How to Analyze Samples for Cancer-Specific Immune Cells

Abstract

The present application provides a method for analyzing a sample from an individual who does not show any pathological symptoms of cancer, comprising contacting the sample with a bait composition having a presentation moiety with a neo-antigenic peptide under conditions sufficient for immune cells to bind to the presentation moiety, and isolating and analyzing the immune cells. Methods for detecting and treating cancer (e.g., residual cancer) are also provided. In some embodiments, the presentation moiety comprises two or more neo-antigenic peptides.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 273,768 (filed October 29, 2021, the contents of which are incorporated by reference in their entirety herein).

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (230062000140SEQLIST.xml; size: 74,317 bytes; creation date: October 25, 2022) are incorporated herein by reference in their entirety.

[0003] Field The present application relates to methods for analyzing a sample for cancer-specific immune cells. [Background technology]

[0004] background The survival prospects for cancer patients are substantially improved if the disease is diagnosed and treated at an early clinical stage. This supports the promise of early detection to improve prognosis. Unfortunately, effective screening tests for early detection do not exist for many cancers.

[0005] On the other hand, a significant proportion of patients who have been successfully treated for cancer have minimal residual disease (MRD) that may progress to metastatic recurrence. MRD is considered to be an essential prognostic factor in predicting the risk of recurrence and in choosing post-remission treatment. However, MRD is often too small to be revealed by even the most sensitive medical imaging modalities, including CT or MRI systems. Therefore, the assessment of minimal residual disease needs to be a very important aspect of tumor monitoring and management in patients, especially those at high risk of disease recurrence.

[0006] The disclosures of all publications, patents, patent applications and published patent applications referenced herein are hereby incorporated by reference in their entirety. Summary of the Invention

[0007] Quick Overview In one aspect, the present application provides a method of analyzing a sample from an individual who does not exhibit any pathological symptoms of cancer, comprising: a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide under conditions sufficient for immune cells to bind to the presentation moiety; b) isolating immune cells associated with the presentation moiety; and c) analyzing the isolated immune cells. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moiety comprises two or more neo-antigenic peptides. In some embodiments, the presentation moiety comprises four neo-antigenic peptides.

[0008] In some embodiments according to any one of the methods described herein, the two or more neo-antigenic peptides in the presentation moiety are the same.

[0009] In some embodiments according to any one of the methods described herein, the neo-antigenic peptide has one or more of the following characteristics: a) has a binding affinity for an MHC molecule 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) has a binding affinity for a cognate TCR molecule (e.g., when bound to an MHC molecule) 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) has a mutation, optionally at the third amino acid position counting from the N-terminus, compared to the wild-type peptide; d) is hydrophobic; and e) has a high content of aromatic residues.

[0010] In some embodiments according to any one of the methods described herein, the neo-antigenic peptide has low immunogenicity.

[0011] In some embodiments according to any one of the methods described herein, the presentation moiety comprises an MHC molecule complexed with the neo-antigenic peptide. In some embodiments, the MHC molecule is 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. In some embodiments, the peptide is about 8 to about 10 amino acids in length. In some embodiments, the MHC is 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 neo-antigenic peptide is about 10 to about 20 amino acids in length.

[0012] In some embodiments according to any one of the methods described herein, the presenting moiety comprises a particle. In some embodiments, the particle is selected from the group consisting of a surface, a nanoparticle, a bead, and a polymer. In some embodiments, the particle is a dextran particle. In some embodiments, the particle is a magnetic nanoparticle or a polystyrene nanoparticle. In some embodiments, the particle is an agarose bead or a sepharose bead.

[0013] In some embodiments according to any one of the methods described herein, the neo-antigenic peptide or MHC is directly attached to the particle.

[0014] In some embodiments according to any one of the methods described herein, the neo-antigenic peptide or MHC is attached to the particle via a binding pair comprising a first binding moiety that is attached to the neo-antigenic peptide and a second binding moiety that is bound to the particle.

[0015] In some embodiments according to any one of the methods described herein, the presentation portion comprises a cell. In some embodiments, the cell comprises a polynucleotide encoding the neo-antigenic peptide. In some embodiments, the polynucleotide encodes a plurality of neo-antigenic peptides.

[0016] In some embodiments of any one of the methods described herein, the presenting moiety further comprises a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS).

[0017] In some embodiments according to any one of the methods described herein, the isolating step comprises separating immune cells associated with the presenting moiety from the remainder of the sample.

[0018] In some embodiments according to any one of the methods described herein, the isolated immune cells are selected from the group consisting of cytotoxic T cells, memory T cells, and tumor infiltrating T cells.

[0019] In some embodiments according to any one of the methods described herein, the isolated immune cell is an isolated single immune cell.

[0020] In some embodiments according to any one of the methods described herein, the isolated immune cells are present in a mixture of immune cells, hi some embodiments, the mixture of immune cells is a mixture that includes T cells, memory T cells, macrophage cells, dendritic cells, or a combination thereof.

[0021] In some embodiments according to any one of the methods described herein, analyzing the isolated immune cells comprises detecting the isolated immune cells.

[0022] In some embodiments according to any one of the methods described herein, analyzing the isolated immune cells comprises quantifying the isolated immune cells in the sample or enriched sample (e.g., including quantifying each of the different types of immune cells collectively or separately).

[0023] In some embodiments according to any one of the methods described herein, analyzing the isolated immune cells comprises sequencing one or more nucleic acids in the isolated immune cells. In some embodiments, analyzing the isolated immune cells further comprises analyzing the sequence of the one or more nucleic acids. In some embodiments, the one or more nucleic acids comprise a nucleic acid sequence that is a TCR sequence. In some embodiments, analyzing the isolated immune cells comprises sequencing a plurality of nucleic acids in the isolated immune cells to obtain a desired nucleic acid profile (e.g., a gene expression profile, a gene mutation profile, or an epigenetic modification profile (e.g., a methylation profile)). In some embodiments, analyzing the isolated immune cells comprises sequencing one or more nucleic acids in a plurality of isolated immune cells. In some embodiments, analyzing the isolated immune cells comprises sequencing a plurality of nucleic acids in a plurality of isolated immune cells to obtain a desired nucleic acid profile (e.g., a gene expression profile, a gene mutation profile, or an epigenetic modification profile (e.g., a methylation profile)).

[0024] In some embodiments according to any one of the methods described herein, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing.

[0025] In some embodiments according to any one of the methods described herein, analyzing the sequence of the one or more nucleic acids comprises RNAseq sequencing.

[0026] In some embodiments according to any one of the methods described herein, analyzing the sequence of the one or more nucleic acids comprises a) obtaining an enriched sample from the isolated immune cells that is enriched for the one or more nucleic acids, and b) sequencing the one or more nucleic acids in the enriched sample.

[0027] In some embodiments according to any one of the methods described herein, analyzing the isolated immune cells further comprises subjecting the isolated immune cells to analysis by mass spectrometry, e.g., to obtain an epigenetic modification profile of the isolated immune cells.

[0028] In some embodiments of any one of the methods described herein, analyzing the isolated immune cells further comprises identifying one or more epigenetic modifications in the isolated immune cells. In some embodiments, the one or more epigenetic modifications comprise DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, and / or glycosylation).

[0029] In some embodiments of any one of the methods described herein, the individual has not been previously diagnosed with cancer. In some embodiments, the individual is at risk of developing cancer. In some embodiments, the individual has previously been treated for cancer and does not show any pathological symptoms of cancer after the treatment.

[0030] In some embodiments according to any one of the methods described herein, the individual is a human. In some embodiments, the individual is at least about 50 years old, at least about 55 years old, at least about 60 years old, at least about 65 years old, at least about 70 years old, at least about 75 years old, or at least about 80 years old.

[0031] In some embodiments according to any one of the methods described herein, the sample is selected from the group consisting of a blood, plasma, and a peripheral blood mononuclear cell (PMBC) sample.

[0032] In some embodiments of any one of the methods described herein, the method further comprises generating a report comprising information regarding the cancer status in the individual. In some embodiments, the information regarding the cancer status comprises: cancer classification; cancer type; cancer nature; cancer origin; cancer stage; cancer progression likelihood; likelihood of developing one or more cancer symptoms; molecular diagnosis; NGS pathology; and / or treatment options for the individual.

[0033] In some embodiments according to any one of the methods described herein, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide. In some embodiments, the plurality of different presentation moieties in the bait composition comprises at least two different presentation moieties each comprising a different MHC molecule. In some embodiments, the plurality of different presentation moieties in the bait composition comprises at least four different presentation moieties each comprising a different MHC molecule. In some embodiments, the plurality of different presentation moieties in the bait composition comprises at least 100 different presentation moieties each comprising a different MHC molecule.

[0034] In some embodiments according to any one of the methods described herein, each of the different presentation moieties comprising different MHC molecules comprises a different detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS).

[0035] In some embodiments according to any one of the methods described herein, the isolating step comprises separating immune cells associated with each of the different presentation moieties that comprise different MHC molecules into different populations.

[0036] In some embodiments of any one of the methods described herein, the method comprises separately contacting each of a plurality of different presentation moieties with a sample from the individual, and isolating the immune cells associated with each of the different presentation moieties.

[0037] In some embodiments, the method comprises: a) analyzing a pre-treatment sample from the individual prior to anti-cancer treatment and a post-treatment sample from the individual according to any one of the methods described herein; and b) identifying differences in a characteristic of the isolated immune cells from the pre-treatment sample and a characteristic of the isolated immune cells from the post-treatment sample.

[0038] In another aspect, the present application provides a method of detecting cancer in an individual, comprising analyzing a sample from the individual according to any one of the methods described herein, wherein a predetermined characteristic of the isolated immune cells is indicative of cancer in the individual. In some embodiments, the predetermined characteristic of the isolated immune cells comprises the presence of the isolated immune cells. In some embodiments, the predetermined characteristic of the isolated immune cells comprises an amount of the isolated immune cells above a threshold level.

[0039] In some embodiments of any one of the methods described herein, the predetermined characteristic of the isolated immune cells comprises a gene expression profile signature, a gene mutation profile signature, and / or an epigenetic modification signature. In some embodiments, the signature epigenetic modification comprises a DNA or RNA methylation, a hydroxymethylation signature, and / or a histone modification signature.

[0040] In some embodiments of any one of the methods of detecting cancer in an individual described herein, wherein the individual has previously been treated with an anti-cancer therapy and does not show any pathological symptoms of cancer after treatment, the method comprises analyzing a post-treatment sample from the individual according to the methods described herein, wherein a predetermined characteristic of the isolated immune cells from the post-treatment sample is indicative of residual cancer in the individual. In some embodiments, the method comprises a) analyzing a pre-treatment sample from the individual prior to anti-cancer therapy and a post-treatment sample from the individual according to the methods described herein, and b) comparing a characteristic of the isolated immune cells from the pre-treatment sample with a characteristic of the isolated immune cells from the post-treatment sample, wherein a predetermined difference in a characteristic of the isolated immune cells from the pre-treatment sample and a characteristic of the isolated immune cells from the post-treatment sample is indicative of residual cancer in the individual.

[0041] In some embodiments, a method of treating cancer in an individual comprising: a) diagnosing the individual as having cancer according to the methods described herein, and b) subjecting the individual to an anti-cancer therapy, in some embodiments, the anti-cancer therapy is not an immunotherapy.

[0042] In some embodiments of any one of the methods described herein, the cancer is a solid tumor.

[0043] In some embodiments of any one of the methods described herein, the cancer is carcinoma, sarcoma, myeloma, leukemia, lymphoma, blastoma, germ cell tumor, or any combination thereof.

[0044] In some embodiments of any one of the methods described herein, the cancer is squamous cell carcinoma or adenocarcinoma. [Brief description of the drawings]

[0045] [Figure 1] FIG. 1 illustrates newly predicted Kras neoantigens (SEQ ID NOs: 57-76) resulting from the six most common Kras mutations (Kras G12V, Kras G12D, Kras G12R, Kras G12C, Kras G12I, Kras G12A).

[0046] [Diagram 2] Figure 2 shows that the four most common HLA-As, including HLA-A:0201, HLA-A:2402, HLA-A:0301, and HLA-A:1101, as well as β2m, were expressed and purified from Escherichia coli (E. coli). These target proteins were expressed in insoluble inclusion bodies (DPE).

[0047] [Diagram 3] Figure 3 shows the construction of five common mutant Kras neoantigen libraries, four types of HLA (HLA-A:0201, HLA-A:2402, HLA-A:0301, HLA-A:1101), and the four most common Kras mutations (Kras G12V, Kras G12D, Kras G12R, Kras G12C). This figure only presents a part of the results of the Kras G12V mutation.

[0048] [Figure 4]Figure 4 reveals 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.

[0049] [Diagram 5] FIG. 5 shows tumor size 12 days after inoculation of 1×105 or 1×106 Pano2-Luc-GFP tumor cells (Panel A) and tumor growth curves, as measured by tumor volume, of Pano2-Luc-GFP tumors in C57BL / 6J mice after subcutaneous challenge with 1×106 tumor cells (Panel B).

[0050] [Figure 6] Figure 6 shows the construction of plasmids overexpressing five common Kras mutations. The band size was approximately 750 bp. Two replicates were loaded, and the bands smaller than 50 bp were non-specific bands.

[0051] [Figure 7A] Figures 7A and 7B show that Kras mutant neoantigen-specific CD8+ T cells were detected in the peripheral blood of mice as early as day 4 after intravenous tumor cell challenge of 4 x 105 or 1 x 106 cells. [Figure 7B] Figures 7A and 7B show that Kras mutant neoantigen-specific CD8+ T cells were detected in the peripheral blood of mice as early as day 4 after intravenous tumor cell challenge of 4 x 105 or 1 x 106 cells.

[0052] [Figure 8] FIG. 8 shows that Kras mutant neoantigen-specific CD8+ T cells were detected in the peripheral blood of mice as early as day 4 after intravenous tumor cell challenge of 1×104 or 1×105 cells.

[0053] [Figure 9A]Figures 9A and 9B show that Kras mutant neoantigen-specific CD8+ T cells were detected in the peripheral blood of mice as early as day 4 after subcutaneous tumor challenge of 4x105 cells. As shown, a tetramer+CD8+ T cell population (range 0.042% to 0.22, median: 0.0866%) was detectable in mice challenged with Pan02-n peptide, but tetramer+CD8+ T cells were not detectable in mice challenged with Pan02-EV cells. [Figure 9B] Figures 9A and 9B show that Kras mutant neoantigen-specific CD8+ T cells were detected in the peripheral blood of mice as early as day 4 after subcutaneous tumor challenge of 4x105 cells. As shown, a tetramer+CD8+ T cell population (range 0.042% to 0.22, median: 0.0866%) was detectable in mice challenged with Pan02-n peptide, but tetramer+CD8+ T cells were not detectable in mice challenged with Pan02-EV cells.

[0054] [Figure 10A] Figures 10A and 10B show tumor growth curves as assessed by bioluminescence of mice inoculated with 4x105 cells of Pan02-EV tumor cells (without neoantigen expressing Kras mutation) and Pan02-n tumor cells (with neoantigen expressing Kras mutation) (Figure 9A), as well as photographs of mice 4 days after tumor challenge (Figure 9B) that evidence no tumors were observed at the inoculation site (red circle). [Figure 10B] Figures 10A and 10B show tumor growth curves as assessed by bioluminescence of mice inoculated with 4x105 cells of Pan02-EV tumor cells (without neoantigen expressing Kras mutation) and Pan02-n tumor cells (with neoantigen expressing Kras mutation) (Figure 9A), as well as photographs of mice 4 days after tumor challenge (Figure 9B) that evidence no tumors were observed at the inoculation site (red circle).

[0055] [Figure 11A] 11A to 11C show the presence or absence of Kras mutation neoantigen-specific CD8+ T cells against a Kras mutation-specific tetramer library in the peripheral blood of pancreatic cancer patients (patients #1 to #13). [Figure 11B] 11A to 11C show the presence or absence of Kras mutation neoantigen-specific CD8+ T cells against a Kras mutation-specific tetramer library in the peripheral blood of pancreatic cancer patients (patients #1 to #13). [Figure 11C] 11A to 11C show the presence or absence of Kras mutation neoantigen-specific CD8+ T cells against a Kras mutation-specific tetramer library in the peripheral blood of pancreatic cancer patients (patients #1 to #13).

[0056] [Figure 12] Figure 12 shows a summary of the presence or absence of CD8+ T cells in 13 confirmed pancreatic patients, along with the presence or absence of Kras mutations and specific HLA-A phenotypes. In this experiment, 20 G12D neoantigenic peptide-related MHC tetramers were included in the bait composition, but only 4 G12R neoantigenic peptide-related tetramers were included. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Detailed Description The present application provides various methods for analyzing samples from individuals who do not show any pathological symptoms of cancer. The present application is based on the unique insight of the inventors that immune cells activated by tumor neoantigens may be detectable in individuals with cancer even before any pathological symptoms of cancer are shown, and thus serve as useful markers for early cancer detection. As shown in the Examples section, exemplary Kras mutation-associated neoantigen-specific T cells express Kras mutation-associated neoantigens (10 4Tumor cells were successfully detected in mice inoculated intravenously or subcutaneously with as few as 1000 cells / ml (see, e.g., FIG. 8) and as early as day 4 after inoculation (see, e.g., FIG. 9A-9B), and prior to any detection via bioluminescence (see, e.g., FIG. 10A-10B), or in pancreatic cancer patients with Kras mutations (see, e.g., FIG. 11A-11C, and FIG. 12). These data revealed that the provided method can be successfully used both for cancer detection, including early cancer detection, as well as to monitor cancer cells in individuals who may have residual microcancer cells.

[0058] The present application provides methods for isolating and analyzing immune cells in individuals who do not show any pathological symptoms of cancer, as well as utilizing the information so obtained for cancer screening in healthy individuals, for diagnosing or aiding in the diagnosis of individuals suspected of having cancer, and for detecting minimal residual disease (MRD) in individuals who have been previously treated with anti-cancer therapy and who do not show any pathological symptoms of cancer after treatment. More detailed analysis of the isolated immune cells can be further used to classify the individual's cancer.

[0059] Thus, in one aspect, the present application provides a method of analyzing a sample from an individual who does not exhibit any pathological symptoms of cancer. In some embodiments, the method comprises: a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide under conditions sufficient for immune cells to bind to the presentation moiety; b) isolating immune cells associated with the presentation moiety; and c) analyzing the isolated immune cells. In some embodiments, the individual has previously been treated for cancer and does not exhibit any pathological symptoms of cancer after the treatment. In some embodiments, the method comprises: a) analyzing a pre-treatment sample from the individual prior to anti-cancer treatment and a post-treatment sample from the individual; and b) identifying differences in characteristics of the isolated immune cells from the pre-treatment sample and the isolated immune cells from the post-treatment sample.

[0060] In another aspect, the present application provides a method for detecting cancer in an individual not exhibiting any pathological symptoms of cancer, comprising analyzing a sample from the individual, wherein a predetermined characteristic of the isolated immune cells is indicative of cancer in the individual.

[0061] In another aspect, the present application provides a method for detecting residual cancer in an individual who has been previously treated with an anti-cancer therapy and who shows no pathological symptoms of cancer after the treatment, comprising analyzing a post-treatment sample from the individual, wherein predetermined characteristic of the isolated immune cells indicate residual cancer in the individual. A post-treatment sample refers to a sample taken from an individual who has undergone cancer treatment. In some embodiments, the post-treatment sample is taken within about 1 week to 3 weeks after the treatment. In some embodiments, the post-treatment sample is taken within about 1 month to 3 months after the treatment. In some embodiments, the post-treatment sample is taken within about 6 months, 9 months, or 12 months after the treatment.

[0062] In another aspect, the application provides a method of treating cancer in an individual, the method comprising: a) diagnosing the individual as having cancer according to the diagnostic methods described herein; and b) administering to the individual an anti-cancer treatment.

[0063] definition In general, the terms used in the claims and the specification are intended to be interpreted as having their plain meaning as understood by a person skilled in the art. Certain terms are defined below to provide further clarity. In the event of a discrepancy between the plain meaning and a provided definition, the provided definition shall prevail.

[0064] As used herein, the term "antigen" is a substance that induces an immune response.

[0065] As used herein, the term "neo-antigen" refers to an antigen that has at least one change that makes it distinct from the corresponding wild-type parent antigen, for example, through a mutation in a tumor cell or a post-translational modification specific to a tumor cell. A neo-antigen may include a polypeptide sequence. A mutation that results in a neo-antigen may include a frameshift or non-frameshift indel, a missense or nonsense substitution, a splice site change, a genomic rearrangement or gene fusion, or any genomic or expression change that creates a neo-ORF. A mutation may also include a splice variant. A post-translational modification that is specific to a tumor cell may include aberrant phosphorylation. A post-translational modification that is specific to a tumor cell may also include a spliced ​​antigen generated by the proteasome. 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.

[0066] As used herein, the term "tumor neo-antigen" or "cancer neo-antigen" is a neo-antigen that is present in tumor cells or tumor tissue of a subject, but is not present in the corresponding normal cells or tissue of said subject.

[0067] As used herein, the term "missense mutation" is a mutation that results in the substitution of one amino acid for another.

[0068] As used herein, the term "nonsense mutation" is a mutation that results in the substitution of an amino acid for a stop codon.

[0069] As used herein, the term "frameshift mutation" is a mutation that causes a change in the frame of a protein.

[0070] As used herein, the term "indel" is an insertion or deletion of one or more nucleic acids.

[0071] As used herein, the term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, have a specified percentage of nucleotides or amino acid residues that are the same as 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. Depending on the application, the percent "identity" may exist over a region of the sequences being compared, e.g., over a functional domain, or alternatively, over the entire length of the two sequences being compared.

[0072] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequence is compared.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence(s) to reference sequence based on designated program parameters.Alternatively, sequence similarity or dissimilarity can be established by the combined presence or absence of specific nucleotides, or for translated sequences, by amino acids (e.g., sequence motifs) at selected sequence positions.

[0073] As used herein, the term "epitope" is the specific portion of an antigen to which an antibody or T-cell receptor typically binds.

[0074] As used herein, the term "immunogenic" is capable of eliciting an immune response, for example, via T cells, B cells, or both.

[0075] As used herein, the terms "HLA binding affinity" and "MHC binding affinity" refer to the affinity of binding between a specific antigen and a specific MHC allele.

[0076] As used herein, the term "bait composition" refers to a composition that contains a molecule (e.g., a neo-antigenic peptide) that is used to enrich for cells that specifically bind to the bait from a sample.

[0077] As used herein, the term "variant" refers to a difference between a nucleic acid of interest and a reference human genome used as a control.

[0078] As used herein, the term "allele" is a version of a gene, or a version of a gene sequence, or a version of a protein.

[0079] As used herein, the term "HLA type" refers to the complement of HLA gene alleles.

[0080] As used herein, the term "exome" is a subset of the genome that codes for proteins. The exome can be a collection of the exons of the genome.

[0081] As used herein, the term "proteome" is the set of all proteins expressed and / or translated by a cell, a group of cells, or an individual.

[0082] As used herein, the term "dextramer" is a dextran-based peptide-MHC multimer used for antigen-specific immune cell staining in flow cytometry.

[0083] As used herein, the term "MHC multimer" is a peptide-MHC complex that contains multiple peptide-MHC monomer units.

[0084] As used herein, the term "MHC tetramer" is a peptide-MHC complex that contains four peptide-MHC monomer units.

[0085] As used herein, a "sample" refers to an aliquot of bodily fluid or tissue obtained from a subject that contains immune cells.

[0086] The term "mammal" encompasses both humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0087] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, improving the disease state, bringing about remission (partial or complete) of the disease, reducing the dose of one or more other medications required to treat the disease, slowing the progression of the disease, improving or improving the quality of life, increasing weight gain, and / or prolonging survival. By "treatment" is also included the reduction of pathological consequences of cancer (e.g., tumor volume, etc.). The methods of the present application contemplate any one or more of these aspects of treatment.

[0088] "Reference" as used herein refers to any sample, standard, or level used for comparison purposes. The reference may be obtained from a healthy sample and / or a non-diseased sample. In some examples, the reference may be obtained from a non-treated sample. In some examples, the reference is obtained from a non-diseased or non-treated sample of an individual. In some examples, the reference is obtained from one or more healthy individuals that are not the individual or patient.

[0089] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, an individual is a human.

[0090] It is understood that embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" various embodiments.

[0091] Reference herein to "about" a value or parameter includes (describes) a variation of the value or parameter itself. For example, a description that refers to "about X" includes the description of "X."

[0092] As used herein, reference to a value or parameter "is not" generally means and describes the value or parameter "unexpected." For example, a method is not used to treat cancer of type X means that the method is used to treat cancers of types other than X.

[0093] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0094] It must be noted that 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.

[0095] Any terms not directly defined herein should be understood to have the meaning generally associated with them as understood in the technical field of the present invention. Certain terms are discussed herein to provide the practitioner with further guidance in describing the compositions, devices, methods, etc. of the various aspects of the present invention and how they are made or used. It is understood that the same thing may be stated in more than one way. As a result, alternative language and synonyms may be used for any one or more of the terms discussed herein. It is not important whether a term is detailed or discussed herein. Some synonyms or substitute methods, materials, etc. are provided. The listing of one or a few synonyms or equivalents does not exclude the use of other synonyms or equivalents unless expressly stated. The use of various examples, including various examples of various terms, is for illustrative purposes and does not limit the scope and spirit of the aspects of the present invention herein.

[0096] Method for analyzing samples from individuals who do not exhibit any pathological symptoms of cancer In some embodiments, a method is provided for analyzing a sample (e.g., a blood, plasma, or PBMC sample) from an individual who does not exhibit any pathological symptoms of cancer. In some embodiments, the method comprises: a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide 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 presentation moiety; b) isolating immune cells associated with the presentation moiety; and c) analyzing the isolated immune cells. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moiety comprises a particle (e.g., a particle selected from the group consisting of a surface, a nanoparticle, a bead, and a polymer). In some embodiments, the presentation moiety further comprises a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0097] In some embodiments, a method is provided for analyzing a sample (e.g., a blood, plasma, or PBMC sample) from an individual who does not exhibit any pathological symptoms of cancer, comprising: a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide 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 presentation moiety, b) isolating immune cells associated with the presentation moiety, and c) analyzing the isolated immune cells, wherein the presentation moiety comprises two or more (e.g., four) neo-antigenic peptides. In some embodiments, the two or more neo-antigenic peptides in the presentation moiety are the same. In some embodiments, the neo-antigenic peptide has one or more of the following characteristics: a) has a binding affinity 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) for an MHC molecule; b) has a binding affinity 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) for a cognate TCR molecule; c) has a mutation, optionally at the third amino acid position counting from the N-terminus, compared to the wild-type peptide; d) is hydrophobic; and e) has a high content of aromatic residues. In some embodiments, the neo-antigenic peptide has low immunogenicity. In some embodiments, the presentation portion comprises an MHC molecule that is complexed with the neo-antigenic peptide. In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, the isolated immune cell is an isolated single immune cell. In some embodiments, the isolated immune cell is present in a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture comprising T cells, memory T cells, macrophage cells, or dendritic cells, or a combination thereof.In some embodiments, analyzing the isolated immune cells comprises detecting and / or quantifying the isolated immune cells. In some embodiments, analyzing the isolated immune cells comprises sequencing one or more nucleic acids in the isolated immune cells, and optionally further comprises analyzing the sequences of the one or more nucleic acids (e.g., TCR-associated sequences). In some embodiments, analyzing the isolated immune cells further comprises identifying one or more epigenetic modifications in the isolated immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further comprises generating a report comprising information regarding a cancer status in the individual. In some embodiments, the information regarding a cancer status comprises a cancer classification; a cancer type; a cancer nature; a cancer origin; a cancer stage; a likelihood of cancer progression; a likelihood of developing one or more cancer symptoms; a molecular diagnosis; an NGS pathology; and / or a treatment option 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 presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprising a different MHC molecule). In some embodiments, each of the different presentation moieties comprising a different MHC molecule comprises a different detectable label (e.g., a fluorophore). In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each of the different presentation moieties comprising a different MHC molecule into different populations, optionally further comprising contacting each of the plurality of different presentation moieties separately with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties.In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presenting moiety comprises a particle (e.g., a particle selected from the group consisting of a surface, a nanoparticle, a bead, and a polymer). In some embodiments, the presenting moiety further comprises a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0098] In some embodiments, a method is provided for analyzing a sample (e.g., a blood, plasma, or PBMC sample) from an individual not exhibiting any pathological symptoms of cancer, comprising: a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide 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 presentation moiety; b) isolating immune cells associated with the presentation moiety; and c) analyzing the isolated immune cells, wherein the presentation moiety comprises an MHC molecule complexed with the neo-antigenic peptide. In some embodiments, the MHC molecule is 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. In some embodiments, the peptide complexed with the MHC class I molecule is about 8 to about 10 amino acids in length. In some embodiments, the MHC is 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 neo-antigenic peptide complexed with the MHC class II molecule is about 10 to about 20 amino acids in length. In some embodiments, the isolated immune cell is an isolated single immune cell. In some embodiments, the isolated immune cell is present in a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture comprising T cells, memory T cells, macrophage cells, or dendritic cells, or a combination thereof. In some embodiments, analyzing the isolated immune cell comprises detecting and / or quantifying the isolated immune cell. In some embodiments, analyzing the isolated immune cell comprises sequencing one or more nucleic acids in the isolated immune cell, and optionally further comprises analyzing the sequence (e.g., a TCR-associated sequence) of the one or more nucleic acids.In some embodiments, analyzing the isolated immune cells further comprises identifying one or more epigenetic modifications in the isolated immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further comprises generating a report comprising information regarding a cancer status in the individual. In some embodiments, the information regarding a cancer status comprises a cancer classification; a cancer type; a cancer nature; a cancer origin; a cancer stage; a likelihood of cancer progression; a likelihood of developing one or more cancer symptoms; a molecular diagnosis; an NGS pathology; and / or a treatment option for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprise a different MHC molecule). In some embodiments, each of the different presentation moieties comprising different MHC molecules comprises a different detectable label (e.g., a fluorophore). In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each of the different presentation moieties comprising different MHC molecules into different populations, and optionally further comprises contacting each of the plurality of different presentation moieties separately with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moiety comprises a particle (e.g., a particle selected from the group consisting of a surface, a nanoparticle, a bead, and a polymer). In some embodiments, the presentation moiety further comprises a detectable label. In some embodiments, the detectable label is a fluorophore.In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0099] In some embodiments, a method is provided for analyzing a sample (e.g., a blood, plasma, or PBMC sample) from an individual who does not exhibit any pathological symptoms of cancer, comprising: a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide 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 presentation moiety; b) isolating immune cells associated with the presentation moiety; and c) analyzing the isolated immune cells, wherein the isolated immune cells comprise a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture comprising T cells, memory T cells, macrophage cells, and / or dendritic cells, or a combination thereof. In some embodiments, analyzing the isolated immune cells comprises detecting and / or quantifying the isolated immune cells. In some embodiments, analyzing the isolated immune cells comprises sequencing one or more nucleic acids in the isolated immune cells, and optionally further comprises analyzing the sequences of the one or more nucleic acids (e.g., TCR-associated sequences). In some embodiments, analyzing the isolated immune cells further comprises identifying one or more epigenetic modifications in the isolated immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further comprises generating a report comprising information regarding a cancer status in the individual. In some embodiments, the information regarding a cancer status comprises a cancer classification; a cancer type; a cancer nature; a cancer origin; a cancer stage; a likelihood of cancer progression; a likelihood of developing one or more cancer symptoms; a molecular diagnosis; an NGS pathology; and / or a treatment option 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 presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprising a different MHC molecule). In some embodiments, each of the different presentation moieties comprising a different MHC molecule comprises a different detectable label (e.g., a fluorophore). In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each of the different presentation moieties comprising a different MHC molecule into different populations, optionally further comprising contacting each of the plurality of different presentation moieties separately with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moieties comprise particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the presentation moieties further comprise a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0100] In some embodiments, a method is provided for analyzing a sample (e.g., a blood, plasma, or PBMC sample) from an individual who does not exhibit any pathological symptoms of cancer, comprising: a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide 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 presentation moiety, b) isolating immune cells associated with the presentation moiety, and c) analyzing the isolated immune cells, wherein analyzing the isolated immune cells comprises detecting and / or quantifying the isolated immune cells. In some embodiments, analyzing the isolated immune cells comprises sequencing one or more nucleic acids in the isolated immune cells, and optionally further comprises analyzing the sequences (e.g., TCR-associated sequences) of the one or more nucleic acids. In some embodiments, analyzing the isolated immune cells further comprises identifying one or more epigenetic modifications in the isolated immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further comprises generating a report comprising information regarding a cancer status in the individual. In some embodiments, the information regarding a cancer status comprises a cancer classification; a cancer type; a cancer nature; a cancer origin; a cancer stage; a likelihood of cancer progression; a likelihood of developing one or more cancer symptoms; a molecular diagnosis; an NGS pathology; and / or a treatment option for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprise a different MHC molecule).In some embodiments, each of the different presentation moieties comprising different MHC molecules comprises a different detectable label (e.g., a fluorophore). In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each of the different presentation moieties comprising different MHC molecules into different populations, and optionally further comprises contacting each of the plurality of different presentation moieties separately with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moieties comprise particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the presentation moieties further comprise a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0101] In some embodiments, a method is provided for analyzing a sample (e.g., a blood, plasma, or PBMC sample) from an individual who does not exhibit any pathological symptoms of cancer, comprising: a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide 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 presentation moiety; b) isolating immune cells associated with the presentation moiety; and c) analyzing the isolated immune cells, wherein analyzing the isolated immune cells comprises sequencing one or more nucleic acids in the isolated immune cells, and optionally analyzing the isolated immune cells further comprises analyzing the sequence of the one or more nucleic acids (e.g., a TCR-associated sequence). In some embodiments, analyzing the sequence of the one or more nucleic acids comprises a) obtaining an enriched sample from the isolated immune cells that is enriched for the one or more nucleic acids, and b) sequencing the one or more nucleic acids in the enriched sample. In some embodiments, analyzing the isolated immune cells further comprises identifying one or more epigenetic modifications in the isolated immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further comprises generating a report comprising information regarding a cancer status in the individual. In some embodiments, the information regarding a cancer status comprises a cancer classification; a type of cancer; a nature of cancer; an origin of cancer; a stage of cancer; a likelihood of cancer progression; a likelihood of developing one or more cancer symptoms; a molecular diagnosis; an NGS pathology; and / or treatment options for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties.In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprising a different MHC molecule). In some embodiments, each of the different presentation moieties comprising a different MHC molecule comprises a different detectable label (e.g., a fluorophore). In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each of the different presentation moieties comprising a different MHC molecule into different populations, optionally further comprising contacting each of the plurality of different presentation moieties separately with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moieties comprise particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the presentation moieties further comprise a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0102] In some embodiments, a method is provided for analyzing a sample (e.g., a blood, plasma, or PBMC sample) from an individual not exhibiting any pathological symptoms of cancer, comprising: a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide 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 presentation moiety; b) isolating immune cells that associate with the presentation moiety; and c) analyzing the isolated immune cells, wherein analyzing the isolated immune cells comprises identifying one or more epigenetic modifications (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)) in the isolated immune cells. In some embodiments, the method further comprises generating a report comprising information regarding the cancer status in the individual. In some embodiments, the information regarding the cancer status includes: cancer classification; cancer type; cancer nature; cancer origin; cancer stage; cancer progression likelihood; likelihood of developing one or more cancer symptoms; molecular diagnosis; NGS pathology; and / or treatment options for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprising a different MHC molecule). In some embodiments, each of the different presentation moieties comprising a different MHC molecule comprises a different detectable label (e.g., a fluorophore). In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each of the different presentation moieties comprising different MHC molecules into different populations, and optionally further comprises contacting each of a plurality of different presentation moieties separately with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties.In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presenting moiety comprises a particle (e.g., a particle selected from the group consisting of a surface, a nanoparticle, a bead, and a polymer). In some embodiments, the presenting moiety further comprises a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0103] In some embodiments, a method is provided for analyzing a sample (e.g., a blood, plasma, or PBMC sample) from an individual who does not exhibit any pathological symptoms of cancer. In some embodiments, the method includes a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide 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 presentation moiety, b) isolating immune cells associated with the presentation moiety, and c) analyzing the isolated immune cells, and further includes generating a report comprising information regarding the cancer status in the individual. In some embodiments, the information regarding the cancer status comprises a cancer classification; a type of cancer; a nature of cancer; an origin of cancer; a stage of cancer; a likelihood of cancer progression; a likelihood of developing one or more symptoms of cancer; a molecular diagnosis; an NGS pathology; and / or a treatment option for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprising a different MHC molecule). In some embodiments, each of the different presentation moieties comprising a different MHC molecule comprises a different detectable label (e.g., a fluorophore). In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each of the different presentation moieties comprising a different MHC molecule into different populations, optionally further comprising contacting each of the plurality of different presentation moieties separately with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moieties comprise particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers).In some embodiments, the display moiety further comprises a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0104] In some embodiments, a method is provided for analyzing a sample (e.g., a blood, plasma, or PBMC sample) from an individual who does not exhibit any pathological symptoms of cancer. In some embodiments, the method comprises: a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide 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 presentation moiety; b) isolating immune cells associated with the presentation moiety; and c) analyzing the isolated immune cells, wherein the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprising a different MHC molecule). In some embodiments, each of the different presentation moieties comprising a different MHC molecule comprises a different detectable label (e.g., a fluorophore). In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS). In some embodiments, the isolating step comprises separating immune cells associated with each of the different presentation moieties comprising different MHC molecules into different populations. In some embodiments, the method further comprises separately contacting each of a plurality of different presentation moieties with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moieties comprise particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the presentation moieties further comprise a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0105] In some embodiments, a method is provided for analyzing a sample (e.g., a blood, plasma, or PBMC sample) from an individual who does not exhibit any pathological symptoms of cancer, comprising: a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide under conditions sufficient for immune cells to bind to the presentation moiety; b) isolating immune cells associated with the presentation moiety; and c) analyzing the isolated immune cells, wherein the isolated immune cells are T cells. In some embodiments, the T cells are cytotoxic T cells. In some embodiments, the T cells are helper T cells. In some embodiments, the T cells are memory T cells. In some embodiments, the T cells are tumor-infiltrating T cells. In some embodiments, the presentation moiety comprises two or more (e.g., four) neo-antigenic peptides. In some embodiments, the neo-antigenic peptide has one or more of the following characteristics: a) has a binding affinity 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) for an MHC molecule; b) has a binding affinity 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) for a cognate TCR molecule; c) has a mutation, optionally at the third amino acid position counting from the N-terminus, compared to the wild-type peptide; d) is hydrophobic; and e) has a high content of aromatic residues. In some embodiments, the neo-antigenic peptide has low immunogenicity. In some embodiments, the presentation portion comprises an MHC molecule that is complexed with the neo-antigenic peptide. In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, analyzing the isolated immune cells comprises detecting and / or quantifying the isolated immune cells.In some embodiments, analyzing the isolated immune cells comprises sequencing one or more nucleic acids in the isolated immune cells, and optionally further comprises analyzing the sequences of the one or more nucleic acids (e.g., TCR-associated sequences). In some embodiments, analyzing the isolated immune cells further comprises identifying one or more epigenetic modifications in the isolated immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further comprises generating a report comprising information regarding a cancer status in the individual. In some embodiments, the information regarding a cancer status comprises a cancer classification; a cancer type; a cancer nature; a cancer origin; a cancer stage; a likelihood of cancer progression; a likelihood of developing one or more cancer symptoms; a molecular diagnosis; an NGS pathology; and / or a treatment option 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 presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprise a different MHC molecule). In some embodiments, each of the different presentation moieties comprising different MHC molecules comprises a different detectable label (e.g., a fluorophore). In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moieties comprise particles (e.g., particles selected from the group consisting of a surface, a nanoparticle, a bead, and a polymer). In some embodiments, the presentation moieties further comprise a detectable label (e.g., a fluorophore). In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to analysis by mass spectrometry.

[0106] In some embodiments, a method of analyzing a sample (e.g., a blood, plasma, or PBMC sample) from an individual not exhibiting any pathological symptoms of cancer comprises: a) contacting the sample with a bait composition comprising a presentation moiety comprising a neo-antigenic peptide under conditions sufficient for immune cells to bind to the presentation moiety; b) isolating immune cells that associate with the presentation moiety; and c) analyzing the isolated immune cells, wherein the presentation moiety comprises an MHC molecule that is complexed with the neo-antigenic peptide, and wherein the MHC molecule is associated with HLA-A. * 24:02, HLA-A * 11:01, HLA-A * 02:01, or HLA-A * 03:01. In some embodiments, the presentation portion is HLA-A * 24:02, HLA-A * 11:01, HLA-A * 02:01 and HLA-A *In some embodiments, the presentation portion comprises two or more (e.g., two, three, and four) different types of MHC class I molecules selected from the group consisting of: 03:01. In some embodiments, the presentation portion further comprises an MHC class II molecule. In some embodiments, the presentation portion does not comprise an MHC class II molecule. In some embodiments, the isolated immune cell is an isolated single immune cell. In some embodiments, the isolated immune cell is present in a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture comprising T cells, memory T cells, macrophage cells, or dendritic cells, or a combination thereof. In some embodiments, the presentation portion comprises two or more (e.g., four) neo-antigenic peptides. In some embodiments, the neo-antigenic peptide has one or more of the following characteristics: a) has a binding affinity 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) for an MHC molecule; b) has a binding affinity 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) for a cognate TCR molecule; c) has a mutation, optionally at the third amino acid position counting from the N-terminus, compared to a wild-type peptide; d) is hydrophobic; and e) has a high content of aromatic residues. In some embodiments, the neo-antigenic peptide has low immunogenicity. In some embodiments, analyzing the isolated immune cells comprises detecting and / or quantifying the isolated immune cells. In some embodiments, analyzing the isolated immune cells comprises sequencing one or more nucleic acids in the isolated immune cells, and optionally further comprises analyzing said sequences (e.g., TCR-associated sequences) of said one or more nucleic acids.In some embodiments, analyzing the isolated immune cells further comprises identifying one or more epigenetic modifications in the isolated immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further comprises generating a report comprising information regarding a cancer status in the individual. In some embodiments, the information regarding a cancer status comprises a cancer classification; a cancer type; a cancer nature; a cancer origin; a cancer stage; a likelihood of cancer progression; a likelihood of developing one or more cancer symptoms; a molecular diagnosis; an NGS pathology; and / or a treatment option for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprise a different MHC molecule). In some embodiments, each of the different presenting moieties comprising different MHC molecules comprises a different detectable label (e.g., a fluorophore). In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presenting moieties comprise a particle (e.g., a particle selected from the group consisting of a surface, a nanoparticle, a bead, and a polymer). In some embodiments, the presenting moieties further comprise a detectable label (e.g., a fluorophore). In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0107] In some embodiments, a method of analyzing a sample (e.g., a blood, plasma, or PBMC sample) from an individual (e.g., an individual not exhibiting any pathological symptoms of cancer) comprises: a) contacting said sample with a bait composition comprising a presentation moiety comprising a plurality of neo-antigenic peptides under conditions sufficient for immune cells to bind to said presentation moiety; b) isolating immune cells that associate with said presentation moiety; and c) analyzing said isolated immune cells, wherein said presentation moiety comprises one or more MHC molecules that are complexed with said plurality of neo-antigenic peptides, and optionally said MHC molecules are HLA-A or HLA-B or HLA-C or HLA-D. * 24:02, HLA-A * 11:01, HLA-A * 02:01, or HLA-A *03:01. In some embodiments, at least one (e.g., each) of the plurality of neo-antigenic peptides comprises one or more known mutations associated with cancer (e.g., Kras G12C, G12D, G12R, G12V, G12I, and / or G12A mutations). In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides, each of which comprises a single known mutation associated with cancer (e.g., Kras G12C, G12D, G12R, G12V, G12I, and / or G12A mutation). See FIG. 1 for an exemplary design of multiple neo-antigenic peptides associated with a single known mutation. In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12C mutations (e.g., as shown in FIG. 1). In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12D mutations (e.g., as shown in FIG. 1). In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12R mutations (e.g., as shown in FIG. 1). In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12V mutations (e.g., as shown in FIG. 1). In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12I mutations (e.g., as shown in FIG. 1). In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12A mutations (e.g., as shown in FIG. 1). In some embodiments, the presentation portion comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12A mutations (e.g., as shown in FIG. 1).* 24:02, HLA-A * 11:01, HLA-A * 02:01 and HLA-A *In some embodiments, the presentation portion comprises two or more (e.g., two, three, and four) different types of MHC class I molecules selected from the group consisting of: 03:01. In some embodiments, the presentation portion further comprises an MHC class II molecule. In some embodiments, the presentation portion does not comprise an MHC class II molecule. In some embodiments, the isolated immune cell is an isolated single immune cell. In some embodiments, the isolated immune cell is present in a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture comprising T cells, memory T cells, macrophage cells, or dendritic cells, or a combination thereof. In some embodiments, the presentation portion comprises two or more (e.g., four) neo-antigenic peptides. In some embodiments, the neo-antigenic peptide has one or more of the following characteristics: a) has a binding affinity 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) for an MHC molecule; b) has a binding affinity 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) for a cognate TCR molecule; c) has a mutation, optionally at the third amino acid position counting from the N-terminus, compared to a wild-type peptide; d) is hydrophobic; and e) has a high content of aromatic residues. In some embodiments, the neo-antigenic peptide has low immunogenicity. In some embodiments, analyzing the isolated immune cells comprises detecting and / or quantifying the isolated immune cells. In some embodiments, analyzing the isolated immune cells comprises sequencing one or more nucleic acids in the isolated immune cells, and optionally further comprises analyzing said sequences (e.g., TCR-associated sequences) of said one or more nucleic acids.In some embodiments, analyzing the isolated immune cells further comprises identifying one or more epigenetic modifications in the isolated immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further comprises generating a report comprising information regarding a cancer status in the individual. In some embodiments, the information regarding a cancer status comprises a cancer classification; a cancer type; a cancer nature; a cancer origin; a cancer stage; a likelihood of cancer progression; a likelihood of developing one or more cancer symptoms; a molecular diagnosis; an NGS pathology; and / or a treatment option for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprise a different MHC molecule). In some embodiments, each of the different presenting moieties comprising different MHC molecules comprises a different detectable label (e.g., a fluorophore). In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presenting moieties comprise a particle (e.g., a particle selected from the group consisting of a surface, a nanoparticle, a bead, and a polymer). In some embodiments, the presenting moieties further comprise a detectable label (e.g., a fluorophore). In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0108] In some embodiments, a method of detecting (or identifying or assessing) the presence of cancer cells (e.g., minimal residual cancer cells, e.g., solid tumor cells) in an individual (e.g., an individual not exhibiting any pathological symptoms of cancer) comprises: a) contacting a sample (e.g., a blood, plasma, or PBMC sample) from the individual with a bait composition comprising a presentation moiety comprising a plurality of neo-antigenic peptides under conditions sufficient for immune cells to bind to said presentation moiety; b) isolating immune cells that associate with said presentation moiety; and c) analyzing said isolated immune cells, wherein said presentation moiety comprises one or more MHC molecules that are complexed with said plurality of neo-antigenic peptides, and optionally said MHC molecules are HLA-A or HLA-B or HLA-C or HLA-D. * 24:02, HLA-A * 11:01, HLA-A * 02:01, or HLA-A *03:01. In some embodiments, the individual has at least one cancer-associated mutation. In some embodiments, at least one (e.g., each) of the plurality of neo-antigenic peptides comprises one or more known mutations associated with cancer (e.g., Kras G12C, G12D, G12R, G12V, G12I, and / or G12A mutations). In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides, each of which comprises a single known mutation associated with cancer (e.g., Kras G12C, G12D, G12R, G12V, G12I, and / or G12A mutations). See FIG. 1 for an exemplary design of multiple neo-antigenic peptides associated with a single known mutation. In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12C mutations (e.g., as shown in FIG. 1). In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12D mutations (e.g., as shown in FIG. 1). In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12R mutations (e.g., as shown in FIG. 1). In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12V mutations (e.g., as shown in FIG. 1). In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12I mutations (eg, as shown in FIG. 1).In some embodiments, the plurality of neo-antigenic peptides comprises at least 5, 10, 15, or 20 distinct neo-antigenic peptides associated with Kras G12A mutations (e.g., as shown in Figure 1). In some embodiments, the presentation portion is HLA-A. * 24:02, HLA-A * 11:01, HLA-A * 02:01 and HLA-A *In some embodiments, the presentation portion comprises two or more (e.g., two, three, and four) different types of MHC class I molecules selected from the group consisting of: 03:01. In some embodiments, the presentation portion further comprises an MHC class II molecule. In some embodiments, the presentation portion does not comprise an MHC class II molecule. In some embodiments, the isolated immune cell is an isolated single immune cell. In some embodiments, the isolated immune cell is present in a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture comprising T cells, memory T cells, macrophage cells, or dendritic cells, or a combination thereof. In some embodiments, the presentation portion comprises two or more (e.g., four) neo-antigenic peptides. In some embodiments, the neo-antigenic peptide has one or more of the following characteristics: a) has a binding affinity 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) for an MHC molecule; b) has a binding affinity 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) for a cognate TCR molecule; c) has a mutation, optionally at the third amino acid position counting from the N-terminus, compared to a wild-type peptide; d) is hydrophobic; and e) has a high content of aromatic residues. In some embodiments, the neo-antigenic peptide has low immunogenicity. In some embodiments, analyzing the isolated immune cells comprises detecting and / or quantifying the isolated immune cells. In some embodiments, analyzing the isolated immune cells comprises sequencing one or more nucleic acids in the isolated immune cells, and optionally further comprises analyzing said sequences (e.g., TCR-associated sequences) of said one or more nucleic acids.In some embodiments, analyzing the isolated immune cells further comprises identifying one or more epigenetic modifications in the isolated immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further comprises generating a report comprising information regarding a cancer status in the individual. In some embodiments, the information regarding a cancer status comprises a cancer classification; a cancer type; a cancer nature; a cancer origin; a cancer stage; a likelihood of cancer progression; a likelihood of developing one or more cancer symptoms; a molecular diagnosis; an NGS pathology; and / or a treatment option for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprise a different MHC molecule). In some embodiments, each of the different presenting moieties comprising different MHC molecules comprises a different detectable label (e.g., a fluorophore). In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presenting moieties comprise a particle (e.g., a particle selected from the group consisting of a surface, a nanoparticle, a bead, and a polymer). In some embodiments, the presenting moieties further comprise a detectable label (e.g., a fluorophore). In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0109] 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 neo-antigenic peptide or MHC is attached directly to the particle. In some embodiments, the neo-antigenic peptide or MHC is attached to the particle via a binding pair comprising a first binding moiety that is attached to the neo-antigenic peptide and a second binding moiety that is bound to the particle. In some embodiments, the presentation moiety comprises a cell. In some embodiments, the cell comprises a polynucleotide that encodes the neo-antigenic peptide. In some embodiments, the polynucleotide encodes multiple neo-antigenic peptides.

[0110] In some embodiments, the individual is a human. In some embodiments, the individual is at least about 50 years old (e.g., at least 50, 60, 70, or 80 years old).

[0111] In some embodiments, the samples described herein (including in this section and any other section of this application) can be any sample from the individual. In some embodiments, the samples described herein are blood samples. In some embodiments, the samples described herein are plasma samples. In some embodiments, the samples described herein comprise peripheral blood mononuclear cells (PMBCs). In some embodiments, the samples described herein are samples obtained from a tissue or organ of the individual (e.g., a biopsy sample). In some embodiments, the sample is obtained from a lymph node of the individual.

[0112] Methods for detecting cancer in an individual In some embodiments, a method of detecting cancer in an individual (e.g., an individual not exhibiting pathological symptoms of cancer, e.g., an individual who has never been diagnosed with cancer, e.g., an individual who is at risk for developing cancer, e.g., an individual who has never been treated for cancer, e.g., an individual who has been treated for cancer and is suspected of having minimal residual cancer cells) is provided, comprising analyzing a sample from the individual according to any of the methods described herein, wherein a predetermined characteristic of the isolated immune cells is indicative of cancer in the individual. In some embodiments, the predetermined characteristic of the isolated immune cells comprises the presence of the isolated immune cells. In some embodiments, the predetermined characteristic of the isolated immune cells comprises an amount of the isolated immune cells above a threshold level. In some embodiments, the predetermined characteristic of the isolated immune cells comprises a gene expression profile signature, a gene mutation profile signature, and / or an epigenetic modification signature. In some embodiments, the signature epigenetic modification comprises a DNA methylation signature and a histone glycosylation signature.

[0113] In some embodiments, methods are provided for detecting cancer in an individual (e.g., an individual who has not been diagnosed with cancer, e.g., an individual who is at risk for developing cancer, e.g., an individual who has been treated for cancer), comprising: a) contacting the sample with a bait composition comprising a presentation moiety that comprises a cancer neo-antigenic peptide 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 presentation moiety; b) isolating immune cells that associate with the presentation moiety; and c) analyzing the isolated immune cells, wherein a predetermined characteristic of the isolated immune cells is indicative of cancer in the individual, wherein the predetermined characteristic of the isolated immune cells comprises the presence of the isolated immune cells, and optionally, the predetermined characteristic of the isolated immune cells comprises an amount of the isolated immune cells above a threshold level. In some embodiments, the predetermined characteristic of the isolated immune cell further comprises a gene expression profile signature, a gene mutation profile signature, and / or an epigenetic modification signature (e.g., a DNA methylation signature and a histone glycosylation signature). In some embodiments, the presentation portion comprises two or more (e.g., four) neo-antigenic peptides. In some embodiments, the two or more neo-antigenic peptides in the presentation portion are the same. In some embodiments, the neo-antigenic peptide has one or more of the following characteristics: a) has a binding affinity 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) for an MHC molecule; b) has a binding affinity 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) for a cognate TCR molecule; c) has a mutation, optionally at the third amino acid position counting from the N-terminus, compared to the wild-type peptide; d) is hydrophobic; and e) has a high content of aromatic residues. In some embodiments, the neo-antigenic peptide has low immunogenicity.In some embodiments, the presentation moiety comprises an MHC molecule complexed with the neo-antigenic peptide. In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, the isolated immune cell is an isolated single immune cell. In some embodiments, the isolated immune cell is present in a mixture of immune cells. In some embodiments, the mixture of immune cells is a mixture comprising T cells, memory T cells, macrophage cells, or dendritic cells, or a combination thereof. In some embodiments, analyzing the isolated immune cell comprises detecting and / or quantifying the isolated immune cell. In some embodiments, analyzing the isolated immune cell comprises sequencing one or more nucleic acids in the isolated immune cell, and optionally further comprises analyzing the sequence (e.g., a TCR-associated sequence) of the one or more nucleic acids. In some embodiments, analyzing the isolated immune cells further comprises identifying one or more epigenetic modifications in the isolated immune cells (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)). In some embodiments, the method further comprises generating a report comprising information regarding a cancer status in the individual. In some embodiments, the information regarding a cancer status comprises a cancer classification; a cancer type; a cancer nature; a cancer origin; a cancer stage; a likelihood of cancer progression; a likelihood of developing one or more cancer symptoms; a molecular diagnosis; an NGS pathology; and / or a treatment option for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprise a different MHC molecule).In some embodiments, each of the different presentation moieties comprising different MHC molecules comprises a different detectable label (e.g., a fluorophore). In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each of the different presentation moieties comprising different MHC molecules into different populations, and optionally further comprises contacting each of the plurality of different presentation moieties separately with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moieties comprise particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the presentation moieties further comprise a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0114] In some embodiments, a method of detecting cancer in an individual (e.g., an individual who has not been diagnosed with cancer, e.g., an individual who is at risk of developing cancer, e.g., an individual who has been treated for cancer) is provided, comprising: a) contacting the sample with a bait composition comprising a presentation moiety comprising a cancer neo-antigenic peptide 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 presentation moiety; b) isolating immune cells associated with the presentation moiety; and c) analyzing the isolated immune cells, wherein a predetermined characteristic of the isolated immune cells is indicative of cancer in the individual, and the predetermined characteristic of the isolated immune cells comprises a gene expression profile signature. In some embodiments, the predetermined characteristic of the isolated immune cells further comprises a gene mutation profile signature, and / or an epigenetic modification signature (e.g., a DNA methylation signature and a histone glycosylation signature). In some embodiments, the presentation portion comprises two or more (e.g., four) neo-antigenic peptides. In some embodiments, the two or more neo-antigenic peptides in the presentation portion are the same. In some embodiments, the neo-antigenic peptide has one or more of the following characteristics: a) has a binding affinity for an MHC molecule 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) has a binding affinity for a cognate TCR molecule 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) has a mutation, optionally at the third amino acid position counting from the N-terminus, compared to the wild-type peptide; d) is hydrophobic; and e) has a high content of aromatic residues. In some embodiments, the neo-antigenic peptide has low immunogenicity. In some embodiments, the presentation moiety comprises an MHC molecule that complexes with the neo-antigenic peptide.In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, the isolated immune cell is an isolated single immune cell. In some embodiments, the isolated 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, macrophage cells, or dendritic cells, or a combination thereof. In some embodiments, analyzing the isolated immune cell includes detecting and / or quantifying the isolated immune cell. In some embodiments, analyzing the isolated immune cell includes sequencing one or more nucleic acids in the isolated immune cell, and optionally further includes analyzing the sequence of the one or more nucleic acids (e.g., TCR-associated sequence). In some embodiments, analyzing the isolated immune cell further includes identifying one or more epigenetic modifications (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)) in the isolated immune cell. In some embodiments, the method further comprises generating a report comprising information regarding the cancer status in the individual. In some embodiments, the information regarding the cancer status comprises: cancer classification; cancer type; cancer nature; cancer origin; cancer stage; cancer progression likelihood; likelihood of developing one or more cancer symptoms; molecular diagnosis; NGS pathology; and / or treatment options for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprising a different MHC molecule). In some embodiments, each of the different presentation moieties comprising a different MHC molecule comprises a different detectable label (e.g., a fluorophore).In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each of the different presentation moieties comprising different MHC molecules into different populations, and optionally further comprising contacting each of the plurality of different presentation moieties separately with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moieties comprise particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the presentation moieties further comprise a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0115] In some embodiments, a method is provided for detecting cancer in an individual (e.g., an individual who has not been diagnosed with cancer, e.g., an individual who is at risk of developing cancer, e.g., an individual who has been treated for cancer), comprising: a) contacting the sample with a bait composition comprising a presentation moiety comprising a cancer neo-antigenic peptide 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 presentation moiety; b) isolating immune cells associated with the presentation moiety; and c) analyzing the isolated immune cells, wherein a predetermined characteristic of the isolated immune cells is indicative of cancer in the individual, and the predetermined characteristic of the isolated immune cells comprises a genetic mutation profile signature and / or an epigenetic modification signature (e.g., a DNA methylation signature and a histone glycosylation signature). In some embodiments, the presentation moiety comprises two or more (e.g., four) neo-antigenic peptides. In some embodiments, the two or more neo-antigenic peptides in the presentation portion are the same. In some embodiments, the neo-antigenic peptide has one or more of the following characteristics: a) a binding affinity 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) for an MHC molecule; b) a binding affinity 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) for a cognate TCR molecule; c) a mutation, optionally at the third amino acid position counting from the N-terminus, compared to the wild-type peptide; d) hydrophobicity; and e) a high content of aromatic residues. In some embodiments, the neo-antigenic peptide has low immunogenicity. In some embodiments, the presentation moiety comprises an MHC molecule complexed with the neo-antigenic peptide, hi some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule.In some embodiments, the isolated immune cell is an isolated single immune cell. In some embodiments, the isolated 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, macrophage cells, or dendritic cells, or a combination thereof. In some embodiments, analyzing the isolated immune cell includes detecting and / or quantifying the isolated immune cell. In some embodiments, analyzing the isolated immune cell includes sequencing one or more nucleic acids in the isolated immune cell, and optionally further includes analyzing the sequence of the one or more nucleic acids (e.g., TCR-associated sequence). In some embodiments, analyzing the isolated immune cell further includes identifying one or more epigenetic modifications (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)) in the isolated immune cell. In some embodiments, the method further includes generating a report including information regarding the cancer status in the individual. In some embodiments, the information regarding the cancer status includes: cancer classification; cancer type; cancer nature; cancer origin; cancer stage; cancer progression likelihood; likelihood of developing one or more cancer symptoms; molecular diagnosis; NGS pathology; and / or treatment options for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprising a different MHC molecule). In some embodiments, each of the different presentation moieties comprising a different MHC molecule comprises a different detectable label (e.g., a fluorophore).In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each of the different presentation moieties comprising different MHC molecules into different populations, and optionally further comprising contacting each of the plurality of different presentation moieties separately with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moieties comprise particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the presentation moieties further comprise a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0116] In some embodiments, a method of detecting residual cancer in an individual who has been previously treated with an anti-cancer therapy and who does not show any pathological symptoms of cancer after treatment is provided, comprising analyzing a post-treatment sample from the individual. In some embodiments, the method further comprises a) analyzing a pre-treatment sample from the individual prior to anti-cancer therapy and a post-treatment sample from the individual according to any of the methods described herein, and b) comparing a characteristic of the isolated immune cells from the pre-treatment sample with a characteristic of the isolated immune cells from the post-treatment sample. In some embodiments, a predetermined difference in the characteristic of the isolated immune cells from the pre-treatment sample and the characteristic of the isolated immune cells from the post-treatment sample indicates residual cancer in the individual. In some embodiments, the predetermined characteristic of the isolated immune cells comprises the presence of the isolated immune cells, and optionally further comprises an amount of the isolated immune cells above a threshold level. In some embodiments, the predetermined characteristic of the isolated immune cells comprises a gene expression profile signature, a gene mutation profile signature, and / or an epigenetic modification signature (e.g., a DNA methylation signature and / or a histone glycosylation signature). In some embodiments, the method comprises: a) contacting the pre-treatment sample and / or the post-treatment sample with a bait composition comprising a presentation moiety comprising a cancer neo-antigenic peptide 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 presentation moiety; b) isolating immune cells associated with the presentation moiety; and c) analyzing the isolated immune cells. In some embodiments, the presentation moiety comprises two or more (e.g., four) neo-antigenic peptides. In some embodiments, the two or more neo-antigenic peptides in the presentation moiety are the same.In some embodiments, the neo-antigenic peptide has one or more of the following characteristics: a) has a binding affinity 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) for an MHC molecule; b) has a binding affinity 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) for a cognate TCR molecule; c) has a mutation, optionally at the third amino acid position counting from the N-terminus, compared to the wild-type peptide; d) is hydrophobic; and e) has a high content of aromatic residues. In some embodiments, the neo-antigenic peptide has low immunogenicity. In some embodiments, the presentation portion comprises an MHC molecule that is complexed with the neo-antigenic peptide. In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, the isolated immune cell is an isolated single immune cell. In some embodiments, the isolated 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, macrophage cells, or dendritic cells, or a combination thereof. In some embodiments, analyzing the isolated immune cell includes detecting and / or quantifying the isolated immune cell. In some embodiments, analyzing the isolated immune cell includes sequencing one or more nucleic acids in the isolated immune cell, and optionally further includes analyzing the sequence of the one or more nucleic acids (e.g., TCR-associated sequence). In some embodiments, analyzing the isolated immune cell further includes identifying one or more epigenetic modifications (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)) in the isolated immune cell. In some embodiments, the method further comprises generating a report comprising information regarding the cancer status in said individual.In some embodiments, the information regarding the cancer status includes: cancer classification; cancer type; cancer nature; cancer origin; cancer stage; cancer progression likelihood; likelihood of developing one or more cancer symptoms; molecular diagnosis; NGS pathology; and / or treatment options for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprising a different MHC molecule). In some embodiments, each of the different presentation moieties comprising a different MHC molecule comprises a different detectable label (e.g., a fluorophore). In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each of the different presentation moieties comprising different MHC molecules into different populations, and optionally further comprising contacting each of the plurality of different presentation moieties separately with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moieties comprise particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the presentation moieties further comprise a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0117] Treatment In some embodiments, methods of treating cancer in an individual (e.g., an individual who has not been diagnosed with cancer, e.g., an individual who is at risk for developing cancer, e.g., an individual who has been treated for cancer) are provided, comprising a) diagnosing the individual as having cancer according to the methods described herein, and b) subjecting the individual to an anti-cancer treatment. In some embodiments, diagnosing the individual comprises a) obtaining a sample from the individual, b) contacting the sample with a bait composition comprising a presentation moiety comprising a cancer neo-antigenic peptide 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 presentation moiety, c) isolating immune cells associated with the presentation moiety, and d) analyzing the isolated immune cells, wherein a predetermined characteristic of the isolated immune cells is indicative of cancer in the individual. In some embodiments, the predetermined characteristic of the isolated immune cells comprises the presence of the isolated immune cells. In some embodiments, the predetermined characteristic of the isolated immune cells comprises an amount of the isolated immune cells above a threshold level. In some embodiments, the predetermined characteristic of the isolated immune cells comprises a gene expression profile signature, a gene mutation profile signature, and / or an epigenetic modification signature (e.g., a DNA methylation signature and / or a histone glycosylation signature). In some embodiments, the presentation portion comprises two or more (e.g., four) neo-antigenic peptides. In some embodiments, the two or more neo-antigenic peptides in the presentation portion are the same.In some embodiments, the neo-antigenic peptide has one or more of the following characteristics: a) has a binding affinity 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) for an MHC molecule; b) has a binding affinity 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) for a cognate TCR molecule; c) has a mutation, optionally at the third amino acid position counting from the N-terminus, compared to the wild-type peptide; d) is hydrophobic; and e) has a high content of aromatic residues. In some embodiments, the neo-antigenic peptide has low immunogenicity. In some embodiments, the presentation portion comprises an MHC molecule that is complexed with the neo-antigenic peptide. In some embodiments, the MHC molecule is an MHC class I molecule and / or an MHC class II molecule. In some embodiments, the isolated immune cell is an isolated single immune cell. In some embodiments, the isolated 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, macrophage cells, or dendritic cells, or a combination thereof. In some embodiments, analyzing the isolated immune cell includes detecting and / or quantifying the isolated immune cell. In some embodiments, analyzing the isolated immune cell includes sequencing one or more nucleic acids in the isolated immune cell, and optionally further includes analyzing the sequence of the one or more nucleic acids (e.g., TCR-associated sequence). In some embodiments, analyzing the isolated immune cell further includes identifying one or more epigenetic modifications (e.g., DNA or RNA methylation, hydroxymethylation, and / or histone modifications (e.g., acetylation, methylation, glycosylation)) in the isolated immune cell. In some embodiments, the method further comprises generating a report comprising information regarding the cancer status in said individual.In some embodiments, the information regarding the cancer status includes: cancer classification; cancer type; cancer nature; cancer origin; cancer stage; cancer progression likelihood; likelihood of developing one or more cancer symptoms; molecular diagnosis; NGS pathology; and / or treatment options for the individual. In some embodiments, the bait composition comprises a plurality of different presentation moieties. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigenic peptide (e.g., at least about 2, 4, 10, 25, 50, 75, or 100 different presentation moieties each comprising a different MHC molecule). In some embodiments, each of the different presentation moieties comprising a different MHC molecule comprises a different detectable label (e.g., a fluorophore). In some embodiments, the isolating step comprises using fluorescence activated cell sorting (FACS) and / or separating immune cells associated with each of the different presentation moieties comprising different MHC molecules into different populations, and optionally further comprising contacting each of the plurality of different presentation moieties separately with a sample from the individual and isolating the immune cells associated with each of the different presentation moieties. In some embodiments, the method further comprises culturing the isolated immune cells prior to the analyzing step. In some embodiments, the presentation moieties comprise particles (e.g., particles selected from the group consisting of surfaces, nanoparticles, beads, and polymers). In some embodiments, the presentation moieties further comprise a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing, RNAseq sequencing, and / or subjecting the isolated immune cells to mass spectrometry analysis.

[0118] In some embodiments, the anti-cancer therapy is a standard or commonly used agent or therapy for treating cancer (e.g., a particular cancer). In some embodiments, the anti-cancer therapy includes a chemotherapeutic agent. In some embodiments, the anti-cancer therapy includes surgery. In some embodiments, the anti-cancer therapy includes radiation therapy. In some embodiments, the anti-cancer therapy includes immunotherapy. In some embodiments, the anti-cancer therapy includes cell therapy (e.g., cell therapy including immune cells (e.g., CAR T cells)). In some embodiments, the anti-cancer therapy includes an angiogenesis inhibitor.

[0119] In some embodiments, the anti-cancer treatment is not an immunotherapy.

[0120] In some embodiments, the individual has minimal residual disease (MRD). In some embodiments, the individual has minimal residual cancer. In some embodiments, the minimal residual cancer is present after the cancer has been surgically removed or cured. In some embodiments, the minimal residual disease is too small to be detected by imaging equipment (e.g., routinely used or standard imaging equipment to detect the cancer). In some embodiments, the location of the minimal residual disease is variable. In some embodiments, the minimal residual cancer is the result of immune evasion or resistance to treatment. In some embodiments, the individual has been previously treated for cancer and does not show any pathological symptoms of cancer after the treatment.

[0121] Bait Compositions, Presentation Moieties, and Neo-Antigen Peptides In some embodiments, the bait compositions described herein comprise one or more presentation moieties comprising one or more neo-antigenic peptides, where immune cells (e.g., T cells) bind (e.g., cognately bind) to the presentation moieties and / or neo-antigenic peptides.

[0122] In some embodiments, the presenting moieties described herein comprise an MHC molecule complexed with a neo-antigenic peptide (eg, a truncal neo-antigenic peptide).

[0123] In some embodiments, the MHC molecule is an MHC class I molecule.

[0124] In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, HLA-C, and HLA-D. In some embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C. In preferred embodiments, the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C. * 24:02, HLA-A * 11:01, HLA-A * 02:01 and HLA-A * In a preferred embodiment, the MHC class I molecule is selected from the group consisting of HLA-A * 24:02, HLA-A * 11:01, HLA-A * 02:01 and HLA-A * Contains multiple types of MHC class I molecules, including 03:01.

[0125] In some embodiments, the neo-antigenic peptide complexed with an MHC I molecule is about 8 to about 10 amino acids in length. In some embodiments, the neo-antigenic peptide is at least 8 (e.g., 8, 9, or 10) amino acids in length.

[0126] In some embodiments, the MHC molecule is a recombinant MHC I molecule.

[0127] In some embodiments, the MHC molecule is an MHC class II molecule.

[0128] In some embodiments, the MHC class II molecule is selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DF. In some embodiments, the MHC class II molecule is selected from the group consisting of HLA-DQ and HLA-DR. In some embodiments, the neo-antigenic peptide complexed with the MHC class II molecule is about 10 to about 20 amino acids in length. In some embodiments, the neo-antigenic peptide is at least 10 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids in length.

[0129] In some embodiments, the MHC molecule is a recombinant MHC II molecule.

[0130] In some embodiments, the MHC molecules include both MHC I and MHC II molecules.

[0131] In a preferred embodiment, the MHC molecule matches at least one HLA type of the individual from whom the sample is obtained. For example, HLA-A * 24:02 or HLA-A * 11:01, the individual has an HLA-A * 24:02 and HLA-A *11:01. Patient-specific NGS data from WGS, WES or RNA-seq can be used to predict HLA types by computational tools (e.g., Optiptype and Polysolver). See, e.g., Szolet et al., Bioinformatics 30, 3310-3316, e.g., Shukla et al., Nat. Biotechnol. 33, 1152-1158. Reads potentially derived from HLA regions can be selected from NGS data and then fully aligned against a full-length genomic library of all known HLA alleles. See, e.g., Nucleic Acids Res. 41, D1222-D1227.

[0132] In some embodiments, the MHC molecule is bound to a chaperone molecule prior to complexing with the neo-antigenic peptide. See, e.g., Overall et al., Nat Commun. 2020 Apr 20;11(1):1909.

[0133] In some embodiments, the presentation portion comprises two or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000, or 100,000) neo-antigen peptides. In some embodiments, the presentation portion comprises four neo-antigen peptides. In some embodiments, the two or more neo-antigen peptides in the presentation portion are the same or similar. In some embodiments, the two or more neo-antigen peptides in the presentation portion are distinct. In some embodiments, at least one of the two or more neo-antigen peptides is a transcal neo-antigen peptide.

[0134] In some embodiments, the presentation moiety comprises an MHC / peptide tetramer. In some embodiments, the MHC / peptide complex is assembled into a tetramer comprising one, two, three, or four MHC / peptide complexes that are bound to the presentation moiety. In some embodiments, the MHC / peptide tetramer further comprises a detectable label. The detectable label is a fluorophore, such as phycoerythrin (PE), allophycocyanin (APC), or any fluorophore known in the art.

[0135] In some embodiments, the MHC / peptide complexes are assembled into multimers (e.g., dimers, trimers, tetramers, pentamers, hexamers, or higher order multimers). In some embodiments, the multimers can include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 MHC / peptide complexes. In some embodiments, high-throughput peptide-MHC (pMHC) tetramer libraries are constructed. See, e.g., Overall et al., Nat Commun. 2020 Apr 20;11(1):1909.

[0136] In some embodiments, the display portion further comprises a barcode (e.g., a DNA barcode). In some embodiments, each of the one or more display portions comprises a unique barcode (e.g., a unique DNA barcode). See, e.g., Overall et al., Nat Commun. 2020 Apr 20; 11(1): 1909.

[0137] In some embodiments, the presentation moiety comprises a particle. In some embodiments, the particle is selected from the group consisting of a surface, a nanoparticle, a bead, and a polymer. In some embodiments, the particle is a magnetic nanoparticle, e.g., a magnetic nanoparticle for isolation using a magnet. See, e.g., Peng et al., Cell Rep. 2019 Sep 3;28(10):2728-2738.e7. In some embodiments, the magnetic particle comprises magnetic iron oxide. In some embodiments, the particle is a polystyrene nanoparticle, e.g., a polystyrene nanoparticle for isolation by gravity. In some embodiments, the particle is an agarose bead. In some embodiments, the particle is a sepharose bead. In some embodiments, the particle is a dextran particle. In some embodiments, the particle is a biotinylated dextran or a streptavidin-coated dextran.

[0138] In some embodiments, the particles are detectable. In some embodiments, the particles are fluorescent. In some embodiments, the particles are directly or indirectly attached to a fluorophore. In some embodiments, the particles are modified with an attachment moiety for attaching additional molecules.

[0139] In some embodiments, the neo-antigenic peptide or MHC is attached directly to the particle. In some embodiments, the neo-antigenic peptide or MHC is attached to the particle via a binding pair comprising a first binding moiety that is attached to the neo-antigenic peptide and a second binding moiety that is attached to the particle. In some embodiments, the binding moiety is any suitable moiety known in the art (e.g., thiol, maleimide, cyclodextrin, amine, adamantine, carboxy, azide, and alkyne).

[0140] In some embodiments, multiple presentation moieties (eg, MHC / peptide complexes) are attached to a single particle.

[0141] In some embodiments, the presentation moiety comprises a cell (e.g., an antigen presenting cell, e.g., a dendritic cell, e.g., a macrophage). In some embodiments, the cell comprises a polynucleotide encoding the neo-antigenic peptide (e.g., a trancal neo-antigenic peptide). In some embodiments, the polynucleotide encodes a plurality of neo-antigenic peptides. In some embodiments, the plurality of neo-antigenic peptides are presented on the surface of the cell. In some embodiments, the plurality of neo-antigenic peptides are presented on the surface of the cell in a complex with an MHC molecule.

[0142] In some embodiments, the cells are obtained from the individual. In some embodiments, the cells have at least one (or two) HLA types that are the same as the HLA type of the individual. For example, the individual has HLA-A * 24:02, then the cells in the presentation portion also have HLA-A * It has 24:02.

[0143] In some embodiments, the presenting moiety further comprises a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, the presenting moiety is itself fluorescent or is directly or indirectly attached to a fluorophore. In some embodiments, the fluorophore is phycoerythrin (PE), allophycocyanin (APC), or any fluorophore known in the art.

[0144] Multiple presentation parts In some embodiments, the bait composition comprises a plurality of different presentation moieties. The use of the plurality of different presentation moieties can facilitate or enhance the identification of heterologous neo-antigen-specific immune cells (e.g., T cells). In some embodiments, the plurality of presentation moieties comprises at least two, three, or four types of MHC molecules. In some embodiments, the plurality of presentation moieties comprises at least two (e.g., two, three, or four) different types of MHC class I molecules, optionally comprising at least two (e.g., two, three, or four) different types of MHC class I molecules that are HLA-A or HLA-B. * 24:02, HLA-A * 11:01, HLA-A * 02:01 and HLA-A * In some embodiments, the presentation moieties are selected from the group consisting of HLA-A * 24:02, HLA-A * 11:01, HLA-A * 02:01 and HLA-A * Including 03:01.

[0145] In some embodiments, at least two of the plurality of different presentation moieties in the bait composition comprise different neo-antigen peptides. In some embodiments, each of the plurality of different presentation moieties in the bait composition comprises a different neo-antigen peptide. In some embodiments, the plurality of different presentation moieties comprises a number (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) of different neo-antigen peptides. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the neo-antigen peptides for a cancer (e.g., a particular cancer) in the neo-antigen database are included in the number of different neo-antigen peptides. In some embodiments, a particular type of MHC molecule (e.g., HLA-A) for a cancer (e.g., a particular cancer) in the neo-antigen database is included in the number of different neo-antigen peptides. * 24:02, HLA-A * 11:01, HLA-A* 02:01, or HLA-A * 03:01) are comprised in said number of different neo-antigenic peptides.

[0146] In some embodiments, the plurality of different presentation portions is HLA-A * 24:02, in which case HLA-A * 24:02 is complexed with at least two different neo-antigen peptides. * 24:02 is complexed with a number (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) of neo-antigenic peptides, optionally where at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the neo-antigenic peptides for a cancer (e.g., a particular cancer) in the neo-antigen database are included in the number of different neo-antigenic peptides. In some embodiments, the plurality of different presentation moieties further comprises HLA-A * 11:01, and if necessary, in this case, HLA-A * In some embodiments, HLA-A 11:01 is complexed with at least two different neo-antigen peptides. * 11:01 is complexed with many (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) neoantigen peptides, optionally in this case with HLA-A for a cancer (e.g., a particular cancer) in the neoantigen database. * In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the neo-antigenic peptides for 11:01 are included in the plurality of different neo-antigenic peptides. * 02:01, if necessary, in this case HLA-A *02:01 is complexed with at least two different neo-antigen peptides. * 02:01 is complexed with a number (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) of neoantigen peptides, optionally in this case with HLA-A for a cancer (e.g., a particular cancer) in the neoantigen database. * In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the neo-antigenic peptides for 02:01 are included in the plurality of different neo-antigenic peptides. * 03:01, if necessary, in this case HLA-A * 03:01 is complexed with at least two different neo-antigen peptides. * 03:01 is complexed with a number (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) of neoantigen peptides, optionally in this case with HLA-A for a cancer (e.g., a particular cancer) in the neoantigen database. * At least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the neo-antigenic peptides for 03:01 are included in said number of different neo-antigenic peptides.

[0147] In some embodiments, the plurality of different presentation portions is HLA-A * 11:01, in which case the MHC molecule is HLA-A * In some embodiments, HLA-A 11:01 is complexed with at least two different neo-antigen peptides. * 11:01 is complexed with many (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) neoantigen peptides, optionally in this case with HLA-A for a cancer (e.g., a particular cancer) in the neoantigen database. *In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the neo-antigenic peptides for 11:01 are included in the plurality of different neo-antigenic peptides. * 02:01, if necessary, in this case HLA-A * 02:01 is complexed with at least two different neo-antigen peptides. * 02:01 is complexed with a number (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) of neoantigen peptides, optionally in this case with HLA-A for a cancer (e.g., a particular cancer) in the neoantigen database. * In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the neo-antigenic peptides for 02:01 are included in the plurality of different neo-antigenic peptides. * 03:01, if necessary, in this case HLA-A * 03:01 is complexed with at least two different neo-antigen peptides. * 03:01 is complexed with a number (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) of neoantigen peptides, optionally in this case with HLA-A for a cancer (e.g., a particular cancer) in the neoantigen database. * At least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the neo-antigenic peptides for 03:01 are included in said number of different neo-antigenic peptides.

[0148] In some embodiments, the plurality of different presentation moieties is HLA-A * 02:01, in which case HLA-A * 02:01 is complexed with at least two different neo-antigen peptides.* 02:01 is complexed with a number (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) of neoantigen peptides, optionally in this case with HLA-A for a cancer (e.g., a particular cancer) in the neoantigen database. * In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the neo-antigenic peptides for 02:01 are included in the plurality of different neo-antigenic peptides. * 03:01, if necessary, in this case HLA-A * 03:01 is complexed with at least two different neo-antigen peptides. * 03:01 is complexed with a number (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) of neoantigen peptides, optionally in this case with HLA-A for a cancer (e.g., a particular cancer) in the neoantigen database. * At least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the neo-antigenic peptides for 03:01 are included in said number of different neo-antigenic peptides.

[0149] In some embodiments, the plurality of different presentation moieties is HLA-A * 03:01, in this case HLA-A * 03:01 is complexed with at least two different neo-antigen peptides. * 03:01 is complexed with a number (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100) of neoantigen peptides, optionally in this case with HLA-A for a cancer (e.g., a particular cancer) in the neoantigen database. *At least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the neo-antigenic peptides for 03:01 are included in said number of different neo-antigenic peptides.

[0150] In some embodiments, the plurality of different presentation moieties comprises at least four types of MHC molecules, wherein the at least four types of MHC molecules are HLA-A * 24:02, HLA-A * 11:01, HLA-A * 02:01 and HLA-A * In some embodiments, each type of MHC molecule (HLA-A * 24:02, HLA-A * 11:01, HLA-A * 02:01) are complexed with at least two different neo-antigenic peptides. In some embodiments, each type of MHC molecule is complexed with a number (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100) of neo-antigenic peptides, optionally in which at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the neo-antigenic peptides for the particular MHC molecule for a cancer (e.g., a particular cancer) in the neo-antigen database are included in the number of different neo-antigenic peptides.

[0151] In some embodiments, the method comprises separately contacting each of a plurality of different presentation moieties separately with a sample from said individual, and separately isolating said immune cells associated with each of said different presentation moieties.

[0152] In some embodiments, the method comprises contacting the plurality of different display moieties with a sample from the individual and analyzing the pool of immune cells.For example, the use of multicolor-labeled MHC tetramers for multiplex flow cytometry, labeled pMHC tetramers for mass cytometry analysis, and DNA-labeled tetramers designed for sequencing analysis has also been reported.See, for example, Andersen et al.Nat Protoc.2012 Apr 12;7(5):891-902.

[0153] Neo-antigen peptides Neoantigens can be formed through various mechanisms. Nonsynonymous somatic mutations that can change the amino acid coding sequence are the main source of neoepitopes. Except for somatic nonsynonymous protein-altering mutations, tumor neoantigens can arise from alternative splicing variations. Numerous computational methods and databases have been developed to identify alternative splicing events from RNA-seq data (e.g., SplAdder and CancerSplicingQTL2). See, for example, Kahles et al., Bioinformatics 32 1840-1847; Tian et al., Nucleic Acids Res. 47 D909-D916. Computational strategies have been developed to identify neoepitopes that generate from intron retention events in tumor transcriptomes, and it has been confirmed that these neoepitopes are processed and presented on MHC-I. See, for example, Smart et al. Nat. Biotechnol. 36 1056-1058.

[0154] Neoantigen detection and screening Various methods are available for detecting and screening neoantigens. Tumor antigens can be successfully identified in serum samples drawn from patients by sandwich immunoassays in miniaturized systems. See, for example, Pollard et al., Proteomics Clin. Appl. 1 934-952 (2007); Yang et al., Biosens. Bioelectron. 40 385-392 (2013). Another tool, called serological proteome analysis (SERPA) or 2D Western blot, consists of isoelectric focusing (IEF) gel migration in the first dimension and SDS-PAGE gel migration in the second dimension. In SERPA, proteins are separated in the gel by their isoelectric point (IP) and molecular weight, and then the proteins are transferred from the gel to a carrier membrane for screening antibodies. Finally, antigen protein spots can be identified by MS. See, for example, Tjalsma et al., Proteomics Clin. Appl. 2 167-180 (2008). This approach has been used to identify antigens in different tumor types. Serological analysis of recombinant cDNA expression libraries (SEREX) is a widely used technique to explore the antigen repertoire of tumors, since it combines serological analysis with antigen cloning technology. In SEREX, first, a cDNA library is constructed from cancer cell lines or fresh tumor samples, then this cDNA library is screened by autologous serum of cancer patients, and finally, immunoreactive clones are sequenced. SEREX has identified various tumor antigens, including CTA, differentiation antigens, mutant antigens, splice variant antigens and overexpressed antigens. See, for example, Chen et al., Proc. Natl. Acad. Sci. USA 94 1914-1918 (1997). In addition, other methods, such as multiplex affinity protein profiling (MAPPing) and nanoplasmonic biosensors, have also been developed to identify tumor antigens.See, for example, Lee et al., Biosens. Bioelectron. 74 341-346 (2015).

[0155] In some embodiments, the one or more neo-antigen peptides described herein are obtained from a neo-antigen database (e.g., any of the neo-antigen databases described herein). For example, Tan et al. constructed a manually curated database for human tumor neo-antigen peptides ("dbPepNeo") based on four criteria: (i) the peptides were isolated from human tumor tissues or human tumor cell lines; (ii) the peptides contained non-synonymous mutations in the amino acid sequence; (iii) the peptides could bind HLA-I molecules; (iv) the peptides could induce CD8+ T cell responses. See Tan et al., Database (Oxford). 2020 Jan 1; 2020: baaa004. Xia et al. constructed another database, NEPdb, using state-of-the-art predictors, that provides pan-cancer predicted HLA-I neo-epitopes derived from 16,745 shared cancer somatic mutations. See Xia et al.,Front Immunol.2021;12:644637. Wu et al. developed a comprehensive tumor-specific neo-antigen database (TSNAdb v1.0) based on pan-cancer immunogenomic analysis of somatic mutation data and human leukocyte antigen (HLA) allele information for 16 tumor types using 7748 tumor samples from The Cancer Genome Atlas (TCGA) and The Cancer Immunome Atlas (TCIA). See Wu et al.,Enomics Proteomics Bioinformatics.2018 Aug;16(4):276-282.

[0156] In some embodiments, the one or more neo-antigenic peptides are obtained from analyzing biological information of the individual (e.g., a patient with cancer). In some embodiments, the neo-antigenic peptides are obtained from computational analysis of the tumor genome of a cancer patient. See, e.g., Roudko et al. Front Immunol. 2020; 11:27. In some embodiments, the neo-antigenic peptides are obtained from computational analysis of the transcriptome of a cancer patient. See, e.g., Caushi et al., Nature. 2021 Aug; 596(7870): 126-132. In some embodiments, the neo-antigenic peptides are obtained from computational analysis of the proteome of a cancer patient. See, e.g., Wen et al. Nat Commun. 2020 Apr 9; 11(1): 1759.

[0157] In some embodiments, the neo-antigenic peptides are selected from patient data. In some embodiments, the patient data is derived from data from a group of patients with a particular type of cancer (e.g., any of the cancers described herein). In some embodiments, the patient data is derived from data from a group of patients with any cancer. In some embodiments, the group of patients are patients of the same gender (e.g., male or female). In some embodiments, the group of patients are patients of the same ethnicity. In some embodiments, the group of patients have one or more biomarkers (e.g., an abnormality in a particular gene, e.g., an abnormality in KRAS, e.g., an abnormality in PTEN).

[0158] In some embodiments, the one or more neo-antigenic peptides are derived from any polypeptide known to contain tumor-specific mutations or polypeptides found to contain tumor-specific mutations. Suitable polypeptides from which the neo-antigenic peptides may be derived can be found, for example, in various databases available in the art (e.g., COSMIC database). These databases curate comprehensive information on somatic mutations in human cancers. In some embodiments, the peptides contain tumor-specific mutations. In some embodiments, the tumor-specific mutations are driver mutations for a particular cancer type.

[0159] In some embodiments, a library of tumor-specific neo-antigenic peptides is synthesized (e.g., based on a patient or group of patients as described above). In some embodiments, the neo-antigenic peptides are obtained by high-throughput sequencing of the exome and pre-screened by epitope prediction algorithms.

[0160] Selection of neo-antigenic peptides In some embodiments, the one or more neo-antigenic peptides used in the bait compositions described herein are further optimized based on one or more selection criteria.

[0161] In some embodiments, the neo-antigenic peptides are further selected based on their potential to be processed and / or presented on cell surface HLA molecules. In some embodiments, an in silico prediction algorithm (e.g., any of the algorithms described herein) is used as the basis for selection. In some embodiments, immunopeptidomic analysis is used as the basis for selection.

[0162] Computational algorithms, such as NetMHC (see, e.g., Andreatta et al., Bioinformatics 32, 511-517, 2016), NetMHCpan (see, e.g., Rammensee et al., Immunogenetics 50, 213-219, 1999), and MHCflurry (O'Donnell et al., Cell Syst. 7, 129-132.e124, 2018), which are trained on large in vitro experimental datasets, can be used to prioritize candidate neoantigens that bind with high affinity to predicted HLA types. For example, Neopepsee and pVAC-Seq are representative analysis pipelines for tumor somatic mutations (Hundal et al., Genome Med. 8, 11, 2016; Kim et al., Ann. Oncol. 29, 1030-1036, 2018). Recently, a new predictive model, EDGE, based on tumor HLA peptide mass spectrometry (MS) datasets, has shown up to a nine-fold increase in positive predictive value (Bulik-Sullivan et al., Nat. Biotechnol. 18:4313).

[0163] In some embodiments, the one or more neo-antigenic peptides are selected based on their binding affinity to a) an MHC molecule and / or b) a cognate TCR molecule.

[0164] In some embodiments, the neo-antigenic peptide has a binding affinity to an MHC molecule that is less than 5000 nM (IC50). In some embodiments, the neo-antigenic peptide has a binding affinity to an MHC molecule that is about 500 nM to 5000 nM (IC50). In some embodiments, the neo-antigenic peptide has a binding affinity to an MHC molecule that is less than 500 nM (IC50). In some embodiments, the neo-antigenic peptide has a binding affinity to an MHC molecule that is about 250 nM to 500 nM IC50. In some embodiments, the neo-antigenic peptide has a binding affinity to an MHC molecule that is less than 250 nM (IC50). In some embodiments, the neo-antigenic peptide has a binding affinity to an MHC molecule that is less than 100 nM (IC50). In some embodiments, the neo-antigenic peptide has a binding affinity to an MHC molecule that is about 50 nM to 500 nM IC50. In some embodiments, the neo-antigenic peptide has a binding affinity to an MHC molecule that is less than 50 nM (IC50). In some embodiments, the neo-antigenic peptide has a binding affinity to an MHC molecule of about 1 nM to 50 nM IC50.

[0165] In some embodiments, the neo-antigenic peptide has a binding affinity to a cognate TCR molecule that is less than 5000 nM (IC50). In some embodiments, the neo-antigenic peptide has a binding affinity to a cognate TCR molecule of about 500 nM-5000 nM (IC50). In some embodiments, the neo-antigenic peptide has a binding affinity to a cognate TCR molecule of about 50 nM-500 nM IC50. In some embodiments, the neo-antigenic peptide has a binding affinity to a cognate TCR molecule of about 1 nM-50 nM IC50.

[0166] In some embodiments, the neo-antigenic peptide is selected based on its mutation status. In some embodiments, the neo-antigenic peptide has a mutation at the third amino acid position from the N-terminus compared to the wild-type peptide. In some embodiments, the neo-antigenic peptide may contain two or more (e.g., at least two, three, four, or five) somatic mutations.

[0167] In some embodiments, the neo-antigenic peptide is selected based on its hydrophobicity. In some embodiments, the neo-antigenic peptide is hydrophobic. In some embodiments, the neo-antigenic peptide has a high content of aromatic residues. In some embodiments, the neo-antigenic peptide has at least about 10%, 20%, 30%, or 40% aromatic residues.

[0168] In some embodiments, the neo-antigen peptide has a binding affinity for an MHC molecule 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) and a binding affinity for a cognate TCR molecule 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), has a mutation compared to a wild-type peptide, optionally at the third amino acid position counting from the N-terminus, is hydrophobic, and has a high content of aromatic residues.

[0169] In some embodiments, the neo-antigen peptide has low immunogenicity.The immunogenicity of the neo-antigen peptide can be predicted by an algorithm developed for this purpose.See, for example, Riley et al., Front Immunol.2019 Aug 28;10:2047;See, for example, Schmidt et al., Cell Rep Med.2021 Feb 6;2(2):100194.

[0170] In some embodiments, the cancer antigen peptide may be flanked by universal sequences or portions thereof, in some embodiments, the universal sequences or portions thereof allow for rapid, high-throughput methods for replacing or inserting nucleotides encoding antigen peptides in a polynucleotide MHC template.

[0171] In some embodiments, the neo-antigen peptides further comprise a unique defined barcode sequence operably associated with the identity of each distinct polypeptide. In some embodiments, the unique defined barcode provides an antigen-specific sequence for identification during analysis of the immune cells. See, e.g., Peng et al., Cell Rep. 2019 Sep 3;28(10):2728-2738.e7.

[0172] The size of the neo-antigen peptides is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about The neo-antigenic peptide molecule may include, but is not limited to, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid residues, and ranges derivable therein. In a preferred embodiment, the neo-antigenic peptide molecule is equal to or less than 50 amino acids.

[0173] In some embodiments, the neo-antigenic peptides complexed with MHC class I molecules have a length of 15 residues or less, typically between about 8 and about 11 residues, and more typically 9 or 10 residues, while in some embodiments, the neo-antigenic peptides complexed with MHC class II molecules have a length of 6 to 30 residues.

[0174] If desired, longer peptides can be designed in several ways. For example, when the presentation potential of the peptides on HLA alleles is predicted or known, the longer peptides can consist of either: (1) individual presented peptides with extensions of 2-5 amino acids toward the N- and C-termini of each corresponding gene product; (2) concatenation of some or all of the presented peptides with their respective extension sequences.

[0175] In some embodiments, the one or more neo-antigenic peptides are about 8-50 amino acids in length. In some embodiments, the neo-antigenic peptide is about 8-10 amino acids in length. In some embodiments, the neo-antigenic peptide is more than 10 amino acids in length, more than 15 amino acids in length, more than 20 amino acids in length, or more than 30 amino acids in length. In some embodiments, the neo-antigenic peptide is about 24-40 amino acids in length.

[0176] In some embodiments, the method involves constructing a plurality of neo-antigenic peptides (e.g., assembling a library such as in Example 1 and FIG. 1). In some embodiments, the plurality of neo-antigenic peptides is based on known mutations (e.g., Kras mutations). In some embodiments, the plurality of neo-antigenic peptides based on a known mutation (e.g., point mutations such as Kras G12D, G12V, G12C, G12R, G12A, or G12I) includes at least 5, 10, 12, 15, 18, or 20 distinct neo-antigenic peptides. In some embodiments, the plurality of neo-antigenic peptides based on one known mutation includes at least about 5, 10, 12, 15, 18, or 20 distinct neo-antigenic peptides that a) have a binding affinity for an MHC molecule of about 1 nM to about 5000 nM, b) have a binding affinity for a cognate TCR molecule of about 1 nM to about 5000 nM, c) have a mutation, optionally at the third amino acid position counting from the N-terminus, compared to a wild-type peptide, d) are hydrophobic, and / or e) have a high content of aromatic residues.

[0177] Exemplary Considerations for Bait Composition Design In some embodiments, the one or more neo-antigenic peptides are selected via one or more steps as described below.

[0178] 1. Determination of a set of peptides covering an optimized number of tumor subclones In some embodiments, the one or more neo-antigenic peptides include truncal peptides. The truncal peptides described herein represent peptides that are presented by all or most tumor subclones and are prioritized for inclusion in the bait composition.

[0179] If there are no truncal peptides predicted to be highly likely to be presented, or if the number of truncal peptides predicted to be highly likely to be presented is sufficiently small that additional non-truncal peptides can be included in the presentation, the peptides can be further prioritized by estimating the number and identity of tumor subclones and selecting peptides to maximize the number of tumor subclones covered.

[0180] 2. Neo-antigen Selection Neo-antigen peptides can be selected through various methods or processes. In some embodiments, candidate neo-antigens are placed in a space having at least one or more of the following axes, and an integrated multi-dimensional model described below is applied, which uses an integrated approach to optimize selection. For example, see International Publication WO2019050994A.

[0181] 1. Probability of sequencing artifacts (a lower probability of artifacts is typically preferred).

[0182] 2. Probability of presentation (higher probabilities of presentation are typically preferred).

[0183] 3. Gene expression (higher expression is typically preferred).

[0184] 4. HLA gene coverage (the greater the number of HLA molecules involved in the presentation of a set of neoantigens, the more preferred they are).

[0185] 5. Coverage of both MHC classes (preferably both MHC-I and MHC-II coverage).

[0186] Exemplary Bait Compositions, Presentation Moieties, and Neo-Antigens (e.g., for Analyzing Immune Cells from an Individual for the Presence of Lung Cancer (e.g., NSCLC)) Exemplary bait compositions were determined using the methodology described herein.

[0187] [Table 1-1] [Table 1-2] [Table 1-3]

[0188] In some embodiments, the one or more neo-antigenic peptides comprise one or more (e.g., at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50) sequences selected from the group consisting of SEQ ID NO:1-SEQ ID NO:56.

[0189] In some embodiments, the one or more neo-antigenic peptides comprise the amino acid sequences set forth in SEQ ID NO:1-SEQ ID NO:5. In some embodiments, the one or more neo-antigenic peptides further comprise one or more (e.g., at least 2, 5, 10, 15, 20, 25, 30, 35, or 40) sequences selected from the group consisting of SEQ ID NO:6-SEQ ID NO:50, and / or one or more (e.g., 1, 2, 3, 4, 5, or 6) sequences selected from the group consisting of SEQ ID NO:51-SEQ ID NO:56.

[0190] In some embodiments, the one or more neo-antigenic peptides comprise one or more (e.g., at least 2, 5, 10, 15, 20, 25, 30, 35, or 40) sequences selected from the group consisting of SEQ ID NO:6-SEQ ID NO:50. In some embodiments, the one or more neo-antigenic peptides comprise the amino acid sequences set forth in SEQ ID NO:6-SEQ ID NO:50. In some embodiments, the one or more neo-antigenic peptides further comprise one or more (e.g., 1, 2, 3, 4, or 5) sequences selected from the group consisting of SEQ ID NO:1-SEQ ID NO:5, and / or one or more (e.g., 1, 2, 3, 4, 5, or 6) sequences selected from the group consisting of SEQ ID NO:51-SEQ ID NO:56.

[0191] In some embodiments, the one or more neo-antigenic peptides comprise the amino acid sequences set forth in SEQ ID NO:51-SEQ ID NO:56. In some embodiments, the one or more neo-antigenic peptides further comprise one or more (e.g., at least 2, 5, 10, 15, 20, 25, 30, 35, or 40) sequences selected from the group consisting of SEQ ID NO:6-SEQ ID NO:50, and / or one or more (e.g., 1, 2, 3, 4, or 5) sequences selected from the group consisting of SEQ ID NO:1-SEQ ID NO:5.

[0192] In some embodiments, the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) neo-antigenic peptides comprise an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:35-37, SEQ ID NO:40, and SEQ ID NO:48.

[0193] In some embodiments, the one or more (e.g., 5, 10, 15, 20, 25, or 30) neo-antigenic peptides comprise an amino acid sequence selected from the group consisting of SEQ ID NO:9-13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28-34, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41-47, SEQ ID NO:49, and SEQ ID NO:50.

[0194] In some embodiments, the one or more (e.g., 1, 2, 3, or 4) neo-antigenic peptides comprise an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:8.

[0195] In some embodiments, the one or more (e.g., 1, 2, 3, 4 or 5) neo-antigenic peptides comprise an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20 and SEQ ID NO:21.

[0196] In some embodiments, the one or more (e.g., 1, 2, 3, or 4) neo-antigenic peptides comprise an amino acid sequence selected from the group consisting of SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:51, and SEQ ID NO:52.

[0197] In some embodiments, the one or more (e.g., one or two) neo-antigenic peptides comprise an amino acid sequence selected from the group consisting of SEQ ID NO:54 and SEQ ID NO:56.

[0198] In some embodiments, the one or more neo-antigenic peptides comprise the amino acid sequences set forth in SEQ ID NO:1-SEQ ID NO:56.

[0199] In some embodiments, the bait composition comprises one or more presentation moieties comprising one or more (e.g., at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50) MHC-peptide complexes (i.e., pMHC) that pair as shown in Table 1. In some embodiments, the bait composition comprises one or more presentation moieties comprising pMHC that comprises one or more neo-antigenic peptides as shown in SEQ ID NO:1-SEQ ID NO:5 that are complexed with an MHC molecule according to Table 1. In some embodiments, the bait composition comprises one or more presentation moieties comprising pMHC that comprises one or more (e.g., at least 2, 5, 10, 15, 20, 25, 30, 35, or 40) neo-antigenic peptides that comprise an amino acid selected from the group consisting of SEQ ID NO:6-SEQ ID NO:50 that are complexed with an MHC molecule according to Table 1. In some embodiments, the bait composition comprises one or more presentation moieties comprising a pMHC comprising one or more (e.g., 1, 2, 3, 4, 5 or 6) neo-antigenic peptides comprising amino acids selected from the group consisting of SEQ ID NO:51 to SEQ ID NO:56, complexed with an MHC molecule according to Table 1.

[0200] In some embodiments, the presentation portion comprises one or more MHC-peptide complexes comprising neo-antigen peptides comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:35-SEQ ID NO:37, SEQ ID NO:40, and SEQ ID NO:48, wherein the MHC molecule is HLA-A. * In some embodiments, the presentation portion comprises one or more MHC-peptide complexes comprising neo-antigen peptides comprising the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:35-SEQ ID NO:37, SEQ ID NO:40, and SEQ ID NO:48, wherein the MHC molecule is HLA-A. *It is 24:02.

[0201] In some embodiments, the presentation portion comprises one or more MHC-peptide complexes comprising neo-antigen peptides comprising an amino acid sequence selected from the group consisting of (e.g., 5, 10, 15, 20, 25, or 30) amino acid sequences selected from the group consisting of SEQ ID NO:9-SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28-SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41-SEQ ID NO:47, SEQ ID NO:49, and SEQ ID NO:50, wherein the MHC molecule is HLA-A. * In some embodiments, the presentation portion comprises one or more MHC-peptide complexes comprising neo-antigen peptides comprising the amino acid sequences set forth in SEQ ID NO:9-SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28-SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41-SEQ ID NO:47, SEQ ID NO:49, and SEQ ID NO:50, wherein the MHC molecule is HLA-A * It is 11:01.

[0202] In some embodiments, the presenting portion comprises one or more MHC-peptide complexes comprising neo-antigenic peptides comprising one or more (e.g., 1, 2, 3, or 4) amino acid sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:8, wherein the MHC molecule is HLA-A. * In some embodiments, the presentation portion comprises one or more MHC-peptide complexes comprising neo-antigen peptides comprising the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:8, wherein the MHC molecule is HLA-A * It is 02:01.

[0203] In some embodiments, the presentation portion comprises one or more MHC-peptide complexes comprising neo-antigenic peptides comprising one or more (e.g., 1, 2, 3, 4 or 5) amino acid sequences selected from the group consisting of SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20 and SEQ ID NO:21, wherein the MHC molecule is HLA-A. * In some embodiments, the presentation portion comprises one or more MHC-peptide complexes comprising neo-antigenic peptides comprising the amino acid sequences set forth in SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20 and SEQ ID NO:21, wherein the MHC molecule is HLA-A * It is 03:01.

[0204] In some embodiments, the presenting portion comprises one or more MHC-peptide complexes comprising neo-antigenic peptides comprising one or more (e.g., 1, 2, 3, or 4) amino acid sequences selected from the group consisting of SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:51, and SEQ ID NO:52, where the MHC molecule is an HLA-B molecule. In some embodiments, the presenting portion comprises one or more MHC-peptide complexes comprising neo-antigenic peptides comprising amino acid sequences set forth in SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:51, and SEQ ID NO:52, where the MHC molecule is an HLA-B molecule.

[0205] In some embodiments, the presentation portion comprises one or more MHC-peptide complexes comprising a neo-antigenic peptide comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 54 and SEQ ID NO: 56, wherein the MHC molecule is an HLA-C molecule. In some embodiments, the presentation portion comprises one or more MHC-peptide complexes comprising a neo-antigenic peptide comprising the amino acid sequences set forth in SEQ ID NO: 54 and SEQ ID NO: 56, wherein the MHC molecule is an HLA-C molecule.

[0206] In some embodiments, the presentation portion comprises an MHC-peptide complex comprising neo-antigen peptides comprising the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO:56, wherein each of the neo-antigen peptides is complexed with an MHC molecule according to Table 1.

[0207] Modification of neoantigen peptides Neo-antigenic peptides with desired activity or properties can be modified to provide certain desired attributes while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind to desired MHC molecules and activate appropriate immune cells (e.g., T cells). For example, the neo-antigenic peptides described herein can be subjected to various changes, e.g., either conservative or non-conservative substitutions, which may provide certain advantages in their use, e.g., improved MHC binding, stability or presentation. By conservative substitution is meant the replacement of an amino acid residue with another that is biologically and / or chemically similar, e.g., replacing one hydrophobic residue with another hydrophobic residue or one polar residue with another polar residue. The substitutions include various combinations, e.g., Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single amino acid substitutions can also be ascertained using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, such as those described in, for example, Merrifield, Science 232:341-347 (1986); Barany & Merrifield, The Peptides, eds. Gross & Meienhofer (NY, Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis (Rockford, 111., Pierce), 2nd ed. (1984).

[0208] The proteins or peptides described herein can be produced by any technique known to those skilled in the art, including protein, polypeptide or peptide expression by standard molecular biology techniques, protein or peptide isolation from natural sources, or protein or peptide chemical synthesis. Nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been previously disclosed, and these sequences can be found in computerized databases known to those skilled in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information at the National Institutes of Health website. The coding regions for known genes can be amplified and / or expressed using the techniques disclosed herein or as would be known to those skilled in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those skilled in the art.

[0209] Isolating, culturing and / or analyzing immune cells that bind the presentation moiety In some embodiments, the methods described herein include isolating immune cells associated with the presenting moiety and analyzing the isolated immune cells. In some embodiments, the methods described herein further include culturing the isolated immune cells prior to the analyzing step.

[0210] In some embodiments, the methods described herein do not include culturing the isolated immune cells prior to the analyzing step.

[0211] In some embodiments, the isolating step comprises separating immune cells associated with the presenting moiety from the remainder of the sample.

[0212] In some embodiments, the isolating step includes using fluorescence-activated cell sorting (FACS). In some embodiments, the presenting moiety may be attached to one or more fluorescent binding moieties, such as a streptavidin core that is attached to or binds to a fluorescent molecule. In some embodiments, the presenting moiety is fluorescent or directly conjugated to a fluorophore. In some embodiments, multiple elements (e.g., particles, binding moieties, binding components) within the presenting moiety may be fluorescent, including each containing a different fluorophore. In some embodiments, the presenting moiety used is magnetic or non-magnetic. In some embodiments, a magnetic separation method may be used in conjunction with FACS (e.g., before FACS, after FACS, or before and after FACS). In some embodiments, magnetic activated cell sorting, affinity chromatography, or any of the various methods of cell sorting known in the art are used.

[0213] In some embodiments, the isolated immune cells are selected from the group consisting of cytotoxic T cells (e.g., CD8+ T cells), helper T cells (e.g., CD4+ T cells), memory T cells, and tumor-infiltrating T cells. In some embodiments, the isolated immune cells are helper T cells.

[0214] In some embodiments, the isolated immune cells are B cells. In some embodiments, the B cells are memory B cells.

[0215] In some embodiments, the isolated immune cell is an isolated single immune cell. In some embodiments, the isolated immune cell is present in a mixture of immune cells. For example, the isolated immune cell (e.g., T cell) may be present in a mixture of immune cells (e.g., a mixture containing both T cells and antigen-presenting cells) when contacted with the presentation moiety. In some embodiments, the mixture of immune cells is a mixture containing T cells, memory T cells, macrophage cells, or dendritic cells, or a combination thereof.

[0216] In some embodiments, culturing the isolated immune cells comprises incubating the isolated immune cells (e.g., T cells) with one or more neo-antigenic peptides described herein. In some embodiments, culturing the isolated immune cells (e.g., T cells) comprises incubating the isolated immune cells (e.g., T cells) with one or more neo-antigenic peptides described herein for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days. In some embodiments, culturing the isolated immune cells (e.g., T cells) comprises incubating the isolated immune cells (e.g., T cells) with one or more neo-antigenic peptides described herein for less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day. In some embodiments, culturing the isolated immune cells comprises incubating the isolated immune cells (e.g., T cells) with one or more cytokines (e.g., IL-2, IL-7, IL-15).

[0217] In some embodiments, analyzing the isolated immune cells comprises detecting the isolated immune cells. In some embodiments, analyzing the isolated immune cells comprises quantifying the isolated immune cells. In some embodiments, analyzing the isolated immune cells comprises sequencing one or more nucleic acids in the isolated immune cells. In some embodiments, analyzing the isolated immune cells further comprises analyzing the sequence of the one or more nucleic acids. In some embodiments, the one or more nucleic acids comprise a nucleic acid sequence selected from the group consisting of TCR sequences. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises whole genome sequencing. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises RNA sequencing. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises single cell sequencing (e.g., scRNAseq, etc.). In some embodiments, analyzing the sequence of the one or more nucleic acids comprises decoding the barcode sequence. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises identifying rearranged genes in the tumor-specific neo-antigen-activated immune cells. In some embodiments, analyzing the sequence of the one or more nucleic acids comprises identifying a rearranged T cell receptor (TCR) gene.

[0218] In some embodiments, analyzing the isolated immune cells includes calculating a number or percentage of immune cells that bind to the bait composition and determining whether the number or percentage of immune cells is above a threshold level, where the number or percentage of immune cells above a threshold level is indicative of cancer in the individual. In some embodiments, the immune cells are T cells (e.g., total T cells). In some embodiments, the immune cells are CD8+ T cells. In some embodiments, the immune cells are CD4+ T cells. In some embodiments, the immune cells are memory T cells. In some embodiments, the threshold is about 1×10 total T cells obtained in the sample (e.g., blood) from the individual. 5 pieces, 5×10 5 pieces, 1×10 6 pieces, 2×10 6 pieces, 6×10 6 pieces, 1×10 7 pieces, 2×10 7 pieces, 5×10 7 pcs or 1×10 8 In some embodiments, at least about 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 500, 1000, 2000, 3000, 4000, or 5000 immune cells bind to the bait composition. In some embodiments, at least about 1×10 5 CD8 cells are analyzed, with a cutoff set at greater than 0.001% (e.g., greater than about 0.001%, greater than 0.002%, greater than 0.003%, greater than 0.004%, or greater than 0.005%) of CD8 cells, and at least greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, or greater than 10 events. In some embodiments, at least about 1 x 10 5 CD8 cells are analyzed and cutoffs are set at greater than 0.005% CD8 cells and at least about 10 events.

[0219] The analysis of the immune repertoire usually involves one or more diversity indices, such as Shannon entropy, clonality, and high expansion clone (HEC) ratio, which are used to evaluate the amplification state of different TCR sequences and determine whether there is a large expansion of a small number of T cell clones. See, for example, Li et al., Cancer Commun (Lond). 2020 Oct; 40 (10): 473-483. It has been found that the greatest diversity was found in peripheral blood samples of healthy populations, consistent with the results of Shannon entropy calculations. Moreover, the TCR repertoire diversity in sentinel lymph nodes from patients with tumors was greater than that in tumor tissues.

[0220] In some embodiments, analyzing immune cells comprises analyzing a repertoire diversity (e.g., Shannon diversity) of T cells obtained from the individual. In some embodiments, analyzing repertoire diversity comprises comparing the repertoire diversity of T cells obtained from the individual to the repertoire diversity of T cells from a reference individual (e.g., a healthy individual, or a group of healthy individuals) and determining whether the repertoire diversity is less than a threshold value, where the repertoire diversity (e.g., Shannon diversity) less than a threshold diversity is indicative of cancer in the individual. In some embodiments, the threshold value is a Shannon diversity less than the Shannon diversity of T cells from a reference individual (e.g., at least about 5%, 10%, 15%, 20%, 25%, or 30%), thereby indicative of cancer in the individual.

[0221] In some embodiments, analyzing the isolated immune cells comprises generating a signature profile. In some embodiments, the signature profile is associated with a particular cancer. In some embodiments, the cancer is hepatocellular carcinoma (HCC), and the signature profile comprises a three-gene signature, where the three genes are CXCR2, CCR2 and EP400. See, for example, Shi et al., Eur J Cancer. 2014 Mar; 50(5): 928-36.

[0222] In some embodiments, analyzing the isolated immune cells includes generating a CD8 and / or CD4 T cell signature profile (e.g., a gene expression profile (RNA-seq), gene rearrangements, 5mC, 5hmC or 5caC profile).

[0223] In some embodiments, analyzing the isolated immune cells comprises sequencing one or more nucleic acids to generate a library. In some embodiments, the one or more nucleic acids are repertoire-related nucleic acids (e.g., VDJ gene-related DNA or RNA). In some embodiments, analyzing the isolated immune cells further comprises analyzing the repertoire of T cells as described above.

[0224] In some embodiments, analyzing the sequence of the one or more nucleic acids comprises a) obtaining an enriched sample from the isolated immune cells that is enriched for the one or more nucleic acids, and b) sequencing the one or more nucleic acids in the enriched sample.

[0225] In some embodiments, analyzing the isolated immune cells further comprises subjecting the isolated immune cells to mass spectrometry analysis. In some embodiments, analyzing the isolated immune cells further comprises subjecting the isolated immune cells to Assay for Transposase-Accessible Chromatin using sequencing (ATAC)-sequencing. In some embodiments, analyzing the isolated immune cells further comprises subjecting the isolated immune cells to chromatin immunoprecipitation (ChIP)-sequencing.

[0226] Epigenetic modifications of tumor-specific immune cells (e.g., T cells, e.g., CD8+ T cells) have been reported. See, e.g., Yang et al., Genome Biol 21, 2(2020); Villanueva et al., Trends Immunol. 2020 Aug; 41(8):676-691. In some embodiments, analyzing the isolated immune cells further comprises identifying one or more epigenetic modifications in a T cell receptor gene of the isolated immune cells. In some embodiments, the one or more epigenetic modifications comprise histone acetylation, histone ubiquitination, and / or histone methylation. In some embodiments, the one or more epigenetic modifications comprise DNA or RNA methylation, hydroxylation, and / or histone glycosylation.

[0227] individual In some embodiments, an individual described herein is a mammal (e.g., a human, a dog, a cat, or a horse). In some embodiments, the individual is a human.

[0228] In some embodiments, the individual has not been previously diagnosed with cancer (eg, any cancer, or a particular type of cancer).

[0229] In some embodiments, the individual has previously been diagnosed with cancer.

[0230] In some embodiments, the individual has minimal residual disease (MRD). In some embodiments, the individual has minimal residual cancer. In some embodiments, the minimal residual cancer is present after the cancer has been surgically removed or cured. In some embodiments, the minimal residual disease is too small to be detected by imaging equipment (e.g., routinely used or standard imaging equipment to detect the cancer). In some embodiments, the location of the minimal residual disease is variable. In some embodiments, the minimal residual cancer is the result of immune evasion or resistance to treatment. In some embodiments, the individual has been previously treated for cancer and does not show any pathological symptoms of cancer after the treatment.

[0231] In some embodiments, the individual is at risk of developing cancer. In some embodiments, the risk of having cancer is based on any one or more factors selected from the group consisting of family history, mutations, environmental factors, and age. In some embodiments, the individual is predicted by a physician to have at least a 20%, 30%, 40%, or 50% risk of developing cancer (e.g., any particular type of cancer) based on any one or more factors selected from the group consisting of family history, mutations, environmental factors, and age.

[0232] In some embodiments, the individual is a human and is at least about 50 years old (eg, at least about 50, 60, 70, or 80 years old).

[0233] In some embodiments, the individual is 14 years of age or younger.

[0234] In some embodiments, the individual is male. In some embodiments, the individual is female.

[0235] cancer The cancers described herein may be general cancers or any type of cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid cancer.

[0236] In some embodiments, the cancer is carcinoma, sarcoma, myeloma, leukemia, lymphoma, blastoma, germ cell tumor, or any combination thereof. In some embodiments, the cancer is squamous cell carcinoma or adenocarcinoma.

[0237] In some embodiments, the cancer is selected from the group consisting of small cell lung cancer, non-small cell lung cancer, nasopharyngeal cancer, colorectal cancer, anal cancer, liver cancer, bladder cancer, testicular cancer, cervical cancer, ovarian cancer, gastric cancer, esophageal cancer, head and neck cancer, pancreatic cancer, prostate cancer, renal cancer, thyroid cancer, melanoma and breast cancer.

[0238] In some embodiments, the cancer is a recurrent cancer.

[0239] Creating reports In some embodiments, the method of analyzing a sample from an individual further comprises generating a report comprising information regarding a cancer status in the individual.

[0240] In some embodiments, the information regarding the cancer status includes the possibility of the presence of cancer, a classification of cancer, a type of cancer, the nature of cancer, the origin of cancer, a stage of cancer, the likelihood of cancer progressing, the likelihood of developing one or more symptoms of cancer, and / or treatment options for the individual.

[0241] The information on the cancer status is based on the results collected from analyzing the immune cells that bind to the bait composition as discussed above. In some embodiments, the likelihood of the presence of cancer is based entirely or at least partially on the number and / or percentage of immune cells (e.g., T cells) that bind to the bait composition. For example, when the number and / or percentage of immune cells (e.g., T cells, e.g., CD8 T cells, e.g., CD4 T cells) that bind to the bait composition significantly exceeds a threshold level (e.g., a threshold level described above), the report generated includes information that the cancer is present in the individual. When the number and / or percentage of immune cells (e.g., T cells, e.g., CD8 T cells, e.g., CD4 T cells) is close to or exceeds a threshold, but does not significantly exceed the threshold, the report generated includes information that the likelihood of the presence of the cancer is medium or high.

[0242] In some embodiments, the likelihood of the presence of cancer is based entirely or at least partially on the repertoire diversity of T cells that bind to the bait composition. For example, when the repertoire diversity (e.g., Shannon diversity) of T cells (e.g., CD8 T cells, e.g., CD4 T cells) that bind to the bait composition is significantly below the threshold, the report generated includes information that the cancer is present in the individual. When the repertoire diversity (e.g., Shannon diversity) of T cells (e.g., CD8 T cells, e.g., CD4 T cells) that bind to the bait composition is close to or below the threshold, but not significantly below the threshold, the report generated includes information that the likelihood of the presence of the cancer is medium or high.

[0243] In some embodiments, the information about the cancer state comprises a gene expression profile signature, a gene mutation profile signature, and / or an epigenetic modification signature. In some embodiments, the signature epigenetic modification comprises a DNA methylation signature and a histone glycosylation signature. EXAMPLES

[0244] The following examples are intended to be purely illustrative of the present invention and therefore should not be construed as limiting the present invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0245] Example 1. A. Method HLA purification HLA-A:02:01, HLA-A:24:02, HLA-A:11:01, HLA-A:03:01 and β-2M were expressed in E. coli. The purified subunits were confirmed by Coomassie blue staining. See, e.g., FIG. 2.

[0246] Kras mutation-specific neoantigen library Neo-antigens containing one of the four most common Kras mutations, including Kras G12V, Kras G12D, Kras G12R, and Kras G12C, were selected to assemble the exemplified neo-antigen library. For each mutation, 20 predicted neo-antigens were designed according to the features of the neo-antigen. These features include binding affinity with MHC molecules and / or T cells, binding stability, sequence of mutations, and published neo-antigen libraries. See, for example, FIG. 1.

[0247] Neoantigen synthesis The neoantigen library was synthesized by Nanjing Peptide Biotech Ltd. The purity of the peptides was >95%.

[0248] Tetramers for specific neoantigens were constructed in vitro. The brief procedure was as follows.

[0249] 1. Place a 100 mL beaker with sterile poles in a 4° C. refrigerator for pre-cooling. Add reduced glutathione to a final concentration of 5 mM; add oxidized glutathione to a final concentration of 0.5 mM; stock concentration is 100 mM (prepared with isopropanol), final concentration is 0.2 mM in a -20° C. refrigerator.

[0250] 2. Take 100 nmol each of MHC heavy chain and 200 nmol each of MHC light chain and thaw them (pre-dissolved in 6 M guanidine hydrochloride). Add 5 mL of injection buffer (see solution composition) and mix homogeneously using a pipette to avoid precipitation caused by high local protein concentration.

[0251] 3. Use 100 ul of DMSO to completely dissolve a mixture of peptides with a purity of 95% or higher (e.g., a mixture of neoantigens containing G12V, G12D, G12R or G12C). Depending on the solubility of the peptide, 30 mg of peptide was dissolved in a minimum amount (e.g., 1 mL) of DMSO.

[0252] 4. Place the folding system (i.e., the system for constructing the MHC-peptide tetramer mixture) in a magnetic stirrer at 4° C. and stir at high speed. Using a 1 mL pipette, slowly add the peptides dropwise to the stirring folding system. Add the peptides dropwise, avoiding excessive local peptide concentrations.

[0253] 5. Place the prepared MHC heavy and MHC light chains on ice to pre-cool them for use. After preparation is complete, use a 1 mL sterile pipette to add 5 mL of the light and heavy chain suspension to the reaction system stirred at high speed. Avoid precipitation caused by excessive local protein concentration. Use the same method when adding the heavy chain to the folding system, adding the folding buffer as close to the mixing rod as possible.

[0254] 6. Return the folding reaction system to 4°C and incubate for 12 hours (overnight). During this period, gently stir using a magnetic stirrer (the rotation speed was controlled at 150-200 rpm / min) to make the folding reaction more complete.

[0255] 7. After incubation (i.e., assembly of the MHC-peptide mixture), add 1 μM MHC heavy chain prepared as in step 2 by the method described in step 5 and incubate thoroughly at 4° C. for 10 hours.

[0256] 8. After incubation (e.g. before leaving for the evening), add an additional 1 μM heavy chain as described above (step 5) and return to 10° C. for at least overnight and at most 5 days.

[0257] 9. Purify the constructed tetramer by anion exchange chromatography. See, for example, Figure 3 for an exemplary successfully constructed MHC tetramer with a neo-antigen peptide containing the G12V mutation. MHC tetramers with neo-antigen peptides containing G12R, G12D or G12C were also generated. Construction of pancreatic tumor cell lines expressing multiple Kras mutation-specific neo-antigens.

[0258] The five most common Kras mutations in pancreatic cancer were co-overexpressed in one plasmid and transfected into cells for stable expression in pancreatic tumor cells Pan02-Luc-GFP or 266-6-luc to produce excess Kras mutation-specific neoantigens. The expression of the transfected Kras mutant sequences was detected by PCR and mass spectrometry. See, for example, Figure 6.

[0259] Animal models of tumor cells co-overexpressing the five most common Kras mutations Subcutaneous tumor model: Tumor cells Pan02-Luc-GFP with different gradient antigen peptide combinations (e.g., 4×10 5C57BL / 6J mice were subcutaneously inoculated with 1000 ng / mL of the 1000 ng / mL IgG1 (1000 ng / mL) ...

[0260] Intravenous model: Tumor cells with different gradient overexpression neoantigen peptide combinations were inoculated via the tail vein. The presence of T cells targeting neoantigens containing Kras mutations was assessed by incubating tetramers targeting specific neoantigen libraries prepared as described above on days 0, 1, 4, 8, 16 and 24, respectively, thus determining the time point of early diagnosis in terms of tumor size and immune stimulation time. The number of residual tumor cells in the peripheral system was monitored in real time by autofluorescence of inoculated cells.

[0261] Collection of peripheral blood from pancreatic cancer patients and capture of T cells Fresh blood was collected from pancreatic cancer patients, and red blood cells were lysed by ACK lysis buffer at room temperature for 5 minutes. Then, the white cell pellet was washed twice with cold PBS on ice before cell staining. Fcblock antibody was used to pretreat the samples for 10 minutes at 4°C, and fluorescently conjugated primary antibodies targeting CD45, CD3e or CD8 were added with tetramer for 30 minutes in the dark at 4°C for cell-specific staining. After staining, cells were washed twice with cold PBS (containing 2% FBS). Finally, cells were immediately detected by flow cytometry, and data was analyzed by Flow Jo V10 software.

[0262] B. Results As shown in Figures 7A-7B, 8, and 9A-9B, Kras mutation-associated neoantigen-specific T cells were expressed in mice inoculated intravenously or subcutaneously with tumor cells expressing Kras mutation-associated neoantigens (10 4As few as 10 cells were successfully detected as early as day 4 after inoculation (see, e.g., FIG. 8), and prior to any detection via bioluminescence (see, e.g., FIG. 10A-B), or in pancreatic cancer patients with Kras mutations (see, e.g., FIG. 11A-C, and FIG. 12). These results demonstrated that the tetramer-based screening platform can be successfully applied for early cancer screening. [Table 2-1] [Table 2-2] [Table 2-3]

Claims

1. A method for analyzing a sample from an individual who does not exhibit any pathological symptoms of cancer, comprising: a) contacting the sample with a bait composition comprising a presentation moiety comprising a cancer neo-antigenic peptide under conditions sufficient for immune cells to bind to the presentation moiety; b) isolating immune cells associated with said presentation moiety; and c) analyzing the isolated immune cells A method comprising:

2. The method described in claim 1, further comprising a step of culturing the isolated immune cells prior to the analyzing step.

3. The method described in claim 1, wherein the presentation portion comprises two or more neo-antigen peptides.

4. The method described in claim 3, wherein the two or more neo-antigen peptides in the presentation portion are the same.

5. The neoantigenic peptide Features include: a) having a binding affinity for an MHC molecule of about 1 nM to about 5000 nM; b) having a binding affinity for its cognate TCR molecule of about 1 nM to about 5000 nM; c) having a mutation, as compared to the wild-type peptide, optionally at the third amino acid position counting from the N-terminus; d) being hydrophobic; and e) having a high content of aromatic residues 10. The method of claim 1, comprising one or more of:

6. The method described in claim 1, wherein the neo-antigen peptide has low immunogenicity.

7. The method described in claim 1, wherein the presentation portion comprises an MHC molecule complexed with the neo-antigen peptide.

8. The method described in claim 7, wherein the MHC molecule is an MHC class I molecule.

9. The method described in claim 8, wherein the MHC class I molecule is selected from the group consisting of HLA-A, HLA-B and HLA-C.

10. The method of claim 9, wherein the peptide is about 8 to about 10 amino acids in length.

11. The method described in claim 7, wherein the MHC is an MHC class II molecule.

12. The method of claim 11, wherein the MHC class II molecule is selected from the group consisting of HLA-DQ and HLA-DR.

13. The method of claim 11, wherein the neo-antigen peptide is about 10 to about 20 amino acids in length.

14. The method described in claim 7, wherein the presentation portion comprises two or more different types of MHC class I molecules selected from the group consisting of HLA-A*24:02, HLA-A*11:01, HLA-A*02:01 and HLA-A*03:

01.

15. The method of claim 1, wherein the presentation portion comprises a particle.

16. The method of claim 1, wherein the particles are selected from the group consisting of surfaces, nanoparticles, beads, and polymers.

17. The method of claim 15, wherein the neo-antigen peptide or MHC is directly attached to the particle.

18. The method described in claim 15, wherein the neo-antigen peptide or MHC is attached to the particle via a binding pair comprising a first binding component attached to the neo-antigen peptide and a second binding component bound to the particle.

19. The method of claim 1, wherein the isolating step includes separating immune cells associated with the presenting portion from the remainder of the sample.

20. The method described in claim 1, wherein the isolated immune cells are selected from the group consisting of cytotoxic T cells, memory T cells, and tumor-infiltrating T cells.

21. The method described in claim 1, wherein the isolated immune cells are present in a mixture of immune cells.

22. The method of claim 21, wherein the mixture of immune cells is a mixture comprising T cells, memory T cells, macrophage cells, or dendritic cells, or a combination thereof.

23. The method described in claim 1, wherein the step of analyzing the isolated immune cells includes detecting the isolated immune cells.

24. The method described in claim 1, wherein the step of analyzing the isolated immune cells includes quantifying the isolated immune cells.

25. The method of claim 1, wherein the step of analyzing the isolated immune cells includes sequencing one or more nucleic acids in the isolated immune cells.

26. The method of claim 1, wherein the individual has not previously been diagnosed with cancer.

27. ​​The method described in claim 26, wherein the individual is at risk of having cancer.

28. The method described in claim 1, wherein the individual has previously been treated for cancer and does not exhibit any pathological symptoms of cancer after the treatment.

29. The method of claim 28, comprising: a) analyzing a pre-treatment sample from said individual prior to anti-cancer treatment and a post-treatment sample from said individual; and b) identifying differences in characteristics of the isolated immune cells from the pre-treatment sample and the isolated immune cells from the post-treatment sample. A method comprising:

30. The method of claim 1, wherein the individual is a human.

31. The method of claim 1, wherein the sample is selected from the group consisting of blood, plasma, and peripheral blood mononuclear cell (PMBC) samples.

32. The method of any one of claims 1 to 31, wherein the bait composition comprises a plurality of different presentation moieties.

33. The method of claim 32, wherein each of the plurality of different presentation portions in the bait composition comprises a different neo-antigen peptide.

34. The method of claim 32, wherein the plurality of different presentation portions in the bait composition comprises at least two different presentation portions, each containing a different MHC molecule.

35. The method of claim 34, wherein the plurality of different presentation portions in the bait composition comprises at least four different presentation portions, each containing a different MHC molecule.

36. A method for obtaining a predetermined characteristic of isolated immune cells as an indicator of cancer in an individual, comprising analyzing a sample from said individual according to claim 1; wherein said predetermined characteristic of said isolated immune cells is indicative of cancer in said individual.

37. a) the predetermined characteristic of the isolated immune cells comprises the presence of the isolated immune cells; b) the predetermined characteristic of the isolated immune cells comprises an amount of the isolated immune cells above a threshold level; c) The method of claim 36, wherein the predetermined characteristics of the isolated immune cells comprise a gene expression profile signature, a gene mutation profile signature, and / or an epigenetic modification signature.

38. A method of obtaining a predetermined characteristic of said isolated immune cells from a post-treatment sample as an indicator of residual cancer in an individual, said individual having previously been treated with an anti-cancer therapy and not exhibiting any pathological symptoms of cancer after treatment, said method comprising analyzing said post-treatment sample from said individual according to the method of claim 28; wherein the predetermined characteristic of the isolated immune cells from the post-treatment sample is indicative of residual cancer in the individual.

39. The method of claim 38, comprising: a) analyzing a pre-treatment sample from the individual prior to anti-cancer treatment and a post-treatment sample from the individual according to the method of claim 28; and b) comparing characteristics of the isolated immune cells from the pre-treatment sample with characteristics of the isolated immune cells from the post-treatment sample. Including, a predetermined difference in a characteristic of the isolated immune cells from the pre-treatment sample and a characteristic of the isolated immune cells from the post-treatment sample is indicative of residual cancer in the individual.

40. A method according to any one of claims 36 to 39, wherein if the indicator indicates that the individual has cancer, it is indicated that the individual should receive anti-cancer treatment.

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