Compositions and methods for analyzing samples with respect to antigen-specific immune cells

Abstract

The present invention provides a method for analyzing samples from one or more individuals to determine the presence or absence of immune cells capable of binding to antigen peptides. It also provides a method for detecting or treating cancer or tumors, pathogenic infections, or autoimmune diseases. Further, it provides a display portion having particles to which multiple MHC-peptide complexes are bound, with at least two of the MHC-peptide complexes being distinct. Additionally, it provides a bait composition having multiple display portions, and a method for analyzing immune cells by using the display portions or the bait composition as a screening tool.

Description

[Technical Field]

[0001] Cross-reference of related applications

[0002] This application claims the interests of U.S. Provisional Application No. 63 / 463,886 filed 3 May 2023, which is incorporated herein by reference in its entirety for any purpose.

[0003] Reference to electronic sequence listings

[0004] The contents of the electronic sequence listing (230062000440SEQLIST.xml, size: 101,021 bytes, creation date: May 1, 2024) are incorporated in their entirety herein by reference.

[0005] This application relates to a composition and method for analyzing a sample with respect to antigen-specific immune cells. [Background technology]

[0006] The recognition of peptide-major histocompatibility complex (pMHC or MHC-peptide complex) class I and II molecules by immune cells (e.g., T cells) plays a crucial role in the efficient control of intracellular pathogens and cancer cells through immune cell (e.g., T cell) stimulation. The affinity of TCRs to pMHC complexes is generally low, and the off-rate of their interaction is rapid. These factors make it difficult to detect antigen-specific T cells using soluble monomeric pMHC complexes. To overcome these drawbacks, MHC multimers have been prepared using antibody dimerization or tetravalent biotin-streptavidin linkage. Such MHC dimers or tetramers exhibit increased affinity for their homologous TCRs. To date, MHC multimers have been successfully used to visualize antigen-specific CD8 T cells ex vivo by flow cytometry.

[0007] Conventional MHC tetramers are constructed by tetramerizing C-terminal biotinylated pMHC monomers using streptavidin molecules. MHC tetramers have been shown to be versatile and useful for detecting αβ T cells by flow cytometry and Insights staining. The well-established characteristics of MHC tetramers and their public and commercial availability have led to their widespread use in T cell research. Techniques for staining MHC tetramers are expanding to encompass new research areas, such as enrichment of rare T cells, construction of large libraries and high-throughput epitope screening, cytometry by time-of-flight mass spectrometry (CyTOF), and combinatorial staining for multiple detection.

[0008] MHC pentamers, consisting of five pMHC complexes arranged in a planar structure, are commercially produced. Because MHC pentamers have a higher valency than dimers or tetramers, they offer increased sensitivity in staining T cells of low-affinity TCRs. More recently, further commercially available pMHC complexes linked to a dextran skeleton (pMHC dextramers) have also been found to be useful for detecting T cells of low-affinity TCRs due to their high valency. See, for example, Mol Cells. 2021 May 31;44(5):328-334.

[0009] A wide variety of assays can be used to characterize immune cell (e.g., T cell) responses in order to determine the state and capabilities of the immune system. Such tests can reveal the fundamental mechanisms underlying immunity, assist in the design of clinical diagnoses, support the development of interventional therapies, and identify the characteristics of effective immune responses. However, the effective detection and analysis of antigen-specific immune cells can be challenging because they often constitute a very small proportion of the cells obtainable from a sample (e.g., a blood sample).

[0010] All publications, patents, patent applications, and published patent application disclosures referenced herein are incorporated herein by reference in their entirety. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Mol Cells.2021 May 31;44(5):328-334 [Overview of the project]

[0012] This application provides, in one embodiment, a method for analyzing a sample from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) immobilizing immune cells from the sample onto a solid carrier, the method comprising: 1) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide before immobilization under conditions sufficient for the immune cells to bind to the display portion; or 2) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide after immobilization under conditions sufficient for the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; and c) detecting immune cells bound to the display portion by detecting the detection agent. In some embodiments, the bait composition containing a display portion containing an antigen peptide and the immune cells are contacted with the immune cells before immobilization under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the bait composition containing a display portion containing an antigen peptide and the immune cells are contacted with the immune cells after immobilization under conditions sufficient for the immune cells to bind to the display portion.

[0013] In another embodiment, this application provides a method for analyzing a sample from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) contacting a bait composition comprising a display portion containing an antigen peptide with immune cells under conditions sufficient for the immune cells to bind to the display portion; b) contacting a detection agent that recognizes the display portion with immune cells under conditions sufficient for the detection agent to bind to the display portion; c) immobilizing immune cells from the sample onto a solid carrier; and d) detecting immune cells bound to the display portion by detecting the detection agent.

[0014] In some embodiments of any of the above methods, the sample is obtained from a single individual.

[0015] In some embodiments of any of the above methods, the sample is a mixture of multiple samples of individuals, each obtained from a different individual. In some embodiments, each of the multiple samples of individuals is processed separately before being combined to form a mixture, to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with molecules containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by washing the immune cells at least once. In some embodiments, the method further includes identifying the sample from which the immune cells originate by identifying the unique barcode associated with the immune cells bound to the display portion.

[0016] In another embodiment, this application provides a method for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) processing each of the multiple samples from an individual separately to associate the immune cells contained therein with a unique sample barcode; b) combining the multiple processed samples to prepare a sample mixture; c) preparing a bait-sample mixture by contacting a bait composition containing a display portion with the sample mixture under conditions sufficient for immune cells to bind to the display portion; d) detecting immune cells bound to the display portion in the bait-sample mixture; and e) identifying the sample from which the immune cells originate by identifying a unique barcode associated with the immune cells bound to the display portion.

[0017] In some embodiments of any of the above methods, the immune cells include T cells, and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the immune cells are selected from the group consisting of cytotoxic T cells, memory T cells, and tumor-infiltrating T cells.

[0018] In some embodiments of any of the above methods, the immune cells include B cells.

[0019] In some embodiments of any of the above methods, the method further includes removing unbound display portions after contacting an agent that recognizes display portions with immune cells, before detecting immune cells bound to the display portions.

[0020] In some embodiments of any of the above methods, the antigen peptide is related to a cancer antigen or tumor antigen, and optionally the antigen peptide is a neoantigen peptide. In some embodiments, the sample is obtained from one or more individuals that do not exhibit pathological symptoms of cancer.

[0021] In some embodiments of any of the above methods, the antigenic peptide is related to a pathogenic antigen, and optionally, the antigenic peptide is related to a viral antigen, a bacterial antigen, or a fungal antigen.

[0022] In some embodiments of any of the above methods, the antigen peptide is an autoantigen peptide related to an autoantigen.

[0023] In some embodiments of any of the above methods, immune cells are immobilized on a solid substrate such as beads. In some embodiments, the beads are bound to antibodies that recognize immune cells, and in some cases, the antibodies are CD8 antibodies.

[0024] In some embodiments of any of the above methods, the display portion contains two or more antigenic peptides, and in some cases, the display portion contains four antigenic peptides. In some embodiments, the two or more antigenic peptides in the display portion are the same.

[0025] In some embodiments of some of the above methods, the display portion includes a library of antigenic peptides for cancer or tumor-related mutations, wherein the library contains at least about 5, 10, 12, 15, 18, or 20 different neoantigen peptides.

[0026] In some embodiments of any of the above methods, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to congener TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues.

[0027] In some embodiments of some of the above methods, the antigen peptide is a neoantigen peptide containing a mutation from the wild-type peptide, and optionally a neoantigen peptide containing a mutation at the third amino acid position from the N-terminus.

[0028] In some embodiments of any of the above methods, the antigen peptide exhibits low immunogenicity.

[0029] In some embodiments of any of the above methods, the display portion includes an MHC molecule complexed with an antigen peptide, which may include an MHC polymer, and may further include an MHC tetramer or MHC dextramer. In some embodiments, the MHC molecule includes an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, which may include an HLA-A molecule, which may further include a mutation that reduces its binding to CD8, and may further include an HLA-A heavy chain having the A245V mutation. In some embodiments, the antigen peptide is about 8 to about 10 amino acids long. In some embodiments, the MHC molecule includes an MHC class II molecule. In some embodiments, the MHC class II molecule is selected from the group consisting of HLA-DQ and HLA-DR. In some embodiments, the antigen peptide is about 10 to about 20 amino acids long. In some embodiments, the display portion contains two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03.

[0030] In some embodiments of any of the above methods, the display portion includes particles. In some embodiments, the particles of the display portion are selected from the group consisting of surfaces, nanoparticles, beads, and polymers. In some embodiments, the particles of the display portion are dextran particles. In some embodiments, the particles of the display portion are magnetic nanoparticles or polystyrene nanoparticles. In some embodiments, the particles of the display portion are agarose beads or Sepharose beads. In some embodiments, the antigen peptide or MHC is directly bound to the particles of the display portion. In some embodiments, the antigen peptide or MHC is bound to the particles of the display portion via a binding pair, the binding pair comprising a first binding component bound to the antigen peptide and a second binding component bound to the particle.

[0031] In some embodiments of any of the above methods, the display portion includes cells. In some embodiments, the cells include polynucleotides encoding antigen peptides. In some embodiments, the polynucleotides encode multiple antigen peptides.

[0032] In some embodiments of any of the above methods, the display portion includes a detectable label. In some embodiments, the detectable label is a fluorophore.

[0033] In some embodiments of any of the above methods, the method further includes separating immune cells bound to the display portion, or further including removing the display portion or immune cells that are not bound to each other.

[0034] In some embodiments of any of the above methods, the method further includes determining the phenotype of immune cells bound to the display portion.

[0035] In some embodiments of any of the above methods, the method further comprises sequencing one or more nucleic acids in immune cells bound to a display portion, optionally comprising analyzing the sequence of one or more nucleic acids, optionally being a TCR sequence, a CD8 sequence, a CD45 sequence, or any specific sequence characteristic of the captured immune cell, optionally comprising analyzing the sequence of one or more nucleic acids by whole-genome sequencing and / or RNA sequencing, or by sequence-specific q-PCR and / or RT-PCR with a set of primers.

[0036] In some embodiments of any of the above methods, the method further includes subjecting immune cells bound to the display portion to mass spectrometry.

[0037] In some embodiments of any of the above methods, the method further comprises identifying one or more epigenetic modifications in immune cells, which may include DNA methylation and / or histone glycosylation.

[0038] In some embodiments of any of the above methods, the individual is human. In some embodiments, the individual is at least about 50 years old.

[0039] In some embodiments of some of the above methods, a) the individual has not been previously diagnosed with cancer and, in some cases, is at risk of having cancer, or b) the individual has previously received treatment for cancer and does not show any pathological symptoms of cancer after treatment.

[0040] In some embodiments of some of the above methods, a) the individual has not been diagnosed with an infection caused by a pathogen, and in some cases the individual is at risk of having an infection, and / or b) the individual does not exhibit any pathological symptoms of an infection caused by a pathogen, and in some cases does not exhibit any pathological symptoms of an infection caused by a pathogen.

[0041] In some embodiments of some of the above methods, a) the individual has not been diagnosed with having an immune response to an autoantigen, and in some cases the individual is at risk of developing an immune response to an autoantigen, and / or b) the individual does not exhibit pathological symptoms of an immune response (e.g., autoimmune disease or autoimmune disorder), and in some cases the individual does not exhibit pathological symptoms of an immune response to an autoantigen.

[0042] In some embodiments of any of the above methods, the sample is selected from the group consisting of blood, plasma, and peripheral blood mononuclear cell (PBMC) samples.

[0043] In some embodiments of any of the above methods, the bait composition comprises a plurality of different display portions. In some embodiments, each of the plurality of different display portions in the bait composition comprises a different antigen peptide. In some embodiments, the plurality of different display portions in the bait composition comprises at least two different display portions, and each of the at least two different display portions comprises a different MHC molecule. In some embodiments, the plurality of different display portions in the bait composition comprises at least four different display portions, and each of the at least four different display portions comprises a different MHC molecule. In some embodiments, the plurality of different display portions in the bait composition comprises at least 100 different display portions, and each of the at least 100 different display portions comprises a different MHC molecule. In some embodiments, each of the different display portions containing different MHC molecules comprises a different detectable label. In some embodiments, the different detectable labels may include fluorophores.

[0044] In some embodiments of any of the above methods, the detection agent includes an antibody that binds to the display portion.

[0045] In another embodiment, the application provides a method for detecting antigen-specific immune cells in an individual, the method comprising analyzing a sample from the individual according to one of the above methods, wherein a predetermined feature of the immune cells indicates a disease or disorder in the individual. In some embodiments, the predetermined feature of the immune cells includes the presence of immune cells. In some embodiments, the presence of immune cells that specifically bind to a bait composition containing one or more neoantigens indicates the presence of cancer. In some embodiments, the presence of immune cells that specifically bind to a bait composition containing one or more antigens of a pathogen (e.g., a virus, bacteria, or fungus) indicates the presence of an infection related to the pathogen. In some embodiments, the presence of immune cells that specifically bind to a bait composition containing one or more autoantigens associated with an autoimmune disease or autoimmune disorder indicates the presence of an immune disease or immune disorder. In some embodiments, the predetermined feature of the immune cells includes a quantity of immune cells above a threshold level. In some embodiments, the predetermined feature of the immune cells includes gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics. In some embodiments, epigenetic modification properties include DNA or RNA methylation, hydroxylation, and histone acetylation, methylation, and / or glycosylation properties.

[0046] In another embodiment, this application provides a method for detecting residual cancer in an individual, wherein the individual has been previously treated with anticancer therapy and has not shown pathological symptoms of cancer after treatment, and the method comprises analyzing a post-treatment sample from the individual according to one of the above methods, wherein predetermined characteristics of immune cells from the post-treatment sample indicate residual cancer in the individual. In some embodiments, the method comprises a) analyzing a pre-treatment sample from an individual prior to anticancer therapy and a post-treatment sample from the individual according to one of the above methods, and b) comparing the characteristics of immune cells from the pre-treatment sample with the characteristics of immune cells from the post-treatment sample, wherein predetermined differences between the characteristics of immune cells from the pre-treatment sample and the characteristics of immune cells from the post-treatment sample indicate residual cancer in the individual.

[0047] In another embodiment, this application provides a method for treating cancer in an individual, the method comprising: a) diagnosing the individual having cancer according to one of the above methods; and b) administering anti-cancer therapy to the individual. In some embodiments, the anti-cancer therapy is not immunotherapy. In some embodiments, the cancer is a solid tumor.

[0048] In another embodiment, this application provides a method for treating an infectious disease in an individual, the method comprising: a) diagnosing the individual as having an infectious disease caused by a pathogen according to some of the above methods; and b) administering treatment to the individual for the infectious disease.

[0049] In another embodiment, this application provides a method for treating an autoimmune disease or autoimmune disorder in an individual, the method comprising: a) diagnosing the individual having an autoimmune disease or autoimmune disorder according to some of the above methods; and b) administering treatment to the individual for the disease or disorder.

[0050] In another embodiment, this application provides a composition comprising one or more display portions comprising particles to which a plurality of MHC-peptide complexes are bound, wherein the plurality of MHC-peptide complexes comprises two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of which comprises at least one MHC molecule forming a complex with a peptide, and at least two or more of the MHC-peptide complexes are different.

[0051] In some embodiments of any of the above-described display portions, one or more of the plurality of MHC-peptide complexes are MHC-peptide monomers, MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, each of the plurality of MHC-peptide complexes is an MHC-peptide monomer, an MHC-peptide dimer, or an MHC-peptide trimer. In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0052] In some embodiments of any of the above-described display portions, the plurality of MHC-peptide complexes on the particle include at least 20 MHC-peptide complexes (e.g., at least 50 or 100 MHC-peptide complexes).

[0053] In some embodiments of any of the above-described display portions, the MHC-peptide complex is bound to the particle via a binding pair, the binding pair comprising a first binding component bound to the MHC-peptide complex or its units (i.e., MHC-peptide monomers) and a second binding component bound to the particle. In some embodiments, the first binding component comprises biotin and the second binding component comprises streptavidin. In some embodiments, the second binding component comprises biotin and the first binding component comprises streptavidin.

[0054] In some embodiments of any of the above-described display components, the particles are solid.

[0055] In some embodiments of any of the above-described display portions, the particle diameter is at least 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 μm. In some embodiments, the particle diameter is about 0.5 μm to about 20 μm, for example, about 0.5 μm to about 10 μm, or about 1 μm.

[0056] In some embodiments of the display portion described above, the particles are approximately 1 μm in size. 2 ~about 20μm 2 It has a surface area of ​​[value].

[0057] In some embodiments of any of the above-described display portions, approximately 10 to 10,000 second binding components are bound to the particles.

[0058] In some embodiments of any of the above-described display components, the particles are spherical. In some embodiments, the particles are beads.

[0059] In some embodiments of any of the above-described display components, the particles are magnetic.

[0060] In some embodiments of any of the above-described display portions, the display portion includes at least two different MHC molecules. In some embodiments, the at least two different MHC molecules include two different MHC class I molecules. In some embodiments, the at least two different MHC molecules include two different MHC class II molecules. In some embodiments, the at least two different MHC molecules include both MHC class I molecules and MHC class II molecules.

[0061] In some embodiments of any of the above-described display portions, the display portion includes an HLA-A molecule. In some embodiments, the HLA-A molecule includes a mutation that reduces its binding to CD8. In some embodiments, the HLA-A molecule includes an HLA A2 heavy chain having the A245V mutation.

[0062] In some embodiments of any of the above-described display portions, the display portion includes at least two MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03.

[0063] In some embodiments of any of the above-described display portions, the display portion includes an MHC class II molecule selected from the group consisting of HLA-DQ and HLA-DR.

[0064] In some embodiments of any of the above-described display portions, the peptide is related to a cancer antigen or tumor antigen. In some embodiments, the peptide is related to a neoantigen.

[0065] In some embodiments of any of the above-described display portions, the peptide is related to an antibody against a pathogen. In some embodiments, the pathogen is a virus, bacteria, or fungus.

[0066] In some embodiments of any of the above-mentioned display portions, the peptide is related to an autoantibody.

[0067] In some embodiments of any of the above-described display portions, the display portion comprises at least two different peptides. In some embodiments, the display portion comprises at least five different peptides. In some embodiments, the display portion comprises at least 10, 20, or 50 different peptides. In some embodiments, at least two different peptides in the display portion are not duplicated. In some embodiments, at least two different peptides in the display portion are duplicated. In some embodiments, at least two different peptides are associated with two different antigens (e.g., derived from two different antigens). In some embodiments, the two different antigens comprise two different mutations in one or more genes associated with a disease. In some embodiments, the disease is cancer and the gene is an oncogene. In some embodiments, the disease is neither cancer nor a tumor (e.g., an infectious disease, e.g., an autoimmune disease).

[0068] In some embodiments of any of the above-described display portions, the display portion does not contain any fluorescent dyes or fluorophores.

[0069] In other embodiments, this application provides a bait composition comprising one or more of the above-mentioned display portions. In some embodiments, the bait composition comprises a plurality of the above-mentioned display portions. In some embodiments, the bait composition comprises at least two different display portions. In some embodiments, the bait composition comprises HLA-A molecules, HLA-B molecules, and HLA-C molecules. In some embodiments, the bait composition comprises HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the bait composition comprises at least 25, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the bait composition contains at least 2, 3, 4, 5, 6, or 7 different duplicate peptides associated with gene mutations. In some embodiments, the genes are oncogenes. In some embodiments, the peptides in the bait composition are associated with mutations in at least 2, 3, 4, 5, or 6 genes. In some embodiments, the genes are oncogenes. In some embodiments, the bait composition contains neither fluorescent dyes nor fluorophores.

[0070] In another embodiment, this application provides a method for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions described herein or a bait composition; and b) detecting or analyzing one or more immune cells bound to one or more display portions. In some embodiments, the immune cells include T cells. In some embodiments, the immune cells are selected from the group consisting of cytotoxic T cells, memory T cells, and tumor-infiltrating T cells. In some embodiments, the T cells are CD4+ T cells and / or CD8+ T cells. In some embodiments, the immune cells include B cells. In some embodiments, the immune cells include a mixture of immune cells including T cells, B cells, macrophages, or dendritic cells, or combinations thereof. In some embodiments, the sample is selected from the group consisting of blood, plasma, and peripheral blood mononuclear cell (PBMC) samples. In some embodiments, the sample is obtained from diseased tissue. In some embodiments, the diseased tissue is cancerous or tumorous tissue. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple individual samples, each obtained from a different individual. In some embodiments, the individuals are human. In some embodiments, the individuals do not have signs of disease. In some embodiments, detecting or analyzing immune cells bound to one or more display portions involves observing aggregation between immune cells and MHC multimer units. In some embodiments, detecting or analyzing immune cells bound to one or more display portions involves 1) immobilizing immune cells on a solid substrate before or after step a), 2) contacting the immobilized immune cells with a detection agent that recognizes the display under conditions sufficient for the detection agent to bind to the display portion, and 3) detecting the immune cells bound to the display portion by detecting the detection agent. [Brief explanation of the drawing]

[0071] [Figure 1]This figure illustrates newly predicted Kras neoantigens (sequence numbers 57-76) derived from the six most common Kras mutations (Kras G12V, Kras G12D, Kras G12R, Kras G12C, Kras G12I, Kras G12A).

[0072] [Figure 2] This figure shows that the four most common HLA-A (including HLA-A:0201, HLA-A:2402, HLA-A:0301, and HLA-A:1101) and β2m were expressed from E. coli and purified. The target proteins were expressed in insoluble inclusion bodies (DPE).

[0073] [Figure 3] This figure shows the assembly of five common variant Kras neoantigen libraries. It includes four HLA types (HLA-A:0201, HLA-A:2402, HLA-A:0301, HLA-A:1101) and four of the most common Kras variants (Kras G12V, Kras G12D, Kras G12R, Kras G12C). The figure shows only a portion of the results for the Kras G12V variant.

[0074] [Figure 4] This figure shows the confirmation of luciferase intensity in the constructed pancreatic tumor cell line. D-luciferin was used as the substrate. The signal was captured 15 minutes after intraperitoneal injection of D-luciferin.

[0075] [Figure 5] This figure shows the tumor size 12 days after inoculation of 1 × 10⁵ or 1 × 10⁶ Pano2-Luc-GFP tumor cells (Panel A), and the tumor growth curve of Pano2-Luc-GFP tumors measured by tumor volume in C57BL / 6J mice after subcutaneous inoculation of 1 × 10⁶ tumor cells (Panel B).

[0076] [Figure 6]This figure shows the structure of a plasmid overexpressing five common Kras mutations. The band size was approximately 750 bp. Two copies were subjected to electrophoresis. Bands smaller than 50 bp were nonspecific bands.

[0077] [Figure 7A] This figure shows that Kras-mutated neoantigen-specific CD8+ T cells were detected in the peripheral blood of mice as early as 4 days after intravenous inoculation of 4 × 10⁵ or 1 × 10⁶ tumor cells. [Figure 7B] This figure shows that Kras-mutated neoantigen-specific CD8+ T cells were detected in the peripheral blood of mice as early as 4 days after intravenous inoculation of 4 × 10⁵ or 1 × 10⁶ tumor cells.

[0078] [Figure 8] This figure shows that Kras-mutated neoantigen-specific CD8+ T cells were detected in the peripheral blood of mice as early as 4 days after intravenous inoculation of 1 × 10⁴ or 1 × 10⁵ tumor cells.

[0079] [Figure 9A] This figure shows that Kras-mutated neoantigen-specific CD8+ T cells were detected in the peripheral blood of mice at an early stage, as early as day 4, after subcutaneous inoculation of 4 × 10⁵ tumor cells. As shown in the figure, tetramer+CD8+ T cell groups (ranging from 0.042% to 0.22%, median: 0.0866%) were detectable in mice inoculated with Pan02-n peptide, but tetramer+CD8+ T cells were not detectable in mice inoculated with Pan02-EV cells. [Figure 9B]This figure shows that Kras-mutated neoantigen-specific CD8+ T cells were detected in the peripheral blood of mice at an early stage, as early as day 4, after subcutaneous inoculation of 4 × 10⁵ tumor cells. As shown in the figure, tetramer+CD8+ T cell groups (ranging from 0.042% to 0.22%, median: 0.0866%) were detectable in mice inoculated with Pan02-n peptide, but tetramer+CD8+ T cells were not detectable in mice inoculated with Pan02-EV cells.

[0080] [Figure 10A] This figure shows tumor growth curves evaluated by bioluminescence (Figure 9A) of mice inoculated with 4 × 10⁵ Pan02-EV (non-Kras mutation-expressing neoantigen) tumor cells and Pan02-n (Kras mutation-expressing neoantigen) tumor cells, as well as a photograph of a mouse showing no tumor at the inoculation site (red circle) four days after tumor inoculation. [Figure 10B] This figure shows tumor growth curves evaluated by bioluminescence (Figure 9A) of mice inoculated with 4 × 10⁵ Pan02-EV (non-Kras mutation-expressing neoantigen) tumor cells and Pan02-n (Kras mutation-expressing neoantigen) tumor cells, as well as a photograph of a mouse showing no tumor at the inoculation site (red circle) four days after tumor inoculation.

[0081] [Figure 11A] This figure shows whether Kras-mutated neoantigen-specific CD8+ T cells containing a Kras mutation-specific tetramer library are present in the peripheral blood of pancreatic cancer patients (patient numbers 1-13). [Figure 11B] This figure shows whether Kras-mutated neoantigen-specific CD8+ T cells containing a Kras mutation-specific tetramer library are present in the peripheral blood of pancreatic cancer patients (patient numbers 1-13). [Figure 11C] This figure shows whether Kras-mutated neoantigen-specific CD8+ T cells containing a Kras mutation-specific tetramer library are present in the peripheral blood of pancreatic cancer patients (patient numbers 1-13).

[0082] [Figure 12] This figure summarizes the presence or absence of CD8+ T cells in 13 confirmed pancreatic cancer patients, some with Kras mutations and others without specific HLA-A phenotypes. In this experiment, MHC tetramers bound to 20 types of G12D neoantigen peptides were included in the bait composition, while only 4 types of G12R neoantigen peptides were bound to the included tetramers.

[0083] [Figure 13] This figure shows an overview and process of MHC class I tetramer (MHC tetramer) synthesis. (A) Components and assembly of MHC peptide monomers. (B) A schematic diagram showing MHC tetramers and targeting strategies for MHC tetramers that bind to specific CD8+ T cells in the blood of cancer patients. Exemplary targets include B2M, streptavidin, biotin, and any labeled tag (e.g., PE, APC, or His).

[0084] [Figure 14]This figure shows the main process of sandwich ELISA. (A) The method is performed step by step in the order shown. The first step is to coat the ELISA plate with capture antibody. Any antigen in the sample (e.g., blood or cell supernatant) is then made to bind to the capture antibody. Any excess sample is washed off the plate. In step 3, detection antibody labeled with horseradish peroxidase or alkaline phosphatase is added, and the detection antibody binds to any target antigen already bound to the capture antibody. Finally, a substrate such as a chromogenic agent is added to the plate to convert the substrate (e.g., TMB or ABTS) into a colored product. The colored product can be measured using a plate reader. (B) A detailed protocol of ELISA is outlined in the figure. (C) Exemplary method. MHC tetramers bind to specific TCRs of CD8+ T cells in the patient's blood or cells. CD8+ cells are then captured by CD8 antibody coated on a plate or CD8 antibody bound to magnetic beads. With HRP detection antibody, the captured CD8 antibody can be identified using a plate reader. Alternatively, cells can be incubated with CD8 antibody coated on a plate or CD8 antibody conjugated to magnetic beads, and then MHC tetramers can be added to the resulting cells. Then, by adding an HRP detection antibody (primarily targeting components of the MHC tetramer), MHC tetramer-bound CD8+ T cells can be detected.

[0085] [Figure 15] This figure shows an overview and process of exemplary methods for capturing specific MHC tetramer-bound CD8+ T cells. (A-B) For example, MHC tetramers with different tags are first added to immune cells. Then, magnetic beads are added as shown in step 2. The presence of MHC tetramers that bind to CD8+ T cells is identified in step 3 using an HRP antibody. (C) CD8 antibody-bound magnetic beads are first added to immune cells. Then, MHC tetramers with different tags are added as shown in step 2. The presence of MHC tetramers that bind to CD8+ T cells is identified in step 3 using an HRP antibody.

[0086] [Figure 16] This figure shows the collection of clinical samples and specific strategies for tumor neoantigen-specific CD8+ T cells using ELISA.

[0087] [Figure 17] This figure shows the main process and results for detecting MHC tetramers that bind to CD8+ T cells using HRP-β2-microglobulin antibody. (A) The main method is shown step by step. Cells were mixed with MHC tetramers and then incubated with CD8 antibody-conjugated magnetic beads. HRP-β2-microglobulin antibody was used as the detection antibody to identify MHC tetramers that bind to CD8+ T cells. (B) Results of detecting MHC tetramers that bind to CD8+ T cells using the method shown in (A).

[0088] [Figure 18] This figure shows the main process and results for detecting MHC tetramers that bind to CD8+ T cells using HRP-PE antibody. (A) The main method is shown step by step. First, cells were mixed with CD8 antibody-conjugated magnetic beads and then incubated with MHC tetramers. HRP-PE antibody was used as the detection antibody to identify MHC tetramers that bind to CD8+ T cells. (B) Results using the method shown in (A).

[0089] [Figure 19] This figure shows the main process and results for detecting MHC tetramers bound to CD8+ T cells using HRP-PE antibody. (A) An improved method for capturing MHC tetramers bound to CD8+ T cells using HRP-PE antibody. In this protocol, after the step of washing the MHC tetramers, the tube was replaced with a new tube. This tube replacement can also be performed in the washing step of CD8-binding beads. (B) Results from the improved method shown in (A). There is a 3-fold difference between group 1 and group 4. A representative photograph (double) of result (B).

[0090] [Figure 20] This diagram shows an example of the scheme.

[0091] [Figure 21] This figure shows a scheme for high-throughput identification of pancreatic neoantigen-specific immune cells (e.g., CD8+ T cells) in cell populations.

[0092] [Figure 22] This figure shows an exemplary list of HLA subtypes and high-frequency tumor mutations used for constructing a tetrameric library for the analysis of early-stage pancreatic cancer.

[0093] [Figure 23] This figure shows a scheme of DNA barcoding antibodies against CD45-specific T cells or CD8-specific T cells.

[0094] [Figure 24] This figure shows a scheme for an exemplary method of identifying DNA barcoded CD8+ T cells by ELISA from multiple pancreatic cancer patients.

[0095] [Figure 25] This figure shows a scheme for another exemplary method for capturing antigen-specific immune cells (e.g., neoantigen-specific CD8+ T cells) using magnetic beads and a DNA barcode-based ELISA assay.

[0096] [Figure 26] This figure shows a scheme for another exemplary method for capturing antigen-specific immune cells (e.g., neoantigen-specific CD8+ T cells) using a tetramer-bead and DNA barcode-based ELISA assay.

[0097] [Figure 27A-1] This figure shows a scheme for another exemplary method for capturing antigen-specific immune cells (e.g., neoantigen-specific CD8+ T cells) using MHC tetramer-beads bound to multiple antigen peptides and a DNA barcode-based ELISA assay. [Figure 27A-2]Same as above.

[0098] [Figure 27B] This figure shows a scheme for another exemplary method for capturing antigen-specific immune cells (e.g., neoantigen-specific CD8+ T cells) using MHC tetramers bound to multiple antigen peptides and DNA barcode-based ELISA assays.

[0099] [Figure 27C] This figure illustrates Kras neoantigens (sequence numbers xx~xx) derived from Kras mutations (Kras G12V, Kras G12D, Kras G12R).

[0100] [Figure 28A] This figure shows the characterization of MHC tetramers and MHC tetramer-beads in PAGE.

[0101] [Figure 28B] This figure shows the detection of Kras mutant neoantigen-specific CD8+ T cells in mouse peripheral blood using MHC tetramer beads in an agglutination assay.

[0102] [Figure 28C] This figure shows the results of detecting Kras mutant neoantigen-specific CD8+ T cells in mouse peripheral blood using MHC tetramer beads in an agglutination assay.

[0103] [Figure 28D] This figure shows the detection of Kras mutant neoantigen-specific CD8+ T cells in mouse spleen using MHC tetramer beads in an agglutination assay.

[0104] [Figure 28E] This figure shows the results of detecting Kras mutant neoantigen-specific CD8+ T cells in mouse spleen using MHC tetramer beads in an agglutination assay.

[0105] [Figure 29A] This figure shows the PAGE characterization of MHC-β-galactosidase beads and Kras G12D MHC monomers.

[0106] [Figure 29B] This figure shows the PAGE characterization of MHC-β-galactosidase beads, as well as Kras G12D MHC monomers and MHC tetramers.

[0107] [Figure 29C] This figure shows the detection of Kras G12D neoantigen-specific CD8+ T cells in mouse peripheral blood using MHC tetramer beads in an agglutination assay.

[0108] [Figure 29D] This figure shows the detection of Kras G12D neoantigen-specific CD8+ T cells in mouse spleen using MHC tetramer beads in an agglutination assay. [Figure 29E] This figure shows the detection of Kras G12D neoantigen-specific CD8+ T cells in mouse spleen using MHC tetramer beads in an agglutination assay.

[0109] [Figure 30A] This figure shows the PAGE characterization of MHC monomers bound to multiple antigen peptides.

[0110] [Figure 30B] This figure shows the PAGE characterization of MHC tetramers bound to multiple antigen peptides.

[0111] [Figure 30C] This figure shows peptide standards used in LC-MS / MS detection.

[0112] [Figure 30D] This figure shows the detection of antigen peptides from PAGE.

[0113] [Figure 30E] This figure shows the characterization of MHC tetramers bound to multiple antigen peptides in LC-MS / MS detection.

[0114] [Figure 30F] This figure shows the characterization of MHC monomers and MHC tetramers using PAGE.

[0115] [Figure 31A] This figure shows the detection of Kras G12V neoantigen-specific CD8+ T cells in mouse spleen using MHC monomer beads.

[0116] [Figure 31B] This figure shows the detection of Kras G12D neoantigen-specific CD8+ T cells in human peripheral blood using MHC monomer beads.

[0117] [Figure 31C] This figure shows the detection of Kras-specific CD8+ T cells in human peripheral blood using MHC monomer beads containing multiple antigen peptides.

[0118] [Figure 31D] This figure shows the detection of stimulated Kras-specific CD8+ T cells in human peripheral blood using MHC monomer beads containing multiple antigen peptides. [Figure 31E] This figure shows the detection of stimulated Kras-specific CD8+ T cells in human peripheral blood using MHC monomer beads containing multiple antigen peptides. [Modes for carrying out the invention]

[0119] This application provides a method for analyzing samples from one or more individuals for the presence or absence of immune cells capable of binding to antigen peptides. In some embodiments, the presence of such immune cells indicates the presence of a disease or illness in the individual. This application is based at least in part on the inventors' own insight that immune cells activated by a specific antigen (e.g., tumor neoantigen) can be detected in an individual even before any pathological symptoms of a disease or illness (e.g., cancer) are shown, and thus serve as a useful marker for the early detection of such a disease or illness.

[0120] As shown in the Examples section, exemplary Kras mutation-associated neoantigen-specific T cells are tumor cells expressing Kras mutation-associated neoantigens (only 10 4 In mice inoculated intravenously or subcutaneously with individual cells (e.g., see Figure 8), successful detection was achieved as early as 4 days post-inoculation (e.g., see Figures 9A-9B), which is earlier than any detection by bioluminescence (e.g., see Figures 10A-10B) or earlier than detection in pancreatic cancer patients carrying Kras mutations (e.g., see Figures 11A-11C and Figure 12). Such data demonstrate that the method provided can be successfully used for cancer detection (e.g., both early cancer detection and monitoring of cancer cells in individuals that may have minimal cancer cells remaining).

[0121] Furthermore, this application provides an innovative method utilizing immunoassays such as ELISA to effectively, economically, and efficiently detect the presence of antigen-specific immune cells even when their abundance in a sample is very low (e.g., less than 1%). The method described in this application has been shown to successfully detect neoantigen-specific immune cells in cancer patients and pathogen-specific immune cells in patients infected with pathogens. Several embodiments of the method exhibit remarkably high sensitivity. Moreover, the method enables high-throughput analysis of mixed samples containing immune cells from multiple individuals (e.g., 10 to 30 individuals) by associating a unique barcode with immune cells from each individual. See, for example, Examples 3 and 4.

[0122] In one embodiment, this application provides a method for analyzing a sample from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) immobilizing immune cells from the sample onto a solid carrier, the method comprising: 1) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide before immobilization under conditions sufficient for the immune cells to bind to the display portion; or 2) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide after immobilization under conditions sufficient for the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; and c) detecting immune cells bound to the display portion by detecting the detection agent.

[0123] In another embodiment, this application provides a method for analyzing a sample from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) contacting a bait composition comprising a display portion containing an antigen peptide with immune cells under conditions sufficient for the immune cells to bind to the display portion; b) contacting a detection agent that recognizes the display portion with immune cells under conditions sufficient for the detection agent to bind to the display portion; c) immobilizing immune cells from the sample onto a solid carrier; and d) detecting immune cells bound to the display portion by detecting the detection agent.

[0124] In another embodiment, this application provides a method for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) processing each of the multiple samples from an individual separately to associate the immune cells contained therein with a unique sample barcode; b) combining the multiple processed samples to prepare a sample mixture; c) preparing a bait-sample mixture by contacting a bait composition containing a display portion with the sample mixture under conditions sufficient for immune cells to bind to the display portion; d) detecting immune cells bound to the display portion in the bait-sample mixture; and e) identifying the sample from which the immune cells originate by identifying a unique barcode associated with the immune cells bound to the display portion.

[0125] Furthermore, the invention also provides methods for detecting antigen-specific immune cells in an individual, methods for detecting residual cancer, and methods for treating cancer, infectious diseases, or autoimmune diseases.

[0126] In another embodiment, this application provides a composition comprising a display portion containing particles bound to a plurality of MHC-peptide complexes. In some embodiments, the plurality of MHC-peptide complexes comprises more than 2, more than 3, more than 4, more than 5, more than 6, more than 8, more than 9, more than 10, or more than 12 MHC-peptide complexes, each of which comprises at least one MHC molecule forming a complex with a peptide, and at least two or more of the MHC-peptide complexes are different. In some embodiments, the MHC-peptide complexes are MHC-peptide monomers. In some embodiments, the particles are solid. In some embodiments, the plurality of MHC-peptide complexes comprises at least 20, 50, or 100 MHC-peptide complexes.

[0127] For example, as shown in the examples, the display portion described herein is highly sensitive and specifically recognizes multiple peptides (e.g., neoantigen peptides), making it a powerful tool that can be used to simultaneously screen multiple antigen-specific immune cells from an individual. Such a display portion, by having multiple heterologous MHC-peptide complexes bound to particles, enables aggregation of antigen-specific immune cells, which can be easily observed under a microscope or with the naked eye. Thus, this provides a powerful, highly sensitive, cost-effective, and convenient platform technology for screening, detecting, and analyzing antigen-specific immune cells in different situations (e.g., cancer, autoimmune diseases, infectious diseases).

[0128] This application also provides a bait composition including a display portion, a method for analyzing or detecting antigen-specific immune cells by using the display portion or the bait composition (for example, those described above), and a method for treating a patient, which involves using the method for detecting antigen-specific immune cells described herein.

[0129] definition

[0130] Generally, terms used in the claims and specification are intended to be interpreted as having the ordinary meaning that would be understood by a person skilled in the art. For specific terms, further definitions are provided below. In the event of any conflict between the ordinary meaning and the definition provided, the definition provided shall prevail.

[0131] As used herein, the term “antigen” refers to a substance that induces an immune response.

[0132] As used herein, the term “neoantigen” refers to an antigen that has become different from its corresponding wild-type parent antigen due to at least one change (e.g., mutation in tumor cells or tumor-cell-specific post-translational modification). Neoantigens may include polypeptide sequences. Mutations resulting in neoantigens may include frameshift or non-frameshift indels, missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, or any genomic or expression changes that produce neoORFs. Mutations may also include splice variants. Tumor-cell-specific post-translational modifications may include abnormal phosphorylation. Tumor-cell-specific post-translational modifications may also include proteasome-generating splice antigens. See Liepe et al., A large fraction of HLA class I ligands are proteasome-generated spliced ​​peptides; Science. 2016 Oct 21;354(6310):354-358.

[0133] As used herein, the terms “tumor neoantigen” or “cancer neoantigen” refer to neoantigens that are present in tumor cells or tumor tissue of the subject but not in the corresponding normal cells or normal tissue of the subject.

[0134] As used herein, the term "missense mutation" refers to a mutation that causes a substitution of one amino acid for another.

[0135] As used herein, the term "nonsense mutation" refers to a mutation that causes a substitution of an amino acid for a stop codon.

[0136] As used herein, the term "frameshift mutation" refers to a mutation that results in a change in the reading frame of a protein.

[0137] As used herein, the term "indel" refers to an insertion or deletion of one or more nucleic acids.

[0138] With respect to two or more nucleic acid or polypeptide sequences, the term "identity" (%) refers to the result of a comparison and maximum alignment such that, when measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection, the two or more sequences or subsequences have the same nucleotides or amino acid residues at a particular percentage. Depending on the application, "identity" (%) may exist over a region of the sequences being compared (e.g., over a functional domain) or over the entire length of the two sequences being compared.

[0139] For array comparison, typically one array serves as the reference array and is compared with a test array. When using an array comparison algorithm, the test array and the reference array are input into a computer, subsequence coordinates are specified as necessary, and array algorithm program parameters are specified. Then, the array comparison algorithm calculates the array identity (%) of the test array(s) relative to the reference array based on the specified program parameters. Alternatively, the similarity or dissimilarity of arrays can be defined by the presence or absence of specific nucleotide combinations, or in the case of translated arrays, by the presence or absence of combinations of amino acids at selected array positions (e.g., array motifs).

[0140] As used herein, the term "epitope" refers to a specific portion of an antigen that is normally bound by an antibody or a T cell receptor.

[0141] As used herein, the term "immunogenicity" refers to the ability to induce an immune response (e.g., via T cells, B cells, or both).

[0142] As used herein, the terms "HLA binding affinity" and "MHC binding affinity" mean the binding affinity between a specific antigen and a specific MHC allele.

[0143] As used herein, the term "bait composition" refers to a composition containing a molecule (e.g., an antigenic peptide) used to enrich cells that specifically bind to a sample in the bait. More specifically, the "antigenic peptides" described herein are not limited to peptides capable of inducing an immune response. "Antigenic peptides" are interchangeable with "peptides" and include any peptide that can be presented by MHC molecules.

[0144] As used herein, the term "mutation" refers to the difference between a subject's nucleic acid and a reference human genome used as a control.

[0145] As used herein, the term "allele" refers to a version of a gene, a version of a gene sequence, or a version of a protein.

[0146] As used herein, the term "HLA type" refers to the entire combination of HLA gene alleles.

[0147] As used herein, the term “exome” refers to a subset of the genome that codes for proteins. An exome can be a collection of exons within a genome.

[0148] As used herein, the term “proteome” refers to the collection of all proteins expressed and / or translated by a cell, a group of cells, or an organism.

[0149] As used herein, the term “dextramer” refers to a dextran-based peptide-MHC polymer used in antigen-specific immunohistochemistry in flow cytometry.

[0150] As used herein, the term "MHC polymer" refers to a peptide-MHC complex comprising multiple peptide-MHC monomer units.

[0151] As used herein, the term "MHC tetramer" refers to a peptide-MHC complex containing four peptide-MHC monomer units. As used herein, the term "MHC monomer" refers to a peptide-MHC complex containing one peptide-MHC monomer unit. As used herein, the term "MHC dimer" refers to a peptide-MHC complex containing two peptide-MHC monomer units. As used herein, the term "MHC trimer" refers to a peptide-MHC complex containing three peptide-MHC monomer units.

[0152] As used herein, the term “latex agglutination test (LAT)” refers to a type of bioanalytical method used to detect the presence (qualitative) of a substance in a sample, and, if applicable, the quantity (quantitative) of the substance in the sample. In the presence of certain substances (e.g., antigens, antibodies, or cells), latex particles agglutinate, forming aggregates or masses of particles.

[0153] As used herein, the term “latex beads” refers to spherical particles, such as polystyrene, poly(methyl methacrylate), polyacrylate, or other similar types of polymers. Such latex beads can vary in size, typically ranging in diameter from nanometers to micrometers. Their surface can be modified or conjugated with functional groups to allow for the binding of biomolecules, compounds, or other substances. Latex beads can be used in a variety of applications, including but not limited to diagnostic assays, bioseparation methods, and drug delivery, and can be used as standards or controls in various analytical techniques.

[0154] As used herein, “sample” refers to an aliquot of bodily fluids or tissues obtained from an object containing immune cells.

[0155] The term “mammal” encompasses both humans and non-humans, including, but is not limited to, humans, non-human primates, dogs, cats, mice, cattle, horses, and pigs.

[0156] As used herein, “treatment” or “to treat” refers to an approach to obtain beneficial or desirable outcomes, including clinical outcomes. For the purposes of this application, beneficial or desirable clinical outcomes include, but are not limited to, one or more of the following: reducing one or more symptoms caused by the disease; reducing the severity of the disease; stabilizing the disease (e.g., preventing or slowing disease progression); preventing or slowing the spread of the disease (e.g., metastasis); preventing or slowing disease recurrence; slowing or slowing disease progression; improving the disease state; achieving remission (partial or total) of the disease; reducing the dosage of one or more other medications required to treat the disease; slowing disease progression; improving or enhancing quality of life; increasing weight gain; and / or extending survival. “Treatment” also includes reducing pathological outcomes of cancer (e.g., tumor volume). The methods of this application are intended to achieve one or more of these forms of treatment.

[0157] As used herein, “reference” means any sample, standard, or level used for comparative purposes. References may be obtained from healthy and / or non-infected samples. In some cases, references may be obtained from untreated samples. In some cases, references may be obtained from uninfected or untreated samples of an individual. In some cases, references may be obtained from one or more healthy individuals that are neither the individual nor the patient.

[0158] The terms “subject,” “individual,” and “patient” are used synonymously herein, as they refer to mammals (for example, humans, cattle, horses, cats, dogs, rodents, or primates, without limitation). In some embodiments, the individual is a human.

[0159] As is apparent, the embodiments of the present application described herein include embodiments "consisting of" and / or embodiments "consisting essentially of".

[0160] As used herein, the term "about" in reference to a value or parameter includes (and describes) that value or parameter as being inclusive of variations. For example, the notation "about X" includes the notation "X".

[0161] As used herein, the expression "not" in reference to a particular value or parameter typically means "other than" and describes a particular value or parameter as being other than that particular value or parameter. For example, a method that is not used to treat cancer of type X means that the method is used to treat cancers other than type X.

[0162] As used herein, the expression "about X to Y" has the same meaning as "about X to about Y".

[0163] Also, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0164] Any term not directly defined herein shall be understood to have the general meaning as understood in the art of the present invention. This specification provides further guidance to practitioners in describing certain terms, including compositions, apparatus, methods, etc., of embodiments of the present invention, and methods of their preparation or use. As is evident, the same content may be expressed in different ways. Therefore, alternative and synonymous terms may be used for one or more of the terms described herein. In this specification, emphasis should not be placed on whether a term is detailed or explained. Synonyms or alternative methods, materials, etc., may be provided. The inclusion of one or more synonyms or equivalents does not preclude the use of other synonyms or equivalents unless explicitly stated. The use of examples, including examples of terminology, is for illustrative purposes only and does not limit the scope or meaning of the embodiments of the invention described herein.

[0165] Method for analyzing samples from one or more individuals

[0166] This application provides a method for analyzing samples from one or more individuals to determine, for example, the presence or absence of immune cells capable of binding to antigen peptides. Such a method can further be used, for example, in the diagnosis of diseases or illnesses (e.g., cancer, infection, autoimmune cells).

[0167] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) immobilizing immune cells from the samples onto a solid carrier, the method comprising: 1) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide before immobilization under conditions sufficient for the immune cells to bind to the display portion; or 2) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide after immobilization under conditions sufficient for the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; and c) detecting immune cells bound to the display portion by detecting the detection agent, wherein the antigen peptide is related to a cancer antigen or tumor antigen, and optionally the antigen peptide is a neoantigen peptide. In some embodiments, the samples are obtained from one or more individuals that do not exhibit pathological symptoms of cancer. In some embodiments, a) the individual has not been previously diagnosed with cancer and, optionally, is at risk of having cancer, or b) the individual has previously received treatment for cancer and does not exhibit any pathological symptoms of cancer after treatment. In some embodiments, the neoantigen peptide contains a mutation from the wild-type peptide and, optionally, a mutation at the third amino acid position from the N-terminus. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples of the individual, each obtained from a different individual. In some embodiments, each of the multiple samples of the individual is processed separately before being combined to form a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with the molecule containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by, for example, washing the immune cells at least once.In some embodiments, before immobilization, the bait composition containing a display portion with antigen peptides is brought into contact with immune cells under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, after immobilization, the bait composition containing a display portion with antigen peptides is brought into contact with immune cells under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptides in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the immune cells are immobilized on a solid substrate such as beads, and optionally, the beads are conjugated with antibodies that recognize the immune cells, and optionally, the antibodies are CD8 antibodies. In some embodiments, the display portion contains two or more antigen peptides, and optionally, the display portion contains four antigen peptides. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to congener TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion includes an MHC molecule that forms a complex with the antigen peptide, and optionally the MHC molecule includes an MHC polymer, and optionally the MHC polymer is an MHC tetramer or MHC dextramer. In some embodiments, the MHC molecule includes an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation.In some embodiments, the display portion includes two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further includes a detectable label (e.g., a fluorophore). In some embodiments, the method further includes separating immune cells bound to the display portion, or further includes removing the display portion or immune cells that are not bound to each other by, for example, washing the immune cells at least once, twice, or three times.

[0168] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) immobilizing immune cells from the samples onto a solid carrier, the method comprising: 1) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide before immobilization under conditions sufficient for the immune cells to bind to the display portion; or 2) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide after immobilization under conditions sufficient for the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; and c) detecting immune cells bound to the display portion by detecting the detection agent, wherein the antigen peptide is related to a pathogen antigen, and optionally the antigen peptide is related to a viral, bacterial, or fungal antigen. In some embodiments, a) the individual has not been diagnosed with an infection by the pathogen, and possibly is at risk of having an infection, and / or b) the individual does not exhibit pathological symptoms of an infection by the pathogen, and possibly does not exhibit pathological symptoms of an infection by the pathogen. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples of the individual, each obtained from a different individual. In some embodiments, each of the multiple samples of the individual is processed separately before being combined to form a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with the molecule containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by the immune cells) containing the barcode by washing the immune cells at least once. In some embodiments, before immobilization, the bait composition containing a display portion containing an antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion.In some embodiments, after immobilization, the bait composition containing a display portion with antigen peptides is brought into contact with immune cells under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptides in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the immune cells are immobilized on a solid substrate such as beads, and optionally, the beads are conjugated with antibodies that recognize the immune cells, and optionally, the antibodies are CD8 antibodies. In some embodiments, the display portion contains two or more antigen peptides, and optionally, the display portion contains four antigen peptides. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to congener TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion includes an MHC molecule that forms a complex with the antigen peptide, and optionally the MHC molecule includes an MHC polymer, and optionally the MHC polymer is an MHC tetramer or MHC dextramer. In some embodiments, the MHC molecule includes an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation.In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0169] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) immobilizing immune cells from the samples onto a solid carrier, the method comprising: 1) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide before immobilization under conditions sufficient for the immune cells to bind to the display portion; or 2) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide after immobilization under conditions sufficient for the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; and c) detecting immune cells bound to the display portion by detecting the detection agent, wherein the antigen peptide is an autoantigen peptide related to an autoantigen. In some embodiments, a) the individual has not been diagnosed with having an immune response to an autoantigen, and possibly the individual is at risk of developing an immune response to an autoantigen, and / or b) the individual does not exhibit pathological symptoms of an immune response (e.g., autoimmune disease or autoimmune disorder), and possibly does not exhibit pathological symptoms of an immune response to an autoantigen. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples of an individual, each obtained from a different individual. In some embodiments, each of the multiple samples of an individual is processed separately before being combined to form a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with the molecule containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by washing the immune cells at least once. In some embodiments, before immobilization, the bait composition containing a display portion containing an antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion.In some embodiments, after immobilization, the bait composition containing a display portion with antigen peptides is brought into contact with immune cells under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptides in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the immune cells are immobilized on a solid substrate such as beads, and optionally, the beads are conjugated with antibodies that recognize the immune cells, and optionally, the antibodies are CD8 antibodies. In some embodiments, the display portion contains two or more antigen peptides, and optionally, the display portion contains four antigen peptides. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to congener TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion includes an MHC molecule that forms a complex with the antigen peptide, and optionally the MHC molecule includes an MHC polymer, and optionally the MHC polymer is an MHC tetramer or MHC dextramer. In some embodiments, the MHC molecule includes an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation.In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0170] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) immobilizing immune cells from the samples onto a solid carrier, the method comprising: 1) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide before immobilization under conditions sufficient for the immune cells to bind to the display portion; or 2) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide after immobilization under conditions sufficient for the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; and c) detecting the immune cells bound to the display portion by detecting the detection agent, wherein the immune cells are immobilized on a solid substrate such as beads, and optionally the beads are bound to antibodies that recognize immune cells, optionally the antibodies being CD8 antibodies. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples from individuals, each obtained from a different individual. In some embodiments, each of several samples of an individual is processed separately before being combined into a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with the molecule containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by the immune cells) containing the barcode by washing the immune cells at least once. In some embodiments, before immobilization, the bait composition containing a display portion containing an antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, after immobilization, the bait composition containing a display portion containing an antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample).In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the display portion includes two or more antigen peptides, and optionally, the display portion includes four antigen peptides. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to homogeneous TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion includes MHC molecules that form a complex with the antigen peptide, and optionally, the MHC molecules include MHC polymers, and optionally, the MHC polymers are MHC tetramers or MHC dextramers. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and optionally the MHC class I molecule comprises an HLA-A molecule, further optionally the HLA-A molecule comprises a mutation that reduces its binding to CD8, and further optionally the HLA-A molecule comprises an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion includes two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further includes a detectable label (e.g., a fluorophore).In some embodiments, the method further includes separating immune cells bound to the display portion, or further including removing the display portion or immune cells that are not bound to each other.

[0171] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to antigen peptides, the method comprising: a) immobilizing immune cells from the samples onto a solid carrier, the method comprising: 1) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide before immobilization under conditions sufficient for the immune cells to bind to the display portion; or 2) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide after immobilization under conditions sufficient for the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; and c) detecting immune cells bound to the display portion by detecting the detection agent, wherein the display portion comprises a library of antigen peptides for mutations related to cancer or tumors, pathogens, or autoantigens, and the library comprises at least 5, 10, 12, 15, 18, or 20 different antigen peptides (e.g., neoantigens). In some embodiments, the samples are obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples of individuals, each obtained from a different individual. In some embodiments, each of the multiple samples of individuals is processed separately before being combined to form a mixture, so that the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the immune cells with the molecules containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by washing the immune cells at least once. In some embodiments, before immobilization, the bait composition containing a display portion containing an antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, after immobilization, the bait composition containing a display portion containing an antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample).In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to homogeneous TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion includes MHC molecules that form a complex with the antigen peptide, and optionally, the MHC molecules include MHC multimers, and further optionally, the MHC multimers are MHC tetramers or MHC dextramers. In some embodiments, the MHC molecules include MHC class I molecules. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0172] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to antigen peptides, the method comprising: a) immobilizing immune cells from the samples onto a solid carrier, wherein 1) before immobilization, the bait composition containing a display portion containing the antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion, or 2) after immobilization, the bait composition containing a display portion containing the antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion, and b) display The method comprises: a) contacting a detection agent that recognizes a display portion with immobilized immune cells under conditions sufficient for the detection agent to bind to the display portion; and c) detecting the immune cells bound to the display portion by detecting the detection agent, wherein the antigen peptide has one or more of the following characteristics: 1) a) binding affinity to MHC molecules of about 1 nM to about 5000 nM; b) binding affinity to homogeneous TCR molecules of about 1 nM to about 5000 nM; c) being hydrophobic; and d) having a high content of aromatic residues; and / or 2) low immunogenicity. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples of individuals, each obtained from a different individual. In some embodiments, each of the multiple samples of individuals is processed separately before being combined to form a mixture, and the immune cells contained therein are associated with a unique sample barcode (e.g., by mixing the molecules containing the unique sample barcode with the immune cells). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) including barcodes by, for example, washing the immune cells at least once. In some embodiments, before immobilization, the bait composition containing the display portion containing the antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, after immobilization, the bait composition containing the display portion containing the antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion.In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the display portion includes an MHC molecule that forms a complex with the antigen peptide, and optionally, the MHC molecule includes an MHC multimer, and even more optionally, the MHC multimer is an MHC tetramer or an MHC dextramer. In some embodiments, the MHC molecule includes an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0173] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) immobilizing immune cells from the samples onto a solid carrier, the method comprising: 1) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide before immobilization under conditions sufficient for the immune cells to bind to the display portion; or 2) contacting the immune cells with a bait composition containing a display portion containing an antigen peptide after immobilization under conditions sufficient for the immune cells to bind to the display portion; b) contacting the immobilized immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; and c) detecting immune cells bound to the display portion by detecting the detection agent, wherein the display portion further comprises a detectable label (e.g., a fluorophore, e.g., PE or APC), and the detection agent binds to the detectable label. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples from individuals, each obtained from a different individual. In some embodiments, each of several samples of an individual is processed separately before being combined into a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with the molecule containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by the immune cells) containing the barcode by washing the immune cells at least once. In some embodiments, before immobilization, the bait composition containing a display portion containing an antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, after immobilization, the bait composition containing a display portion containing an antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample).In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the display portion includes an MHC molecule that forms a complex with the antigen peptide, and optionally, the MHC molecule includes an MHC multimer, and optionally, the MHC multimer is an MHC tetramer or MHC dextramer. In some embodiments, the MHC molecule includes an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and optionally, the MHC class I molecule includes an HLA-A molecule, and optionally, the HLA-A molecule includes a mutation that reduces its binding to CD8, and optionally, the HLA-A molecule includes an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the method further comprises isolating immune cells bound to the display portion, or further comprising isolating the display portion or immune cells not bound to each other.

[0174] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) contacting immune cells with a bait composition containing a display portion comprising an antigen peptide under conditions sufficient for the immune cells to bind to the display portion; b) contacting immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; c) immobilizing immune cells from the samples onto a solid carrier; and d) detecting immune cells bound to the display portion by detecting the detection agent, where the antigen peptide is related to a cancer antigen or tumor antigen, and optionally the antigen peptide is a neoantigen peptide. In some embodiments, the samples are obtained from one or more individuals that do not exhibit pathological symptoms of cancer. In some embodiments, a) the individuals have not been previously diagnosed with cancer and optionally the individuals are at risk of having cancer, or b) the individuals have previously received cancer treatment and do not exhibit pathological symptoms of cancer after treatment. In some embodiments, the neoantigen peptide comprises a mutation from the wild-type peptide and optionally the mutation at the third amino acid position from the N-terminus. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples of individuals, each obtained from a different individual. In some embodiments, each of the multiple samples of individuals is processed separately before being combined to form a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with the molecules containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by washing the immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less.In some embodiments, immune cells are immobilized on a solid substrate such as beads, and optionally the beads are bound to antibodies that recognize the immune cells, and optionally the antibodies are CD8 antibodies. In some embodiments, the display portion comprises two or more antigen peptides, and optionally the display portion comprises four antigen peptides. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to homogeneous TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion comprises MHC molecules that form a complex with the antigen peptides, and optionally the MHC molecules comprises MHC polymers, and optionally the MHC polymers are MHC tetramers or MHC dextramers. In some embodiments, the MHC molecules comprise MHC class I molecules. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion includes two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further includes a detectable label (e.g., a fluorophore).In some embodiments, the method further includes separating immune cells bound to the display portion, or further including removing the display portion or immune cells that are not bound to each other.

[0175] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) contacting immune cells with a bait composition comprising a display portion containing an antigen peptide under conditions sufficient for the immune cells to bind to the display portion; b) contacting immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; c) immobilizing immune cells from the samples onto a solid carrier; and d) detecting immune cells bound to the display portion by detecting the detection agent, where the antigen peptide is related to a pathogen antigen, and optionally, the antigen peptide is related to a viral, bacterial, or fungal antigen. In some embodiments, a) the individual has not been diagnosed with an infection by the pathogen, optionally, the individual is at risk of having an infection, and / or b) the individual does not exhibit pathological symptoms of an infection by the pathogen, optionally, does not exhibit pathological symptoms of an infection by the pathogen. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples from individuals, each obtained from a different individual. In some embodiments, each of several samples of an individual is processed separately before being combined into a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with the molecules containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by washing the immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the immune cells are immobilized on a solid substrate such as beads, and optionally, the beads are bound to antibodies that recognize the immune cells, and optionally, the antibodies are CD8 antibodies.In some embodiments, the display portion comprises two or more antigen peptides, and optionally, four antigen peptides. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to congener TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion comprises an MHC molecule that forms a complex with the antigen peptide, and optionally, the MHC molecule comprises an MHC polymer, and optionally, the MHC polymer is an MHC tetramer or an MHC dextramer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0176] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) contacting immune cells with a bait composition containing a display portion containing an antigen peptide under conditions sufficient for the immune cells to bind to the display portion; b) contacting immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; c) immobilizing immune cells from the samples onto a solid carrier; and d) detecting immune cells bound to the display portion by detecting the detection agent, where the antigen peptide is an autoantigen peptide related to an autoantigen. In some embodiments, a) the individual has not been diagnosed with having an immune response to an autoantigen, and possibly the individual is at risk of developing an immune response to an autoantigen, and / or b) the individual does not exhibit pathological symptoms of an immune response (e.g., autoimmune disease or autoimmune disorder), and possibly does not exhibit pathological symptoms of an immune response to an autoantigen. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples from individuals, each obtained from a different individual. In some embodiments, each of several samples of an individual is processed separately before being combined into a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with the molecules containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by washing the immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the immune cells are immobilized on a solid substrate such as beads, and optionally, the beads are bound to antibodies that recognize the immune cells, and optionally, the antibodies are CD8 antibodies.In some embodiments, the display portion comprises two or more antigen peptides, and optionally, four antigen peptides. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to congener TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion comprises an MHC molecule that forms a complex with the antigen peptide, and optionally, the MHC molecule comprises an MHC polymer, and optionally, the MHC polymer is an MHC tetramer or an MHC dextramer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0177] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) contacting immune cells with a bait composition comprising a display portion containing an antigen peptide under conditions sufficient for the immune cells to bind to the display portion; b) contacting immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; c) immobilizing immune cells from the sample onto a solid carrier; and d) detecting immune cells bound to the display portion by detecting the detection agent, where the immune cells are immobilized on a solid substrate such as beads, and optionally the beads are bound to antibodies that recognize the immune cells, optionally the antibodies being CD8 antibodies. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples of individuals, each obtained from a different individual. In some embodiments, each of the multiple samples of individuals is processed separately before being combined into a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing immune cells with a molecule containing a unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) including barcodes by washing the immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the display portion includes two or more antigen peptides, and optionally, the display portion includes four antigen peptides.In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to congener TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion includes an MHC molecule that forms a complex with the antigen peptide, and optionally the MHC molecule includes an MHC polymer, and optionally the MHC polymer is an MHC tetramer or MHC dextramer. In some embodiments, the MHC molecule includes an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0178] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to antigen peptides, the method comprising: a) contacting immune cells with a bait composition comprising a display portion containing antigen peptides under conditions sufficient for the immune cells to bind to the display portion; b) contacting immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; c) immobilizing immune cells from the samples onto a solid carrier; and d) detecting immune cells bound to the display portion by detecting the detection agent, wherein the display portion comprises a library of antigen peptides for mutations related to cancer or tumors, pathogens, or autoantigens, and the library comprises at least 5, 10, 12, 15, 18, or 20 different antigen peptides (e.g., neoantigens). In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples from individuals, each obtained from a different individual. In some embodiments, each of several samples of an individual is processed separately before being combined into a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with the molecules containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by washing the immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to congener TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues.In some embodiments, the antigen peptide is of low immunogenicity. In some embodiments, the display portion comprises an MHC molecule complexed with the antigen peptide, optionally comprising an MHC multimer, and possibly the MHC multimer being an MHC tetramer or MHC dextramer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, optionally comprising an HLA-A molecule, possibly comprising a mutation that reduces its binding to CD8, and possibly comprising an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0179] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) contacting immune cells with a bait composition containing a display portion containing an antigen peptide under conditions sufficient for the immune cells to bind to the display portion; b) contacting immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; c) immobilizing immune cells from the samples onto a solid carrier; and d) detecting immune cells bound to the display portion by detecting the detection agent, wherein the antigen peptide has one or more of the following characteristics: 1) a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules; b) having a binding affinity of about 1 nM to about 5000 nM to homogeneous TCR molecules; c) being hydrophobic; and d) having a high content of aromatic residues; and / or 2) having low immunogenicity. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples from individuals, each obtained from a different individual. In some embodiments, each of several samples of an individual is processed separately before being combined into a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with the molecules containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by washing the immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the display portion includes an MHC molecule that forms a complex with the antigen peptide, and optionally, the MHC molecule includes an MHC multimer, and further optionally, the MHC multimer is an MHC tetramer or an MHC dextramer.In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and optionally the MHC class I molecule comprises an HLA-A molecule, further optionally the HLA-A molecule comprises a mutation that reduces its binding to CD8, and further optionally the HLA-A molecule comprises an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0180] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) contacting immune cells with a bait composition comprising a display portion containing an antigen peptide under conditions sufficient for the immune cells to bind to the display portion; b) contacting immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; c) immobilizing immune cells from the samples onto a solid carrier; and d) detecting immune cells bound to the display portion by detecting the detection agent, wherein the display portion further comprises a detectable label (e.g., a fluorophore, e.g., PE or APC), and the detection agent binds to the detectable label. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples of individuals, each obtained from a different individual. In some embodiments, each of the multiple samples of individuals is processed separately before being combined to form a mixture, to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing immune cells with a molecule containing a unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) including barcodes by, for example, washing immune cells at least once. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the display portion includes MHC molecules that form a complex with the antigen peptide, and optionally, the MHC molecules include MHC multimers, and even more optionally, the MHC multimers are MHC tetramers or MHC dextramers. In some embodiments, the MHC molecules include MHC class I molecules.In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the method further comprises isolating immune cells bound to the display portion, or further comprising isolating the display portion or immune cells not bound to each other.

[0181] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) processing each of the multiple samples from an individual separately to associate the immune cells contained therein with a unique sample barcode; b) combining the multiple processed samples to prepare a sample mixture; c) preparing a bait-sample mixture by contacting a bait composition containing a display portion with the sample mixture under conditions sufficient for immune cells to bind to the display portion; d) detecting immune cells bound to the display portion in the bait-sample mixture; and e) identifying the sample from which the immune cells originate by identifying a unique barcode associated with the immune cells bound to the display portion, where the antigen peptide is related to a cancer antigen or tumor antigen, and optionally the antigen peptide is a neoantigen peptide. In some embodiments, the samples are obtained from one or more individuals that do not exhibit pathological symptoms of cancer. In some embodiments, a) the individual has not been previously diagnosed with cancer and, optionally, is at risk of having cancer, or b) the individual has previously received cancer treatment and does not exhibit cancer pathological symptoms after treatment. In some embodiments, the neoantigen peptide contains a mutation from the wild-type peptide and, optionally, a mutation at the third amino acid position from the N-terminus. In some embodiments, before immobilization, the bait composition containing the display portion containing the antigen peptide and immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, after immobilization, the bait composition containing the display portion containing the antigen peptide and immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells) and, optionally, the immune cells contain T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1%.In some embodiments, immune cells are immobilized on a solid substrate such as beads, and optionally the beads are bound to antibodies that recognize the immune cells, and optionally the antibodies are CD8 antibodies. In some embodiments, the display portion comprises two or more antigen peptides, and optionally the display portion comprises four antigen peptides. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to homogeneous TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion comprises MHC molecules that form a complex with the antigen peptides, and optionally the MHC molecules comprises MHC polymers, and optionally the MHC polymers are MHC tetramers or MHC dextramers. In some embodiments, the MHC molecules comprise MHC class I molecules. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion includes two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further includes a detectable label (e.g., a fluorophore).In some embodiments, the method further includes separating immune cells bound to the display portion, or further including removing the display portion or immune cells that are not bound to each other.

[0182] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) processing each of the multiple samples from an individual separately to associate the immune cells contained therein with a unique sample barcode; b) combining the multiple processed samples to prepare a sample mixture; c) preparing a bait-sample mixture by contacting a bait composition containing a display portion with the sample mixture under conditions sufficient for immune cells to bind to the display portion; d) detecting immune cells bound to the display portion in the bait-sample mixture; and e) identifying the sample from which the immune cells originate by identifying a unique barcode associated with the immune cells bound to the display portion, where the antigen peptide is related to a pathogen antigen, and optionally, the antigen peptide is related to a viral, bacterial, or fungal antigen. In some embodiments, a) the individual has not been diagnosed with a pathogen infection, and optionally, the individual is at risk of having an infection, and / or b) the individual does not exhibit, and optionally, does not exhibit, pathological symptoms of a pathogen infection. In some embodiments, before immobilization, the bait composition containing the antigen peptide display portion and immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, after immobilization, the bait composition containing the antigen peptide display portion and immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the immune cells are immobilized on a solid substrate such as beads, and optionally, the beads are conjugated with antibodies that recognize the immune cells, and optionally, the antibodies are CD8 antibodies.In some embodiments, the display portion comprises two or more antigen peptides, and optionally, four antigen peptides. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to congener TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion comprises an MHC molecule that forms a complex with the antigen peptide, and optionally, the MHC molecule comprises an MHC polymer, and optionally, the MHC polymer is an MHC tetramer or an MHC dextramer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0183] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) processing each of the multiple samples from an individual separately to associate the immune cells contained therein with a unique sample barcode; b) combining the multiple processed samples to prepare a sample mixture; c) preparing a bait-sample mixture by contacting a bait composition containing a display portion with the sample mixture under conditions sufficient for immune cells to bind to the display portion; d) detecting immune cells bound to the display portion in the bait-sample mixture; and e) identifying the sample from which the immune cells originate by identifying a unique barcode associated with the immune cells bound to the display portion, wherein the antigen peptide is an autoantigen peptide related to an autoantigen. In some embodiments, a) the individual has not been diagnosed with having an immune response to an autoantigen, and possibly the individual is at risk of developing an immune response to an autoantigen, and / or b) the individual does not exhibit pathological symptoms of an immune response (e.g., autoimmune disease or autoimmune disorder), and possibly does not exhibit pathological symptoms of an immune response to an autoantigen. In some embodiments, before immobilization, the bait composition containing the antigen peptide display portion and immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, after immobilization, the bait composition containing the antigen peptide display portion and immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1% or less. In some embodiments, the immune cells are immobilized on a solid substrate such as beads, and optionally, the beads are conjugated with antibodies that recognize the immune cells, and optionally, the antibodies are CD8 antibodies.In some embodiments, the display portion comprises two or more antigen peptides, and optionally, four antigen peptides. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to congener TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion comprises an MHC molecule that forms a complex with the antigen peptide, and optionally, the MHC molecule comprises an MHC polymer, and optionally, the MHC polymer is an MHC tetramer or an MHC dextramer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0184] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) processing each of the multiple samples from an individual separately to associate the immune cells contained therein with a unique sample barcode; b) combining the multiple processed samples to prepare a sample mixture; c) preparing a bait-sample mixture by contacting a bait composition containing a display portion with the sample mixture under conditions sufficient for immune cells to bind to the display portion; d) detecting immune cells bound to the display portion in the bait-sample mixture; and e) identifying the sample from which the immune cells originate by identifying a unique barcode associated with the immune cells bound to the display portion, wherein the immune cells are immobilized on a solid substrate (e.g., by beads), and optionally the beads are bound with antibodies that recognize the immune cells, and optionally the antibodies are CD8 antibodies. In some embodiments, prior to immobilization, the bait composition containing a display portion with an antigen peptide and the immune cells are contacted under conditions sufficient for immune cells to bind to the display portion. In some embodiments, after immobilization, the bait composition containing a display portion with an antigen peptide is brought into contact with immune cells under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells contain T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the display portion contains two or more antigen peptides, and optionally, the display portion contains four antigen peptides. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to homogeneous TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues.In some embodiments, the antigen peptide is of low immunogenicity. In some embodiments, the display portion comprises an MHC molecule complexed with the antigen peptide, optionally comprising an MHC multimer, and possibly the MHC multimer being an MHC tetramer or MHC dextramer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, optionally comprising an HLA-A molecule, possibly comprising a mutation that reduces its binding to CD8, and possibly comprising an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0185] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to antigen peptides, the method comprising: a) processing each of the multiple samples from an individual separately to associate the immune cells contained therein with unique sample barcodes; b) combining the multiple processed samples to prepare a sample mixture; c) preparing a bait-sample mixture by contacting a bait composition containing a display portion containing antigen peptides with the sample mixture under conditions sufficient for immune cells to bind to the display portion; d) detecting immune cells bound to the display portion in the bait-sample mixture; and e) identifying the sample from which the immune cells originate by identifying a unique barcode associated with the immune cells bound to the display portion, wherein the display portion comprises a library of antigen peptides for mutations related to cancer or tumors, pathogens, or autoantigens, and the library comprises at least about 5, 10, 12, 15, 18, or 20 different antigen peptides (e.g., neoantigens). In some embodiments, before immobilization, the bait composition containing the display portion with the antigen peptide is brought into contact with immune cells under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, after immobilization, the bait composition containing the display portion with the antigen peptide is brought into contact with immune cells under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells contain T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules, b) having a binding affinity of about 1 nM to about 5000 nM to homogeneous TCR molecules, c) being hydrophobic, and d) having a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity.In some embodiments, the display portion comprises an MHC molecule complexed with an antigen peptide, the MHC molecule optionally comprising an MHC multimer, and further optionally the MHC multimer being an MHC tetramer or MHC dextramer. In some embodiments, the MHC molecule comprises an MHC class I molecule. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, the MHC class I molecule optionally comprising an HLA-A molecule, further optionally the HLA-A molecule comprising a mutation that reduces its binding to CD8, and further optionally the HLA-A molecule comprising an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0186] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) processing each of the multiple samples from an individual separately to associate the immune cells contained therein with a unique sample barcode; b) combining the multiple processed samples to prepare a sample mixture; c) preparing a bait-sample mixture by contacting a bait composition containing a display portion with the sample mixture under conditions sufficient for immune cells to bind to the display portion; d) detecting immune cells bound to the display portion in the bait-sample mixture; and e) identifying the sample from which the immune cells originate by identifying a unique barcode associated with the immune cells bound to the display portion, wherein the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules; b) having a binding affinity of about 1 nM to about 5000 nM to homogeneous TCR molecules; c) being hydrophobic; and d) having a high content of aromatic residues; and / or 2) having low immunogenicity. In some embodiments, before immobilization, the bait composition containing the display portion with the antigen peptide is brought into contact with immune cells under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, after immobilization, the bait composition containing the display portion with the antigen peptide is brought into contact with immune cells under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the display portion includes an MHC molecule that forms a complex with the antigen peptide, and optionally, the MHC molecule includes an MHC multimer, and even more optionally, the MHC multimer is an MHC tetramer or an MHC dextramer. In some embodiments, the MHC molecule includes an MHC class I molecule.In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the display portion further comprises a detectable label (e.g., a fluorophore). In some embodiments, the method further comprises isolating immune cells bound to the display portion, or isolating the display portion or immune cells not bound to each other.

[0187] In some embodiments, a method is provided for analyzing multiple samples from one or more individuals for the presence or absence of immune cells capable of binding to an antigen peptide, the method comprising: a) processing each of the multiple samples from an individual separately to associate the immune cells contained therein with a unique sample barcode; b) combining the multiple processed samples to prepare a sample mixture; c) preparing a bait-sample mixture by contacting a bait composition containing a display portion containing an antigen peptide with the sample mixture under conditions sufficient for immune cells to bind to the display portion; d) detecting immune cells bound to the display portion in the bait-sample mixture; and e) identifying the sample from which the immune cells originate by identifying a unique barcode associated with the immune cells bound to the display portion, wherein the display portion further comprises a detectable label (e.g., a fluorophore, e.g., PE or APC), and the detection agent is bound to the detectable label. In some embodiments, the bait composition containing the display portion containing an antigen peptide with immune cells is contacted with immune cells under conditions sufficient for immune cells to bind to the display portion before immobilization. In some embodiments, after immobilization, the bait composition containing a display portion with an antigen peptide is brought into contact with immune cells under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, the sample is a blood sample (e.g., a PBMC sample). In some embodiments, the immune cells include T cells (e.g., CD8 T cells), and optionally, the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in amounts of about 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the display portion includes an MHC molecule that forms a complex with the antigen peptide, and optionally, the MHC molecule includes an MHC multimer, and even more optionally, the MHC multimer is an MHC tetramer or an MHC dextramer. In some embodiments, the MHC molecule includes an MHC class I molecule.In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C, and the MHC class I molecule may include an HLA-A molecule, and further may the HLA-A molecule include a mutation that reduces its binding to CD8, and further may the HLA-A molecule include an HLA A2 heavy chain having the A245V mutation. In some embodiments, the display portion comprises two or more different MHC class I molecules selected from the group consisting of a) HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:03. In some embodiments, the method further comprises isolating immune cells bound to the display portion, or further comprising isolating the display portion or immune cells not bound to each other.

[0188] In some embodiments, the “conditions” in the phrase “under conditions sufficient for immune cells to bind to the display portion” refers to the conditions under which specific binding occurs between immune cells and the display portion. In some embodiments, this refers to conditions under which incubation of the immune cells and the display portion (e.g., in culture medium, at room temperature) is performed for about 5 minutes or more, about 10 minutes or more, about 15 minutes or more, about 20 minutes or more, about 25 minutes or more, or about 30 minutes or more. In some embodiments, this refers to conditions under which at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the maximum binding between immune cells and the display portion occurs. Maximum binding can be evaluated, for example, by exposing immune cells to the display portion for a suitable time (e.g., 30 minutes) under suitable conditions (e.g., room temperature).

[0189] In some embodiments, immune cells are immobilized on a solid substrate by an immune cell-binding agent that is coated on or bound to the solid substrate. In some embodiments, the agent is an antibody that specifically recognizes surface markers of immune cells. In some embodiments, the agent is an anti-CD8 antibody and the immune cells are CD8 T cells. In some embodiments, the agent is an anti-CD4 antibody and the immune cells are CD4 T cells. In some embodiments, the agent is an anti-CD3 antibody and the immune cells are T cells. Immobilization can be performed by mixing the immune cells with the agent that specifically binds to the immune cells (or by incubating the immune cells with the agent). In some embodiments, the agent is pre-coated or bound to a solid substrate such as a plate or magnetic beads. The immune cells can be brought into contact with the agent for a certain period of time, after which a washing step can be applied to remove cells that are not bound to the agent (e.g., using a magnet). See, for example, Examples 2-4.

[0190] In some embodiments, the solid carrier (or "solid substrate" which can be used synonymously) includes the surface of a plate or particles (e.g., the surface of a particle, e.g., the inner surface of a porous particle). In some embodiments, the solid carrier includes any of the following: beads (e.g., magnetic beads), porous beads, porous matrices, arrays, glass surfaces, silicon surfaces, plastic surfaces, filters, membranes, nylon, silicon wafer chips, flow-through chips. In some embodiments, the solid carrier includes any of the following: biochips including electronic devices, microtiter wells, ELISA plates, spin interference disks, nitrocellulose membranes, nitrocellulose polymer surfaces, nanoparticles, or microspheres.

[0191] In some embodiments, the sample is a mixture of multiple samples of an individual. In some embodiments, the multiple samples of an individual originate from a single individual. In some embodiments, the multiple samples of an individual originate from multiple individuals.

[0192] In some embodiments, the sample is a mixture of multiple samples of an individual, each of which is obtained from a different individual. In some embodiments, the method involves processing each of the multiple samples of the individual separately before combining them into a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with a molecule containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by washing the immune cells at least once. For example, association can be achieved by mixing immune cells with a molecule containing the unique barcode that binds to immune cells and causes them to link together (e.g., an antibody that binds to immune cells, e.g., a CD45 antibody). For example, a unique barcode can be associated with cells by an antibody bound to or fused with a barcode that specifically binds to cells. In some embodiments, the barcode is a nucleic acid barcode (e.g., a DNA barcode). In some embodiments, the unique DNA barcode is a randomized barcode having a nucleic acid length of about 10–20 (e.g., 12–18). In some embodiments, the barcode is a peptide barcode. In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by, for example, washing immune cells at least once, twice, or three times.

[0193] In some embodiments, the method further includes identifying the sample from which the immune cells originate by identifying a unique barcode associated with the immune cells bound to the display portion. For example, nucleic acids can be purified and sequencing can be used to identify the immune cells associated with the unique DNA barcode. See, for example, Example 4.

[0194] In some embodiments, the particles described herein are dextran particles. In some embodiments, the particles are magnetic nanoparticles or polystyrene nanoparticles. In some embodiments, the particles are agarose beads or Sepharose beads. In some embodiments, the antigen peptide or MHC is directly bound to the particle. In some embodiments, the antigen peptide or MHC is bound to the particle via a binding pair, the binding pair comprising a first binding component bound to the antigen peptide and a second binding component bound to the particle. In some embodiments, the display portion comprises cells. In some embodiments, the cells comprise polynucleotides encoding antigen peptides. In some embodiments, the polynucleotides encode multiple antigen peptides.

[0195] In some embodiments, the individual is a human. In some embodiments, the individual is approximately 50 years of age or older (e.g., 50 years or older, 60 years or older, 70 years or older, or 80 years or older).

[0196] In some embodiments, the sample described herein (including this section and any other sections of this application) may be any sample from an individual. In some embodiments, the sample described herein is a blood sample. In some embodiments, the sample described herein is a plasma sample. In some embodiments, the sample described herein includes peripheral blood mononuclear cells (PMBCs). In some embodiments, the sample described herein is a sample obtained from the tissue or organ of an individual (e.g., a biopsy sample). In some embodiments, the sample is obtained from the lymph nodes of an individual.

[0197] The methods described herein (e.g., methods for analyzing immune cells and methods for detecting antigen-specific immune cells described in the following sections) have high specificity and sensitivity, and enable the detection of antigen-specific immune cells even when the number of antigen-specific immune cells in the sample is low (e.g., less than 1%). Sensitivity is calculated as the percentage of true positives / (true positives + false negatives). Specificity is calculated as the percentage of true negatives / (true negatives + false positives). In some embodiments, the methods described herein exhibited a specificity of greater than about 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, or greater than 100%. In some embodiments, the methods described herein exhibited a sensitivity of greater than about 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, or greater than 80%. See Figure 12. In some embodiments, the sample comprises PBMCs, and the immune cells comprise or are T cells or B cells contained in the PBMCs. In some embodiments, prior to contacting the immune cells with the bait composition, usually no enrichment step is performed on the immune cells, or no enrichment step is performed on the immune cells that specifically bind to the antigen peptide. In some embodiments, throughout the method, usually no enrichment step is performed on the immune cells, or no enrichment step is performed on the immune cells that specifically bind to the antigen peptide. In some embodiments, the sample contains PBMCs of 1x10 8 or less, 5x10 7 or less, 2x10 7 or less, 1x10 7 or less, 5x10 6 or less, 2x10 6 or less, or 1x10 6 or less. In some embodiments, the sample contains PBMCs of 1x10 6 or less, 5x10 5 or less, 2x10 5 or less, 1x10 5 or less, 5x10 4 or less, 2x10 4 or less, 1x10 4 or less, 7x10 3 or less, 5x10 3 or less, 2x10 3 or less, 1x10 3Below, 5x10 2 Below, 2x10 2 The following, or 1x10 2 The following immune cells are included (e.g., CD8 T cells, e.g., immune cells that bind to antigen peptides in the bait composition, e.g., CD4 T cells or CD8 T cells that bind to antigen peptides in the bait composition).

[0198] Methods for analyzing immune cells

[0199] This application includes a method for analyzing immune cells in a sample from an organism, the method comprising: a) contacting a plurality of immune cells with one or more display portions or bait compositions described herein; and b) detecting or analyzing one or more immune cells bound to one or more display portions.

[0200] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions or bait compositions described herein; and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual does not exhibit pathological symptoms of cancer, infection, or autoimmune disease. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of the one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which a plurality of MHC-peptide complexes are bound, where the plurality of MHC-peptide complexes comprises at least 12 or more MHC-peptide complexes, where each MHC-peptide complex is an MHC-peptide monomer comprising an MHC molecule that forms a complex with a peptide, and at least two of the MHC-peptide complexes are different. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the bait composition comprises at least 2, 3, 4, 5, 6, or 7 different duplicate peptides associated with gene mutations. In some embodiments, the gene is an oncogene.In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).

[0201] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions or bait compositions described herein; and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual does not exhibit pathological symptoms of cancer, infection, or autoimmune disease. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of the one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which multiple MHC-peptide complexes are bound, where the multiple MHC-peptide complexes comprise two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of the MHC-peptide complexes comprises an MHC molecule that forms a complex with a peptide, at least two of the MHC-peptide complexes are different, and the multiple MHC-peptide complexes comprise at least 20, 50, 100, 500, 1,000, 5,000, 10,000, 20,000, 50,000, 100,000, 15,000, or 200,000 MHC-peptide complexes. In some embodiments, the one or more display portions are contained in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises HLA-DM molecules, HLA-DO molecules, HLA-DP molecules, HLA-DQ molecules, or HLA-DR molecules. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules.In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the bait composition contains at least 2, 3, 4, 5, 6, or 7 different duplicate peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least 1, 2, 3, 4, 5, or 6 HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0202] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions or bait compositions described herein; and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual does not exhibit pathological symptoms of cancer, infection, or autoimmune disease. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 105 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4 The following applies to some embodiments: In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which multiple MHC-peptide complexes are bound, where the multiple MHC-peptide complexes comprise two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each MHC-peptide complex comprises an MHC molecule that forms a complex with a peptide, at least two of the MHC-peptide complexes are different, and the particle is at least about 1 μm in size. 2It has a surface area or a diameter of at least 0.5 μm. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises HLA-DM molecules, HLA-DO molecules, HLA-DP molecules, HLA-DQ molecules, or HLA-DR molecules. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the bait composition comprises at least 2, 3, 4, 5, 6, or 7 different duplicate peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0203] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions or bait compositions described herein; and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual does not exhibit pathological symptoms of cancer, infection, or autoimmune disease. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which a plurality of MHC-peptide complexes are bound, where the plurality of MHC-peptide complexes comprises two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of which comprises an MHC molecule that forms a complex with a peptide, at least two of the MHC-peptide complexes are different, and the plurality of MHC-peptide complexes comprises a) at least two different MHC molecules and b) at least two different peptides. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens.In some embodiments, the bait composition contains at least two, three, four, five, six, or seven different duplicate peptides related to the gene mutation. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0204] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions or bait compositions described herein; and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual does not exhibit pathological symptoms of cancer, infection, or autoimmune disease. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which a plurality of MHC-peptide complexes are bound, where the plurality of MHC-peptide complexes comprises two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of which comprises an MHC molecule that forms a complex with a peptide, and at least two of the MHC-peptide complexes are different, and the display portion comprises at least two different MHC class I molecules. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens.In some embodiments, the bait composition contains at least two, three, four, five, six, or seven different duplicate peptides related to the gene mutation. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0205] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions or bait compositions described herein; and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual does not exhibit pathological symptoms of cancer, infection, or autoimmune disease. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which a plurality of MHC-peptide complexes are bound, where the plurality of MHC-peptide complexes comprises two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of which comprises an MHC molecule that forms a complex with a peptide, and at least two of the MHC-peptide complexes are different, and the display portion comprises at least two different MHC class II molecules. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens.In some embodiments, the bait composition comprises at least 2, 3, 4, 5, 6, or 7 different overlapping peptides related to gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display moiety or the bait composition does not contain either a fluorescent dye or a fluorophore. In some embodiments, the MHC molecule matches at least 1, 2, 3, 4, 5, or 6 HLA types of the individual from which the sample is derived. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the plurality of MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, MHC-peptide trimer, or MHC-peptide tetramer.

[0206] In some embodiments, provided is a method for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display moieties or bait compositions described herein; and b) detecting or analyzing one or more immune cells that bind to one or more of the display moieties, wherein the immune cells are obtained from a sample (e.g., PBMC) of the individual, and the individual is at risk of developing a disease or disorder (e.g., cancer, an infectious disease, or an autoimmune disease or disorder) (e.g., the individual has a family history of such a disease or has been exposed to a pathogen but has not received treatment for the disease or disorder). In some embodiments, the number of cells (e.g., PBMC) in the sample is about 5x10 6 or less, 2x10 6 or less, 10 6 or less, 5x10 5 or less, 2x10 5 or less, 10 5Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises HLA-DM molecules, HLA-DO molecules, HLA-DP molecules, HLA-DQ molecules, or HLA-DR molecules. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the bait composition comprises at least 2, 3, 4, 5, 6, or 7 different duplicate peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).

[0207] In some embodiments, a method for analyzing immune cells in a sample from an individual is provided, the method comprising: a) contacting a plurality of immune cells with one or more display portions; and b) detecting or analyzing one or more immune cells that bind to one or more of the display portions, wherein the immune cells are obtained from a sample of the individual (e.g., PBMC), and the individual is at risk of developing a disease or disorder (e.g., cancer, an infection, or an autoimmune disease or disorder) (e.g., the individual has a family history of such a disease or has been exposed to a pathogen but has not received treatment for the disease or disorder). In some embodiments, the number of cells (e.g., PBMC) in the sample is about 5x10 6 or less, 2x10 6 or less, 10 6 or less, 5x10 5 or less, 2x10 5 or less, 10 5 or less, 5x10 4 or less, 4x10 4 or less, 2x10 4 or less, or 10 4The following applies: In some embodiments, each of the one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which a plurality of MHC-peptide complexes are bound, where the plurality of MHC-peptide complexes comprises two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, where each MHC-peptide complex is an MHC-peptide monomer comprising an MHC molecule that forms a complex with a peptide, and at least two of the MHC-peptide complexes are different. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the bait composition comprises at least 2, 3, 4, 5, 6, or 7 different duplicate peptides associated with gene mutations.In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cell is a T cell (e.g., a CD8+ T cell or a CD4+ T cell).

[0208] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions; and b) detecting or analyzing one or more immune cells bound to one or more of the display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual is at risk of developing a disease or illness (e.g., cancer, infection, or autoimmune disease or autoimmune disorder) (e.g., the individual has a family history of such a disease or has been exposed to a pathogen but has not received treatment for the disease or illness). In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of the one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which multiple MHC-peptide complexes are bound, where the multiple MHC-peptide complexes comprise two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of the MHC-peptide complexes comprises an MHC molecule that forms a complex with a peptide, at least two of the MHC-peptide complexes are different, and the multiple MHC-peptide complexes comprise at least 20, 50, 100, 500, 1,000, 5,000, 10,000, 20,000, 50,000, 100,000, 15,000, or 200,000 MHC-peptide complexes. In some embodiments, the one or more display portions are contained in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises HLA-DM molecules, HLA-DO molecules, HLA-DP molecules, HLA-DQ molecules, or HLA-DR molecules. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules.In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the bait composition contains at least 2, 3, 4, 5, 6, or 7 different duplicate peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least 1, 2, 3, 4, 5, or 6 HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0209] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions; and b) detecting or analyzing one or more immune cells bound to one or more of the display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual is at risk of developing a disease or illness (e.g., cancer, infection, or autoimmune disease or autoimmune disorder) (e.g., the individual has a family history of such a disease or has been exposed to a pathogen but has not received treatment for the disease or illness). In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 106 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4 The following applies to some embodiments: In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which multiple MHC-peptide complexes are bound, where the multiple MHC-peptide complexes comprise two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each MHC-peptide complex comprises an MHC molecule that forms a complex with a peptide, at least two of the MHC-peptide complexes are different, and the particle is at least about 1 μm in size. 2It has a surface area or a diameter of at least 0.5 μm. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises HLA-DM molecules, HLA-DO molecules, HLA-DP molecules, HLA-DQ molecules, or HLA-DR molecules. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the bait composition comprises at least 2, 3, 4, 5, 6, or 7 different duplicate peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0210] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions; and b) detecting or analyzing one or more immune cells bound to one or more of the display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual is at risk of developing a disease or illness (e.g., cancer, infection, or autoimmune disease or autoimmune disorder) (e.g., the individual has a family history of such a disease or has been exposed to a pathogen but has not received treatment for the disease or illness). In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which a plurality of MHC-peptide complexes are bound, where the plurality of MHC-peptide complexes comprises two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of which comprises an MHC molecule that forms a complex with a peptide, at least two of the MHC-peptide complexes are different, and the plurality of MHC-peptide complexes comprises a) at least two different MHC molecules and b) at least two different peptides. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens.In some embodiments, the bait composition contains at least two, three, four, five, six, or seven different duplicate peptides related to the gene mutation. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0211] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions; and b) detecting or analyzing one or more immune cells bound to one or more of the display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual is at risk of developing a disease or illness (e.g., cancer, infection, or autoimmune disease or autoimmune disorder) (e.g., the individual has a family history of such a disease or has been exposed to a pathogen but has not received treatment for the disease or illness). In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which a plurality of MHC-peptide complexes are bound, where the plurality of MHC-peptide complexes comprises two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of which comprises an MHC molecule that forms a complex with a peptide, and at least two of the MHC-peptide complexes are different, and the display portion comprises at least two different MHC class I molecules. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens.In some embodiments, the bait composition contains at least two, three, four, five, six, or seven different duplicate peptides related to the gene mutation. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0212] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions; and b) detecting or analyzing one or more immune cells bound to one or more of the display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual is at risk of developing a disease or illness (e.g., cancer, infection, or autoimmune disease or autoimmune disorder) (e.g., the individual has a family history of such a disease or has been exposed to a pathogen but has not received treatment for the disease or illness). In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which a plurality of MHC-peptide complexes are bound, where the plurality of MHC-peptide complexes comprises two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of which comprises an MHC molecule that forms a complex with a peptide, and at least two of the MHC-peptide complexes are different, and the display portion comprises at least two different MHC class II molecules. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens.In some embodiments, the bait composition contains at least two, three, four, five, six, or seven different duplicate peptides related to the gene mutation. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0213] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions or bait compositions described herein; and b) detecting or analyzing one or more immune cells bound to one or more display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual has minimal residual disease (MRD). In some embodiments, the individual has minimal residual carcinoma. In some embodiments, minimal residual carcinoma is present after cancer has been surgically removed or treated. In some embodiments, minimal residual disease is too small to be detected by imaging equipment (e.g., routinely used or standard imaging equipment for cancer detection). In some embodiments, the location of minimal residual disease is diverse. In some embodiments, minimal residual carcinoma is a result of immune evasion or resistance to treatment. In some embodiments, the individual has previously received cancer treatment and has not shown pathological symptoms of cancer after treatment. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises HLA-DM molecules, HLA-DO molecules, HLA-DP molecules, HLA-DQ molecules, or HLA-DR molecules. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the bait composition comprises at least 2, 3, 4, 5, 6, or 7 different duplicate peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0214] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions; and b) detecting or analyzing one or more immune cells bound to one or more of the display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual has minimal residual disease (MRD). In some embodiments, the individual has minimal residual carcinoma. In some embodiments, minimal residual carcinoma is present after cancer has been surgically removed or treated. In some embodiments, the individual has previously received cancer treatment and has not shown any pathological symptoms of cancer after treatment. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of the one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which a plurality of MHC-peptide complexes are bound, where the plurality of MHC-peptide complexes comprises two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, where each MHC-peptide complex is an MHC-peptide monomer comprising an MHC molecule that forms a complex with a peptide, and at least two of the MHC-peptide complexes are different. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the bait composition comprises at least 2, 3, 4, 5, 6, or 7 different duplicate peptides associated with gene mutations.In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecule matches at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cell is a T cell (e.g., a CD8+ T cell or a CD4+ T cell).

[0215] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions; and b) detecting or analyzing one or more immune cells bound to one or more of the display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual has minimal residual disease (MRD). In some embodiments, the individual has minimal residual carcinoma. In some embodiments, minimal residual carcinoma is present after cancer has been surgically removed or treated. In some embodiments, the individual has previously received cancer treatment and has not shown any pathological symptoms of cancer after treatment. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of the one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which multiple MHC-peptide complexes are bound, where the multiple MHC-peptide complexes comprise two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of the MHC-peptide complexes comprises an MHC molecule that forms a complex with a peptide, at least two of the MHC-peptide complexes are different, and the multiple MHC-peptide complexes comprise at least 20, 50, 100, 500, 1,000, 5,000, 10,000, 20,000, 50,000, 100,000, 15,000, or 200,000 MHC-peptide complexes. In some embodiments, the one or more display portions are contained in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises HLA-DM molecules, HLA-DO molecules, HLA-DP molecules, HLA-DQ molecules, or HLA-DR molecules. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules.In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the bait composition contains at least 2, 3, 4, 5, 6, or 7 different duplicate peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least 1, 2, 3, 4, 5, or 6 HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0216] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions; and b) detecting or analyzing one or more immune cells bound to one or more of the display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual has minimal residual disease (MRD). In some embodiments, the individual has minimal residual carcinoma. In some embodiments, minimal residual carcinoma is present after cancer has been surgically removed or treated. In some embodiments, the individual has previously received cancer treatment and has not shown any pathological symptoms of cancer after treatment. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶.6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4 The following applies to some embodiments: In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which multiple MHC-peptide complexes are bound, where the multiple MHC-peptide complexes comprise two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each MHC-peptide complex comprises an MHC molecule that forms a complex with a peptide, at least two of the MHC-peptide complexes are different, and the particle is at least about 1 μm in size. 2It has a surface area or a diameter of at least 0.5 μm. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises HLA-DM molecules, HLA-DO molecules, HLA-DP molecules, HLA-DQ molecules, or HLA-DR molecules. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens. In some embodiments, the bait composition comprises at least 2, 3, 4, 5, 6, or 7 different duplicate peptides associated with gene mutations. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells).In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0217] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions; and b) detecting or analyzing one or more immune cells bound to one or more of the display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual has minimal residual disease (MRD). In some embodiments, the individual has minimal residual carcinoma. In some embodiments, minimal residual carcinoma is present after cancer has been surgically removed or treated. In some embodiments, the individual has previously received cancer treatment and has not shown any pathological symptoms of cancer after treatment. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which a plurality of MHC-peptide complexes are bound, where the plurality of MHC-peptide complexes comprises two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of which comprises an MHC molecule that forms a complex with a peptide, at least two of the MHC-peptide complexes are different, and the plurality of MHC-peptide complexes comprises a) at least two different MHC molecules and b) at least two different peptides. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens.In some embodiments, the bait composition contains at least two, three, four, five, six, or seven different duplicate peptides related to the gene mutation. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0218] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions; and b) detecting or analyzing one or more immune cells bound to one or more of the display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual has minimal residual disease (MRD). In some embodiments, the individual has minimal residual carcinoma. In some embodiments, minimal residual carcinoma is present after cancer has been surgically removed or treated. In some embodiments, the individual has previously received cancer treatment and has not shown any pathological symptoms of cancer after treatment. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 105 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which a plurality of MHC-peptide complexes are bound, where the plurality of MHC-peptide complexes comprises two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of which comprises an MHC molecule that forms a complex with a peptide, and at least two of the MHC-peptide complexes are different, and the display portion comprises at least two different MHC class I molecules. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens.In some embodiments, the bait composition contains at least two, three, four, five, six, or seven different duplicate peptides related to the gene mutation. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0219] In some embodiments, a method is provided for analyzing immune cells in a sample from an individual, the method comprising: a) contacting a plurality of immune cells with one or more display portions; and b) detecting or analyzing one or more immune cells bound to one or more of the display portions, wherein the immune cells are obtained from a sample of an individual (e.g., PBMCs), and the individual has minimal residual disease (MRD). In some embodiments, the individual has minimal residual carcinoma. In some embodiments, minimal residual carcinoma is present after cancer has been surgically removed or treated. In some embodiments, the individual has previously received cancer treatment and has not shown any pathological symptoms of cancer after treatment. In some embodiments, the number of cells (e.g., PBMCs) in the sample is approximately 5 x 10⁻¹⁶. 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 105 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4The following applies: In some embodiments, each of one or more display portions comprises a particle (e.g., spherical particle, e.g., solid particle, e.g., bead) to which a plurality of MHC-peptide complexes are bound, where the plurality of MHC-peptide complexes comprises two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of which comprises an MHC molecule that forms a complex with a peptide, and at least two of the MHC-peptide complexes are different, and the display portion comprises at least two different MHC class II molecules. In some embodiments, one or more display portions are included in the bait composition. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC I molecules, and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises a) at least two, three, or four HLA-A molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides, where the two, three, or four HLA-A molecules are selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01. In some embodiments, the bait composition comprises a) at least two different (or at least three different) MHC II molecules and b) at least about 20, 50, 100, 150, 200, or 250 different peptides. In some embodiments, the bait composition comprises an HLA-DM molecule, an HLA-DO molecule, an HLA-DP molecule, an HLA-DQ molecule, or an HLA-DR molecule. In some embodiments, the bait composition comprises HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. In some embodiments, the peptides in the bait composition are associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens.In some embodiments, the bait composition contains at least two, three, four, five, six, or seven different duplicate peptides related to the gene mutation. In some embodiments, the gene is an oncogene. In some embodiments, the display portion or bait composition does not contain any fluorescent dyes or fluorophores. In some embodiments, the MHC molecules match at least one, two, three, four, five, or six HLA types of the individual from which the sample originates. In some embodiments, the immune cells are T cells (e.g., CD8+ T cells or CD4+ T cells). In some embodiments, one or more of the multiple MHC-peptide complexes are MHC-peptide dimers, MHC-peptide tetramers, MHC-peptide pentamers, MHC-peptide octamers, MHC-peptide dextramers, or MHC-peptide dodecamers. In some embodiments, at least one of the multiple MHC-peptide complexes has two different MHC-peptide monomers. In some embodiments, at least one MHC complex is an MHC-peptide dimer, an MHC-peptide trimer, or an MHC-peptide tetramer.

[0220] In some embodiments, immune cells are immobilized on a solid substrate by an immune cell-binding agent that is coated on or bound to the solid substrate. In some embodiments, the agent is an antibody that specifically recognizes surface markers of immune cells. In some embodiments, the agent is an anti-CD8 antibody and the immune cells are CD8 T cells. In some embodiments, the agent is an anti-CD4 antibody and the immune cells are CD4 T cells. In some embodiments, the agent is an anti-CD3 antibody and the immune cells are T cells. Immobilization can be performed by mixing the immune cells with the agent that specifically binds to the immune cells (or by incubating the immune cells with the agent). In some embodiments, the agent is pre-coated or bound to a solid substrate such as a plate or magnetic beads. The immune cells can be brought into contact with the agent for a certain period of time, after which a washing step can be applied to remove cells that are not bound to the agent (e.g., using a magnet). See, for example, the examples.

[0221] In some embodiments, the number of immune cells is about 10 8 Below, 5x10 7 The following 10 7 Below, 5x10 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4 The following applies:

[0222] In some embodiments, the solid substrate includes a plate or the surface of particles (e.g., the surface of particles different from those used as carriers in the display portion, e.g., the inner surface of porous particles). In some embodiments, the solid substrate includes any of the following: beads (e.g., magnetic beads), porous beads, porous matrices, arrays, glass surfaces, silicon surfaces, plastic surfaces, filters, membranes, nylon, silicon wafer chips, or flow-through chips. In some embodiments, the solid substrate includes any of the following: biochips containing electronic devices, microtiter wells, ELISA plates, spin interference disks, nitrocellulose membranes, nitrocellulose polymer surfaces, nanoparticles, or microspheres.

[0223] In some embodiments, contact of multiple immune cells with a display portion or bait composition is carried out under conditions sufficient for the immune cells to bind to the display portion. In some embodiments, “conditions” in the phrase “under conditions sufficient for the immune cells to bind to the display portion” refers to conditions under which specific binding occurs between the immune cells and the display portion. In some embodiments, this refers to conditions under which incubation of the immune cells and the display portion (e.g., in culture medium, at room temperature) is carried out for about 5 minutes or more, about 10 minutes or more, about 15 minutes or more, about 20 minutes or more, about 25 minutes or more, or about 30 minutes or more. In some embodiments, this refers to conditions under which at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the maximum binding between the immune cells and the display portion occurs. Maximum binding can be evaluated, for example, by contacting the immune cells with the display portion for a suitable time (e.g., 30 minutes) under suitable conditions (e.g., room temperature).

[0224] In some embodiments, the sample is a mixture of multiple samples of an individual. In some embodiments, the multiple samples of an individual originate from a single individual. In some embodiments, the multiple samples of an individual originate from multiple individuals.

[0225] In some embodiments, the sample is a mixture of multiple samples of an individual, each of which is obtained from a different individual. In some embodiments, the method involves processing each of the multiple samples of the individual separately before combining them into a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with a molecule containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by washing the immune cells at least once. For example, association can be achieved by mixing immune cells with a molecule containing the unique barcode that binds to immune cells and causes them to link together (e.g., an antibody that binds to immune cells, e.g., a CD45 antibody). For example, a unique barcode can be associated with cells by an antibody bound to or fused with a barcode that specifically binds to cells. In some embodiments, the barcode is a nucleic acid barcode (e.g., a DNA barcode). In some embodiments, the unique DNA barcode is a randomized barcode having a nucleic acid length of about 10–20 (e.g., 12–18). In some embodiments, the barcode is a peptide barcode. In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by, for example, washing immune cells at least once, twice, or three times.

[0226] In some embodiments, the method further includes identifying the sample from which the immune cells originate by identifying a unique barcode associated with the immune cells bound to the display portion. For example, nucleic acids can be purified and sequencing can be used to identify the immune cells associated with the unique DNA barcode.

[0227] In some embodiments, the individual is a human. In some embodiments, the individual is approximately 50 years of age or older (e.g., 50 years or older, 60 years or older, 70 years or older, or 80 years or older).

[0228] In some embodiments, the sample described herein (including this section and any other sections of this application) may be any sample from an individual. In some embodiments, the sample described herein is a blood sample. In some embodiments, the sample described herein is a plasma sample. In some embodiments, the sample described herein includes peripheral blood mononuclear cells (PMBCs). In some embodiments, the number of PBMCs is about 10 8 Below, 5x10 7 The following 10 7 Below, 5x10 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4 The following applies: In some embodiments, the sample described herein is a sample obtained from the tissue or organ of an individual (e.g., a biopsy sample). In some embodiments, the sample is obtained from the lymph node of an individual.

[0229] The methods described herein have high specificity and sensitivity that enable the detection of antigen-specific immune cells even when antigen-specific immune cells are present in small amounts (e.g., less than 1%) in the sample. In some embodiments, the sample comprises PBMCs, and the immune cells comprise T cells or B cells contained in the PBMCs, or are T cells or B cells contained in the PBMCs. In some embodiments, the immune cells are typically not enriched before contacting the immune cells with the bait composition, or the immune cells specifically bound to the peptide are not enriched. In some embodiments, the immune cells are typically not enriched from the beginning to the end of the method, or the immune cells specifically bound to the peptide are not enriched. In some embodiments, the sample comprises 1 x 10⁻⁶ 8 Below, 5x10 7 Below, 2x107 Below, 1x10 7 Below, 5x10 6 Below, 2x10 6 The following, or 1x10 6 The following PBMCs are included. In some embodiments, the sample is 1 x 10 6 Below, 5x10 5 Below, 2x10 5 Below, 1x10 5 Below, 5x10 4 Below, 2x10 4 Below, 1x10 4 Below, 7x10 3 Below, 5x10 3 Below, 2x10 3 Below, 1x10 3 Below, 5x10 2 Below, 2x10 2 The following, or 1x10 2 The following immune cells are included (e.g., CD8 T cells, e.g., immune cells that bind to peptides in the bait composition, e.g., CD4 T cells or CD8 T cells that bind to peptides in the bait composition).

[0230] Method for detecting antigen-specific immune cells in an individual

[0231] In some embodiments, a method is provided for detecting antigen-specific immune cells in an individual (for example, an individual that does not exhibit pathological symptoms of cancer or tumor, pathological symptoms of an infection caused by a specific pathogen, or pathological symptoms of an autoimmune disease or autoantigen-related disease; for example, an individual at risk of developing such a disease or illness (e.g., cancer, infection, or autoimmune disease or autoimmune disorder); for example, an individual that has never received treatment for such a disease or illness; for example, an individual that has received treatment for a disease or illness such as cancer and is suspected of having minimal residual cancer cells), the method comprising analyzing a sample from the individual according to one of the methods described herein, wherein predetermined characteristics of the immune cells indicate a disease or illness in the individual. In some embodiments, predetermined characteristics of the immune cells include the presence of immune cells. In some embodiments, the presence of immune cells that specifically bind to a bait composition containing one or more neoantigens indicates the presence of cancer. In some embodiments, the presence of immune cells that specifically bind to a bait composition containing one or more antigens of a pathogen (e.g., virus, bacteria, fungus) indicates the presence of an infection related to the pathogen. In some embodiments, the presence of immune cells that specifically bind to a bait composition containing one or more autoantigens associated with an autoimmune disease or autoimmune disorder indicates the presence of an immune disease or disorder. In some embodiments, predetermined characteristics of immune cells include a quantity of immune cells above a threshold level. In some embodiments, predetermined characteristics of immune cells include gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics. In some embodiments, epigenetic modification characteristics include DNA methylation characteristics and histone glycosylation characteristics.

[0232] In some embodiments, a method is provided for detecting antigen-specific immune cells in an individual (for example, an individual that does not show pathological symptoms of cancer or tumor, pathological symptoms of an infection caused by a specific pathogen, or pathological symptoms of an autoimmune disease or autoantigen-related disease, for example, an individual at risk of developing such a disease or illness (e.g., cancer, infection, or autoimmune disease or autoimmune disorder), for example, an individual that has never received treatment for such a disease or illness, for example, an individual that has received treatment for a disease or illness such as cancer and is suspected of having minimal residual cancer cells), the method comprising a) immobilizing immune cells from a sample onto a solid carrier The method comprises: 1) before immobilization, contacting a bait composition containing a display portion containing an antigen peptide with immune cells under conditions sufficient for the immune cells to bind to the display portion; or 2) after immobilization, contacting a bait composition containing a display portion containing an antigen peptide with immune cells under conditions sufficient for the immune cells to bind to the display portion; b) contacting a detection agent that recognizes the display portion with the immobilized immune cells under conditions sufficient for the detection agent to bind to the display portion; and c) detecting immune cells bound to the display portion by detecting the detection agent. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples of individuals, each obtained from a different individual. In some embodiments, each of the multiple samples of individuals is processed separately before being combined to form a mixture to associate the immune cells contained therein with a unique sample barcode (e.g., by mixing the immune cells with a molecule containing the unique sample barcode). In some embodiments, the method further includes removing unbound molecules (molecules not bound by immune cells) containing the barcode by washing the immune cells at least once. In some embodiments, prior to immobilization, the bait composition containing the display portion with the antigen peptide is brought into contact with immune cells under conditions sufficient to allow the immune cells to bind to the display portion.In some embodiments, after immobilization, the bait composition containing the display portion with the antigen peptide is brought into contact with immune cells under conditions sufficient to allow the immune cells to bind to the display portion.

[0233] In some embodiments, a method is provided for detecting antigen-specific immune cells in an individual (for example, an individual that does not exhibit pathological symptoms of cancer or tumor, pathological symptoms of an infection caused by a specific pathogen, or pathological symptoms of an autoimmune disease or autoantigen-related disease, for example, an individual at risk of developing such a disease or illness (e.g., cancer, infection, or autoimmune disease or autoimmune disorder), for example, an individual that has never received treatment for such a disease or illness, for example, an individual that has received treatment for a disease or illness such as cancer and is suspected of having minimal residual cancer cells), the method comprising: a) contacting immune cells with a bait composition comprising a display portion containing an antigen peptide under conditions sufficient for the immune cells to bind to the display portion; b) contacting immune cells with a detection agent that recognizes the display portion under conditions sufficient for the detection agent to bind to the display portion; c) immobilizing immune cells from the sample onto a solid carrier; and d) detecting immune cells bound to the display portion by detecting the detection agent. In some embodiments, the sample is obtained from a single individual. In some embodiments, the sample is a mixture of multiple samples of individuals, each obtained from a different individual. In some embodiments, each of several samples of an individual is processed separately before being combined into a mixture, and the immune cells contained therein are associated with a unique sample barcode.

[0234] In some embodiments, a method is provided for detecting antigen-specific immune cells in multiple individuals (e.g., a) multiple individuals who do not exhibit pathological symptoms of cancer or tumor, pathological symptoms of infection by a specific pathogen, or pathological symptoms of autoimmune disease or autoantigen-related disease; b) multiple individuals at risk of developing such disease or illness (e.g., cancer, infection, or autoimmune disease or autoimmune disorder); c) multiple individuals who have never received treatment for such disease or illness; or d) multiple individuals who have received treatment for a disease or illness such as cancer and are suspected of having minimal residual cancer cells), wherein the method detects antigen-specific immune cells in a) individual The method includes: a) processing each of several samples separately and associating the immune cells contained therein with a unique sample barcode; b) combining the several processed samples to prepare a sample mixture; c) preparing a bait-sample mixture by contacting a bait composition containing an antigen peptide display portion with the sample mixture under conditions sufficient for immune cells to bind to the display portion; d) detecting immune cells bound to the display portion in the bait-sample mixture; and e) identifying the sample from which the immune cells originate by identifying a unique barcode associated with the immune cells bound to the display portion.

[0235] In some embodiments, a method is provided for detecting antigen-specific immune cells in an individual (for example, an individual that does not exhibit pathological symptoms of cancer or tumor, pathological symptoms of an infection caused by a specific pathogen, or pathological symptoms of an autoimmune disease or autoantigen-related disease, for example, an individual at risk of developing such a disease or illness (e.g., cancer, infection, or autoimmune disease or autoimmune disorder), for example, an individual that has never received treatment for such a disease or illness, for example, an individual that has received treatment for a disease or illness such as cancer and is suspected of having minimal residual cancer cells), the method comprising: a) contacting a bait composition comprising a display portion containing an antigen peptide with a sample under conditions sufficient for immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; b) separating the immune cells bound to the display portion; and c) analyzing the immune cells, wherein a predetermined characteristic of the immune cells indicates a disease or illness in the individual, and the predetermined characteristic of the immune cells includes the presence of immune cells, and optionally includes a quantity of immune cells above a threshold level. In some embodiments, predetermined characteristics of immune cells include gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and histone glycosylation characteristics). In some embodiments, the display portion contains two or more (e.g., four) antigen peptides. In some embodiments, the two or more antigen peptides in the display portion are identical.In some embodiments, the antigen peptide has one or more of the following characteristics: a) binding affinity to MHC molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) binding affinity to congener TCR molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) mutations relative to the wild-type peptide, and optionally mutations at the third amino acid position from the N-terminus relative to the wild-type peptide; d) hydrophobicity; and e) a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion includes MHC molecules that form a complex with the antigen peptide. In some embodiments, the MHC molecules are MHC class I molecules and / or MHC class II molecules. In some embodiments, the immune cells are isolated single immune cells. In some embodiments, the immune cells are present in a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture comprising T cells, memory T cells, macrophages, or dendritic cells, or a combination thereof. In some embodiments, further analysis of immune cells includes quantifying the immune cells. In some embodiments, further analysis of immune cells includes sequencing one or more nucleic acids within the immune cells, and optionally further includes analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences). In some embodiments, further analysis of immune cells further includes identifying one or more epigenetic modifications in the immune cells (e.g., methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further includes generating a report containing information about the cancer status in an individual.In some embodiments, information regarding the state of cancer includes cancer classification, cancer type, cancer nature, cancer origin, cancer stage, likelihood of cancer progression, likelihood of developing one or more cancer symptoms, molecular diagnostics, NGS pathology, and / or treatment options for the individual. In some embodiments, the bait composition comprises multiple different display portions. In some embodiments, each of the multiple different display portions in the bait composition (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display portions, each containing a different MHC molecule) contains a different antigenic peptide. In some embodiments, each of the different display portions containing different MHC molecules contains a different detectable label (e.g., a fluorophore). In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells that bind to each of the different display portions containing different MHC molecules into different populations, optionally including contacting each of the multiple different display portions with a sample from the individual and separating the immune cells that bind to each of the different display portions. In some embodiments, the method further includes culturing immune cells prior to the analytical step. In some embodiments, the display portion includes particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the display portion further includes a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of one or more nucleic acids includes whole-genome sequencing, RNA sequencing, and / or subjecting the immune cells to mass spectrometry.

[0236] In some embodiments, a method is provided for detecting antigen-specific immune cells in an individual (for example, an individual that does not exhibit pathological symptoms of cancer or tumor, pathological symptoms of an infection caused by a specific pathogen, or pathological symptoms of an autoimmune disease or autoantigen-related disease, for example, an individual at risk of developing such a disease or illness (e.g., cancer, infection, or autoimmune disease or autoimmune disorder), for example, an individual that has never received treatment for such a disease or illness, for example, an individual that has received treatment for a disease or illness such as cancer and is suspected of having minimal residual cancer cells), the method comprising: a) contacting a bait composition comprising a display portion containing an antigen peptide with a sample under conditions sufficient for immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; b) separating the immune cells bound to the display portion; and c) analyzing the immune cells, wherein predetermined characteristics of the immune cells indicate cancer in the individual, and predetermined characteristics of the immune cells include gene expression profile characteristics. In some embodiments, the predetermined characteristics of immune cells further include gene mutation profile characteristics and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and histone glycosylation characteristics). In some embodiments, the display portion comprises two or more (e.g., four) antigen peptides. In some embodiments, the two or more antigen peptides in the display portion are identical. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) having a binding affinity of about 1 nM to about 5000 nM to congeneral TCR molecules (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) having a mutation relative to the wild-type peptide, and in some cases having a mutation at the third amino acid position from the N-terminus relative to the wild-type peptide; d) being hydrophobic; and e) having a high content of aromatic residues.In some embodiments, the antigen peptide is of low immunogenicity. In some embodiments, the display portion includes an MHC molecule that forms a complex with the antigen peptide. In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, the immune cell is an isolated single immune cell. In some embodiments, the immune cell is present in a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture including T cells, memory T cells, macrophages, or dendritic cells, or a combination thereof. In some embodiments, further analysis of the immune cell includes detecting and / or quantifying the immune cell. In some embodiments, further analysis of the immune cell includes sequencing one or more nucleic acids within the immune cell, and optionally further includes analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences). In some embodiments, further analysis of the immune cell includes identifying one or more epigenetic modifications in the immune cell (e.g., methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further includes generating a report containing information about the cancer status of an individual. In some embodiments, the information about the cancer status includes cancer classification, cancer type, cancer nature, cancer origin, cancer stage, likelihood of cancer progression, likelihood of developing one or more cancer symptoms, molecular diagnostics, NGS pathology, and / or treatment options for the individual. In some embodiments, the bait composition comprises a plurality of different display moieties. In some embodiments, each of the plurality of different display moieties in the bait composition (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display moieties, each containing a different MHC molecule) contains a different antigenic peptide. In some embodiments, each of the different display moieties containing different MHC molecules contains a different detectable label (e.g., a fluorophore).In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells bound to each of different display portions containing different MHC molecules into different populations, and optionally including contacting each of a plurality of different display portions with a sample from an organism and separating the immune cells bound to each of the different display portions. In some embodiments, the method further includes culturing the immune cells before the analysis step. In some embodiments, the display portion includes particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the display portion further includes a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of one or more nucleic acids includes whole-genome sequencing, RNA sequencing, and / or subjecting the immune cells to mass spectrometry.

[0237] In some embodiments, a method is provided for detecting cancer in an individual (e.g., an individual who has never been diagnosed with cancer, an individual at risk of developing cancer, an individual who has previously received cancer treatment), the method comprising: a) contacting a bait composition containing a display moiety comprising cancer antigen peptides with a sample under conditions sufficient to allow immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display moiety; b) separating the immune cells bound to the display moiety; and c) analyzing the immune cells, wherein predetermined characteristics of the immune cells indicate cancer in the individual, and these predetermined characteristics of the immune cells include gene mutation profile characteristics and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and histone glycosylation characteristics). In some embodiments, the display moiety comprises two or more (e.g., four) antigen peptides. In some embodiments, the two or more antigen peptides in the display moiety are identical. In some embodiments, the antigen peptide has one or more of the following characteristics: a) binding affinity to MHC molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) binding affinity to congener TCR molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) mutations relative to the wild-type peptide, and optionally mutations at the third amino acid position from the N-terminus relative to the wild-type peptide; d) hydrophobicity; and e) a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion includes MHC molecules that form a complex with the antigen peptide. In some embodiments, the MHC molecules are MHC class I molecules and / or MHC class II molecules. In some embodiments, the immune cells are isolated single immune cells. In some embodiments, the immune cells are present in a mixture of immune cells.In some embodiments, the immune cell mixture is a mixture comprising T cells, memory T cells, macrophages, or dendritic cells, or a combination thereof. In some embodiments, further analysis of immune cells includes detecting and / or quantifying immune cells. In some embodiments, further analysis of immune cells includes sequencing one or more nucleic acids within immune cells, and optionally further includes analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences). In some embodiments, further analysis of immune cells includes identifying one or more epigenetic modifications in immune cells (e.g., methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further includes generating a report containing information about the cancer status of an individual. In some embodiments, the information about the cancer status includes cancer classification, cancer type, cancer nature, cancer origin, cancer stage, likelihood of cancer progression, likelihood of developing one or more cancer symptoms, molecular diagnostics, NGS pathology, and / or treatment options for the individual. In some embodiments, the bait composition comprises several different display portions. In some embodiments, each of the multiple different display portions in the bait composition (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display portions, each containing a different MHC molecule) contains a different antigenic peptide. In some embodiments, each of the different display portions containing a different MHC molecule contains a different detectable label (e.g., a fluorophore). In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells bound to each of the different display portions containing different MHC molecules into different populations, optionally including contacting each of the multiple different display portions with a sample from an organism and separating the immune cells bound to each of the different display portions. In some embodiments, the method further includes culturing the immune cells before the analysis step.In some embodiments, the display portion includes particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the display portion further includes a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of one or more nucleic acids includes whole-genome sequencing, RNA sequencing, and / or subjecting immune cells to mass spectrometry.

[0238] In some embodiments, a method is provided for detecting residual cancer in an individual, wherein the individual has been previously treated with anticancer therapy and has not shown pathological symptoms of cancer after treatment, and the method includes analyzing a post-treatment sample from the individual. In some embodiments, the method includes a) analyzing a pre-treatment sample from an individual prior to anticancer therapy and a post-treatment sample from the individual according to one of the methods described herein, and b) comparing the characteristics of immune cells from the pre-treatment sample with the characteristics of immune cells isolated from the post-treatment sample. In some embodiments, a predetermined difference between the characteristics of immune cells from the pre-treatment sample and the characteristics of immune cells from the post-treatment sample indicates residual cancer in the individual. In some embodiments, the predetermined characteristics of immune cells include the presence of immune cells and, optionally, further, an amount of immune cells above a threshold level. In some embodiments, the predetermined characteristics of immune cells include gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and / or histone glycosylation characteristics). In some embodiments, the method includes a) contacting a pre-treatment sample and / or a post-treatment sample with a bait composition comprising a display portion containing cancer antigen peptides under conditions sufficient to allow immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; b) separating the immune cells bound to the display portion; and c) analyzing the immune cells. In some embodiments, the display portion contains two or more (e.g., four) antigen peptides. In some embodiments, the two or more antigen peptides in the display portion are identical.In some embodiments, the antigen peptide has one or more of the following characteristics: a) binding affinity to MHC molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) binding affinity to congener TCR molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) mutations relative to the wild-type peptide, and optionally mutations at the third amino acid position from the N-terminus relative to the wild-type peptide; d) hydrophobicity; and e) a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion includes MHC molecules that form a complex with the antigen peptide. In some embodiments, the MHC molecules are MHC class I molecules and / or MHC class II molecules. In some embodiments, the immune cells are isolated single immune cells. In some embodiments, the immune cells are present in a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture comprising T cells, memory T cells, macrophages, or dendritic cells, or a combination thereof. In some embodiments, further analysis of immune cells includes detecting and / or quantifying the immune cells. In some embodiments, further analysis of immune cells includes sequencing one or more nucleic acids within the immune cells, and optionally further includes analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences). In some embodiments, further analysis of immune cells further includes identifying one or more epigenetic modifications in the immune cells (e.g., methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further includes generating a report containing information about the cancer status in an individual.In some embodiments, information regarding the state of cancer includes cancer classification, cancer type, cancer nature, cancer origin, cancer stage, likelihood of cancer progression, likelihood of developing one or more cancer symptoms, molecular diagnostics, NGS pathology, and / or treatment options for the individual. In some embodiments, the bait composition comprises multiple different display portions. In some embodiments, each of the multiple different display portions in the bait composition (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display portions, each containing a different MHC molecule) contains a different antigenic peptide. In some embodiments, each of the different display portions containing different MHC molecules contains a different detectable label (e.g., a fluorophore). In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells that bind to each of the different display portions containing different MHC molecules into different populations, optionally including contacting each of the multiple different display portions with a sample from the individual and separating the immune cells that bind to each of the different display portions. In some embodiments, the method further includes culturing immune cells prior to the analytical step. In some embodiments, the display portion includes particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the display portion further includes a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of one or more nucleic acids includes whole-genome sequencing, RNA sequencing, and / or subjecting the immune cells to mass spectrometry.

[0239] Aggregation test

[0240] In some embodiments, the methods described herein (e.g., methods for analyzing immune cells or detecting antigen-specific immune cells) involve performing an agglutination test (e.g., including). In some embodiments, the agglutination test involves observing the aggregation of cells under a microscope or with the naked eye. In some embodiments, the number of immune cells is about 10 8 Below, 5x10 7 The following 10 7 Below, 5x10 6 Below, 2x10 6 The following 10 6 Below, 5x10 5 Below, 2x10 5 The following 10 5 Below, 5x10 4 Below, 4x10 4 Below, 2x10 4 The following, or 10 4 The following applies:

[0241] Agglutination testing is a type of bioanalytical method used to detect the presence (qualitative) of a substance in a sample, and, in some cases, the quantity (quantitative) of that substance. The process typically involves the agglutination or aggregation of particles that respond to specific substances, such as antigens, antibodies, or cells. In some embodiments, agglutination reactions occur with specially designed particles and are often used in a variety of diagnostic applications, including blood typing, pathogen detection, and antibody level measurement.

[0242] In some embodiments, the agglutination test involves mixing a sample potentially containing the antigen of interest with an antibody or other binder. Upon the presence of the antigen, it binds to the antibody, causing particles to agglutinate or aggregate. This agglutination can be directly observed or quantified using various detection methods. In some embodiments, the reaction is controlled to avoid nonspecific agglutination, which may lead to false-positive results. In some embodiments, various buffers and additives can be used to achieve desired reaction conditions, including pH, ionic strength, and temperature. In some embodiments, the test can be performed in various forms (e.g., slide agglutination, tube agglutination, or microplate agglutination) depending on the type of sample, target concentration, and required processing volume.

[0243] In some embodiments, latex bead agglutination tests (LBATs) are used, with latex beads being used as carrier particles for binders. In some embodiments, such latex beads offer several advantages, such as uniform size, shape, and surface properties that can be precisely controlled to optimize test performance. In some embodiments, latex beads can be functionalized with various chemical groups and further bound to suitable reagents such as antibodies or antigens, or the latex beads can be passively bound to such reagents.

[0244] In some embodiments, LBAT includes bead functionalization. Latex beads are functionalized with the required binder. This may include steps of chemical activation, coupling chemistry, and purification. In some embodiments, LBAT includes sample preparation. The sample is prepared according to the specific requirements of the test. This may include dilution, filtration, or other pretreatment steps. In some embodiments, LBAT includes a mixing reaction. That is, the functionalized beads are mixed with the sample in a controlled environment. Buffers, stabilizers, or other additives may be used to ensure optimal reaction conditions. In some embodiments, LBAT includes incubation. The reaction mixture is incubated to allow time for aggregation to occur. Temperature, time, and stirring may be adjusted to achieve the desired reaction rate. In some embodiments, LBAT includes detection. Aggregation is observed or quantified using a suitable detection method. Such detection methods may include visual inspection, microscopy, or instrumental analysis such as spectrophotography.

[0245] In some embodiments, using latex beads in agglutination tests makes it possible to customize the beads to meet the specific requirements of various applications, including infectious disease diagnosis, immunological research, and food testing.

[0246] Latex beads, common particles developed in the mid-20th century, have played a significant role in advancements in diagnostics and molecular biology. In some embodiments, such beads are synthesized by emulsion polymerization, in which monomers are polymerized in an aqueous phase to form polymer beads. In some embodiments, such processes often involve the use of stabilizers and surfactants to control the size and shape of the particles. In some embodiments, further purification in the synthesis method allows for improved uniformity and control of the physical properties of the particles, such as size, shape, and surface charge.

[0247] In some embodiments, latex beads have a core made from a synthetic polymer, with polystyrene being the most common core material. In some embodiments, latex beads are best known for their structural strength and, furthermore, for their chemical and physical inertness when interacting with biological systems. In some embodiments, other types of latex beads are synthesized using polymers such as poly(methyl methacrylate) (PMMA) and polylactic acid-glycolic acid copolymer (PLGA), which are biodegradable polymers known for their use in drug delivery applications. In some embodiments, the size of the latex beads can be precisely controlled, typically in the range of about 20 nanometers to about 100 micrometers in diameter. In some embodiments, the core of the beads often incorporates a crosslinking agent such as divinylbenzene (DVB). In some embodiments, crosslinking imparts a three-dimensional network within the polymer, significantly enhancing the structural integrity and solvent resistance of the beads.

[0248] In some embodiments, the latex beads have a diameter of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 μm. In some embodiments, the latex beads have a diameter of about 0.01 to about 150 μm, or 0.1 to about 100 μm (e.g., about 0.1 to about 50 μm, e.g., about 0.5 μm to about 20 μm, e.g., about 0.5 μm to about 10 μm, e.g., about 0.5 μm to about 5 μm, e.g., about 0.5 μm to about 2.5 μm). In some embodiments, the latex beads have a diameter of about 0.1 to about 10 μm. In some embodiments, the latex beads have a diameter of about 0.5 to about 2 μm. In some embodiments, the latex beads have a diameter of about 1 μm.

[0249] In some embodiments, surface modification of latex beads is central to their usefulness in various applications. Such surface modification allows the beads to be tailored to specific test requirements. In some embodiments, beads can be designed to bind to various biological targets such as antibodies, proteins, carbohydrates, DNA, or RNA by introducing various surface functional groups. In some embodiments, carboxylated beads provide a reactive surface for covalent bonding of biomolecules, for example, and are ideal for the immobilization of proteins or antibodies by carbodiimide chemistry. In some embodiments, amine-functionalized beads have primary amine groups that can react with various target molecules and are commonly used for the binding of oligonucleotides and peptides. In some embodiments, aldehyde-functionalized beads have the ability to react with amine groups to form Schiff bases, which are suitable for protein coupling. Hydroxyl beads, on the other hand, can be used for further modification or as a blank control. In some embodiments, special functionalization options include magnetic beads, fluorescently labeled beads, and biotinylated beads that can be bound to streptavidin or avidin, which improve the adaptability and responsiveness of latex beads in a wide range of applications.

[0250] Treatment method

[0251] In some embodiments, a method is provided for treating cancer in an individual (e.g., an individual who has never been diagnosed with cancer, an individual at risk of developing cancer, an individual who has previously received cancer treatment), the method comprising: a) diagnosing the individual with cancer according to the method herein; and b) administering anticancer therapy to the individual. In some embodiments, diagnosing the individual comprises: a) obtaining a sample from the individual; b) contacting the sample with a bait composition comprising a display portion containing a cancer antigen peptide under conditions sufficient to allow immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; c) separating the immune cells bound to the display portion; and d) analyzing the immune cells, where a predetermined characteristic of the immune cells indicates cancer in the individual. In some embodiments, a predetermined characteristic of the immune cells includes the presence of immune cells. In some embodiments, a predetermined characteristic of the immune cells includes a quantity of immune cells above a threshold level. In some embodiments, predetermined characteristics of immune cells include gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and / or histone glycosylation characteristics). In some embodiments, the display portion contains two or more (e.g., four) antigen peptides. In some embodiments, the two or more antigen peptides in the display portion are identical. In some embodiments, the antigen peptide has one or more of the following characteristics: a) having a binding affinity of about 1 nM to about 5000 nM to MHC molecules (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) having a binding affinity of about 1 nM to about 5000 nM to congeneral TCR molecules (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) having a mutation relative to the wild-type peptide, and in some cases having a mutation at the third amino acid position from the N-terminus relative to the wild-type peptide; d) being hydrophobic; and e) having a high content of aromatic residues.In some embodiments, the antigen peptide is of low immunogenicity. In some embodiments, the display portion includes an MHC molecule that forms a complex with the antigen peptide. In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, the immune cell is an isolated single immune cell. In some embodiments, the immune cell is present in a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture including T cells, memory T cells, macrophages, or dendritic cells, or a combination thereof. In some embodiments, further analysis of the immune cell includes detecting and / or quantifying the immune cell. In some embodiments, further analysis of the immune cell includes sequencing one or more nucleic acids within the immune cell, and optionally further includes analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences). In some embodiments, further analysis of the immune cell includes identifying one or more epigenetic modifications in the immune cell (e.g., methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further includes generating a report containing information about the cancer status of an individual. In some embodiments, the information about the cancer status includes cancer classification, cancer type, cancer nature, cancer origin, cancer stage, likelihood of cancer progression, likelihood of developing one or more cancer symptoms, molecular diagnostics, NGS pathology, and / or treatment options for the individual. In some embodiments, the bait composition comprises a plurality of different display moieties. In some embodiments, each of the plurality of different display moieties in the bait composition (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display moieties, each containing a different MHC molecule) contains a different antigenic peptide. In some embodiments, each of the different display moieties containing different MHC molecules contains a different detectable label (e.g., a fluorophore).In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells bound to each of different display portions containing different MHC molecules into different populations, and optionally including contacting each of a plurality of different display portions with a sample from an organism and separating the immune cells bound to each of the different display portions. In some embodiments, the method further includes culturing the immune cells before the analysis step. In some embodiments, the display portion includes particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the display portion further includes a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of one or more nucleic acids includes whole-genome sequencing, RNA sequencing, and / or subjecting the immune cells to mass spectrometry.

[0252] In some embodiments, anticancer therapy is a standard or commonly used drug or therapy for treating cancer (e.g., a specific type of cancer). In some embodiments, anticancer therapy involves chemotherapeutic agents. In some embodiments, anticancer therapy involves surgery. In some embodiments, anticancer therapy involves radiotherapy. In some embodiments, anticancer therapy involves immunotherapy. In some embodiments, anticancer therapy involves cell therapy (e.g., cell therapy involving immune cells (e.g., CAR T cells)). In some embodiments, anticancer therapy involves angiogenesis inhibitors.

[0253] In some embodiments, anti-cancer therapy is not immunotherapy.

[0254] In some embodiments, the individual has minimal residual disease (MRD). In some embodiments, the individual has minimal residual carcinoma. In some embodiments, minimal residual carcinoma is present after cancer has been surgically removed or treated. In some embodiments, minimal residual disease is too small to be detected by imaging equipment (e.g., routinely used or standard imaging equipment for cancer detection). In some embodiments, the location of minimal residual disease is diverse. In some embodiments, minimal residual carcinoma is a result of immune evasion or resistance to treatment. In some embodiments, the individual has previously received cancer treatment and has not shown any pathological symptoms of cancer after treatment.

[0255] In some embodiments, a method is provided for treating an infection in an individual (e.g., an individual not diagnosed with an infection by a pathogen (e.g., a virus, bacteria, or fungus), e.g., an individual at risk of developing an infection, e.g., an individual who has previously received treatment for an infection), the method comprising: a) diagnosing the individual with an infection according to the method herein; and b) administering therapy for the infection to the individual. In some embodiments, diagnosing the individual comprises: a) obtaining a sample from the individual; b) contacting the sample with a bait composition comprising a display portion containing an antigenic peptide of the infection under conditions sufficient to allow immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; c) separating the immune cells bound to the display portion; and d) analyzing the immune cells, where predetermined characteristics of the immune cells indicate an infection in the individual. In some embodiments, predetermined characteristics of the immune cells include the presence of immune cells. In some embodiments, predetermined characteristics of the immune cells include a quantity of immune cells above a threshold level. In some embodiments, predetermined characteristics of immune cells include gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and / or histone glycosylation characteristics). In some embodiments, the display portion contains two or more (e.g., four) antigen peptides. In some embodiments, the two or more antigen peptides in the display portion are identical.In some embodiments, the antigen peptide has one or more of the following characteristics: a) binding affinity to MHC molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) binding affinity to congener TCR molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) mutations relative to the wild-type peptide, and optionally mutations at the third amino acid position from the N-terminus relative to the wild-type peptide; d) hydrophobicity; and e) a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion includes MHC molecules that form a complex with the antigen peptide. In some embodiments, the MHC molecules are MHC class I molecules and / or MHC class II molecules. In some embodiments, the immune cells are isolated single immune cells. In some embodiments, the immune cells are present in a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture comprising T cells, memory T cells, macrophages, or dendritic cells, or a combination thereof. In some embodiments, further analysis of immune cells includes detecting and / or quantifying the immune cells. In some embodiments, further analysis of immune cells includes sequencing one or more nucleic acids within the immune cells, and optionally further includes analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences). In some embodiments, further analysis of immune cells further includes identifying one or more epigenetic modifications in the immune cells (e.g., methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further includes generating a report containing information about the infectious disease status in the individual.In some embodiments, information regarding the state of an infection includes the classification of the infection, the type of infection, the nature of the infection, the origin of the infection, the stage of the infection, the likelihood of the infection progressing, the likelihood of developing symptoms of one or more infections, molecular diagnostics, NGS pathology, and / or treatment options for the individual. In some embodiments, the bait composition comprises a plurality of different display portions. In some embodiments, each of the plurality of different display portions in the bait composition (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display portions, each containing a different MHC molecule) contains a different antigenic peptide. In some embodiments, each of the different display portions containing a different MHC molecule contains a different detectable label (e.g., a fluorophore). In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells that bind to each of the different display portions containing different MHC molecules into different populations, optionally including contacting each of the plurality of different display portions with a sample from an individual and separating the immune cells that bind to each of the different display portions. In some embodiments, the method further includes culturing immune cells prior to the analytical step. In some embodiments, the display portion includes particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the display portion further includes a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of one or more nucleic acids includes whole-genome sequencing, RNA sequencing, and / or subjecting the immune cells to mass spectrometry.

[0256] In some embodiments, a method is provided for treating an autoimmune disease or autoimmune illness in an individual (e.g., an individual not diagnosed with an autoimmune disease or autoimmune illness, e.g., an individual at risk of developing an autoimmune disease or autoimmune illness, e.g., an individual who has previously received treatment for an autoimmune disease or autoimmune illness), the method comprising: a) diagnosing the individual with an autoimmune disease or autoimmune illness according to the method described herein; and b) administering therapy to the individual for the autoimmune disease or autoimmune illness. In some embodiments, diagnosing the individual comprises: a) obtaining a sample from the individual; b) contacting the sample with a bait composition containing a display portion containing an antigenic peptide of the autoimmune disease or autoimmune illness under conditions sufficient to allow immune cells (e.g., T cells, cytotoxic T cells, helper T cells, memory T cells, and / or tumor-infiltrating T cells) to bind to the display portion; c) separating the immune cells bound to the display portion; and d) analyzing the immune cells, where predetermined characteristics of the immune cells indicate an autoimmune disease or autoimmune illness in the individual. In some embodiments, predetermined characteristics of the immune cells include the presence of immune cells. In some embodiments, a predetermined characteristic of immune cells includes a quantity of immune cells above a threshold level. In some embodiments, a predetermined characteristic of immune cells includes gene expression profile characteristics, gene mutation profile characteristics, and / or epigenetic modification characteristics (e.g., DNA methylation characteristics and / or histone glycosylation characteristics). In some embodiments, the display portion includes two or more (e.g., four) antigen peptides. In some embodiments, the two or more antigen peptides in the display portion are identical.In some embodiments, the antigen peptide has one or more of the following characteristics: a) binding affinity to MHC molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); b) binding affinity to congener TCR molecules of about 1 nM to about 5000 nM (e.g., about 1 nM to about 50 nM, about 50 nM to about 500 nM, about 500 nM to about 5000 nM); c) mutations relative to the wild-type peptide, and optionally mutations at the third amino acid position from the N-terminus relative to the wild-type peptide; d) hydrophobicity; and e) a high content of aromatic residues. In some embodiments, the antigen peptide has low immunogenicity. In some embodiments, the display portion includes MHC molecules that form a complex with the antigen peptide. In some embodiments, the MHC molecules are MHC class I molecules and / or MHC class II molecules. In some embodiments, the immune cells are isolated single immune cells. In some embodiments, the immune cells are present in a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture comprising T cells, memory T cells, macrophages, or dendritic cells, or a combination thereof. In some embodiments, further analysis of immune cells includes detecting and / or quantifying the immune cells. In some embodiments, further analysis of immune cells includes sequencing one or more nucleic acids within the immune cells, and optionally further includes analyzing the sequences of one or more nucleic acids (e.g., TCR-related sequences). In some embodiments, further analysis of immune cells further includes identifying one or more epigenetic modifications in the immune cells (e.g., methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further includes generating a report containing information about the autoimmune disease or autoimmune disorder status in an individual.In some embodiments, information regarding the state of an autoimmune disease or autoimmune illness includes the classification of the autoimmune disease or autoimmune illness, the type of autoimmune disease or autoimmune illness, the nature of the autoimmune disease or autoimmune illness, the origin of the autoimmune disease or autoimmune illness, the stage of the autoimmune disease or autoimmune illness, the likelihood of progression of the autoimmune disease or autoimmune illness, the likelihood of developing symptoms of one or more autoimmune diseases or autoimmune illnesses, molecular diagnostics, NGS pathology, and / or treatment options for the individual. In some embodiments, the bait composition comprises multiple different display moieties. In some embodiments, each of the multiple different display moieties in the bait composition (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different display moieties, each containing a different MHC molecule) contains a different antigenic peptide. In some embodiments, each of the different display moieties containing different MHC molecules contains a different detectable label (e.g., a fluorophore). In some embodiments, the separation step includes using fluorescence-activated cell sorting (FACS) and / or separating immune cells bound to each of different display portions containing different MHC molecules into different populations, and optionally including contacting each of a plurality of different display portions with a sample from an organism and separating the immune cells bound to each of the different display portions. In some embodiments, the method further includes culturing the immune cells before the analysis step. In some embodiments, the display portion includes particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the display portion further includes a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of one or more nucleic acids includes whole-genome sequencing, RNA sequencing, and / or subjecting the immune cells to mass spectrometry.

[0257] Bait composition, display portion, MHC-peptide complex, and antigen peptide

[0258] In some embodiments, this application provides compositions or bait compositions comprising one or more display moieties. The bait compositions described herein comprise one or more display moieties comprising one or more antigen peptides (e.g., comprising one or more MHC-peptide complexes). In the bait composition, immune cells (e.g., T cells) bind to the display moieties and / or antigen peptides. Either such bait composition or display moiety can be used in the sample analysis methods, antigen-specific immune cell detection methods, and therapeutic methods described herein.

[0259] Display portion (i.e., MHC polymer unit)

[0260] In some embodiments, the display portion described herein includes an MHC molecule that forms a complex with an antigen peptide (e.g., a neoantigen peptide, e.g., a truncal neoantigen peptide).

[0261] In one embodiment, this application provides a display portion comprising a carrier (e.g., a particle) to which a plurality of MHC-peptide complexes (e.g., MHC-peptide monomers) are bound, wherein at least two of the MHC-peptide complexes are different.

[0262] In some embodiments, the display portion comprises a carrier (e.g., a particle) to which multiple MHC-peptide complexes are bound, where the multiple MHC-peptide complexes comprise two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of which comprises at least one MHC molecule that forms a complex with a peptide, and at least two or more of the MHC-peptide complexes are different.

[0263] In some embodiments, the display portion does not contain either fluorescent dyes or fluorophores.

[0264] In some embodiments, the present application provides a display portion comprising parti...

Claims

1. A method for analyzing samples from one or more individuals to determine the presence or absence of immune cells capable of binding to antigen peptides, a) Immobilizing immune cells from the sample onto a solid carrier, 1) Before immobilization, the bait composition containing the display portion containing the antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion, or 2) After immobilization, the bait composition containing the display portion containing the antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion, b) Bringing the detection agent that recognizes the display portion and the immobilized immune cells into contact under conditions sufficient for the detection agent to bind to the display portion, c) detecting the immune cells bound to the display portion by detecting the detection agent, The method further comprises, depending on the circumstances, separating immune cells bound to the display portion, or removing the display portion or immune cells not bound to each other, before step c).

2. A method for analyzing samples from one or more individuals to determine the presence or absence of immune cells capable of binding to antigen peptides, a) Bringing the bait composition containing the antigen peptide display portion and the immune cells into contact under conditions sufficient for the immune cells to bind to the display portion, b) Bringing the detection agent that recognizes the display portion and the immune cells into contact under conditions sufficient for the detection agent to bind to the display portion, c) Immobilizing the immune cells from the sample onto a solid carrier, d) detecting the immune cells bound to the display portion by detecting the detection agent, The method further comprises, depending on the circumstances, separating immune cells bound to the display portion, or removing the display portion or immune cells not bound to each other, before step d).

3. The method according to claim 1 or claim 2, wherein the sample is obtained from a single individual.

4. The method according to claim 1 or 2, wherein the sample is a mixture of multiple samples of individuals obtained from different individuals, and optionally further comprises processing each of the multiple samples of individuals separately before combining them to form the mixture to associate the immune cells contained therein with a unique sample barcode, optionally removing unbound molecules including the barcode, optionally further comprising identifying the sample from which the immune cells originate by identifying the unique barcode associated with the immune cells bound to the display portion.

5. a) Before immobilization, the bait composition containing the display portion containing the antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion, or b) After immobilization, the bait composition containing the display portion containing the antigen peptide and the immune cells are brought into contact under conditions sufficient for the immune cells to bind to the display portion.

6. A method for analyzing multiple samples from one or more individuals to determine the presence or absence of immune cells capable of binding to antigen peptides, a) Processing each of the multiple samples of the individual separately and associating the immune cells contained therein with a unique sample barcode, b) Combining the above-mentioned multiple processed samples to prepare a sample mixture, c) Preparing a bait-sample mixture by contacting a bait composition containing an antigen peptide display portion with the sample mixture under conditions sufficient for immune cells to bind to the display portion, d) To detect immune cells bound to the display portion in the bait-sample mixture, e) Identifying the sample from which the immune cells originate by identifying the unique barcode associated with the immune cells bound to the display portion, The method further comprises, depending on the circumstances, separating immune cells bound to the display portion, or removing the display portion or immune cells not bound to each other, before step d).

7. The method according to any one of claims 1 to 6, wherein the immune cells include T cells, and optionally the immune cells include T cells that specifically bind to the antigen peptide in the bait composition in an amount of about 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, and optionally the immune cells are selected from the group consisting of cytotoxic T cells, memory T cells, and tumor-infiltrating T cells.

8. The method according to any one of claims 1 to 7, further comprising bringing the agent that recognizes the display portion into contact with the immune cells, and then removing the unbound display portion before detecting the immune cells bound to the display portion.

9. The method according to any one of claims 1 to 8, wherein the antigen peptide is related to a cancer antigen or tumor antigen, optionally the antigen peptide is a neoantigen peptide, and optionally the sample is obtained from one or more individuals that do not exhibit pathological symptoms of cancer.

10. The method according to any one of claims 1 to 5 and 7 to 9, wherein the immune cells are immobilized on a solid substrate such as beads, and optionally, an antibody that recognizes the immune cells is bound to the beads, and optionally, the antibody is a CD8 antibody.

11. The method according to any one of claims 1 to 10, wherein the display portion comprises two or more antigen peptides, and optionally comprises four antigen peptides, and optionally the two or more antigen peptides of the display portion are the same.

12. The method according to any one of claims 9 to 11, wherein the display portion comprises a library of antigen peptides for cancer or tumor-related mutations, and the library comprises at least about 5, 10, 12, 15, 18, or 20 different neoantigen peptides.

13. The method according to any one of claims 1 to 12, wherein the display portion includes an MHC molecule that forms a complex with the antigen peptide (i.e., an MHC-peptide complex), the MHC molecule includes an MHC class I molecule, optionally the MHC molecule includes an MHC polymer, and optionally the MHC polymer is an MHC dimer, an MHC trimer, an MHC tetramer, or an MHC dextramer.

14. The display portion is a) A group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) The method according to claim 13, comprising two or more different types of MHC class I molecules selected from the group consisting of HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and HLA-A*33:

03.

14. The method according to any one of claims 1 to 13, wherein the display portion comprises particles, and optionally the particles of the display portion are selected from the group consisting of surfaces, nanoparticles, beads, and polymers.

15. The method according to claim 14, wherein the particles include beads, and the beads have a diameter of at least about 0.1 μm, 0.25 μm, 0.5 μm, or 1 μm.

16. The method according to any one of claims 14 to 15, wherein the antigen peptide or MHC is a) directly bound to the particle of the display portion, or b) bound to the particle of the display portion via a binding pair, the binding pair comprising a first binding component bound to the antigen peptide or MHC and a second binding component bound to the particle.

17. a) separating immune cells bound to the display portion, or removing immune cells not bound to the display portion or to each other, and / or b) determining the phenotype of the immune cells bound to the display portion, the method according to any one of claims 1 to 16.

18. The method according to any one of claims 1 to 17, wherein the individual is a human.

19. a) The individual has not been previously diagnosed with cancer, and may be at risk of developing cancer, or b) The method according to any one of claims 9 to 18, wherein the individual has previously received treatment for cancer and does not exhibit any pathological symptoms of cancer after such treatment.

20. The method according to any one of claims 1 to 19, wherein the sample is selected from the group consisting of blood, plasma, and peripheral blood mononuclear cell (PBMC) samples.

21. The method according to any one of claims 1 to 20, wherein the bait composition comprises a plurality of different display portions, and optionally the plurality of different display portions in the bait composition comprises at least two different display portions, and each of the at least two different display portions comprises a different MHC molecule.

22. A method for detecting antigen-specific immune cells in an individual, comprising analyzing a sample from the individual according to any one of claims 1 to 21, The method wherein predetermined characteristics of the immune cells indicate a disease or disorder in the individual.

23. The method according to claim 22, wherein the predetermined characteristics of the immune cells include the presence of the immune cells, and optionally a) the presence of immune cells that specifically bind to a bait composition containing one or more neoantigens indicates the presence of cancer, b) the presence of immune cells that specifically bind to a bait composition containing one or more antigens of a pathogen (e.g., a virus, a bacterium, a fungus) indicates the presence of a pathogen infection, or c) the presence of immune cells that specifically bind to a bait composition containing one or more autoantigens associated with an autoimmune disease or autoimmune disorder indicates the presence of an immune disease or immune disorder.

24. A method for detecting residual cancer in an individual that has been previously treated with anticancer therapy and does not show pathological symptoms of cancer after treatment, comprising analyzing a post-treatment sample from the individual according to the method described in any one of claims 9 to 21, The method wherein predetermined characteristics of the immune cells from the post-treatment sample indicate residual cancer in the individual.

25. A method for treating cancer in an individual, a) Diagnosing the individual having cancer according to the method of claim 23, b) The method comprising administering anticancer therapy to the individual.

26. A method for treating infectious diseases in individuals, a) Diagnosing the individual having an infectious disease caused by a pathogen according to the method of claim 23, b) The method comprising administering treatment to the individual against the pathogen.

27. A method for treating an autoimmune disease or autoimmune disorder in an individual, a) Diagnosing the individual having an autoimmune disease or autoimmune disorder according to the method of claim 23, b) The method comprising administering treatment to the individual for the disease or disorder.

28. A display portion comprising particles to which multiple MHC-peptide complexes are bound, wherein the multiple MHC-peptide complexes comprise two or more, three or more, four or more, five or more, six or more, eight or more, nine or more, ten or more, or twelve or more MHC-peptide complexes, each of the MHC-peptide complexes comprises at least one MHC molecule that forms a complex with a peptide, and at least two or more of the MHC-peptide complexes are different.

29. The display portion according to claim 28, wherein one or more of the plurality of MHC-peptide complexes is an MHC-peptide monomer, an MHC-peptide dimer, an MHC-peptide trimer, an MHC-peptide tetramer, an MHC-peptide pentamer, an MHC-peptide octamer, an MHC-peptide dextramer, or an MHC-peptide dodecamer.

30. The display portion according to claim 28 or 29, wherein at least one of the plurality of MHC-peptide complexes has two different MHC-peptide monomers, and optionally the at least one MHC complex is an MHC-peptide dimer, a peptide trimer, or an MHC-peptide tetramer.

31. The display portion according to any one of claims 28 to 30, wherein the plurality of MHC-peptide complexes comprises at least 20 MHC-peptide complexes, and optionally, the plurality of MHC-peptide complexes comprises at least 50 or 100 MHC-peptide complexes.

32. The display portion according to any one of claims 28 to 31, wherein the MHC-peptide complex is bound to the particle via a binding pair, and the binding pair comprises a first binding component bound to the MHC-peptide complex or a unit thereof, and a second binding component bound to the particle, wherein the first binding component optionally comprises biotin, and the second binding component comprises streptavidin.

33. a) The particle is solid, b) The particles have a diameter of about 0.1 μm to about 50 μm (for example, at least 0.1 μm, 0.2 μm, 0.25 μm, 0.5 μm, 0.75 μm, or 1 μm), and optionally the particles have a diameter of about 0.5 μm to about 20 μm, optionally the particles have a diameter of about 0.5 μm to about 10 μm, and optionally the particles have a diameter of about 1 μm. c) The particles are approximately 1 μm ２ ~about 20μm ２ It has a surface area, d) The particle has about 10 to about 10,000 second binding components bound thereto, e) The particles are spherical, and in some cases the particles are beads, and / or f) The display portion according to any one of claims 28 to 32, wherein the particles are magnetic.

34. The display portion contains at least two different MHC molecules, and optionally, a) The at least two different MHC molecules include two different MHC class I molecules, b) The at least two different MHC molecules comprise two different MHC class II molecules, and / or c) The display portion according to any one of claims 28 to 33, wherein the at least two different MHC molecules include both MHC class I molecules and MHC class II molecules.

35. a) The display portion comprises an HLA-A molecule, and optionally the HLA-A molecule comprises a mutation that reduces its binding to CD8, and optionally the HLA-A molecule comprises an HLA-A2 heavy chain having the A245V mutation. b) The display portion comprises at least two MHC class I molecules selected from a) the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, or b) the group consisting of HLA-A*69:01, HLA-A*31:01, HLA-A*29:01, HLA-A*33:02, HLA-A*02:06, HLA-A*02:07, HLA-A*30:01, HLA-A*01:01, HLA-A*02:03, and / or c) The display portion according to any one of claims 28 to 34, wherein the display portion comprises an MHC class II molecule selected from the group consisting of HLA-DQ and HLA-DR.

36. a) The peptide is related to a cancer antigen or tumor antigen, and optionally, the peptide is related to a neoantigen. b) The peptide is related to an antigen of a pathogen, and the pathogen may be a virus, bacterium, or fungus. c) The peptide is related to an autoantigen, d) The display portion comprises at least two different peptides, optionally comprising at least five different peptides, optionally comprising at least two, five, or ten different peptides, and / or e) The display portion according to any one of claims 28 to 35, wherein the display portion does not contain a fluorescent dye or a fluorophore.

37. A bait composition comprising the display portion according to any one of claims 28 to 36.

38. a) Includes at least two different display sections, b) comprising HLA-A molecules, HLA-B molecules, and HLA-C molecules, c) Including HLA-A*24:02, HLA-A*11:01, HLA-A*02:01, and HLA-A*03:01, d) comprising HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules, e) comprising at least 25, 50, 100, 150, 200, or 250 different peptides, f) The peptide in the bait composition is associated with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 different antigens, g) comprising at least two, three, four, five, six, or seven different duplicate peptides related to gene mutations, wherein the gene is an oncogene, h) The peptide in the bait composition is associated with mutations in at least two, three, four, five, or six genes, and optionally the genes are oncogenes, and / or i) The bait composition according to claim 37, which does not contain a fluorescent dye or a fluorophore.

39. A method for analyzing immune cells in a sample from an individual, a) Contacting a plurality of immune cells with the display portion described in any one of claims 28 to 36 or the bait composition described in claim 37 or claim 38, b) The method comprising detecting or analyzing one or more immune cells bound to one or more of the display portions.

40. a) The immune cells include T cells, b) The immune cells are selected from the group consisting of cytotoxic T cells, memory T cells, and tumor-infiltrating T cells, and optionally the T cells are CD4+ T cells and / or CD8+ T cells. c) The immune cells include B cells and / or d) The method according to claim 39, wherein the immune cells include a mixture of immune cells including T cells, B cells, macrophages, or dendritic cells, or a combination thereof.

41. a) The sample is selected from the group consisting of blood, plasma, and peripheral blood mononuclear cell (PBMC) samples, and / or b) The method according to claim 39 or 40, wherein the sample is obtained from diseased tissue, and optionally the diseased tissue is cancerous tissue or tumor tissue.

42. a) The method according to any one of claims 39 to 41, wherein the sample is obtained from a single individual, or b) the sample is a mixture of multiple samples of individuals obtained from different individuals, respectively.

43. a) the individual is a human, and / or b) the individual does not have signs of disease, and optionally the disease is cancer, the method according to any one of claims 39 to 42.

44. The method according to any one of claims 39 to 43, wherein detecting or analyzing the immune cells bound to one or more display portions includes observing the aggregation of immune cells with the one or more display portions.

45. The method according to any one of claims 39 to 44, wherein detecting or analyzing the immune cells bound to one or more display portions comprises: 1) immobilizing the immune cells on a solid substrate before or after step a); 2) bringing a detection agent that recognizes the display and the immobilized immune cells into contact under conditions sufficient for the detection agent to bind to the display portion; and 3) detecting the immune cells bound to the display portion by detecting the detection agent.

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