 comprise (a) sequences encoding different recombinant HLA class I α chain alleles, (b) sequences encoding an affinity acceptor peptide, and optionally (c) sequences encoding β2 microglobulin, wherein sequences (a) and (b), and optionally (c), are operably linked.

[0298] Provided herein are compositions comprising recombinant polynucleic acids comprising two or more sequences, each comprising a sequence encoding an affinity acceptor-tagged HLA, wherein the affinity acceptor-tagged HLA-encoding sequences comprise (a) a sequence encoding a recombinant 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, the recombinant polynucleic acid is isolated. 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 class II HLA is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP.

[0299] In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the sequence encoding 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, the sequence encoding the affinity acceptor peptide is operably linked to the C-terminus of the sequence encoding the recombinant HLA allele.

[0300] In some embodiments, the sequence encoding the affinity acceptor peptide is operably linked to the sequence encoding the recombinant HLA allele by a linker.

[0301] In some embodiments, two or more sequences encoding affinity acceptor-tagged HLA are expressed from the same polynucleotide, hi some embodiments, two or more sequences encoding affinity acceptor-tagged HLA are expressed from different polynucleotides.

[0302] In some embodiments, the encoded affinity acceptor peptide specifically binds to the affinity acceptor peptide binding molecule.

[0303] In some embodiments, the two or more affinity acceptor-tagged HLA-encoding sequences comprise two or more affinity acceptor peptides. In some embodiments, the two or more affinity acceptor-tagged HLA-encoding sequences comprise three or more affinity acceptor-tagged HLA-encoding sequences, wherein at least two of the three or more affinity acceptor-tagged HLA-encoding sequences comprise the same affinity acceptor peptide. In some embodiments, the two or more affinity acceptor peptides are unique for each of the two or more affinity acceptor-tagged HLA-encoding sequences. In some embodiments, the encoded affinity acceptor peptide is a biotin acceptor peptide (BAP), a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin binding peptide (SPB) tag, a Strep tag, a Strep tag II, an albumin binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag (B-tag), a calmodulin binding peptide (CBP) tag ... c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, a c-myc tag, Choramphenicol 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), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1,The affinity acceptor peptide may be selected from the group consisting of Softag3, TC-tag, VSV-tag, Xpress-tag, Isopeptag, SpyTag, SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag, biotin-carboxy 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, and combinations thereof, and optionally, the first or second affinity acceptor peptide comprises two or more repeats of the tag sequence. In some embodiments, the affinity acceptor peptide binding molecule is an antibody specific for biotin or the affinity acceptor 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 affinity acceptor peptide binding molecule does not specifically interact with the amino acid sequence of recombinant class I HLA or class II HLA.

[0304] In some embodiments, for two or more of the recombinant polynucleic acids, the sequence encoding the affinity acceptor-tagged HLA is stably integrated into the genome of the cell.

[0305] In some embodiments, the β2 microglobulin-encoding sequence or the HLA class II β chain-encoding sequence is connected to the second affinity acceptor peptide-encoding sequence. In some embodiments, the second affinity acceptor peptide comprises an HA tag. In some embodiments, the β2 microglobulin-encoding sequence or the HLA class II β chain-encoding sequence is connected to the recombinant HLA and affinity acceptor peptide-encoding sequence by a linker. In some embodiments, the linker comprises a polynucleic acid sequence encoding a cleavable linker. In some embodiments, the cleavable linker is a ribosome skipping site or an internal ribosome entry site (IRES) element. In some embodiments, the ribosome skipping site or IRES is cleaved when expressed in a cell. In some embodiments, the ribosome skipping 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.

[0306] Provided herein are compositions comprising two or more isolated polypeptide molecules encoded by the polynucleic acids of the compositions described herein. Provided herein are compositions comprising cell populations comprising two or more polypeptide molecules encoded by the polynucleic acids of the compositions described herein. Provided herein are compositions comprising cell populations comprising the compositions described herein. Provided herein are compositions comprising cell populations comprising one or more cells comprising the compositions described herein.

[0307] In some embodiments, the cell population comprises one or more endogenous class I or class II In some embodiments, the cell population is engineered to lack one or more endogenous HLA class I alleles. In some embodiments, the cell population is engineered to lack one or more endogenous HLA class II alleles. In some embodiments, the cell population is engineered to lack one or more endogenous HLA class II alleles. In some embodiments, the cell population is engineered to lack one or more endogenous HLA class I alleles and one or more endogenous HLA class II alleles. In some embodiments, the cell population is engineered to lack 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 population of low cell surface HLA class I or class II expressing cells. In some embodiments, the composition is formulated with a peptide or a polynucleic acid encoding a peptide specific for the patient's HLA type.

[0308] Provided herein are methods of producing cells, the methods comprising transducing or transfecting two or more cells with two or more polynucleic acids of the compositions described herein. Provided herein are peptides identified according to the methods described herein.

[0309] Provided herein is a method for inducing an anti-tumor response in a mammal, the method comprising administering to the mammal an effective amount of a polynucleic acid comprising the sequence of a peptide described herein. Provided herein is a method for inducing an anti-tumor response in a mammal, the method comprising administering to the mammal an effective amount of a peptide comprising the sequence of a peptide described herein. Provided herein is a method for inducing an anti-tumor response in a mammal, the method comprising administering to the mammal cells comprising a peptide comprising the sequence of a peptide described herein. Provided herein is a method for inducing an anti-tumor response in a mammal, the method comprising administering to the mammal cells comprising an effective amount of a polynucleic acid comprising a sequence encoding a peptide comprising the sequence of a peptide described herein. In some embodiments, the cells present the peptide as an HLA-peptide complex. Provided herein is a method for inducing an immune response in a mammal, the method comprising administering to the mammal an effective amount of a polynucleic acid comprising a sequence encoding a peptide described herein. Provided herein is a method for inducing an immune response in a mammal, the method comprising administering to the mammal an effective amount of a peptide comprising the sequence of a peptide described herein. Provided herein is a method for inducing an immune response in a mammal, the method comprising administering to the mammal an effective amount of cells comprising a peptide comprising the sequence of a peptide described herein. Provided herein is a method for inducing an immune response in a mammal, the method comprising administering to the mammal an effective amount of cells comprising a polynucleic acid comprising a sequence encoding a peptide comprising the sequence of a peptide described herein.

[0310] In some embodiments, 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.

[0311] Provided herein is a method for treating a mammal having a disease, the method comprising administering to the mammal an effective amount of a polynucleic acid comprising a sequence encoding a peptide described herein. Provided herein is a method for treating a mammal having a disease, the method comprising administering to the mammal an effective amount of a peptide comprising the sequence of a peptide described herein. Provided herein is a method for treating a mammal having a disease, the method comprising administering to the mammal an effective amount of cells comprising a peptide comprising the sequence of a peptide described herein. Provided herein is a method for treating a mammal having a disease, the method comprising administering to the mammal an effective amount of cells comprising a polynucleic acid comprising a sequence encoding a peptide comprising the sequence of a peptide described herein.

[0312] In some embodiments, the disease is cancer. In some embodiments, the disease is an infection by an infectious agent. In some embodiments, the infectious agent is a pathogen, optionally a virus or bacterium, or a parasite. In some embodiments, the virus is selected from the group consisting of BK virus (BKV), dengue virus (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 lymphotropic virus (HTLV-1), influenza virus, RSV, HPV, rabies, mumps, rubella virus, poliovirus, yellow fever, hepatitis A, hepatitis B, rotavirus, varicella 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 fever, Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus B, Bacillus anthracis, tetanus toxoid, Neisseria meningitidis, BCG, cholera, and any combination thereof. In some embodiments, the parasite is a parasitic helminth or protozoan. In some embodiments, the parasite 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.

[0313] Provided herein are methods for enriching immunogenic peptides, the methods comprising: providing a cell population comprising one or more cells expressing affinity acceptor-tagged HLA, the affinity acceptor peptide operably linked to a recombinant HLA encoded by a recombinant HLA allele; and enriching HLA-peptide complexes comprising the affinity acceptor-tagged HLA. In some embodiments, the method further comprises determining the sequence of the immunogenic peptide isolated from the HLA-peptide complex. In some embodiments, the determining step comprises using LC-MS / MS.

[0314] Provided herein are methods of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a polynucleic acid comprising a sequence encoding a peptide described herein. Provided herein are methods of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a peptide comprising the sequence of a peptide described herein. Provided herein are methods of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of cells comprising a peptide comprising the sequence of a peptide described herein. Provided herein are methods of treating a disease or disorder in a subject, the method comprising administering to the subject cells comprising an effective amount of a polynucleic acid comprising a sequence encoding a peptide comprising the sequence of a peptide described herein.

[0315] Enrichment of HLA-peptide complexes The genes encoding HLA class I and class II glycoproteins are the most polymorphic coding sequences in the human genome. However, there are relatively constant or non-variable regions on the HLA class I heavy chain and HLA class II α and β chains, which can be targeted by antibodies that selectively capture either the HLA class I heavy chain or the HLA class II α or β chain. However, because the α and β chains usually associate with each other in vivo, immunopurification of the α chain of intact soluble HLA can simultaneously precipitate the β chain, and vice versa. Anti-HLA class II antibodies for enriching HLA-associated polypeptides can recognize conserved epitopes presented on either the α or β chain.

[0316] Enrichment methods using HLA allele-specific antibodies or non-HLA-specific reagents are well known in the art.For example, HLA-C polypeptides are typically expressed by individuals at lower levels than HLA-A and HLA-B.Therefore, in order to enhance the detection of HLA-C using antibodies, it may be advantageous to provide specific immunopurification of HLA-C using HLA-C-specific antibodies in addition to other purification methods.Many examples of monoclonal or polyclonal antibodies that specifically bind to individual HLA chains are commercially available.

[0317] A universal immunopurification (IP) pipeline for enriching one or more single-allele HLA polypeptide complexes is provided herein. An example of such a method for enriching HLA-associated polypeptides is a method that includes an immunopurification step. The universal IP pipeline includes a universal IP construct consisting of a DNA construct encoding an affinity-tagged HLA class I or class II allele that is expressed from an expression vector by cell transfection or transduction.

[0318] Cells transfected or transduced with the universal IP construct were 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 a single HLA class I allele, class II-deficient cell lines expressing a pair of HLA class II alleles, or class I- and class II-deficient cell lines expressing a single HLA class I and / or a pair of class II alleles. In an exemplary embodiment, the class I-deficient B cell line is B721.221. In some embodiments, the cells are A375, HEK293T, HeLa, or expi293. However, it will be apparent to those skilled in the art that other cell populations that are class I- and / or class II-deficient can be generated. Methods for generating class I and / or class II deficient cells and cell lines are known in the art, and an exemplary method for deleting / inactivating endogenous class I or class II genes includes, for example, CRISPR-Cas9-mediated genome editing in THP-1 cells. In some embodiments, the cell population is a professional antigen-presenting cell, such as a macrophage, B cell, or dendritic cell. The cell may be a B cell or a dendritic cell. In some embodiments, the cell is a tumor cell or a cell derived from a tumor cell line. In some embodiments, the cell is a cell isolated from a patient. In some embodiments, the cell contains an infectious agent or a portion thereof.

[0319] In some embodiments, the universal IP construct comprises a class I or class II HLA construct comprising an affinity acceptor tag and an affinity molecule. In some embodiments, the universal IP construct comprises at least one specifically binding affinity acceptor tag and affinity molecule.In some embodiments, the affinity acceptor tag is a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin binding peptide (SPB) tag, a Strep tag, a Strep tag II, an albumin binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag (B-tag), a calmodulin binding peptide (CBP) tag, a chloramphenicol acetyltransferase (CAT) tag, a 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), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity These are the eXact tag, protein C tag, S1-tag, S-tag, biotin-carboxy 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, the VSV-G epitope tag derived from the vesicular stomatitis virus glycoprotein, or the V5 tag derived from a small epitope (Pk) found on the P and V proteins of the paramyxovirus simian virus 5 (SV5).In some embodiments, the affinity acceptor tag may comprise multiple repeats of a tag sequence (e.g., a 3x polyhistidine tag, a 3x FLAG tag). In some embodiments, the affinity acceptor tag may comprise multiple repeats of a tag sequence (e.g., a 3x polyhistidine tag, a 3x FLAG tag). In some embodiments, the affinity acceptor tag is an "epitope tag," a type of peptide tag that adds a recognizable epitope (antibody binding site) to an HLA-protein to provide binding of the corresponding antibody, thereby enabling identification or affinity purification of the tagged protein. Non-limiting examples of epitope tags are protein A or protein G, which bind to IgG. In some embodiments, the affinity acceptor tag comprises a biotin acceptor peptide (BAP) or a human influenza hemagglutinin (HA) peptide sequence. Numerous other tag moieties are known to and can be envisioned by those skilled in the art and are contemplated herein. Any peptide tag can be used, as long as the peptide tag can be expressed as an element of an affinity acceptor-tagged HLA-peptide complex.

[0320] The affinity tag can be placed on the N-terminus or C-terminus of the HLA allele. A cleavage sequence such as F2A or an internal ribosome entry site (IRES) can be placed between the alpha chain and beta2 microglobulin (class I) or between the alpha chain and beta chain (class II). In some embodiments, a single class I HLA allele is HLA-A * 02:01, HLA-A * 23:01 and HLA-B * 14:02, or HLA-E * 01:01, and class II HLA alleles are 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 is EGRGSL LTCGDVEENPGP(T2A), ATNFSLLKQAGDVEENPGP(P2A), QCTN YALLKLAGDVESNPGP(E2A), or VKQTLNFDLLKLAGDVESNP It may be GP(F2A).

[0321] In some embodiments, HLA-peptide complex immunopurification is biotin-based. In some embodiments, HLA-peptide complex immunopurification is streptavidin- or NeutrAvidin-based. In some embodiments, HLA-peptide complexes can also be enriched from biological samples by chromatographic techniques such as HPLC. In some embodiments, depletion of highly abundant serum proteins can be used to enrich HLA-peptide complexes. In some embodiments, methods for removing abundant serum proteins include dye ligands (for albumin), proteins A and G (for γ-globulin), or specific antibodies that bind with high affinity to and selectively deplete these species from the sample (Govorukhina, Reijmers et al., 2006). Such strategies increases the number of HLA-derived peptide sequences that can be identified in a single mass spectrometry analysis.

[0322] The degree of enrichment desired to optimize the resolution of specific HLA sequences from a biological sample will depend on the initial concentration of HLA sequences in the biological sample, as well as the concentration and nature of other non-HLA proteins in the sample.

[0323] Classical protein purification techniques can be used alone or in combination with the universal IP pipeline method provided herein to enrich HLA-peptide complexes within biological samples. Classical protein separation (purification) techniques are based on size differences (ultrafiltration, gel filtration, or size exclusion chromatography); charge differences (anion / cation exchange chromatography or hydrophobic interaction chromatography); and a combination of size and charge differences (1D or 2D electrophoresis). Immunopurification options include the use of monoclonal or polyclonal antibodies that specifically bind to HLA proteins. Other protein affinity purification options include the use of proteins known to bind to HLA, such as 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 with high affinity to HLA (computer modeling algorithms can be used to predict peptide / HLA binding characteristics). Any of these high HLA affinity protein options can be immobilized on an insoluble solid support to prepare an affinity matrix that can be used to capture HLA from a liquid biological sample. Appropriate elution conditions will result in concentration and purification (isolation) of the HLA content of the sample.

[0324] In some embodiments, the enriching step comprises enriching for intact cells expressing the affinity acceptor-tagged HLA-peptide complexes. In some embodiments, the method does not comprise lysing the cells prior to the enriching step. In some embodiments, the method further comprises lysing one or more cells prior to the enriching step. In some embodiments, the enriching step comprises contacting the affinity acceptor-tagged HLA-peptide complexes with an affinity acceptor peptide-binding molecule that specifically binds to the affinity acceptor peptide. In some examples, the enriching step does not comprise the use of a tetramer reagent.

[0325] disease specific antigen 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, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid residues, and any range derivable therein.

[0326] In some embodiments, the antigenic peptide molecule is equal to or less than 50 amino acids. In some embodiments, the antigenic peptide molecule is equal to about 20 to about 30 amino acids. Longer peptides can be designed in several ways. For example, if the HLA-binding regions are predicted or known, the longer peptide can consist of either individual binding peptides containing extensions of 0 to 10 amino acids toward the N- and C-termini of the respective corresponding gene products. The longer peptide can also consist of a concatenation of some or all of the binding peptides, each containing the extended sequence. Alternatively, if sequencing reveals a long (more than 10 residues) epitope sequence present in diseased tissue (e.g., due to frameshift, readthrough, or intron inclusion resulting in a new peptide sequence), the longer peptide can consist of the entire new disease-specific amino acid extension. In both cases, the use of longer peptides requires endogenous processing by professional antigen-presenting cells, such as dendritic cells, and can result in more effective antigen presentation and induction of T cell responses. In some embodiments, the extended sequence is altered to improve the biochemical properties of the polypeptide (such as solubility or stability) or to improve the likelihood of efficient proteasomal processing of the peptide.

[0327] Antigenic peptides and polypeptides can bind to HLA proteins. In some embodiments, antigenic peptides can bind to HLA proteins with higher affinity than corresponding natural / wild-type peptides. The antigenic peptides 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 autoimmune responses and / or induce immune tolerance when administered to a subject.

[0328] The present disclosure also provides compositions comprising multiple antigenic peptides. Reference to an antigenic peptide includes any suitable delivery modality (e.g., nucleic acid) that can result in the introduction of the peptide into a subject's cells. In some embodiments, the composition comprises at least three 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 composition contains at least 20 different peptides. In some embodiments, the composition contains up to 20 different peptides. According to the present disclosure, two or more of the different peptides may be derived from the same polypeptide. For example, if an antigenic variant encodes a polypeptide, two or more of the antigenic peptides may be derived from that polypeptide. In one embodiment, the two or more antigenic peptides derived from a polypeptide may comprise a tiled array spanning the polypeptide (e.g., the antigenic peptides may comprise a series of overlapping antigenic peptides spanning part or all of the polypeptide). The antigenic peptides may be derived from mutations in human cancers or infectious agents or autoimmune diseases.

[0329] Antigenic peptides, polypeptides, and analogs can be further modified to contain additional chemical moieties not normally part of the protein. These derivatized moieties can improve solubility, biological half-life, absorption, or binding affinity of the protein. The moieties can also reduce or eliminate any undesirable side effects of the protein, etc. A review of these moieties can be found in Remington's Pharmaceutical Sciences, Vol. 20th ed., Mack Publishing Co., Easton, PA (2000). For example, antigenic peptides and polypeptides with desired activity can be modified as needed to provide certain desirable attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide in binding to desired MHC molecules and activating appropriate T cells. For example, antigenic peptides and polypeptides can undergo various changes, such as conservative or non-conservative substitutions, which can provide certain advantages in their use, such as improved MHC binding. Such conservative substitutions can involve replacing one amino acid residue with another that is biologically and / or chemically similar, e.g., replacing one hydrophobic residue with another, or one polar residue with another. The effects of single amino acid substitutions can also be probed using D-amino acids. Such modifications can be made using well-known peptide synthesis procedures, such as those described in, for example, Merrifield, Science 232:341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer (eds., NY, Academic Press), pp. 1-284 (1979); and Stewart and Young, Solid Phase Peptide Synthesis (Rockford, III., Pierce), 2nd ed. (1984).

[0330] For example, antigenic peptides can be modified by adding or deleting amino acids to extend or reduce the amino acid sequence of the compound. Antigenic peptides, polypeptides, or analogs can also be modified by changing the order or composition of certain residues. Those skilled in the art will understand that certain amino acid residues essential for biological activity, such as those at critical contact sites or conserved residues, generally cannot be altered without adverse effects on biological activity. Non-essential amino acids need not be limited to those naturally occurring in proteins, such as La-amino acids or their D-isomers, but can also include unnatural amino acids such as β-γ-δ-amino acids, as well as many derivatives of La-amino acids.

[0331] Antigen peptides can be optimized by using a series of peptides with single amino acid substitutions to determine the effect of charge, hydrophobicity, and the like on MHC binding. For example, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions can be made along the length of the peptide to exhibit different patterns of sensitivity to various MHC molecules and T cell receptors. Additionally, multiple substitutions using small, relatively neutral moieties such as Ala, Gly, Pro, or similar residues can be used. Substitutions can be homo- or hetero-oligomeric. The number and type of residues substituted or added depend on the spacing required between essential contact points and certain functional attributes (e.g., hydrophobicity vs. hydrophilicity) desired. Such substitutions can also achieve increased binding affinity for MHC molecules or T cell receptors compared to the affinity of the parent peptide. In any event, such substitutions should use amino acid residues or other molecular fragments selected to avoid, for example, steric and charge interference that could disrupt binding. Amino acid substitutions are typically of single residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at the final peptide.

[0332] Antigenic peptides can be modified to provide desired attributes. For example, the ability of a peptide to induce CTL activity can be enhanced by linking it to a sequence containing at least one epitope capable of inducing a helper T cell response. In some embodiments, the immunogenic peptide / T helper conjugate is linked by a spacer molecule. In some embodiments, the spacer comprises a relatively small, neutral molecule, such as an amino acid or amino acid mimetic that is substantially uncharged under physiological conditions. The spacer can be selected from, for example, Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that, if desired, the spacer need not contain the same residues and thus may be a hetero- or homo-oligomer. The antigenic peptide can be linked to the T helper peptide directly at the amino or carboxy terminus of the peptide or via a spacer. The amino terminus of the antigenic peptide or T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378-389.

[0333] Monoallelic HLA cell lines Monoallelic cell lines expressing a single class I HLA allele, a pair of class II HLA alleles, or a single class I HLA allele and a pair of class II HLA alleles can be generated by transducing or transfecting a suitable cell population with a polynucleic acid, such as a vector, encoding a single HLA allele. Suitable cell populations include, for example, class I-deficient cell lines expressing a single HLA class I allele, class II-deficient cell lines expressing a pair of HLA class II alleles, or class I and class II-deficient cell lines expressing a single HLA class I and / or a pair of class II alleles. In an exemplary embodiment, the class I-deficient B cell line is B721.221. However, it is clear to those skilled in the art that other cell populations that are class I and / or class II-deficient can be generated. An exemplary method for deleting / inactivating endogenous class I or class II genes includes, for example, CRISPR-Cas9-mediated genome editing in THP-1 cells. In some embodiments, the cell population is a professional antigen-presenting cell, such as a macrophage, a B cell, or a dendritic cell. The cell may be a B cell or a dendritic cell. In some embodiments, the cell is a tumor cell or a cell derived from a tumor cell line. In some embodiments, the cell is a cell isolated from a patient. In some embodiments, the cell contains an infectious agent or a portion thereof. In some embodiments, the cell population is at least 10 7The cell population comprises cells. In some embodiments, the cell population is further modified, such as by increasing or decreasing the expression and / or activity of at least one gene. In some embodiments, the gene encodes a member of the immunoproteasome. The immunoproteasome is known to be involved in processing HLA class I-bound peptides and includes the LMP2 (β1i), MECL-1 (β2i), and LMP7 (β5i) subunits. The immunoproteasome can also be induced by interferon-gamma. Thus, in some embodiments, the cell population can be contacted with one or more cytokines, growth factors, or other proteins. The 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 subjected to various environmental conditions, such as stress (heat stress, oxygen deprivation, glucose starvation, DNA-damaging agents, etc.). In some embodiments, the cells are contacted with one or more of a chemotherapeutic agent, radiation, targeted therapy, or immunotherapy. Thus, the methods disclosed herein can be used to study the effects 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 in which the HLA-peptide population is being identified.

[0334] The single HLA-alleles of the present disclosure can be encoded and expressed using a virus-based system (e.g., an adenovirus system, an adeno-associated virus (AAV) vector, a poxvirus, or a lentivirus). Plasmids that can be used for adeno-associated virus, adenovirus, and lentivirus delivery have been previously described (see, for example, U.S. Patent Nos. 6,955,808 and 6,943,019, and U.S. Patent Application No. 20080254008, which are incorporated herein by reference). Of the vectors that can be used in implementing the present disclosure, integration into the host genome of cells is possible using retroviral gene transfer methods, which often result in long-term expression of the inserted transgene. In an exemplary embodiment, the retrovirus is a lentivirus. Furthermore, high transduction efficiency has been observed in many different cell types and target tissues. The tropism of retroviruses can be altered by incorporating foreign envelope proteins and expanding the potential target population of target cells. Retroviruses can also be engineered to infect only certain cell types, allowing for conditional expression of the inserted transgene. Cell-type-specific promoters can be used to target expression in specific cell types. Lentiviral vectors are retroviral vectors (thus, both lentiviruses and retroviral vectors can be used in the practice of the present disclosure). Furthermore, lentiviral vectors can transduce or infect non-dividing 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 Figure 3.

[0335] The choice of retroviral gene transfer system may depend on the target tissue. Retroviral vectors contain cis-acting long terminal repeats that have packaging capacity for up to 6-10 kb of foreign sequence. A minimal set of cis-acting LTRs is sufficient for replication and packaging of the vector, which is then used to integrate the desired nucleic acid into the target cell to provide persistent expression. Widely used retroviral vectors that can be used in the practice of the present disclosure include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al. (1992), J. Virol. 66:2731-2739; Johann et al. (1993)). 1992), J. Virol. 66:1635-1640; Sommnerfelt et al. (1990 (1998), Virol. 176:58-59; Wilson et al. (1998), J. Virol. 63:2374-2378; Miller et al. (1991), J. Virol. 65:2220-2224; see PCT / US94 / 05700. Also useful in the practice of the present disclosure are minimal non-primate lentiviral vectors, such as lentiviral vectors based on equine infectious anemia virus (EIAV) (see, e.g., Wiley InterScience See Balagaan (2006) J Gene Med; 8:275-285, published online November 21, 2005 at DOI: 10.1002 / jgm.845. The vector may have a cytomegalovirus (CMV) promoter that drives expression of the target gene. Accordingly, the present disclosure particularly contemplates vectors useful in the practice of the present disclosure: viral vectors, including retroviral vectors and lentiviral vectors.

[0336] Any HLA allele can be expressed in the cell population. In exemplary embodiments, the HLA allele is a class I HLA allele. In some embodiments, the class I HLA allele is an HLA-A allele or an HLA-B allele. In some embodiments, the HLA allele is a class II HLA allele. Sequences of class I and class II HLA alleles 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 is an example.

[0337] In some embodiments, HLA alleles are selected to match the genotype of interest.In some embodiments, HLA alleles are mutated HLA alleles, and can be non-naturally occurring alleles or naturally occurring alleles in affected patients.The method disclosed herein has the additional advantage of identifying HLA binding peptides for HLA alleles associated with various disorders and alleles that exist at low frequencies.Therefore, in some embodiments, HLA alleles exist at a frequency of less than 1% in a population, such as in a Caucasian population.

[0338] In some embodiments, the nucleic acid sequence encoding the HLA allele further comprises an affinity acceptor tag that can be used to immunopurify the HLA-protein. Suitable tags are well known in the art.In some embodiments, the affinity acceptor tag is a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin binding peptide (SPB) tag, a Strep tag, a Strep tag II, an albumin binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag (B-tag), a calmodulin binding peptide (CBP) tag, a chloramphenicol acetyltransferase (CAT) tag, a 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), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity These are the eXact tag, protein C tag, S1-tag, S-tag, biotin-carboxy 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, the VSV-G epitope tag derived from the vesicular stomatitis virus glycoprotein, or the V5 tag derived from a small epitope (Pk) found on the P and V proteins of the paramyxovirus simian virus 5 (SV5).In some embodiments, the affinity acceptor tag is an "epitope tag," a type of peptide tag that adds a recognizable epitope (antibody binding site) to an HLA-protein to provide binding of the corresponding antibody, thereby enabling identification or affinity purification of the tagged protein. Non-limiting examples of epitope tags are Protein A or Protein G, which bind to IgG. In some embodiments, the affinity acceptor tag comprises a biotin acceptor peptide (BAP) or a human influenza hemagglutinin (HA) peptide sequence. Numerous other tag moieties are known to and can be envisioned by those skilled in the art and are contemplated herein. Any peptide tag can be used as long as the peptide tag can be expressed as an element of an affinity acceptor-tagged HLA-peptide complex.

[0339] The methods provided herein include isolating HLA-peptide complexes from cells transfected or transduced with a universal IP HLA construct. In some embodiments, the complexes can be isolated using standard immunoprecipitation techniques known in the art using commercially available antibodies. The cells can first be lysed. HLA class I-peptide complexes can be isolated using an HLA class I-specific antibody, such as the W6 / 32 antibody, while HLA class II-peptide complexes can be isolated using an HLA class II-specific antibody, such as the M5 / 114.15.2 monoclonal antibody. In some embodiments, a single (or pair of) HLA alleles is expressed as a fusion protein with a peptide tag, and the HLA-peptide complex is isolated using a binding molecule that recognizes the peptide tag.

[0340] The method further includes isolating peptides from the HLA-peptide complexes and sequencing the peptides. The peptides are isolated from the complexes by any method known to those skilled in the art, such as acid elution. While any sequencing method can be used, in some embodiments, a mass spectrometry method is used, 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 reviewed in Medzihradszky KF and Chalkley RJ. Mass Spectrom Rev. 2015 January-February; 34(1):43-63.

[0341] 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 endogenous HLA class I alleles and endogenous HLA class II alleles. In some embodiments, the cell population comprises cells enriched or sorted by, for example, fluorescence-activated cell sorting (FACS). In some embodiments, the cell population is sorted using fluorescence-activated cell sorting (FACS). In some embodiments, the cell population is previously sorted by FACS for cell surface expression of either class I or class II HLA, or both class I and class II HLA. For example, FACS can be used to sort cell populations for cell surface expression of HLA class I alleles, HLA class II alleles, or a combination thereof.

[0342] Library of affinity acceptor-tagged HLA constructs The term "library," as used herein, refers to a collection of nucleic acid molecules (circular or linear). In one embodiment, a library may contain a plurality (i.e., two or more) of nucleic acid molecules, which may be derived from a common source organism, organ, tissue, or cell. In some embodiments, the library is representative of all, a portion, or a significant portion of the nucleic acid content of an organism (a "genomic" library), or a set of nucleic acid molecules (a cDNA library or segments derived therefrom) that represent all, a portion, or a significant portion of the expressed nucleic acid molecules in a cell, tissue, organ, or organism. A library may also contain random sequences, such as those generated by de novo synthesis, mutagenesis of one or more sequences, or the like. Such libraries may be contained in one or more vectors. The library of affinity acceptor-tagged HLA constructs provided herein contains DNA sequences encoding elements of HLA alleles, affinity acceptor peptides, or linkers. Suitable molecular biology techniques can be found in Sambrook et al. (Molecular Cloning; A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989). Cloning of nucleic acid segments can be easily performed. Several methods for this purpose are described, for example, in the following references: Ferguson, J. et al., Gene 16 191 (1981) and Hashimoto-Gotoh, T. et al., Gene 41:125 (1986). As used herein, other terms used in recombinant nucleic acid technology and the field of molecular and cell biology will be commonly understood by those of ordinary skill in the applicable art.

[0343] The various elements or domains of a recombinant HLA allele can be arranged in any order between the N-terminus and C-terminus of the recombinant HLA allele. An element or domain that is closer to the N-terminus of a recombinant polypeptide encoded from a recombinant HLA allele than another element or domain is said to be "N-terminal" to that other element or domain. Similarly, an element or domain that is closer to the C-terminus of a recombinant polypeptide encoded from a recombinant HLA allele than another element or domain is said to be "C-terminal" to that other element or domain. Unless explicitly stated otherwise, different elements or domains of a recombinant polypeptide encoded from a recombinant HLA allele need not be contiguous (i.e., without one or more intervening elements or domains). In some embodiments, different elements or domains of a recombinant polypeptide encoded from a recombinant HLA allele may be contiguous.

[0344] Recombinant polypeptides encoded from recombinant HLA alleles may include one or more optional elements, such as one or more linkers, peptide tags (such as epitope tags), or protease recognition sites. In some embodiments, the peptide tag is an affinity acceptor peptide. A linker is a relatively short stretch of amino acids that separates other elements or domains of the recombinant protein. In some embodiments, the linker is 1 to 100 amino acids in length; e.g., 5 to 75, 10 to 60, 15 to 50, 15 to 40, or 1 to 50 amino acids in length.

[0345] 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 (recombinant HLA class I or class II affinity acceptor-tagged peptide) is obtained using a method such as that described herein. The nucleic acid sequence encoding the protein is cloned into an expression vector suitable for a specific host cell of interest using standard recombinant DNA procedures. The expression vector includes (among other elements) a regulatory sequence (e.g., a promoter) that can be operably linked to the nucleic acid molecule encoding the desired protein to cause the expression of such a nucleic acid molecule in the host cell. Together, the regulatory sequence and the nucleic acid sequence encoding the protein are an "expression cassette." The expression vector may also include an origin of replication, a marker gene that provides phenotypic selection in transformed cells, one or more other promoters, and a polylinker region containing several restriction sites for the insertion of heterologous nucleic acid sequences.

[0346] Expression vectors useful for expressing heterologous proteins in many host cells are well known in the art, and some specific examples are provided herein. The host cells are transfected with the expression vector (or infected with a virus containing it) using any method suitable for the particular host cell. Such transfection methods are also well known in the art, and non-limiting exemplary methods are described herein. The transfected or transduced host cell can express the protein encoded by the corresponding nucleic acid sequence in the expression cassette.

[0347] In some embodiments, the class I or class II HLA construct comprises an affinity acceptor tag and an affinity molecule at the N-terminus or C-terminus, hi some embodiments, the class I or class II HLA construct comprises at least one specifically binding affinity acceptor tag and affinity molecule.In some embodiments, the affinity acceptor tag is a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin binding peptide (SPB) tag, a Strep tag, a Strep tag II, an albumin binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag (B-tag), a calmodulin binding peptide (CBP) tag, a chloramphenicol acetyltransferase (CAT) tag, a 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), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) 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, Softag1, Softag3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity These are the eXact tag, protein C tag, S1-tag, S-tag, biotin-carboxy 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, the VSV-G epitope tag derived from the vesicular stomatitis virus glycoprotein, or the V5 tag derived from a small epitope (Pk) found on the P and V proteins of the paramyxovirus simian virus 5 (SV5).In some embodiments, the affinity acceptor tag may comprise multiple repeats of a tag sequence (e.g., a 3x polyhistidine tag, a 3x FLAG tag). In some embodiments, the affinity acceptor tag may comprise multiple repeats of a tag sequence (e.g., a 3x polyhistidine tag, a 3x FLAG tag). In some embodiments, the affinity acceptor tag is an "epitope tag," a type of peptide tag that adds a recognizable epitope (antibody binding site) to an HLA-protein to provide binding of the corresponding antibody, thereby allowing identification or affinity purification of the tagged protein. Non-limiting examples of epitope tags are Protein A or Protein G, which bind to IgG.

[0348] In some embodiments, the affinity acceptor tag comprises a biotin acceptor peptide (BAP) or a human influenza hemagglutinin (HA) peptide sequence. Many other tag moieties are known to those skilled in the art, can be envisioned by those skilled in the art, and are contemplated herein. Any peptide tag can be used as long as the peptide tag can be expressed as an element of an affinity acceptor-tagged HLA-peptide complex.

[0349] The affinity tag can be placed on the N-terminus or C-terminus of the HLA allele. In some embodiments, the affinity tag is placed on the C-terminus of the HLA allele to allow HLA-peptide localization to the cell surface relative to the ER. In some embodiments, the affinity tag is placed on the N-terminus of the HLA allele to allow isolation of a single HLA from a cell line expressing multiple endogenous HLA alleles. In yet another embodiment, the affinity tag is added to the variable β chain to immunopurify specific class II HLA heterodimers.

[0350] 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, a single Class I HLA allele is HLA-A *02:01, HLA-A * 23:01 and HLA-B * 14:02, or HLA-E * 01:01, and class II HLA alleles are HLA-DRB * 01:01, HLA-DRB * 01:02 and HLA-DRB * 11:01, HLA-DRB * 15:01, or HLA-DRB * It is 07:01.

[0351] Non-limiting exemplary affinity acceptor-tagged HLA constructs are shown in Figures 2, 6C and 7C.

[0352] Treatment method Personalized immunotherapy using tumor-specific peptides has been described (Ott et al., Hematol. Oncol. Clin. N. Am. 28 (2014), 559-569). Efficient selection of tumor-specific peptides for use as immunogens requires the ability to predict which tumor-specific peptides will efficiently bind to the HLA alleles present in the patient. One of the critical barriers to developing curative and tumor-specific immunotherapy is the identification and selection of highly specific and defined tumor antigens to avoid autoimmunity. Tumor neoantigens, which arise as a result of genetic alterations within malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.), represent the most tumor-specific class of antigens. Neoantigens have rarely been used in cancer vaccines or immunogenic compositions due to the technical difficulties of their identification, optimized antigen selection, and generation of neoantigens for use in vaccines or immunogenic compositions. These challenges can be addressed by identifying mutations in neoplasms / tumors that are present at the DNA level in the tumor but not in matched germline samples from subjects with a high proportion of cancer; analyzing the identified mutations using one or more peptide-MHC binding prediction algorithms to generate multiple neo-antigenic T cell epitopes that are expressed within the neoplasm / tumor and bind to a high proportion of patient HLA alleles; and synthesizing multiple neo-antigenic peptides selected from the set of all neo-antigenic peptides and predicted binding peptides for use in cancer vaccines or immunogenic compositions suitable for treating subjects with a high proportion of cancer (Figures 18A and 18B).

[0353] For example, translating peptide sequencing information into therapeutic vaccines may involve predicting mutant peptides that can bind to HLA molecules in a high percentage of individuals. Efficient selection of specific mutations for use as immunogens requires the ability to predict which mutant peptides will efficiently bind to a high percentage of patients' HLA alleles. Recently, neural network-based learning techniques using validated binding and non-binding peptides have improved the accuracy of prediction algorithms for the major HLA-A and -B alleles. However, even with improved neural network-based algorithms that encode HLA-peptide binding rules, several factors limit the ability to predict peptides presented on HLA alleles.

[0354] For example, translating peptide sequencing information into therapeutic vaccines may involve formulating drugs as long, multi-epitope vaccines. Targeting many variant epitopes, which effectively exploits the immune system's vast capabilities, prevents opportunities for immune evasion through downregulation of immune-targeted gene products and offsets the known inaccuracy of epitope prediction methods. Synthetic peptides provide a useful means for efficiently preparing multiple immunogens and rapidly translating the identification of variant epitopes into effective vaccines. Peptides can be easily chemically synthesized and purified using reagents free of contaminating bacteria or animal material. Their small size allows for clear focus on the variant region of the protein and also reduces irrelevant antigenic competition from other components (non-variant proteins or viral vector antigens).

[0355] For example, translating peptide sequencing information into a therapeutic vaccine may involve combining it with a potent vaccine adjuvant. Effective vaccines require a potent adjuvant to initiate an immune response. For example, poly-ICLC, an agonist of TLR3 and the RNA helicase domains of MDA5 and RIG3, has demonstrated several desirable properties for a vaccine adjuvant. These properties include inducing local and systemic activation of immune cells in vivo, stimulating chemokine and cytokine production, and stimulating antigen presentation by DCs. Furthermore, poly-ICLC can induce sustained CD4+ and CD8+ responses in humans. Importantly, striking similarities in the upregulation of transcriptional and signaling pathways were observed in subjects vaccinated with poly-ICLC and volunteers who had previously received a highly effective replicative yellow fever vaccine. Furthermore, more than 90% of ovarian cancer patients immunized with poly-ICLC in combination with the NYESO-1 peptide vaccine (in addition to Montanide) demonstrated induction of CD4+ and CD8+ T cells and antibody responses to the peptide in a recent phase 1 trial. At the same time, poly-ICLC has been extensively tested in over 25 clinical trials to date and has shown a relatively benign toxicity profile.

[0356] In some embodiments, immunogenic peptide can be identified from cells derived from subjects with disease or condition.In some embodiments, immunogenic peptide can be specific to subjects with disease or condition.In some embodiments, immunogenic peptide can bind to HLA that matches the HLA haplotype of subjects with disease or condition.

[0357] In some embodiments, a library of peptides can be expressed in a cell. In some embodiments, the cell comprises an identified or characterized peptide. In some embodiments, the identified or characterized peptide is an endogenous peptide. In some embodiments, the peptide is an exogenous peptide. For example, the identified or characterized peptide can be expressed from a plurality of sequences encoding a library of peptides.

[0358] Prior to this disclosure, the majority of LC-MS / MS studies of the HLA peptidome used cells expressing multiple HLA molecules, which required the use of native bioinformatics predictors or "deconvolution" to assign peptides to one of up to six class I alleles (Bassani-Sternberg and Gfeller, 2016). Thus, peptides that did not exactly match known motifs could not be unambiguously reported as binders for a given HLA allele.

[0359] Provided herein is a method for predicting peptides, such as mutant peptides, that can bind to an individual's HLA molecules. In some embodiments, the application provides a method for identifying the most suitable peptide for preparing an immunogenic composition for a subject from a given set of peptides comprising an antigen, comprising selecting from the given set of peptides a plurality of peptides that can bind to the subject's HLA protein, wherein the ability to bind to the HLA protein is determined by analyzing the peptide sequences using a machine trained with a peptide sequence database corresponding to specific HLA-binding peptides for each of the subject's HLA alleles. Provided herein is a method for identifying the most suitable peptide for preparing an immunogenic composition for a subject from a given set of peptides comprising an antigen, comprising selecting from the given set of peptides a plurality of peptides that have been determined to be capable of binding to the subject's HLA protein, wherein the ability to bind to the HLA protein is determined by analyzing the peptide sequences using a machine trained with a peptide sequence database obtained by performing the method described above. Thus, in some embodiments, the present disclosure provides a method for identifying a plurality of subject-specific peptides for preparing a subject-specific immunogenic composition, wherein the subject has a tumor, and the subject-specific peptides are specific to the subject and the subject's tumor, the method comprising the steps of sequencing a sample from the subject's tumor and a non-tumor sample from the subject; determining, based on the nucleic acid sequencing, non-silent mutations present in the genome of the subject's cancer cells but not present in the subject's normal tissues, 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 that is an epitope specific to the subject's tumor, and each ide...

Claims

[Claim 1] The invention as described in the drawings.

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