Compositions and methods for identifying antigen-specific T cells

Abstract

Disclosed herein are antigenic peptide-MHC complexes, referred to as comPACT polypeptides and comPACT polynucleotides, and methods for generating such complexes. Also disclosed herein are methods for generating libraries of comPACT polynucleotides and polypeptides and their exemplary use in the high-precision capture of cancer neoepitope-reactive T cells. Dual-particle detection approaches for detecting neoantigen-specific T cells with improved sensitivity and specificity are provided. Also provided are signal-to-noise ratio analyses of isolated T cells for the detection of neoantigen-specific T cells with improved T cell detection.

Description

[Technical field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to, and incorporates by reference in their entireties, U.S. Provisional Application No. 62 / 804,649, filed February 12, 2019, U.S. Provisional Application No. 62 / 826,823, filed March 29, 2019, U.S. Provisional Application No. 62,876,380, filed July 19, 2019, and U.S. Provisional Application No. 62 / 867,165, filed June 26, 2019.

[0002] [Sequence table] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on January 21, 2020, is named 087520_0125_SL.txt and is 292725 bytes in size. [Background technology]

[0003] T cells are the primary mediators of adaptive immunity. Directed by the specificity of each T cell's unique T cell receptor (TCR), T cells regulate autoimmunity, aid in the activation of B cells and innate effectors, and directly kill infected and cancer cells in a precisely targeted manner. Each TCR recognizes a ligand presented by a major histocompatibility complex (MHC) molecule on a target cell. Identification of relevant peptide-MHC complex ligands plays a role in understanding immune responses to tumors and pathogens. MHC complex ligands are also useful for understanding responses to self and dietary antigens. This understanding allows for clinically beneficial immunotherapies (e.g., TCR gene transfer and vaccines) that initiate, amplify, or attenuate immune responses to target antigens.

[0004] Mutant "neoepitopes" are important targets of endogenous and altered immune responses against cancer. Neoepitope-reactive tumor-infiltrating leukocytes (TILs) are present in the endogenous repertoire and regress tumors upon adoptive transfer. Similarly, tumor gene mutation counts predict the clinical efficacy of CTLA-4 or PD-1 blockade, suggesting that these checkpoint inhibition strategies affect tumor regression by liberating neoepitope-reactive T cells. Because neoepitopes arise from somatic mutations in tumor cells, they are not commonly presented by thymic epithelial cells to induce central immune tolerance. Thus, T cell responses directed against these neoepitopes are tumor-specific, likely high affinity, and patient-specific (i.e., personal). From a clinical standpoint, this presents an opportunity and a challenge: neoepitopes are excellent targets for immunotherapy, but TCR isolation methods must be high-throughput enough to enable therapeutic application on a clinically useful scale.

[0005] There is an unmet need for rapid and robust TCR ligand discovery technologies for both basic and translational research. Peptide-MHC multimers allow sorting of T cells according to the antigen specificity of their TCR, which is a critical step in isolating tumor-specific TCRs for gene therapy. A typical current peptide-MHC production protocol begins with solid-phase synthesis of the peptide ligand of interest. In parallel, universal β2-microglobulin and associated MHC class I molecules are heterologously expressed in E. coli, and misfolded inclusion bodies are generated. Each peptide is added to a refolding reaction containing β2-microglobulin and associated MHC class I molecules. Finally, a portion of the ternary complex that refolds correctly can be purified and formulated for use in peptide-MHC multimer generation. To facilitate parallel production of specific MHC molecules with many different peptide ligands, Schumacher and colleagues devised a photocleavable peptide that binds to a specific MHC molecule as a conditional ligand. A single refolding reaction is performed to generate the MHC molecule bound to its conditional ligand. Upon exposure to UV light, the conditional ligand is cleaved and exchanged with the desired peptide present in excess. Many such exchange reactions can be performed in parallel, allowing the construction of a pMHC library for that particular MHC allele. Yet, this state-of-the-art technology has challenging limitations. First, the generation, purification, and refolding of MHC molecules expressed in E. coli inclusion bodies is laborious, resulting in low yields of correctly folded peptide-MHC complexes. Second, the turnaround time (weeks) for commercial peptide synthesis is at odds with optimal timescales in the context of personalized on-demand TCR gene therapy directed at patient-specific neoepitopes. Third, many predicted ligands cannot be used to screen T cells with this approach, because the biophysical properties of the peptide (e.g., hydrophobicity) prevent its synthesis or exchange. Fourth, exchange efficiencies are generally low (<50% exchange efficiency for the majority of predicted HLA-binding peptides).The resulting mixture of correctly folded exchanged MHC and misfolded unliganded MHC results in multimer staining with low signal-to-noise ratios, a problem that is exacerbated when screening T cells with multiplexed peptide-MHC reagent pools. Fifth, the design and validation of conditional ligands for each novel MHC allele is a laborious and non-robust task. As the MHC locus is the most multiallelic locus in the human genome, this is a major obstacle to implementing neoepitope-targeted gene therapy in patients with various MHC haplotypes. Taken together, these constraints highlight the need for novel technologies in this field. Disclosed herein are various compositions and methods for generating peptide-MHC multimers that address these constraints. Summary of the Invention

[0006] The present disclosure provides compositions and methods for identifying neoepitopes, identifying and isolating T cell receptors, modifying primary cells to express specific T cell receptors, expanding the modified T cells, and treating disorders using cellular therapy. In various embodiments, the present invention provides improved cellular therapy methods and compositions for identifying neoepitopes, identifying and isolating T cell receptors, modifying primary cells to express specific T cell receptors, expanding the modified T cells, and treating proliferative diseases, disorders, and conditions.

[0007] In one aspect, provided herein is a method for detecting a marker comprising: providing two or more distinct particle sets, each distinct particle set comprising at least one defined barcode operably associated with a unique antigenic peptide and an identity of the antigenic peptide, each set comprising a first particle comprising a first identification label and a second particle comprising a second identification label different from the first identification label; 11. A method for identifying antigen specificity of a T cell, comprising: providing a sample known or suspected to contain one or more T cells; contacting the sample with two or more sets of particles, where the contacting comprises providing conditions sufficient for a single T cell to bind to a unique antigen of at least one of the set of particles; isolating one or more T cells bound to the set of particles by their associated first and second identifying labels; performing an assay to identify one or more barcodes bound to the set of particles that are bound to the isolated T cells; determining a ratio of barcodes bound to the isolated T cells, where the ratio is calculated by identifying a first copy number of a predominant barcode and a second copy number of a distinct barcode and dividing the first copy number by the second copy number; and identifying the antigen specificity of the T cell based on the ratio.

[0008] In one aspect, provided herein is a method for identifying antigen specificity of a T cell, comprising obtaining or having obtained at least one antigen-specific T cell bound to two or more distinct sets of particles, each distinct set of particles comprising at least one defined barcode operably associated with a unique antigenic peptide and an identity of the antigenic peptide, each set comprising a first particle comprising a first identifying label and a second particle comprising a second identifying label distinct from the first identifying label; performing or having performed at least one assay to identify one or more barcodes detectably associated with the set of particles that is bound to the T cell; and determining or having determined a ratio of barcodes bound to the T cell, identifying the antigen specificity of the T cell, the ratio being calculated by distinguishing between a first copy number of a predominant barcode and a second copy number of a distinct barcode and dividing the first copy number by the second copy number.

[0009] In one aspect, provided herein is a method for identifying antigen specificity of a T cell, comprising: obtaining or having obtained a dataset comprising data relating to one or more barcodes detectably bound to a set of particles that directly or indirectly bind to a T cell, wherein the one or more barcodes are each operatively associated with a unique antigenic peptide; and determining or having determined a ratio of barcodes bound to the T cell that identifies the antigen specificity of the T cell, the ratio being calculated by identifying a first copy number of a predominant barcode and a second copy number of a distinct barcode and dividing the first copy number by the second copy number.

[0010] In some embodiments, the dataset comprises one or more barcodes and one or more barcode copy numbers.

[0011] In some embodiments, the unique antigenic peptide is the same for each distinct set of particles.

[0012] In some embodiments, the first particle comprises a first barcode and the second particle comprises a second barcode that is different from the first barcode, and the first and second barcodes are associated with an identity of an antigen.

[0013] In some embodiments, the ratio of barcodes corresponds to the antigen specificity of the isolated T cells.

[0014] In some embodiments, if the ratio of barcodes exceeds a threshold, the isolated T cells are identified as antigen-specific T cells.

[0015] In some embodiments, the threshold is at least 2 or greater.

[0016] In some embodiments, the threshold is at least 5 or greater.

[0017] In some embodiments, the threshold is at least 10 or greater.

[0018] In some embodiments, the threshold is between 2 and 5.

[0019] In some embodiments, the threshold is between 5 and 10.

[0020] In some embodiments, the threshold is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-5, 3-6, 4-7, 5-8, 5-10, 7-10 or more, or greater than 10.

[0021] In some embodiments, the assay is a nucleotide-based assay.

[0022] In some embodiments, the nucleotide-based assay is a PCR assay, an RT-PCR assay, a sequencing assay, or a hybridization assay.

[0023] In some embodiments, the assay determines the sequence of one or more barcodes.

[0024] In some embodiments, the assay determines the sequence and copy number of one or more barcodes.

[0025] In some embodiments, the method further comprises obtaining T cell receptor (TCR) CDR sequences.

[0026] In some embodiments, the method further comprises obtaining TCR alpha and beta chain sequences.

[0027] In some embodiments, the antigen specificity of the T cell comprises each of (a) the sequence of the antigenic peptide and (b) the TCR sequence of the bound T cell.

[0028] In some embodiments, the first identification label of each first particle is the same in each set.

[0029] In some embodiments, the second identification label of each second particle is the same in each set.

[0030] In some embodiments, the first identification label of each first particle is the same in each set, and the second identification label of each second particle is the same in each set.

[0031] In some embodiments, the first and second identifying labels are fluorophores.

[0032] In some embodiments, the first fluorophore is allophycocyanin (APC).

[0033] In some embodiments, the second fluorophore is phycoerythrin (PE).

[0034] In some embodiments, the set of particles includes a third particle that includes a third barcode that is different from the first and second barcodes, and the first, second, and third barcodes are associated with an identity of an antigen.

[0035] In some embodiments, the unique antigenic peptide is selected from the group consisting of a tumor antigenic peptide, a neo-antigenic peptide, a tumor neo-antigenic peptide, a viral antigenic peptide, a bacterial antigenic peptide, a phospho-antigenic peptide, and a microbial antigenic peptide.

[0036] In some embodiments, the unique antigenic peptide is a neo-antigenic peptide.

[0037] In some embodiments, neoantigens are derived from tumor sequencing data from a subject that is used to identify one or more somatic mutations present in the data compared to wild type.

[0038] In some embodiments, neoantigens are determined using in silico prediction algorithms.

[0039] In some embodiments, the predictive algorithm further comprises an MHC binding algorithm for predicting binding between a neo-antigen and an MHC peptide.

[0040] In some embodiments, the sample is selected from a blood sample, a bone marrow sample, a tissue sample, a tumor sample, or a peripheral blood mononuclear cell (PBMC) sample.

[0041] In some embodiments, the sample is a PBMC sample.

[0042] In some embodiments, the sample is a tumor sample.

[0043] In some embodiments, the sample is a bone marrow sample.

[0044] In some embodiments, the T cells are human T cells.

[0045] In some embodiments, the T cells are CD8+ T cells.

[0046] In some embodiments, the CD8+ T cells are human CD8+ T cells.

[0047] In some embodiments, the methods include a library of distinct particle sets.

[0048] In some embodiments, the library comprises between 2 and 500 distinct particle sets.

[0049] In some embodiments, the library comprises at least 60 distinct particle sets.

[0050] In some embodiments, each particle comprises an MHC peptide.

[0051] In some embodiments, the MHC peptide is a mammalian MHC peptide.

[0052] In some embodiments, the MHC peptide is a human MHC peptide.

[0053] In some embodiments, the MHC peptide is a class I HLA peptide.

[0054] In some embodiments, the HLA peptide comprises an HLA-A, HLA-B, or HLA-C peptide.

[0055] In some embodiments, the HLA peptides are HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:0 1, HLA-A*33:01, HLA-A*68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA- A*29:02, HLA-A*68:02, HLA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:0 2, HLA-B*46:01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA- B*40:02, HLA-B*42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:0 3, HLA-B*37:01, HLA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA- C*03:04, HLA-C*05:01, HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:0 3. Contains HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, or HLA-C*17:01.

[0056] In some embodiments, the HLA peptide comprises a Y84A or Y84C mutation.

[0057] In some embodiments, each particle comprises an HLA I peptide and a β2M peptide.

[0058] In some embodiments, the β2M peptide is a mammalian β2M peptide.

[0059] In some embodiments, the β2M peptide is a human β2M peptide.

[0060] In some embodiments, the β2M peptide contains a mutation that allows or increases binding to a thiol dye.

[0061] In some embodiments, the mutation is S88C.

[0062] In some embodiments, each particle comprises a polypeptide that comprises, from amino terminus to carboxyl terminus, (i) an antigenic peptide, (ii) a β2M peptide, and (iii) an MHC peptide.

[0063] In some embodiments, the polypeptide further comprises a first universal target peptide before the antigenic peptide and a second universal target peptide between the antigenic peptide and the β2M peptide that is different from the first universal target peptide.

[0064] In some embodiments, each particle comprises a polypeptide comprising, from the amino terminus to the carboxyl terminus, (i) a first universal target peptide, (ii) an antigenic peptide, (iii) a second universal target peptide that is different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide.

[0065] In some embodiments, the antigenic peptide is 7-15 amino acids, 7-10, 8-9, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

[0066] In some embodiments, the polypeptide comprising the unique antigenic peptide is biotinylated.

[0067] In some embodiments, each particle in a distinct set of particles comprises a streptavidin core and at least one copy of a unique antigenic peptide.

[0068] In some embodiments, the particles comprise one, two, three, or four copies of a unique antigenic peptide.

[0069] In one aspect, provided herein is a library comprising two or more distinct sets of particles, each comprising a unique antigenic peptide and a defined barcode operably associated with the identity of the antigenic peptide, each set comprising a first particle comprising a first identifying label and a second particle comprising a second identifying label different from the first identifying label.

[0070] In some embodiments, the identifying label is a fluorophore.

[0071] In some embodiments, the fluorophore is APC or PE.

[0072] In one aspect, provided herein is a particle comprising a tetrameric solid support bound to a unique barcode and three or less attached polypeptide molecules, the polypeptide molecules comprising, in amino-terminal to carboxyl-terminal direction, (i) an antigenic peptide, (ii) a β2M peptide, and (iii) an MHC peptide, wherein the barcode is operably associated with the identity of the antigenic peptide.

[0073] In some embodiments, the polypeptide further comprises a first universal target peptide before the antigenic peptide and a second universal target peptide between the antigenic peptide and the β2M peptide that is different from the first universal target peptide.

[0074] In some embodiments, the solid support is a streptavidin core.

[0075] In some embodiments, the polypeptide molecule is biotinylated.

[0076] In some embodiments, the polypeptide molecule is attached to a streptavidin core via a biotin-streptavidin interaction.

[0077] In some embodiments, the particles further comprise an identifying label.

[0078] In some embodiments, the identifying label is a fluorophore.

[0079] In some embodiments, the fluorophore is APC or PE.

[0080] In one aspect, provided herein is a library comprising particles, the library comprising two or more distinct particles, each distinct particle comprising a unique antigenic peptide.

[0081] In one aspect, provided herein is a method for detecting a T cell in a subject comprising the steps of: providing two or more distinct particle sets, each distinct particle set comprising at least one defined barcode operably associated with a unique antigenic peptide and an identity of the antigenic peptide, each set comprising a first particle comprising a first identifying label and a second particle comprising a second identifying label different from the first identifying label; providing a sample known or suspected to comprise one or more T cells, the samples being obtained from a subject over time; contacting the sample with the two or more particle sets, the contacting providing conditions sufficient for a single T cell to bind to a unique antigen of at least one of the particle sets. performing an assay to identify one or more barcodes bound to the isolated T cells; determining a ratio of barcodes bound to the isolated T cells, the ratio being calculated by identifying a first copy number of a predominant barcode from step (e) and a second copy number of a distinct barcode and dividing the first copy number by the second copy number; identifying an antigen specificity of the T cells based on the ratio; and monitoring changes in the antigen-specific T cells identified by the method in the subject.

[0082] In some embodiments, the first and second identifying labels are fluorophores.

[0083] In some embodiments, the fluorophore is APC or PE.

[0084] In one aspect, provided herein is a method of monitoring an immune repertoire in a subject, comprising the steps of: obtaining or having obtained a dataset comprising data relating to one or more barcodes detectably bound, directly or indirectly, to a T cell, wherein each of the one or more barcodes is operatively associated with a unique antigenic peptide; determining or having determined a ratio of barcodes bound to the T cell that identifies the antigen specificity of the T cell, wherein the ratio is calculated by distinguishing a first copy number of a predominant barcode from step (a) and a second copy number of a distinct barcode and dividing the first copy number by the second copy number; identifying the sequence of the unique antigenic peptide bound to the antigen-specific T cell; and monitoring changes in the antigen-specific T cells identified by the method in the subject.

[0085] In some embodiments, the dataset includes one or more barcodes and one or more barcode copy numbers.

[0086] In some embodiments, the unique antigenic peptide is the same for each distinct set of particles.

[0087] In some embodiments, monitoring changes in T cells comprises administering to the subject a soluble, labeled, antigen-specific TCR.

[0088] In some embodiments, the subject is administered immunotherapy based on the identified antigen-specific T cell alterations.

[0089] In some embodiments, the immunotherapy is a checkpoint inhibitor.

[0090] In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the anti-PD-1 antibody is selected from the group including pembrolizumab, nivolumab, and cemiplimab. In some embodiments, the anti-PD-LI antibody is selected from the group including atezolizumab, avelumab, and durvalumab. In some embodiments, the anti-CTLA4 antibody is ipilimumab. In some embodiments, the checkpoint inhibitor is an anti-TIGIT antibody. In some embodiments, the anti-TIGIT antibody is selected from the group including AB154 (Arcus), tiragolumab (Genentech), BMS-986297 (BMS), MK-7684 (Merck), and etigilimab (OncoMed).

[0091] In one aspect, provided herein is a method for detecting a T cell in a subject comprising the steps of: providing two or more distinct particle sets, each distinct particle set comprising at least one defined barcode operably associated with a unique antigenic peptide and an identity of the antigenic peptide, each set comprising a first particle comprising a first identifying label and a second particle comprising a second identifying label different from the first identifying label; providing a sample known or suspected to contain one or more T cells; contacting the sample with the two or more particle sets, the contacting comprising sufficient time for a single T cell to bind to the unique antigen of at least one of the particle sets. providing conditions comprising: isolating one or more T cells associated with a first and a second identifying marker; performing an assay to identify one or more barcodes bound to the isolated T cells; determining a ratio of barcodes bound to the isolated T cells, the ratio being calculated by identifying a first copy number of a predominant barcode from step (e) and a second copy number of a distinct barcode and dividing the first copy number by the second copy number; and identifying a unique antigen peptide sequence bound to the antigen-specific T cells.

[0092] In some embodiments, the first and second identifying labels are fluorophores.

[0093] In some embodiments, the fluorophore is APC or PE.

[0094] In one aspect, provided herein is a method of identifying an antigen.

[0095] The method includes the steps of obtaining or having obtained a dataset comprising data relating to one or more barcodes directly or indirectly bound to T cells, where the one or more barcodes are each operatively associated with a unique antigenic peptide; determining or having determined a proportion of barcodes bound to T cells, where the proportion is calculated by identifying a first copy number of a predominant barcode from step (a) and a second copy number of a distinct barcode and dividing the first copy number by the second copy number; and identifying the sequence of the unique antigenic peptide bound to the antigen-specific T cells.

[0096] In some embodiments, step (a) comprises obtaining a T cell-based sample and assaying it to obtain a dataset.

[0097] In some embodiments, the dataset includes one or more barcodes and one or more barcode copy numbers.

[0098] In some embodiments, the unique antigenic peptide is the same for each distinct set of particles.

[0099] In one aspect, provided herein is a method for detecting T cells comprising the steps of: providing two or more distinct particle sets, each distinct particle set comprising a unique antigenic peptide and at least one defined barcode operably associated with the identity of the antigenic peptide, each set comprising a first particle comprising a first identifying label and a second particle comprising a second identifying label different from the first identifying label; providing a sample known or suspected to contain one or more T cells; contacting the sample with the two or more particle sets, wherein the contacting provides conditions sufficient for a single T cell to bind to the unique antigen of at least one of the particle sets. performing an assay to identify one or more barcodes bound to the isolated T cells; determining a ratio of barcodes bound to the isolated T cells, the ratio being calculated by identifying a first copy number of the predominant barcode from step (e) and a second copy number of a distinct barcode and dividing the first copy number by the second copy number; and identifying HLA and peptide complexes bound to the antigen-specific T cells.

[0100] In some embodiments, the first and second identifying labels are fluorophores.

[0101] In some embodiments, the fluorophore is APC or PE.

[0102] In one aspect, provided herein is a method of identifying HLA and peptide complexes, comprising: obtaining or having obtained a dataset comprising data relating to one or more barcodes detectably bound directly or indirectly to a T cell, wherein each of the one or more barcodes is operably associated with a unique antigenic peptide; determining or having determined a proportion of barcodes bound to the T cell, wherein the proportion is calculated by identifying a first copy number of a predominant barcode from step (a) and a second copy number of a distinct barcode and dividing the first copy number by the second copy number; and identifying HLA and peptide complexes bound to antigen-specific T cells.

[0103] In some embodiments, step (a) comprises obtaining a T cell-based sample and assaying it to obtain a dataset.

[0104] In some embodiments, the dataset includes one or more barcodes and one or more barcode copy numbers.

[0105] In some embodiments, the unique antigenic peptide is the same for each distinct set of particles.

[0106] In one aspect, provided herein is a method of identifying subjects for immunotherapeutic treatment comprising the steps of: providing two or more distinct particle sets, each distinct particle set comprising a unique antigenic peptide and at least one defined barcode operably associated with the identity of the antigenic peptide, each set comprising a first particle comprising a first identification label and a second particle comprising a second identification label distinct from the first identification label; providing a sample known or suspected to comprise one or more T cells; contacting the sample with the two or more particle sets, wherein the contacting provides conditions sufficient for a single T cell to bind to the unique antigen of at least one of the particle sets; isolating one or more T cells associated with the first and second identification labels; performing an assay to identify one or more barcodes bound to the isolated T cells; determining a ratio of barcodes bound to the isolated T cells, wherein the ratio is calculated by distinguishing between a first copy number of a predominant barcode from step (e) and a second copy number of a distinct barcode and dividing the first copy number by the second copy number.

[0107] In some embodiments, the first and second identifying labels are fluorophores.

[0108] In some embodiments, the fluorophore is APC or PE.

[0109] In one aspect, provided herein is a method of identifying subjects for immunotherapy treatment comprising the steps of: obtaining or having obtained a dataset comprising data relating to one or more barcodes detectably bound, directly or indirectly, to a T cell, wherein each of the one or more barcodes is operably associated with a unique antigenic peptide; and determining or having determined a ratio of barcodes bound to the T cell, wherein the ratio is calculated by identifying a first copy number of a predominant barcode from step (a) and a second copy number of a distinct barcode and dividing the first copy number by the second copy number.

[0110] In some embodiments, step (a) comprises obtaining a T cell-based sample and assaying it to obtain a dataset.

[0111] In some embodiments, the dataset includes one or more barcodes and one or more barcode copy numbers.

[0112] In some embodiments, the unique antigenic peptide is the same for each distinct set of particles.

[0113] In some embodiments, the immunotherapy comprises a T cell vaccine, a dendritic cell vaccine, a nucleic acid vaccine, a peptide vaccine, a viral vaccine, a soluble TCR, a TCR-drug conjugate, an antibody, or an antibody-drug conjugate.

[0114] In some embodiments, the antibody comprises a monoclonal antibody.

[0115] In one aspect, provided herein is a method of identifying unique TCR sequences comprising the steps of: providing two or more distinct particle sets, each distinct particle set comprising a unique antigenic peptide and at least one defined barcode operably associated with the identity of the antigenic peptide, each set comprising a first particle comprising a first identifying label and a second particle comprising a second identifying label distinct from the first identifying label; providing a sample known or suspected to comprise one or more T cells; contacting the sample with the two or more particle sets, the contacting comprising providing conditions sufficient for a single T cell to bind to the unique antigen of at least one of the particle sets; isolating one or more T cells associated with the first and second identifying labels; performing an assay to identify one or more barcodes bound to the isolated T cells; determining a ratio of barcodes bound to the isolated T cells, the ratio being calculated by distinguishing between a first copy number of a predominant barcode from step (e) and a second copy number of a distinct barcode and dividing the first copy number by the second copy number; and identifying the unique TCR sequences.

[0116] In some embodiments, the first and second identifying labels are fluorophores.

[0117] In some embodiments, the fluorophore is APC or PE.

[0118] In one aspect, provided herein is a method of identifying a unique TCR sequence, comprising: obtaining or having obtained a dataset comprising data relating to one or more barcodes detectably bound, directly or indirectly, to a T cell, wherein the one or more barcodes are each operatively associated with a unique antigenic peptide; determining or having determined a proportion of barcodes bound to the T cell, wherein the proportion is calculated by identifying a first copy number of a predominant barcode from step (a) and a second copy number of a distinct barcode and dividing the first copy number by the second copy number; and identifying the unique TCR sequence.

[0119] In some embodiments, step (a) comprises obtaining a T cell-based sample and assaying it to obtain a dataset.

[0120] In some embodiments, the dataset comprises one or more barcode sequences and one or more barcode copy numbers.

[0121] In some embodiments, the unique antigenic peptide is the same for each distinct set of particles.

[0122] In some embodiments, the method further comprises producing a soluble TCR polypeptide comprising the identified unique TCR sequence.

[0123] In some embodiments, the soluble TCR polypeptide is linked to a label or a drug.

[0124] In some embodiments, the method is repeated to identify at least two unique TCR sequences.

[0125] In some embodiments, the method further comprises producing a library comprising at least two unique TCR sequences.

[0126] In one aspect, provided herein is a method of treating cancer in a subject, comprising the steps of: obtaining or having obtained a dataset comprising data relating to one or more barcodes detectably associated, directly or indirectly, to a T cell, wherein each of the one or more barcodes is operatively associated with a unique antigenic peptide; determining or having determined a proportion of barcodes associated with the T cell, identifying the antigen specificity of the T cell, wherein the proportion is calculated by distinguishing a first copy number of a predominant barcode from step (a) and a second copy number of a distinct barcode and dividing the first copy number by the second copy number; identifying at least one or both of the TCR sequences of the T cell and generating an engineered T cell comprising at least one or both of the TCR sequences; and administering the engineered T cell to the subject.

[0127] In some embodiments, the method further comprises administering immunotherapy.

[0128] In some embodiments, the immunotherapy is a checkpoint inhibitor.

[0129] In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0130] In some embodiments, the T cells are autologous.

[0131] In some embodiments, the modified T cells are autologous.

[0132] In some embodiments, the unique antigenic peptide is presented by HLA class I on the cell surface of the subject's cancer.

[0133] In certain embodiments, the presently disclosed subject matter provides a method for treating T cells. In certain embodiments, the method includes: (a) contacting a sample with a plurality of distinct particle sets; (b) isolating one or more T cells bound to the particles; (c) identifying the barcodes of the particles bound to the isolated T cells; and (d) determining the ratio of each barcode. In certain embodiments, each particle includes a unique antigenic peptide, an operably associated barcode, and at least one identifying label. In certain embodiments, the sample includes T cells. In certain embodiments, the contacting includes providing conditions suitable for a single T cell to bind to the unique antigenic peptide of at least one of the particle sets.

[0134] In certain embodiments, the ratio is calculated by identifying the number of copies of the first barcode and the number of copies of the second barcode and dividing the number of copies of the first barcode by the number of copies of the second barcode. In certain embodiments, the unique antigenic peptide is the same for each distinct set of particles. In certain embodiments, each distinct set of particles comprises at least one or more barcodes, each barcode being associated with an identity of an antigenic peptide. In certain embodiments, the ratio of each barcode corresponds to the antigen specificity of the isolated T cell. In certain embodiments, the isolated T cell is identified as an antigen-specific T cell if the ratio of the first barcode exceeds a threshold value. In certain embodiments, the threshold value is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-5, 3-6, 4-7, 5-8, 5-10, 7-10 or more, or greater than 10.

[0135] In certain embodiments, identifying the barcodes comprises a nucleotide-based assay. In certain embodiments, the nucleotide-based assay is a PCR, RT-PCR, sequencing, or hybridization assay. In certain embodiments, the nucleotide-based assay determines the sequence of each barcode. In certain embodiments, the nucleotide-based assay determines the copy number of each barcode. In certain embodiments, the nucleotide-based assay determines (a) the sequence of each barcode and / or (b) the copy number of each barcode.

[0136] In certain embodiments, the method further comprises obtaining a T cell receptor (TCR) CDR sequence. In certain embodiments, the method further comprises obtaining a TCR gene sequence. In certain embodiments, the TCR gene sequence is a TCR alpha or TCR beta chain sequence.

[0137] In certain embodiments, the method includes identifying the antigen specificity of the T cell, hi certain embodiments, the antigen specificity of the T cell comprises a TCR sequence of the T cell combined with a sequence of an antigenic peptide.

[0138] In certain embodiments, at least one distinguishing label is the same in each distinct particle set. In certain embodiments, the method includes at least two different distinguishing labels. In certain embodiments, at least one distinguishing label is a fluorophore. In certain embodiments, the fluorophore is selected from the group including allophycocyanin (APC) and phycoerythrin (PE). In certain embodiments, the at least two different distinguishing labels are fluorophores, and the fluorophore is selected from the group including allophycocyanin (APC) and phycoerythrin (PE).

[0139] In certain embodiments, the antigenic peptide is selected from the group consisting of tumor antigens, neo-antigens, tumor neo-antigens, viral antigens, bacterial antigens, phosphoantigens, and microbial antigens. In certain embodiments, the neo-antigens are identified from tumor sequencing data from the subject. In certain embodiments, an in silico prediction algorithm is used to determine the neo-antigens. In certain embodiments, the prediction algorithm further comprises an MHC binding algorithm for predicting binding between the neo-antigen and the MHC peptide.

[0140] In certain embodiments, the sample is selected from a blood sample, a bone marrow sample, a tissue sample, a tumor sample, or a peripheral blood mononuclear cell (PBMC) sample. In certain embodiments, the T cells are human T cells. In certain embodiments, the T cells are CD8 + T cells.

[0141] In certain embodiments, the method comprises a library of distinct particle sets. In certain embodiments, the library comprises between 2 and 500 distinct particle sets. In certain embodiments, each particle comprises an MHC peptide. In certain embodiments, the MHC peptide is a human MHC peptide. In certain embodiments, the MHC peptide is a class I HLA peptide. In certain embodiments, the HLA peptide comprises an HLA-A, HLA-B, or HLA-C peptide.In certain embodiments, the HLA peptides are HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01 , HLA-A*33:01, HLA-A*68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A *29:02, HLA-A*68:02, HLA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02 , HLA-B*46:01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B *40:02, HLA-B*42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03 , HLA-B*37:01, HLA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C *03:04, HLA-C*05:01, HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:03 , HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, or HLA-C*17:01.

[0142] In certain embodiments, each particle comprises an HLA peptide and a β2M peptide. In certain embodiments, the β2M peptide is a human β2M peptide. In certain embodiments, the β2M peptide comprises a mutation. In certain embodiments, the mutation is S88C.

[0143] In certain embodiments, each particle comprises a polypeptide comprising, from amino terminus to carboxyl terminus, (i) an antigenic peptide, (ii) a β2M peptide, and (iii) an MHC peptide. In certain embodiments, the antigenic peptide is 7-15 amino acids, 7-10, 8-9, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In certain embodiments, the polypeptide is biotinylated. In certain embodiments, the particle is selected from the group consisting of magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles. In certain embodiments, the particle is coated with streptavidin.

[0144] In certain embodiments, the presently disclosed subject matter provides a method for monitoring immune repertoire in a subject. In certain embodiments, the method includes monitoring changes in antigen-specific T cells in the subject. In certain embodiments, the method includes administering immunotherapy to the subject. In certain embodiments, the immunotherapy is adoptive cell transfer or checkpoint inhibitors. In certain embodiments, any of the methods disclosed herein are used to monitor immune repertoire in a subject.

[0145] In certain embodiments, the presently disclosed subject matter provides a method for identifying at least one TCR sequence. In certain embodiments, the at least one TCR sequence is a TCR alpha sequence, a TCR beta sequence, or a combination thereof. In certain embodiments, the method further comprises producing a soluble TCR polypeptide. In certain embodiments, any of the methods disclosed herein are used to identify at least one TCR sequence.

[0146] In certain embodiments, the presently disclosed subject matter provides a library of particles. In certain embodiments, the library comprises at least two particle sets. In certain embodiments, each particle set comprises an antigenic peptide, a barcode operably associated with the identity of the antigenic peptide, and at least one identifying label. In certain embodiments, the at least one identifying label is the same in each particle set. In certain embodiments, there are at least two different identifying labels in each distinct particle set. In certain embodiments, the at least one identifying label is a fluorophore. In certain embodiments, the fluorophore is selected from the group including allophycocyanin (APC) and phycoerythrin (PE). In certain embodiments, the at least two different identifying labels are fluorophores, and the fluorophore is selected from the group including allophycocyanin (APC) and phycoerythrin (PE).

[0147] In certain embodiments, the presently disclosed subject matter further provides a particle. In certain embodiments, the particle comprises at least one polypeptide, a barcode, and an identification label. In certain embodiments, the polypeptide comprises an antigenic peptide, a β2M peptide, and an MHC peptide. In certain embodiments, the barcode is operatively associated with the identity of the antigenic peptide. In certain embodiments, the particle is selected from the group consisting of magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles. In certain embodiments, the identification label is a fluorophore. In certain embodiments, the particle is coated with streptavidin. In certain embodiments, the polypeptide is labeled.

[0148] In certain embodiments, the subject matter of the present disclosure further discloses a method of treating cancer in a subject. In certain embodiments, the method includes: (a) preparing a plurality of particles, each of which comprises a plurality of labeled polypeptides; (b) contacting the plurality of particles with a plurality of T cells from a subject under conditions suitable for antigen-specific binding of the T cells to the particles; (c) isolating the T cells bound to the particles and identifying the TCR gene sequences of the isolated T cells; (d) preparing a polynucleotide comprising a homology arm and at least one TCR gene sequence; (e) recombining the polynucleotide into an endogenous locus of the T cells of the subject; (f) culturing the modified T cells to produce a population of T cells; and (g) administering a therapeutically effective number of the modified T cells to the subject, thereby treating cancer. In certain embodiments, the polypeptide comprises an antigen peptide, a β2M sequence, an HLA sequence, and a detectable label. In certain embodiments, the TCR gene sequence is patient-specific. In certain embodiments, the TCR gene sequence is located between the homology arms.

[0149] In certain embodiments, the presently disclosed subject matter further discloses a method of modifying a cell. In certain embodiments, the method comprises: (a) introducing a homologous recombination (HR) template nucleic acid sequence into a cell; and (b) recombining the HR template nucleic acid into an endogenous locus of the cell. In certain embodiments, the HR template nucleic acid comprises: (a) first and second homology arms that are homologous to a first and second endogenous sequence of the cell; (b) a T cell receptor (TCR) gene sequence obtained by any of the methods disclosed herein; and (c) a first 2A coding sequence located upstream of the TCR gene sequence and a second 2A coding sequence located downstream of the TCR gene sequence, the first and second 2A coding sequences encoding the same amino acid sequence, but differing from each other by codons. In certain embodiments, the first and second endogenous sequences are homologous to the first and second homology arms of the HR template nucleic acid. In certain embodiments, the TCR gene sequence is located between the first HR arm and the second HR arm. In certain embodiments, the 2A coding sequence is a P2A coding sequence. In certain embodiments, the HR template further comprises a sequence encoding a flexible linker. In certain embodiments, the sequence encoding the flexible linker is located immediately upstream of the 2A coding sequence. In certain embodiments, the flexible linker has a Gly Ser Gly amino acid sequence. In certain embodiments, the HR template further comprises a sequence encoding a protease cleavage sequence. In certain embodiments, the protease cleavage sequence is a furin sequence. In certain embodiments, the protease cleavage sequence is a TEV sequence. In certain embodiments, the protease cleavage sequence is upstream of a second 2A coding sequence.

[0150] In certain embodiments, the presently disclosed subject matter further discloses compositions comprising modified cells. In certain embodiments, the modified cells comprise an exogenous nucleic acid sequence integrated into an endogenous locus. In certain embodiments, the exogenous nucleic acid sequence comprises: (a) a TCR gene sequence; and (b) a first 2A coding sequence located upstream of the TCR gene sequence and a second 2A coding sequence located downstream of the TCR gene sequence. In certain embodiments, the TCR gene sequence is identified by any of the methods disclosed herein. In certain embodiments, the first and second 2A coding sequences encode the same amino acid sequence, differing by codons from each other. In certain embodiments, the 2A coding sequence is a P2A coding sequence. In certain embodiments, the exogenous nucleic acid sequence further comprises a sequence encoding a flexible linker. In certain embodiments, the sequence encoding the flexible linker is located immediately upstream of the 2A coding sequence. In certain embodiments, the flexible linker has a Gly Ser Gly amino acid sequence. In certain embodiments, the exogenous nucleic acid further comprises a sequence encoding a protease cleavage sequence. In certain embodiments, the protease cleavage sequence is a furin sequence. In certain embodiments, the protease cleavage sequence is a TEV sequence. In certain embodiments, the protease cleavage sequence is upstream of the second 2A coding sequence. [Brief description of the drawings]

[0151] These and other features, aspects, and advantages of the disclosed compositions and methods will become better understood with regard to the following description and the accompanying drawings.

[0152] [Figure 1]Figure 1 shows an exemplary comPACT minigene design. SS refers to an optional signal sequence; US1 refers to the first universal target site; NeoE refers to the antigenic peptide sequence site, neoepitope; US2 refers to the second universal target site; L1 refers to the optional first linker sequence; Beta2m refers to the beta-2-microglobulin domain sequence; L2 refers to the optional second linker sequence; MHC heavy chain refers to the MHC heavy chain allele; L3 refers to the optional third linker sequence; purification cluster refers to an optional purification cluster including a biotinylation sequence, a protease cleavage site, and an affinity tag sequence. While FIG. 1 shows His6 (SEQ ID NO:34) (6-His tag (SEQ ID NO:34)) as the affinity tag, any other suitable affinity tag can be used, including, but not limited to, histidine tags of different lengths (polyHis tags), HAT tags, FLAG tags (or FLAG epitopes), epitopes specific for any antibody used for purification, galactose binding protein tags, fluorescent tags, GST tags, HA tags, Halo tags, MBP tags, Myc tags, polyAsp tags, polyPhe tags, protein C, streptavidin / biotin tags, Strep tags, protein G, or any other protein purification tags capable of purifying comPACT polypeptides.

[0153] Additionally, while Figure 1 shows that nickel resin (see "Ni" in the figure) was used to purify the His6-tagged (SEQ ID NO:34) comPACT polypeptide, other His6 (SEQ ID NO:34) affinity resins have also been used. In particular, zinc resin has been used to successfully purify the His6-tagged (SEQ ID NO:34) comPACT polypeptide from solution. Cobalt and calcium resins are two other exemplary His6 (SEQ ID NO:34) affinity resins that can be used.

[0154] [Diagram 2]Figure 2 shows a diagram of an exemplary modular, off-the-shelf platform for rapidly constructing libraries of antigenic peptide ligands complexed with selected MHC alleles. Figure 2 discloses, in order of appearance, SEQ ID NOs: 9, 11, and 13, respectively.

[0155] [Diagram 3] Figure 3 is a schematic diagram of an exemplary restriction digestion cloning reaction for replacing a dummy insert in an MHC template with a selected neoepitope sequence. The dummy insert (underlined, bold; top set of sequences in the figure) contains four stop codons in different frames and unique restriction sites for destruction of the uncut or religated template. Restriction sites on either side of the insert are indicated by boxes. The bottom set of sequences shows the NeoE insert after the dummy insert has been cut and ligated with the correct NeoE sequence. Figure 3 discloses SEQ ID NOs:270-274, respectively, in order of appearance.

[0156] [Figure 4] FIG. 4 is a schematic diagram of an exemplary restriction digestion cloning reaction for inserting a selected neoepitope sequence into an MHC template. The neoepitope sequence (underlined, bold) is synthesized as a primer flanked by two different restriction sites (boxed). A universal primer with the reverse complement of the 3' restriction site is used in a PCR reaction to form a double stranded primer dimer of the neoepitope sequence. Restriction digestion of both the neoepitope and MCH template vector allows for the insertion of the selected neoepitope sequence into the MHC template sequence by a ligation reaction. The ligation reaction is transformed into E. coli and the plasmid prepared from the transformed E. coli is used in a mammalian producer cell transfection reaction. FIG. 4 discloses SEQ ID NOs: 275-276, 271-274 and 277, respectively, in order of appearance.

[0157] [Diagram 5]Figure 5 is a schematic diagram of an exemplary alternative restriction digest cloning reaction for inserting a selected neoepitope sequence into an MHC template. Two complementary NeoE-encoding primers are synthesized with portions of the 5' and 3' restriction sites. These primers are annealed to simulate an overhang from a restriction digest. A precut vector (which crucially retains a 5' phosphate at its overhanging end) is then ligated with the annealed NeoE insert, and the ligation product is transformed into E. coli for plasmid production. Figure 5 discloses SEQ ID NOs: 278-279, 271-274, and 277, respectively, in order of appearance.

[0158] [Figure 6] 6 is a schematic diagram of an exemplary PCR-based method for inserting a selected neoepitope sequence into an MHC template. Two complementary NeoE-encoding primers are synthesized: a forward primer with the 3' sequence of the second universal site of the MHC template; and a reverse primer with the 3' sequence of the complementary sequence of the first universal site of the MHC template. These primers are mixed together with the 5' fragment of the MHC template with the first universal sequence site and the second fragment of the MHC template with the second universal site and the remainder of the comPACT minigene. A first PCR amplification cycle generates two nucleotide fragments, one fragment encoding the first universal site region including the downstream neoepitope, and the other encoding the neoepitope followed by the remainder of the comPACT gene. These two fragments, overlapping with the unique neoepitope sequence, are then assembled, and the complete assembly is amplified and cleaned up for transfection.

[0159] [Figure 7] FIG. 7 shows a Western blot using NTA-HRP reagent detecting total protein expression and His-tags of 30 mL of mammalian cells transfected with the comPACT gene (Neo12) over a 7-day time course.

[0160] [Figure 8] Figure 8 shows a gel of Ni-NTA affinity chromatography purification of Neo12 comPACT protein, where Pre represents crude lysate, FT represents flow-through, W represents wash, and E represents elution.

[0161] [Figure 9] Figure 9 shows the size exclusion chromatography spectrum of purified Neo12 protein, where the major peak is Neo12 protein and the minor peak is ATP added in the biotinylation step.

[0162] [Figure 10] FIG. 10 shows a purification experiment similar to that shown in FIG. 8, using 0.7 cell culture volumes.

[0163] [Figure 11] Figure 11 shows the crude and purified proteins of eight different NeoE comPACT proteins, each with a different antigen sequence. Figure 11 discloses SEQ ID NOs: 11 and 13, respectively, in order of appearance.

[0164] [Figure 12] FIG. 12 shows size-exclusion chromatography spectra of the eight NeoE comPACT proteins of FIG.

[0165] [Figure 13] Figure 13A shows the NeoE comPACT protein (specifically the Neo12 comPACT protein) generated using the PCR assembly method described in Figure 6 (linear amplicon) compared to the NeoE comPACT protein (specifically the Neo12 comPACT protein) generated from a plasmid (plasmid). Figure 13B shows a DNA gel of the linear amplicons generated by the PCR assembly method. Each lane contains a comPACT minigene (specifically the Neo12 comPACT minigene) with a different neoepitope sequence.

[0166] [Figure 14] Figure 14 shows a streptavidin bead pull-down assay to test complete biotinylation of comPACT protein. Figure 14 discloses "(His6)" as SEQ ID NO:34.

[0167] [Figure 15] Figure 15 shows biotinylation of different comPACT proteins in crude cell lysates visualized via Western blot using streptavidin-HRP. Figure 15 discloses "His6" as SEQ ID NO: 34. Figure 15 also discloses SEQ ID NOs: 280, 280-281 and 281, respectively, in order of appearance.

[0168] [Figure 16] Figures 16A, 16B, and 16C show the production and purification of the BirA enzyme (Figures 16B and 16C) and the TEV protease (Figure 16A) in E. coli.

[0169] [Figure 17] FIG. 17 shows biotinylation of comPACT protein using BirA (lane 2) and cleavage of the His6 tag (SEQ ID NO:34) using TEV protease (lane 3).

[0170] [Figure 18] FIG. 18 shows cell sorting of BirA and V5 transduced cells based on V5 expression.

[0171] [Figure 19] FIG. 19 shows antigen-specific capture of T cells using multimerized comPACT protein.

[0172] [Figure 20] FIG. 20 shows comPACT NTAmer production using S88Cβ2M comPACT protein.

[0173] [Figure 21] FIG. 21 shows the coupling of Cy5 to S88C comPACT protein monomer.

[0174] [Figure 22] Figure 22A shows an exemplary diagram of the cloning strategy for producing comPACT polynucleotides. Figure 22B provides sequence validation statistics obtained from 824 individual comPACT polynucleotides.

[0175] [Figure 23] FIG. 23 provides an exemplary diagram of the workflow for producing comPACT polynucleotides and proteins.

[0176] [Figure 24] 24A and 24B show the percent of patients covered by the top HLA I alleles in the United States compared to comPACT HLA repertoire size.

[0177] [Figure 25A] FIG. 25A shows the monodispersity of comPACT proteins for a representative selection of comPACT proteins. [Figure 25B] FIG. 25B shows comPACT protein yields for a representative selection of comPACT proteins. [Figure 25C] FIG. 25C shows expression of comPACT proteins for a representative selection of comPACT proteins.

[0178] [Figure 26] Figure 26 provides a schematic diagram of the imPACT signal-noise neo-antigen T cell isolation process.

[0179] [Figure 27]Figure 27A provides an illustration of non-specific barcode signal strength for distinguishing signal from noise, and Figure 27B provides an example of specific barcode signal strength for distinguishing signal from noise.

[0180] [Figure 28] FIG. 28 provides an illustration of single versus dual barcodes.

[0181] [Figure 29] Figure 29A provides a schematic of the dual tetramer staining process. Figure 29B provides data showing imPACT tetramer staining of MART5 antigen-specific T cells. Figure 29C provides data showing imPACT tetramer staining of neo12 antigen-specific T cells.

[0182] [Diagram 30] Figure 30 provides an example of the sensitivity of isolated neo-antigen T cells via neo-ID signal-to-noise ratio.

[0183] [Diagram 31] FIG. 31 provides an example of the specificity of the impact neoantigen isolation process (i.e., the imPACT isolation technology).

[0184] [Diagram 32] Figure 32A provides a FACS plot of gene-edited specific T cells (full rectangles) showing a NeoID signal-to-noise ratio greater than 10. Non-specific T cells (circles) show an S / N<10. Figure 32B shows quantification of the signal-to-noise ratio and the average of specific and non-specific T cells.

[0185] [Diagram 33] Figure 33A provides an example of imPACT analysis of a PBMC sample using a single barcode method and validation of the imPACT isolated TCR. Figure 33A discloses SEQ ID NOs: 282-284, respectively, in order of appearance. Figure 33B provides an example of imPACT analysis of a PBMC sample using a single barcode method and validation of the imPACT isolated TCR.

[0186] [Fig. 34A-C] Figure 34A provides a FACS plot of dual stained T cells using imPACT analysis of PBMC samples using a single barcode method. Figure 34A discloses SEQ ID NOs: 285-292, respectively, in order of appearance. Figure 34B provides a FACS plot of CD45RA and CD95 stained T cells after dual tetramer staining. Figure 34B discloses SEQ ID NOs: 285-292, respectively, in order of appearance. Figure 34C provides a table summarizing the TRA (SEQ ID NOs: 293-300, respectively, in order of appearance), TRB (SEQ ID NOs: 285-292, respectively, in order of appearance), gene, and neo-antigen peptide (SEQ ID NOs: 204, 203, 203, 203, 205, 203, and 206-207, respectively, in order of appearance) sequences of isolated T cells after imPACT analysis. [Fig. 34D-E] Figure 34D provides a summary of the neoantigen-specific T cells isolated. Figure 34E provides a summary of the number of lymphocytes isolated for each TCR identified using the imPACT method.

[0187] [Figure 35A] Figure 35A provides an example of a validation screen of the imPACT analysis of the PBMC samples used in Figure 34. Figure 35A discloses SEQ ID NOs: 207, 301, 206, and 302, respectively, in order of appearance. [Figure 35B] FIG. 35B provides an example of a validation screen of the imPACT analysis of the PBMC samples used in FIG.

[0188] [Diagram 36] FIG. 36 provides an illustration of mutant targeted T cells of the PBMC sample used in FIG.

[0189] [Figure 37A-C]Figure 37A provides a FACS plot of dual stained T cells using imPACT analysis of a PBMC sample using dual barcoding. Figure 37B provides a FACS plot of CD45RA and CD95 stained T cells after dual tetramer staining. Figure 37B discloses SEQ ID NOs: 303-305, respectively, in order of appearance. Figure 37C provides a table summarizing the TRA, TRB, gene, and neo-antigen peptide sequences of isolated T cells after imPACT analysis. FIG. 37C discloses "top1.NeoAg" sequences as SEQ ID NOs: 306, 306, 208, 208, 208, 208, 208, 208 and 208; "top2.NeoAg" sequences as SEQ ID NOs: 307, 306, 208, 208, 208, 208, 208, 208 and 208; "tra.CDR3" sequences as SEQ ID NOs: 308-310, 310-312, 312, 312 and 312; "trb.CDR3" sequences as SEQ ID NOs: 313-314, 304, 304, 303, 305, 305, 305 and 305; and "peptide tumor" sequences as SEQ ID NOs: 306, 306, 208, 208, 208, 208, 208, 208 and 208, all in order of appearance, respectively. [Figure 37D] FIG. 37D provides an example of a validation screen of an imPACT assay using comPACT dextramers.

[0190] [Figure 38A] FIG. 38A provides a summary of neoantigen-specific TCRs isolated from patient samples. [Figure 38B] FIG. 38B provides a table summarizing the HAL type, cancer, number of targets, and number of TCRs found in TILs.

[0191] [Figure 39] Figure 39A shows the antigen specificity of HCMV and EBV T cells. Figure 39B shows the number of TCR hits.

[0192] [Figure 40A] FIG. 40A shows a comparison of tetramer, trimer, and dextramer isolation methods using F5 T cells. [Figure 40B] FIG. 40B shows a comparison of tetramer, trimer, and dextramer isolation methods using neo12T cells.

[0193] [Diagram 41] Figure 41A shows a comparison of trimer and dextramer isolation methods using PBMC samples and neo12, CMV, and M1W T cells. Figure 41B shows the average barcode signal-to-noise ratio when using trimer or dextramer.

[0194] [Figure 42A] Figure 42A shows the time course of neoantigen-specific T cells in the peripheral blood of patient PACT157. Figure 42A discloses SEQ ID NOs: 315-317, respectively, in order of appearance. [Figure 42B] Figure 42B shows the time course of neoantigen-specific T cells in the peripheral blood of patient PACT132. Figure 42B discloses SEQ ID NOs: 318-319, respectively, in order of appearance. [Figure 42C] Figure 42C shows the time course of neoantigen-specific T cells in the peripheral blood of patient PACT131. Figure 42C discloses SEQ ID NOs: 320-323, respectively, in order of appearance.

[0195] [Diagram 43] FIG. 43 shows the phenotypic characterization of neoantigen-specific T cells from one patient.

[0196] [Diagram 44] Figure 44A shows the functional characterization of TCR clones isolated against the PIK3CA neo-antigen target. Figure 44B shows the functional characterization of TCR clones isolated against the PIK3CA neo-antigen target.

[0197] [Diagram 45]Figure 45 provides a summary of the number of neo-antigen-specific T cells per CD8 T cell in each T cell sample collected during the course of anti-PD-1 antibody treatment in patient PACT135. Figure 45 discloses "KTYFKPFHPK" as SEQ ID NO:256 and "YFKPFHPKF" as SEQ ID NO:227.

[0198] [Figure 46] Figure 46 shows the potent T cell gene editing efficiency of 14 neoTCRs in both CD4 and CD8 T cells.

[0199] [Figure 47] FIG. 47 shows that neoTCR was internalized upon co-culture of neoTCR T cell cognate comPACT-Dextramare and melanoma-matched cell line M489 with or without iFNγ preincubation.

[0200] [Figure 48] FIG. 48 shows that neoTCR T cells derived from patient PACT135 expressed the activation marker 4-1BB.

[0201] [Figure 49] FIG. 49 shows that neoTCR T cells derived from patient PACT135 expressed the activation marker OX40.

[0202] [Figure 50] FIG. 50 provides a graph of the percentage of tumor cell confluency following co-culture with all neoTCR T cells identified from PACT135.

[0203] [Figure 51A] FIG. 51A provides individual graphs of the percentage of tumor cell confluency following co-culture with each neoTCR T cell. [Figure 51B] FIG. 51B provides individual graphs of the percentage of tumor cell confluency following co-culture with each neoTCR T cell.

[0204] [Figure 52] FIG. 52 shows IFNγ, IL2, and TNFα secretion by TCR218 T cells after co-culture with M489 cells.

[0205] [Figure 53] FIG. 53 shows IFNγ, IL2, and TNFα secretion by TCR221 and TCR227 T cells after co-culture with M489 cells.

[0206] [Figure 54] FIG. 54 shows IFNγ and TNFα secretion by TCR222 T cells after co-culture with M489 cells.

[0207] [Figure 55] Figure 55 shows IFNγ secretion by TCR219, TCR220, TCR223, TCR224, TCR225, TCR228, TCR229, TCR232, TCR240, TCR241 T cells after co-culture with M489 cells with or without pre-treatment with IFNγ.

[0208] [Figure 56] FIG. 56 provides a summary of the numbers of neo-antigen-specific T cells isolated from patient PACT035.

[0209] [Figure 57] Figure 57A shows HLA-A2 expression in KV1858 cell line. Figure 57B shows HLA-A2 expression in KV1832 cell line. Figure 57C shows HLA-A2 expression in SW620 cell line. Figure 57D shows GFP expression in transfected SW620 cells.

[0210] [Figure 58] FIG. 58 shows Nur77 expression in TCR089neoTCR T cells co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation.

[0211] [Figure 59] Figure 59A shows that T cells expressing neoTCR killed SW620 homozygous tumor cells, and Figure 59B shows no killing of wild type SW620 cells.

[0212] [Figure 60] FIG. 60 shows that TCR089 killed SW620 cells homozygous for the COX6C-R20Q mutation, but not wild-type SW620 cells.

[0213] [Figure 61] FIG. 61 shows the secretion of IFNγ in TCR089 T cells after co-culture with SW620 cells homozygous for the COX6C-R20Q mutation.

[0214] [Figure 62] FIG. 62 shows the methodology of the imPACT isolation technology: NeoE-specific TCRs were isolated from patients treated with checkpoint inhibitors, sequencing was performed, tumor antigens were identified, an algorithm was used to select neoepitopes, and screened using comPACT polypeptides and the imPACT isolation technology to capture neoepitope-specific T cells.

[0215] [Figure 63] Figure 63 shows a patient sample from a patient who did not respond to anti-PD-1 therapy, with the patient's neoE-HLA complex and the resulting identified TCR disassembly. Figure 63 discloses SEQ ID NO:324.

[0216] [Figure 64] FIG. 64 shows that neoTCR-T cells kill autologous melanoma tumor cells.

[0217] [Figure 65]Figure 65A shows the ability of neoTCR-T cells to kill autologous tumor cells. Figure 65B shows that neoTCR-T cells express activation markers when co-cultured with autologous tumor cells. Figure 65C shows that neoTCR-T cells secrete interferon gamma when co-cultured with autologous tumor cells.

[0218] [Figure 66] Figure 66A shows that neoTCR T cell therapy eradicated tumors implanted in mice, and Figure 66B shows the number of human CD8 T cells / mL present in mouse blood 4 days after neoTCR T cell infusion and 35 days after infusion.

[0219] [Figure 67A] Figures 67A and 67B show the design of the neo-antigen-specific TCR construct (neoTCR) used to integrate the neo-antigen-specific TCR construct into the TCR alpha locus. Figure 67A shows the target TCR alpha locus (endogenous TRAC, top panel) and its CRISPR Cas9 target site (horizontal stripes, cleavage site indicated by arrows), and the circular plasmid homologous recombination (HR) template (bottom panel) containing the polynucleotide encoding the neoTCR, located between the left and right homology arms ("LHA" and "RHA", respectively) prior to integration. RNP: CRISPR / Cas9 complex. [Figure 67B] FIG. 67B shows neoTCR integrated at the TCR alpha locus (top panel), transcribed and spliced ​​neoTCR mRNA (middle panel), and translation and processing of the expressed neoTCR (bottom panel). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0220] [Definition] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0221] As used herein, the term "antigen-specific T cells" refers to cells that are distinguished from one another by their T cell receptor (TCR) that confers their antigen specificity.

[0222] Embodiments of the compositions and methods disclosed herein include recombinant antigen-MHC complexes capable of pairing with cognate T cells. As used herein, "antigen-MHC," "antigen-MHC complex," "recombinant antigen-MHC complex," "peptide MHC," and "p / MHC" are used interchangeably to refer to a major histocompatibility complex that contains a peptide in its antigen binding groove.

[0223] As used herein, "antigen" includes any antigen, including patient-specific antigens.

[0224] "Antigenic peptide", "antigenic peptide", "neoepitope" and "NeoE" are used interchangeably and refer to a peptide derived from an antigen identified on a cell of interest (e.g., an antigen expressed by a tumor cell, if it is a tumor cell) that is incorporated into a comPACT polypeptide using the molecular biology techniques described herein. Furthermore, as explicitly provided in the Examples, the terms "neo-antigenic sequence" and "neo-antigenic insert" may have the same meaning as "antigenic peptide", "antigenic peptide", "neoepitope" and "NeoE". The terms also refer to a peptide or peptide fragment capable of binding to an MHC molecule.

[0225] "Antigen-MHC complex", "antigen-MHC", "recombinant antigen-MHC complex", "peptide MHC", "p / MHC" and "neoantigen-MHC complex" are all used interchangeably and refer to the ternary complex consisting of an HLA / MHC heavy chain, a β2 M chain, and an antigenic peptide.

[0226] "Anti-CTLA4 antibodies" are antibodies that bind to CTLA-4 and stop it from functioning. This can boost the body's immune response against cancer cells and include ipilimumab. AB154 (Arcus), tiragolumab (Genentech / Roche), BMS-986297 (BMS), MK-7684 (Merck), and etigilimab (OncoMed). In addition to anti-CTLA4 antibodies, CTLA4 inhibitors (both large and small molecules) can be used in combination with any neoTCR product.

[0227] "Anti-PD-1 antibody," "antibody that binds to PD-1," and "anti-PD-1 therapy" refer to an antibody that binds and is capable of binding to PD-1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting PD-1. In certain embodiments, an antibody that binds or is capable of binding to PD-1 can block the interaction of PD-1 with PD-L1 and enhance the immune response against cancer cells. Anti-PD-1 antibodies include, but are not limited to, pembrolizumab, nivolumab, and cemiplimab. In addition to anti-PD1 antibodies, PD1 inhibitors (both large and small molecules) can be used in combination with any neoTCR product.

[0228] "Anti-PD-L1 antibody," "antibody that binds to PD-L1," and "anti-PD-L1 therapy" refer to an antibody that binds to and is capable of binding to PD-L1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting PD-L1. In certain embodiments, an antibody that binds to or is capable of binding to PD-L1 can block the interaction of PD-1 with PD-L1 and enhance the immune response against cancer cells. Anti-PD-L1 antibodies include, but are not limited to, atezolizumab, avelumab, and durvalumab. In addition to anti-PD-L1 antibodies, PD-L1 inhibitors (both large and small molecules) can be used in combination with any neoTCR product.

[0229] "Attachment moiety" means any chemical or biological moiety that can be used to attach a polynucleotide or polypeptide to a chemical or biological substrate. As used herein, an attachment moiety is used to attach a polynucleotide or polypeptide to a particle.

[0230] "Checkpoint inhibitors" refers to a type of drug that blocks certain proteins made by some types of immune system cells, such as T cells, and some cancer cells. These proteins help to suppress the immune response and can prevent T cells from killing cancer cells. When these proteins are blocked, the "brakes" on the immune system are released and T cells can better kill cancer cells. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2. Some immune checkpoint inhibitors are used to treat cancer.

[0231] "Barcode", "barcode sequence", "nucleotide barcode", "barcoded polynucleotide", "neoID" and "neoID barcode" may be used interchangeably and refer to a nucleotide sequence used to tag and identify a particular peptide.

[0232] "Barcoded particle" means a particle that has a barcode attached to it. "Beta-2-microglobulin", "β-2-microglobulin", and "β2M" are used interchangeably and have the same meaning.

[0233] "comPACT" and "comPACT construct" are used interchangeably and refer to either polynucleotides or polypeptides, depending on the context of how the term is used, including neoantigens and MCH complexes. comPACT may further include signal sequences, universal targeting sites, linkers, and purification clusters. Figure 1 shows a non-limiting depiction of comPACT.

[0234] "comPACT library" and "comPACT-neoID library" are used interchangeably and refer to one or more comPACT libraries.

[0235] "comPACT minigene" or "comPACT polynucleotide" or "comPACT gene" or "comPACT polynucleotide molecule" are used interchangeably and refer to a nucleic acid sequence that encodes a comPACT protein.

[0236] "comPACT protein" or "comPACT polypeptide" or "comPACT polypeptide molecule" refers to an MHC molecule expressed as a single polypeptide fusion of a universal target sequence, an antigenic peptide, a second universal target sequence, β2-microglobulin, and an MHC class I heavy chain comprising the α1, α2, and α3 domains that form the MHC display portion. The comPACT polypeptides described herein may further optionally include linker sequences between any or all of the individual components of the comPACT polypeptide. An example of the placement of an optional linker sequence within a comPACT polypeptide is shown in the comPACT minigene in FIG. 1.

[0237] By "effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0238] "Epitope" or "epitope tag" refers to an affinity tag where a peptide sequence is genetically engineered into a polypeptide and an antibody can bind to the peptide sequence. Epitope tags include, but are not limited to, V5 tags, Myc tags, HA tags, Spot tags, NE tags, and all other epitopes that can be used as affinity tags. Epitope tags can be formed from contiguous amino acid stretches (linear epitopes) or can contain non-contiguous amino acids that are in spatial proximity, e.g., due to antigen folding (conformational epitopes). "Linker" refers to any amino acid sequence (or a nucleic acid sequence encoding such an amino acid sequence) used to link components of a fusion protein. When applied to comPACT proteins (fusion proteins), linkers can be used, for example, to link NeoE to β2M, or β2M to MHC heavy chains, or MHC heavy chains to purification clusters.

[0239] Both "host cell" and "producer cell" refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. A host cell includes the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell.

[0240] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0241] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0242] "MHC complex" refers to a complex containing β2-microglobulin and an MHC heavy chain. The MHC complex can be a polypeptide or a polynucleotide encoding such a polypeptide, and an MHC complex is included in all comPACT proteins, and such polynucleotides encoding β2-microglobulin and an MHC heavy chain are included in all comPACT minigenes. Figures 1 and 2 show two examples of MHC complexes included in comPACT minigenes. For example, Figure 11 shows a Western blot of comPACT proteins containing an MHC class I heavy chain complex.

[0243] "MHC display portion" means an MHC class I heavy chain comprising the α1, α2, and α3 domains.

[0244] "MHC" means major histocompatibility complex, a set of genes that encode cell surface proteins essential for the adaptive immune system to recognize foreign molecules. The primary function of MHC molecules is to bind foreign antigens (including antigens presented on endogenous cells that are harmful to an organism such as humans) and display them on the cell surface for recognition by appropriate T cells. The three subgroups of the MHC family are class I, class II, and class III.

[0245] "MHC class I" refers to the subgroup of the MHC family that contains the beta-2-microglobulin subunit.

[0246] "Neoantigen" refers to an antigen that has at least one alteration, e.g., a mutation in the polypeptide sequence, a difference in post-translational modification, or a difference in expression level, that makes the neoantigen or presentation of the neoantigen different from its corresponding wild-type antigen. "Neoantigen" and "tumor neoantigen" refer to a specific antigen on a cell that can be used as an identification target for killing. When applied to cancer and tumors, a neoantigen is an antigen specific to the tumor or cancer. When applied to pathogens and pathogen-infected cells, a neoantigen is an antigen specific to the pathogen or pathogen-infected cell. "Tumor neoantigen" refers to a neoantigen derived from a tumor or cancer, e.g., a tumor in a patient.

[0247] "neoTCR product," "neoTCR T cell therapy," "neoTCR T cell treatment," and "neoTCR T cells" are used interchangeably and all refer to genetically engineered T cells expressing a TCR that recognizes a neoepitope identified and designed using comPACT polypeptide and polynucleotide and imPACT isolation technology.

[0248] "Neo12" and "Neo12 protein" refer to exemplary neoepitopes.

[0249] "NTAmer" refers to a complex comprising a comPACT polypeptide.

[0250] "Prefabricated" refers to a comPACT minigene that includes beta-2-microglobulin, an MHC heavy chain allele, and a location for inserting a neoepitope within such a construct, with respect to the design of the comPACT polynucleotide and the comPACT polypeptide produced therefrom. In certain embodiments, the order of the construct from 5' to 3' is 1) neoepitope, 2) beta-2-microglobulin, and 3) MHC heavy chain allele. In certain embodiments, a signal sequence, a universal target site (e.g., a restriction enzyme site), a flexible linker, and a purification cluster are also incorporated into the construct. In certain embodiments, the structure of the construct, including the additional elements, is the construct disclosed in FIG. 2.

[0251] "Operably associated" means, with respect to particle construction, that each particle constructed using a given comPACT (including a particular neo-antigen expressed therein) is associated with one or more barcodes unique to that particle. In this way, downstream sequencing of which barcode is bound to a particular cell can be used to determine which comPACT (and therefore which neo-antigen) is responsible for that binding.

[0252] "Particle," "particle set," "particle pair," and "distinct particle set" all refer to the comPACT core that contains a substrate that can be specifically sorted or isolated and to which components of comPACT (and additional polypeptides, polynucleotides, and chemical entities) can be attached, with reference to the term "particle." In certain embodiments, a "particle set" refers to a plurality of particles.

[0253] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredients contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is administered.

[0254] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, additives, stabilizers, or preservatives.

[0255] As used herein, "polynucleotide" or "nucleic acid" are used interchangeably and include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by the sequence of bases, where the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. Polynucleotide refers to any DNA (including but not limited to cDNA, ssDNA, and dsDNA) and any RNA (including but not limited to ssRNA, dsRNA, and mRNA), and further includes synthetic forms of DNA and RNA, as well as mixed polymers comprising two or more of these molecules. One of skill in the art will know which form is being referred to based, for example, on the context in which polynucleotide is being used. Polynucleotides can be linear or circular. In addition, the term polynucleotide includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Polynucleotides can include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases, including derivatized sugar or phosphate backbone linkages or chemically modified residues. Polynucleotides include DNA and RNA molecules suitable as vectors to directly express the polypeptides of the invention in vitro and / or in vivo.

[0256] "Proliferative disorder" refers to excessive cell proliferation of one or more subsets of cells in a multicellular organism that results in harm (i.e., discomfort or reduced life expectancy) to the multicellular organism. Cell proliferative disorders can occur in a variety of animals and humans. As used herein, proliferative disorders include neoplastic disorders.

[0257] "Protein" and "polypeptide" are used interchangeably herein.

[0258] "Purification cluster" means an optional portion of comPACT that contains genetically encoded elements that allow for purification of comPACT.

[0259] "Signal sequence" means a short peptide present at the N-terminus of newly synthesized proteins destined for the secretory pathway. Signal sequences can be included in the design and production of comPACT.

[0260] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, prevention of metastasis, slowing the rate of disease progression, amelioration or palliation of the disease state, and remission or improvement of prognosis. In some aspects, the antibodies of the invention are used to delay the onset of disease or to slow the progression of a disease.

[0261] "Universal target site," "universal target sequence," and "universal sequence" may be used interchangeably and refer to a polynucleotide sequence that can be cleaved by a restriction enzyme or a primer binding site that can be used for binding a primer and amplifying a desired sequence.

[0262] "Vector," "expression vector," and "expression construct" can be used interchangeably and refer to discrete elements that are used to introduce heterologous DNA into cells for either its expression or replication. As used herein, vectors can be engineered and used for the in vivo or in vitro expression of a polypeptide gene product encoded by a coding sequence inserted into the vector.

[0263] When referring to T cells, "young" or "younger" refers to memory stem cells (T MSC) and central memory cells (T CM ) These cells exhibit T cell proliferation upon specific activation and are competent for multiple cell divisions. They also have the capacity to engraft after reinfusion and rapidly differentiate into effector T cells upon exposure to their cognate antigen, not only to target and kill tumor cells but also to persist for surveillance and control of ongoing cancer.

[0264] As used herein, the terms "barcoded T cell," "paired T cell," "T cell-binding nanoparticle," and "T cell paired antigen-MHC complex" refer to a complex of a T cell that has a T cell receptor that binds to an antigenic peptide presented by an MHC molecule on a barcoded NP-antigen-MHC complex (i.e., a particle-comPACT complex).

[0265] As used herein, "antibody" or "antibodies" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments.

[0266] The term "in vivo" refers to a process that takes place in an organism that contains a cell.

[0267] The term "mammal" as used herein includes both humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0268] The term "percent sequence identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have a certain percentage of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence, as determined 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 sequence identity" can 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 to be compared.

[0269] In sequence comparison, typically, one sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters.

[0270] Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0271] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0272] Please note that as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0273] [Other interpretative conventions] Ranges described herein are understood to be abbreviated to all values ​​within the range, including the endpoints described. For example, a range of 1 to 50 is understood to include any number or fraction thereof, combination of numbers or fractions thereof, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, including any number or fraction thereof.

[0274] [Preface] T cell-mediated immunity can be characterized by the activation of antigen-specific cytotoxic T cells that can induce death in cells that display antigen in the major histocompatibility complex (MHC) on their surface. These cells that display antigen-containing MHC complexes include virus-infected cells, cells that contain intracellular bacteria, cells that have internalized or phagocytosed extracellular sources of protein, and cancer cells that display tumor antigens.

[0275] Native class I MHC heavy chains contain about 350 amino acids; native β2-microglobulin contains about 100 amino acids, and class I antigenic peptides typically have a length of about 7 to about 15 amino acids. Class I heavy chains are encoded by genes of the major histocompatibility complex, designated HLA-A, -B, and -C in humans and H-2K, D, and L in mice. Class I heavy chains and β2-microglobulin are separately encoded on different chromosomes. Antigenic peptides are usually processed by cells from a protein source, such as a virus, bacteria, or cancer cell. A variety of variants have been identified for the polypeptides encoded by the HLA-A, -B, and -C MHC genes in humans and the H-2K, D, and L MHC genes in mice.

[0276] An embodiment of the method disclosed herein relates to a method for producing a single molecule in which a selected neo-antigen is bound to an MHC complex comprising β2-microglobulin (β2M) and an MHC heavy chain. Different MHC heavy chains can be bound to the β2M molecule to form a different number of MHC templates. By utilizing universal target sequences flanking the neo-epitope insertion site (also called neo-antigen insertion site), the disclosed method of inserting neo-antigens into the MHC template via restriction digestion or PCR-based assembly provides the ability to build a library of different neo-antigen-MHC complexes in a high-throughput manner that can be personalized for a given patient. These complexes are called "comPACT proteins" and can then be bound to particles, barcoded particles, or surfaces for use in, for example, isolating and identifying patient-specific T cell populations that target patient-specific neo-antigens. Methods for binding antigen-MHC complexes and the use of such complexes are disclosed in PCT / US2018 / 21611, filed March 8, 2018, which is incorporated herein by reference in its entirety.

[0277] [Nucleotide and peptide composition] [MHC complex] Briefly, as used herein, a comPACT polypeptide refers to an MHC molecule expressed as a single fusion polypeptide of a universal target sequence, an antigenic peptide, a second universal target sequence, a β2-microglobulin, and an MHC class I heavy chain comprising the α1, α2, and α3 domains forming the MHC display portion. The comPACT polypeptides described herein may further optionally comprise linker sequences between any or all of the individual components of the comPACT polypeptide. An example of the placement of an optional linker sequence within a comPACT polypeptide is provided in the comPACT minigene in FIG. 1. The MHC display portion may comprise a recombinant MHC molecule. The design and production of individual comPACT polypeptides and libraries of comPACT polypeptide molecules are described in International Application PCT / US2019 / 025415, filed April 2, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, comPACT polypeptides may include disulfide traps, as described in U.S. Publication Nos. 2009 / 0117153 and 2008 / 0219947, each of which is incorporated herein by reference. The antigen-MHC complexes formed by comPACT proteins result in the display of the antigen for recognition by a cognate TCR molecule. In some embodiments, the MHC complexes may be MHC class I (MHC I) complexes that are paired with CD8 positive (CD8+) T "killer" cells. In some embodiments, the MHC complexes may be MHC class II (MHC II) complexes that are paired with CD4 positive (CD4+) T cells.

[0278] In some embodiments, the comPACT MHC class I heavy chain sequence may include a single amino acid substitution, addition, and / or deletion, for example, a substitution of Tyr-84 with a non-aromatic amino acid other than proline. In these embodiments, the amino acid substitution may be any amino acid encoded by the standard genetic code, such as leucine, isoleucine, valine, serine, threonine, alanine, histidine, glutamine, asparagine, lysine, aspartic acid, glutamic acid, cysteine, arginine, serine, or glycine, or may be a modified or unusual amino acid. In one embodiment, the comPACT MHC class I heavy chain sequence includes a substitution of tyrosine-84 to alanine. In another embodiment, the comPACT MHC class I heavy chain sequence includes a substitution of tyrosine-84 to cysteine.

[0279] β2-microglobulin (β2M) may comprise a recombinant β2M molecule. In some embodiments, the β2M sequence may comprise a single amino acid substitution, addition, and / or deletion, as described above. In one embodiment, the substitution comprises a serine-88 to cysteine. In one embodiment, the substitution may be a substitution of any naturally occurring non-cysteine ​​amino acid of β2M to cysteine, where the substitution does not adversely affect the function of β2M within the comPACT polypeptide, and where the substitution allows for the conjugation of a thiol-reactive moiety. Such substitutions can be achieved, for example, by cysteine ​​screening of the protein using mutagenesis techniques known to those of skill in the art. Such thiol-reactive moieties can be used to detect β2M or the entire comPACT polypeptide. In certain embodiments, the thiol-reactive moiety is a thiol-reactive dye (fluorophore) conjugate, which allows the use of comPACT to measure kinetic parameters of TCR-comPACT binding (see, for example, Example 8). In certain embodiments, the thiol-reactive moiety is a dye (fluorophore) that contains a sulfhydryl-reactive crosslinker reactive group, including, but not limited to, maleimide, iodoacetamide or derivatives thereof, haloacetyl, pyridyl disulfide, and all other thiol-reactive binding partners (see, e.g., Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

[0280] [Universal Array] The antigenic peptide is generally flanked by a universal sequence or a portion thereof. These sequences allow for a rapid, high-throughput method for replacing or inserting nucleotides encoding antigenic peptides into a polynucleotide MHC template. The universal sequence may include restriction sites for restriction digest-based cloning. Exemplary restriction sites include, but are not limited to, Nco1, BamHI, BlpI, BspEI, BstBI, Xbal, HindIII, EcoRI, ApaI, NotI, β2M, MHC heavy chain, NeoE, any restriction site not present in the signal sequence (if present), purification cluster (if present), or fusions of any of their components (including optional linker sequences), and any combination thereof. Alternatively, the universal sequence may be a primer binding site. Universal primer sequences known in the art may be used in the compositions and methods disclosed herein, or the sequences may be different from the universal primer sequences described above and designed to promote specific binding / amplification and eliminate non-specific binding / amplification. The universal sequence can be 4-50, 4-15, 15-40, 15-35, 15-30, 20-40, 25-40, or 30-40 nucleotides in length. The universal sequence can be at least 4, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides in length. In some embodiments, the universal target sequence is 4-8 nucleotides in length. In other embodiments, the universal target sequence is 9-25 nucleotides in length. In other embodiments, the universal target sequence is 25-35 nucleotides in length. In other embodiments, the universal target sequence is at least about 15 nucleotides in length. In certain aspects, the one or more universal target sequences are absent from the genetic material that has been modified, e.g., to reduce or eliminate off-target effects and / or increase specificity.

[0281] [Linker] In various embodiments, comPACT can include a first flexible linker inserted between the antigenic peptide segment and the β2-microglobulin segment. Such a linker can extend from the carboxyl terminus of the antigenic peptide segment to link the amino terminus of the β2-microglobulin segment. In a non-limiting example, when comPACT is expressed, the linked peptide ligand can fold into the binding groove to generate a functional comPACT protein. In various embodiments, the linker is at least about 10 amino acids and up to about 15 amino acids. In various embodiments, the linker is between 4 and 32 amino acids.

[0282] In various embodiments, comPACT may include a second flexible linker inserted between the β2-microglobulin segment and the MHC heavy chain segment. Such a linker may extend from the carboxyl terminus of the β2-microglobulin segment to and link the amino terminus of the MHC heavy chain segment. In a non-limiting example, expression of comPACT may cause the β2-microglobulin and the MHC heavy chain to fold into a binding groove to generate a molecule that may function in promoting T cell proliferation. In various embodiments, the linker may include at least about 15 amino acids and up to about 20 amino acids. In various embodiments, the linker is at least about 10 amino acids and up to about 15 amino acids. In various embodiments, the linker is between 4 and 32 amino acids.

[0283] In various embodiments, comPACT may include a third flexible linker inserted between the MHC heavy chain segment and the purification cluster. Such a linker may extend from the carboxyl terminus of the MHC heavy chain segment to the amino terminus of the purification cluster, linking them. In various embodiments, the linker is at least about 10 amino acids and up to about 15 amino acids. In various embodiments, the linker is between 4 and 32 amino acids. In various embodiments, the linker is as few as two or three amino acids.

[0284] In certain embodiments, the same linker can be used for the first and second linkers, and optionally for the third linker, if present. In certain embodiments, the same linker is used for the first, second, and third linkers. In certain embodiments, all three linkers are (G4S)4 linkers (SEQ ID NO:19). In certain embodiments, all three linkers are (G3S)n linkers (SEQ ID NO:201). In certain embodiments, all three linkers are (GSGGS)n linkers (SEQ ID NO:11). In certain embodiments, all three linkers are (GCGGS)n linkers (SEQ ID NO:13).

[0285] In certain embodiments, different sequences are used for each of the first and second linkers, and optionally also for the third linker, if present.

[0286] In certain embodiments, two of the first, second, and optionally third linkers are the same and one is different.

[0287] Any suitable flexible linker sequence known in the art may be used. Suitable linker sequences include, but are not limited to, GGGGS (G4S) (SEQ ID NO:9), GGGS (G3S) (SEQ ID NO:201), GSGGS (SEQ ID NO:11), or GCGGS (SEQ ID NO:13) sequence motifs. In certain embodiments, a cleavable linker may be used in either the first, second, or third linker. In certain embodiments, a cleavable linker is used only in the first linker. In certain embodiments, a cleavable linker is used only in the second linker. In certain embodiments, a cleavable linker is used only in the third linker.

[0288] In certain embodiments, the linkers (first, second, and / or third) can be selected from the group comprising rigid or low flexibility linkers.

[0289] [Signal sequence] In various embodiments, comPACT polynucleotides and polypeptides include a signal sequence and a signal peptide. In one embodiment, the signal sequence is a signal sequence from human growth hormone (hGH). Additional signal sequences can also be used, including but not limited to a signal sequence from β2M, or any other eukaryotic or prokaryotic signal sequence known in the art. Any signal sequence that directs the comPACT protein into the secretory pathway (for secretion of comPACT from the cell) can be used.

[0290] In certain embodiments, the signal sequence comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the signal sequence comprises the nucleic acid sequence of SEQ ID NO: 1.

[0291] The signal sequence can be 70 to 80 nucleotides in length. The signal sequence can be 40-90, 40-60, 45-70, 50-80, 60-90, 55-70, 60-80, or 70-80 nucleotides in length. The signal peptide can be 10-30, 10-20, 15-30, or 20-30 amino acids in length.

[0292] [promoter] The comPACT polynucleotide composition may further comprise a promoter for transcribing the encoded polynucleotide into an mRNA transcript that can be translated by a host cell. The promoter may be derived from a prokaryotic, viral, or eukaryotic organism (e.g., but not limited to, mammalian). Any suitable promoter for gene transcription in a cell may be used. In certain embodiments, a eukaryotic promoter may be used. In certain embodiments, the type of eukaryotic promoter is a constitutive promoter, an inducible promoter, or a specific promoter. In certain embodiments, the eukaryotic promoter is an EF1α, cytomegalovirus (CMV), CAG, PGK, RE, U6, or UAS promoter. In certain embodiments, a prokaryotic promoter may be used. In certain embodiments, the type of prokaryotic promoter is a constitutive promoter, a constitutive promoter that requires the presence of a specific polymerase (e.g., T7 or Sp6 RNA polymerase), a promoter that is constitutive in the absence of a repressor and inducible in the presence of an inducer (e.g., but not limited to, the lac promoter, which is constitutive in the absence of the lac repressor but can be induced by IPTG or lactose), an inducible promoter, a repressible promoter, or a regulatable promoter. In certain embodiments, the prokaryotic promoter is a T7, Sp6, lac, araBad, trp, or Ptac promoter. In certain embodiments, a viral promoter can be used. In certain embodiments, the type of viral promoter is an AAV promoter or an SV40 promoter.

[0293] In some embodiments, the comPACT polynucleotide comprises an SV40 or any viral promoter. In certain embodiments, a strong viral promoter may be beneficial depending on the cell line and reagents.

[0294] In some embodiments, the comPACT polynucleotide comprises a CMV promoter.

[0295] [Affinity Tag] The comPACT polynucleotide composition may further comprise at least one sequence encoding an affinity tag or epitope tag. In some embodiments, the comPACT polynucleotide comprises at least two affinity tag or epitope tag sequences. Any suitable affinity tag or epitope tag can be used in the comPACT polynucleotide or polypeptide. Such epitope tags include, but are not limited to, AviTag (or any avidin / streptavidin tag), strep tag, polyhistidine (His6) tag (SEQ ID NO:34), FLAG tag, HA tag, and Myc tag. The sequence of the polynucleotide comPACT gene is translated into a peptide of the comPACT polypeptide. These epitope tags can be used for affinity chromatography purification or quantification of the expressed comPACT polypeptide. For example, the His6 tag (SEQ ID NO:34) can be used to purify the comPACT protein by HA tag binding affinity chromatography. In certain embodiments, a metal ion resin can be used to purify HA-tagged proteins. In certain embodiments, Ni2+ (nickel) resin, Co2+ (cobalt) resin, Cu2+ (copper) resin, Ca2+ (calcium) resin, Zn2+ (zinc) resin, or any combination thereof can be used to purify HA-tagged proteins. In certain embodiments, Ni2+ resin is used to purify HA-tagged comPACT proteins. In certain embodiments, a mixture of Ni2+ and Zn2+ resins is used to purify HA-tagged comPACT proteins. In certain embodiments, the resin is an immobilized metal affinity chromatography resin (IMAC). In certain embodiments, the metal ion is bound to the resin matrix with a chelating ligand. In certain embodiments, the metal ion is bound to the resin matrix with nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA).

[0296] Additionally, AviTag encodes a known biotinylation site recognized by the BirA enzyme. Inclusion of this peptide sequence in a protein allows for biotinylation of the sequence via enzymatic modification by BirA. Thus, a comPACT polypeptide comprising an AviTag (or any avidin / streptavidin tag) sequence and a His6 tag (SEQ ID NO:34) can be biotinylated and purified via the His6 tag (SEQ ID NO:34) by metal affinity chromatography (e.g., Ni-NTA affinity chromatography or any other metal affinity resin described herein), and the purity or quantity of the purified protein can be assessed via biotin visualization with streptavidin or other avidin reagents. In some embodiments, a comPACT polynucleotide comprises an AviTag (or any avidin / streptavidin tag) sequence. In some embodiments, a comPACT polypeptide comprises an AviTag (or any avidin / streptavidin) epitope. In some embodiments, a comPACT polynucleotide comprises a His6 sequence (SEQ ID NO:34). In some embodiments, a comPACT polypeptide comprises a His6 epitope (SEQ ID NO:34). In some embodiments, a comPACT polynucleotide comprises an AviTag (or any avidin / streptavidin) sequence and a His6 sequence (SEQ ID NO:34). In some embodiments, a comPACT polypeptide comprises an AviTag (or any avidin / streptavidin) epitope and a His6 epitope (SEQ ID NO:34).

[0297] [Protease cleavage site] The comPACT polynucleotide composition may further comprise a sequence encoding a protease cleavage site in the purification cluster. This cleavage site may be encoded between the first and second affinity tag sequences, allowing for cleavage of the second affinity tag from the comPACT protein after comPACT has been expressed and subjected to a purification round. Any suitable protease cleavage site known in the art may be used, including, but not limited to, cleavage sites recognized by TEV, thrombin, factor Xa, enteropeptidase, and rhinovirus 3C protease, among others. In one embodiment, the protease cleavage site nucleotide sequence encodes a TEV cleavage site. In another embodiment, the comPACT polypeptide comprises a TEV protease cleavage site.

[0298] [Poly A tail] The comPACT polynucleotide composition may further comprise a polyadenylation (polyA) tail. Eukaryotic (including mammalian) or prokaryotic polyA sequence motifs may be used. This sequence may be included when the comPACT polynucleotide is constructed via PCR (e.g., not in the context of an expression construct or vector) for direct transfection into a host cell. Any suitable polyA tail and sequence motif may be used in the comPACT polynucleotide, including, but not limited to, the polyA tails of SV40, hGH, bGG, and rbGlob sequences. Such sequences include the sequence motif AAUAA. In one embodiment, the comPACT polynucleotide comprises a BHG polyA tail sequence.

[0299] [Antigen sequence] The antigenic sequence (i.e., the sequence of the neo-antigen to which the neoepitope portion of the comPACT polypeptide is designed to bind) can be between 20-60, 20-30, 25-35, 20-45, 30-45, 40-60, or 45-60 nucleotides in length. The antigenic peptide can be or be derived from an exogenous antigen, an endogenous antigen (including heterologous, autologous, and homologous antigens), or an autoantigen. The antigenic peptide can be or be derived from an antigen that originates as an exogenous antigen and then becomes an endogenous antigen (e.g., an intracellular virus). The antigenic peptide can be or be derived from a tumor antigen, a neo-antigen, a tumor neo-antigen, a viral antigen, a bacterial antigen, a phosphoantigen, or a microbial antigen. In one embodiment, the antigenic peptide is a neo-antigen. The antigenic peptide can be selected from patient data and can include one or more somatic mutations.

[0300] To generate a comprehensive comPACT library containing multiple neoepitopes and thus multiple comPACT polypeptides, antigen sequences need to be predicted and identified. Antigenic peptide prediction can include a predictive algorithm, which can be designed to predict the binding of antigenic peptides or neoantigens to MHC alleles. Antigenic peptide prediction is discussed further below.

[0301] In some embodiments, the nucleotide sequence encoding the antigenic peptide is between 20-60, between 20-30, between 25-35, between 20-45, between 30-45, between 40-60, or between 45-60 nucleotides in length. In other embodiments, the nucleotide sequence encoding the antigenic peptide is 20-30 nucleotides in length. In some embodiments, the antigenic peptide is 7-15 amino acids, 7-10, 8-9, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

[0302] [Biotinylation] The comPACT proteins described herein can be further biotinylated via any suitable method. One such method utilizes BirA biotin-protein ligase, which is commercially available. A specific amino acid sequence known as the AviTag sequence (GLNDIFEAQKIEWHE (SEQ ID NO:30)) is encoded by the protein of interest. BirA ligase, d-biotin and ATP are added to a reaction mixture containing the protein of interest. BirA covalently attaches biotin to the lysine of the AviTag sequence, thereby biotinylating the protein of interest. The newly biotinylated protein can then be purified and used for downstream applications. Other methods known in the art for biotinylating proteins can also be utilized. For clarity, any applicable avidin / streptavidin sequence can be used in comPACT protein preparations.

[0303] [Expression constructs and vectors] The comPACT polynucleotide molecules can be inserted into an expression construct or expression vector, for example, a plasmid (to increase the number of expression constructs or expression vectors encoding comPACT polynucleotides for protein production) and for protein production. The expression construct or expression vector can be a eukaryotic, prokaryotic, or viral expression vector. Any suitable expression construct or expression vector known in the art can be used, including bacterial expression plasmids such as E. coli or Bacillus subtilis plasmids; eukaryotic expression vectors such as mammalian expression vectors or yeast expression vectors; or viral vectors such as adenovirus expression vectors, lentivirus expression vectors, vaccinia expression vectors, or baculovirus expression vectors. Mammalian expression constructs or expression vectors can be used (e.g., transfected) in cultured mammalian cell lines, such as Chinese Hamster Ovary (CHO), J558, NSO, SP2-O, HEK293, HECK293T, Expi293, HeLa, or any derivative or variant of the CHO, HEK293, Expi293, or HeLa cell lines, and any other suitable mammalian cell lines. Mammalian expression constructs or expression vectors can be used in primary mammalian cell lines, such as immune cells or tumor cells, either obtained directly from an organism (e.g., a human) or harvested (e.g., from a human), frozen, and then thawed as needed. In addition to mammalian expression vectors and expression constructs, eukaryotic expression vectors and expression constructs can be used (e.g., transfected) in insect cell lines, such as Sf9 or Sf12 (or any derivative or variant thereof), or yeast cell lines, such as Pichia pastoris (or any derivative or variant thereof), as appropriate. In addition, the expression construct or expression vector may include a nucleotide barcode. The nucleotide barcode may be unique for each expression construct or vector. In some embodiments, nucleotide sequences encoding a signal sequence, beta-2-microglobulin, and an MHC allele may be ligated into an expression construct or expression vector having a non-coding or dummy antigen insert.This non-coding antigen insert can then be removed by a suitable cloning technique, such as restriction digestion, and the desired antigen sequence (for clarity in this example, antigen sequence refers to neo-antigen sequence) inserted via ligation or any other suitable cloning technique.

[0304] In some aspects, provided herein are comPACT libraries comprising two or more comPACT polypeptides. Such libraries are generated by encoding two or more comPACT polypeptides in an expression construct or expression vector. In certain embodiments, each expression construct or expression vector comprises a single comPACT polynucleotide. In certain embodiments, the number of expression constructs or expression vectors (each expression construct or expression vector can be the same expression construct or expression vector) is the same number as the number of distinct comPACT polynucleotides. In certain embodiments, the comPACT polynucleotides are inserted into an expression construct or expression vector using the same or different universal target sites. In other aspects, provided herein are MHC libraries comprising two or more MHCs. Such libraries are generated by encoding two or more MHC polypeptides in an expression construct or expression vector. In other aspects, provided herein are HLA libraries comprising two or more HLAs. Such libraries are generated by encoding two or more HLA polypeptides in an expression construct or expression vector.

[0305] [Host cell] In another aspect, provided herein is a host cell comprising a polynucleotide molecule or expression construct as described herein. The host cell may be any suitable host cell known in the art, including, but not limited to, bacterial cells such as E. coli or Bacillus subtilis, or eukaryotic host cells such as Chinese Hamster Ovary (CHO), J558, NSO, SP2-O, HEK293, HEK293T, Expi293, HeLa, insect cell lines such as Sf9 or Sf12, yeast cells such as Pichia pastoris, other suitable eukaryotic or prokaryotic cell lines that are scientifically rational based on the selection of the construct and vector, or any derivative or modification of any such cell line. The host cell may also stably express the biotinylation enzyme BirA. The host cell may be a primary cell or an immortalized cell line.

[0306] In some embodiments, the polynucleotide is integrated into the cell genome. In some embodiments, the polynucleotide is extrachromosomal. In some embodiments, the host cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is selected from the group consisting of a stem cell, a tumor cell, an immortalized cell, and an embryonic cell. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the cell is an E. coli cell. In some embodiments, the cell expresses a BirA protein or a fragment thereof.

[0307] In certain embodiments, any of the expression constructs or expression vectors described herein may be inserted into a host cell for polypeptide production (e.g., inserted through transfection, transformation, or a similar process based on the host cell type). In certain embodiments, an expression construct or expression vector encoding a library of comPACT polypeptides, MHC, or HLA described above may be inserted into a host cell for polypeptide production and purification (e.g., inserted through transfection, transformation, or a similar process based on the host cell type). In certain embodiments, an expression construct or expression vector encoding a library of comPACT polypeptides as described below may be inserted into a host cell for polypeptide production and purification (e.g., inserted through transfection, transformation, or a similar process based on the host cell type).

[0308] [Library] In certain embodiments, the library comprises two or more distinct comPACT polynucleotide molecules. In certain embodiments, the library comprises two or more distinct polypeptide molecules. In certain embodiments, the library comprises two or more distinct comPACT polypeptide molecules attached to particles.

[0309] In certain embodiments, either 1) a comPACT polynucleotide library, 2) a comPACT polypeptide library, or 3) a comPACT polypeptide attached to a particle library, comprises more than two of each molecule in each such library. In certain embodiments, either 1) a comPACT polynucleotide library, 2) a comPACT polypeptide library, or 3) a comPACT polypeptide attached to a particle library, has no upper limit to the number of each molecule in each such library, and thus comprises many each comPACT polypeptide, comPACT polypeptide attached to a particle, or comPACT polynucleotide. In certain embodiments, the upper limit is determined by the number of tumor neo-antigens detected. In certain embodiments, the upper limit is determined by the number of potential neo-epitopes identified based on the tumor neo-antigens detected. In certain embodiments, the upper limit is determined by an algorithm.

[0310] The library may contain 2-1000 comPACT polypeptides, comPACT polypeptides attached to particles, or comPACT polynucleotides. In some embodiments, the library comprises 2-900, 2-800, 2-700, 2-600, 2-500, 2-480, 2-400, 2-300, 2-200, 2-100, 2-50, 2-66, 2-48, 2-30, 2-20, 2-19, 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-480, 10-400, 10-300, 10-200, 10-100, 10-50, 10-66, 10-48, 10-30, 10-20, 20-1000, 20-900 , 20-800, 20-700, 20-600, 20-500, 20-480, 20-400, 20-300, 20-200, 20-100, 20-50, 20-50, 20-66, 20-48, 20-30, 30-1000, 30-900, 30-800, 30-700, 30-600, 30-500, 30-480, 30-400, 30-300, 30-200, 30-100, 30-50, 30-50, 30-66, 30-48, 30-40, 40-1000, 40-900, 40-800, 40-700, 4 0-600, 40-500, 40-480, 40-400, 40-300, 40-200, 40-100, 40-60, 40-50, 40-66, 40-48, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-480, 50-400, 50-300, 50-200, 50-100, 50-60, 50-66, 60-1000, 60-900, 60-800, 60-700, 60-600, 60-500, 60-480, 60-400, 60-300, 60-200 , 60-100, 70-1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70-480, 70-400, 70-300, 70-200, 70-100, 70-80, 70-90, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500, 80-480, 80-400, 80-300, 80-200, 8 ...In some embodiments, the library comprises between 2-19, 48-480, 48-66, 66-480, 220-240, 40-60, 48-66, 50-70, or 60-80 comPACT polypeptides, comPACT polypeptides attached to particles, or comPACT polynucleotides. In some embodiments, the library comprises at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 48, 50, 55, 60, 65, 66, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375 , 400, 425, 450, 475, 500, 525, 550, 600, 562, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 comPACT polypeptides, comPACT polypeptides attached to particles, or comPACT polynucleotides. In some embodiments, the library comprises 2, 10, 15, 20, 24, 48, 66, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 comPACT polypeptides, comPACT polypeptides attached to particles, or comPACT polynucleotides. In some embodiments, two or more comPACT polypeptides, comPACT polypeptides attached to particles, or comPACT polynucleotides in a library have distinct neoepitope sequences and distinct MHC sequences.

[0311] In certain embodiments, the library comprises two or more comPACT polynucleotides, each comPACT polynucleotide in the library comprising a neoepitope sequence and an MHC heavy chain sequence corresponding to a neoantigen detected in a patient sample.

[0312] In some embodiments, the library comprises two or more distinct polynucleotide molecules, where each distinct polynucleotide molecule comprises (i) a first universal sequence, (ii) a nucleotide sequence encoding an antigenic peptide, where the nucleotide sequence is not the same for each of the two or more polynucleotide molecules, (iii) a second universal target sequence, (iv) a β2M sequence, and (v) an MHC allele sequence. In some embodiments, the MHC allele sequence is not the same for each of the two or more polynucleotide molecules.

[0313] HLA-A*01:01 HLA-A*02:01 and HLA-A*03:01 were also expressed 、HLA-A*24:02、HLA-A*30:02、HLA-A*31:01、HLA-A*32:01、HLA -A*33:01、HLA-A*68:01、HLA-A*11:01、HLA-A*23:01、HLA-A*30:01、HLA-A*33:03、HLA-A*25:01、:HL2*A-A*26 、HLA-A*68:02、HLA-B*07:02、HLA-B*14:02、HLA-B*18:01、HLA-B*27:02、HLA-B*39:01、HLA-B*40:01-B*B、HLA.4HL 6:01、HLA-B*50:01、HLA-B*57:01、HLA-B*58:01、HLA-B*08:01、HLA-B*15:01、HLA-B*15:03、HLA-B*45:01-、HLA-B*02:01- -B*42:01、HLA-B*44:03、HLA-B*51:01、HLA-B*53:01、HLA-B*13:02、HLA-B*15:07、HLA-B*27:05、7:HLA-3B 、3 1、HLA-B*38:01、HLA-B*41:02、HLA-B*44:05、HLA-B*49:01、HLA-B*52:01、HLA-B*55:01、HLA-C*02:04-C*0、HL 05:01、HLA-C*07:01、HLA-C*01:02、HLA-C*04:01、HLA-C*06:02、HLA-C*07:02、HLA-C*16:01、HLA-C*03:03 A-C*08:01、HLA-C*08:02、HLA-C*12:02、HLA-C*12:03、HLA-C* 14:02 HLA-C*15:02 and HLA-C*17:01 have also been reported to be associated with HLA-C*17:01.In one embodiment, the library includes at least the HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*68:02, HLA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, HLA-B*46:01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40:02, HLA-B*42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA-B*37:01, HLA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA-C*05:01, HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:03, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, and HLA-C*17:01 alleles.

[0314] In certain embodiments, the HLA library contains HLA-A*01:01、HLA-A*02:01、HLA-A*03:01、HLA-A*11:01、HLA-A*23:01、HLA-A*24:02、HLA-A*25: 01、HLA-A*26:01、HLA-A*29:02、HLA-A*30:01、HLA-A*30:02、HLA-A*31:01、HLA-A*32:01、HLA-A*33:01、HLA-A*33:03、HLA -A*68:01、HLA-A*68:02、HLA-B*07:02、HLA-B*08:01、HLA-B*13:02、HLA-B*14:02、HLA-B*15:01、HLA-B*15:03、HLA-B*15: 07、HLA-B*18:01、HLA-B*27:02、HLA-B*27:05、HLA-B*35:01、HLA-B*35:03、HLA-B*37:01、HLA-B*38:01、HLA-B*39:01、HLA -B*40:01、HLA-B*40:02、HLA-B*41:02、HLA-B*42:01、HLA-B*44:02、HLA-B*44:03、HLA-B*44:05、HLA-B*46:01、HLA-B*49: 01、HLA-B*50:01、HLA-B*51:01、HLA-B*52:01、HLA-B*53:01、HLA-B*55:01、HLA-B*57:01、HLA-B*58:01、HLA-C*01:02、HLA -C*02:02, HLA-C*03:03, HLA-C*03:04, HLA-C*04:01, HLA-C*05:01, HLA-C*06:02, HLA-C*07:01, HLA-C*07:02, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, HLA-C*16:01, HLA-C*17:01.In certain embodiments, the HLA library contains HLA-A*01:01、HLA-A*02:01、HLA-A*03:01、HLA-A*11:01、HLA-A*23:01、HLA-A*24:02、HLA-A*25: 01、HLA-A*26:01、HLA-A*29:02、HLA-A*30:01、HLA-A*30:02、HLA-A*31:01、HLA-A*32:01、HLA-A*33:01、HLA-A*33:03、HLA -A*68:01、HLA-A*68:02、HLA-B*07:02、HLA-B*08:01、HLA-B*13:02、HLA-B*14:02、HLA-B*15:01、HLA-B*15:03、HLA-B*15: 07、HLA-B*18:01、HLA-B*27:02、HLA-B*27:05、HLA-B*35:01、HLA-B*35:03、HLA-B*37:01、HLA-B*38:01、HLA-B*39:01、HLA -B*40:01、HLA-B*40:02、HLA-B*41:02、HLA-B*42:01、HLA-B*44:02、HLA-B*44:03、HLA-B*44:05、HLA-B*46:01、HLA-B*49: 01、HLA-B*50:01、HLA-B*51:01、HLA-B*52:01、HLA-B*53:01、HLA-B*55:01、HLA-B*57:01、HLA-B*58:01、HLA-C*01:02、HLA -C*02:02, HLA-C*03:03, HLA-C*03:04, HLA-C*04:01, HLA-C*05:01, HLA-C*06:02, HLA-C*07:01, HLA-C*07:02, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, HLA-C*16:01, HLA-C*17:01.In certain embodiments, the HLA library comprises at least 50%, 60%, 70%, 80%, or 90% or more of the following HLA alleles: HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-A*23:01, HLA-A*24:02, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*30:01, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*33:03, HLA-A*68:01, HLA-A*68:02, HLA-B*07:02, HLA-B*08:01, HLA-B*13:02, HLA-B*14:02, HLA-B*15:01, HLA-B*15:03, HLA-B*15:07, HLA-B*18:01, HLA-B*27:02, HLA-B*27:05, HLA-B*35:01, HLA-B*35:03, HLA-B*37:01, HLA-B*38:01, HLA-B*39:01, HLA-B*40:01, HLA-B*40:02, HLA-B*41:02, HLA-B*42:01, HLA-B*44:02, HLA-B*44:03, HLA-B*44:05, HLA-B*46:01, HLA-B*49:01, HLA-B*50:01, HLA-B*51:01, HLA-B*52:01, HLA-B*53:01, HLA-B*55:01, HLA-B*57:01, HLA-B*58:01, HLA-C*01:02, HLA-C*02:02, HLA-C*03:03, HLA-C*03:04, HLA-C*04:01, HLA-C*05:01, HLA-C*06:02, HLA-C*07:01, HLA-C*07:02, HLA-C*07:04, HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, HLA-C*16:01, HLA-C*17:01.

[0315] In some embodiments, the library comprises two or more distinct polypeptide molecules, wherein the antigenic peptide is not the same for each of the two or more polypeptide molecules, and each distinct polypeptide is attached to a particle. In some embodiments, the library further comprises a unique defined barcode sequence operably associated with the identity of each distinct polypeptide.

[0316] The embodiment includes a barcoded polynucleotide comprising a defined barcode sequence. The barcoded polynucleotide can be a polynucleotide that provides a unique antigen-specific sequence for identification after T cell isolation. Thus, each unique comPACT is attached to a particle with a unique defined barcode sequence. This allows for an operational association between a given antigen and a pairwise unique given barcode.

[0317] The barcoded polynucleotide can be ssDNA or dsDNA. The polynucleotides containing the barcode can be modified at their 5' end to include an attachment moiety for attachment to a particle. For example, the polynucleotides containing the barcode sequence are conjugated to a biotin molecule to bind to a streptavidin core attached to a particle such as dextran. However, any suitable attachment moiety can be used to attach the polynucleotide to the particle. Suitable attachment moiety pairs are known in the art, as described herein and understood by those skilled in the art. Non-limiting examples of attachment moieties include thiol, maleimide, adamantane, cyclodextrin, amine, carboxy, azide, and alkyne.

[0318] [particle] As used herein, "nanoparticles" or alternatively referred to as "particles" refer to substrates that can be specifically sorted or separated and to which other entities can be attached. In certain embodiments, the "entities" attached to the particles are comPACT and a barcode. In certain embodiments, in addition to comPACT and a barcode, additional entities (e.g., fluorophores or other imaging agents) can be attached to the particles. In certain embodiments, in addition to comPACT and a barcode, additional proteins can be attached to the particles. For example, additional proteins can be attached to the particles to promote T cell binding or increase the stability of comPACT. In certain embodiments, a comPACT protein, a barcode, an imaging agent, and additional proteins can be attached to the particles. In certain embodiments, multiple comPACT proteins are attached to the particles.

[0319] In some embodiments, the particles are magnetic, e.g., for separation using a magnet. In some embodiments, the magnetic particles include magnetic iron oxide. Examples of magnetic particles include, but are not limited to, Dynabeads (Thermo Fisher). In some embodiments, the particles are polystyrene particles, e.g., for separation by gravity. In other embodiments, the particles can be surfaces, beads, or polymers. Examples of beads include, but are not limited to, agarose beads and sepharose beads. In certain embodiments, the particles can be fluorescent or directly or indirectly attached to a fluorophore.

[0320] In certain embodiments, the particles are modified with attachment moieties for attachment of additional molecules. Particle modifications include attachment moieties that can pair (e.g., covalently bind) with corresponding cognate (e.g., complementary) attachment moieties bound to polynucleotides. Any suitable pair of attachment moieties can be used to modify the particles and polynucleotide detection tags for attachment. Non-limiting examples of attachment moiety pairs include streptavidin / biotin systems, thiol groups (e.g., cysteine) and cysteine-reactive moieties (e.g., maleimide, adamantane, and cyclodextrin), amino groups and carboxy groups, and azide groups and alkyne groups. In some embodiments, the attachment moiety can include a cleavage moiety. In other embodiments, the attachment moiety bound to the complementary cognate attachment moiety can be reversible, such as a reducible thiol group. In an exemplary embodiment, the modified particles are streptavidin-coated magnetic nanoparticles, such as 1 μm particles (e.g., Dynabeads MyOn Streptavidin T1 beads from ThermoFisher Scientific), and the polynucleotides can be biotinylated for attachment to the modified particles.

[0321] The particle can be a dextran, such as biotinylated dextran or streptavidin-coated dextran. Modified dextran is described in further detail in Bethune et al., BioTechniques 62:123-130 Mar. 2017 and U.S. Publication No. 2015 / 0329617, which is incorporated herein by reference in its entirety. Biotinylated comPACT can be attached to streptavidin-coated dextran.

[0322] comPACT proteins can also be assembled into tetramers containing one, two, three, or four biotinylated comPACT proteins bound to a streptavidin core. Tetramers can also contain a fluorophore, such as phycoerythrin (PE) or allophycocyanin (APC), bound to the streptavidin core. MHC class I and II tetramers are well known in the art. MHC class I tetramers are described in further detail in Burrow SR et al., J Immunol December 1, 2000, 165 (11) 6229-6234, and MHC class II tetramers are described in further detail in Nepom GT, J Immunol March 15, 2012, 188 (6) 2477-2482, both of which are incorporated herein by reference in their entireties.

[0323] comPACT proteins can also be assembled into multimers. In some embodiments, comPACT protein multimers can be dimers, trimers, tetramers, pentamers, hexamers, or higher order multimers. In some embodiments, multimers can include at least two or more comPACT proteins. In some embodiments, multimers can include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 comPACT proteins.

[0324] [Particle Sets and Libraries] Also contemplated are distinct particle sets, each distinct particle set comprising a unique antigenic peptide (as used herein referred to as comPACT) and at least one defined barcode operably associated with the identity of the antigenic peptide. The particle set comprises at least two particles, each individual particle comprising a unique antigenic peptide and at least one defined barcode operably associated with the identity of the antigenic peptide. In some embodiments, the distinct particle set comprises at least two particles. In some embodiments, the distinct particle set comprises at least three particles. In some embodiments, the distinct particle set comprises at least four particles. In some embodiments, the unique antigenic peptide (as used herein referred to as comPACT) comprises a comPACT polynucleotide molecule or polypeptide molecule.

[0325] Also contemplated are libraries of distinct particle sets. A library of distinct particle sets can include between 2 and 1000 particle sets.In some embodiments, the library comprises 2-900, 2-800, 2-700, 2-600, 2-500, 2-480, 2-400, 2-300, 2-200, 2-100, 2-50, 2-66, 2-48, 2-30, 2-20, 2-19, 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-480, 10-400, 10-300, 10-200, 10-100, 10-50, 10-66, 10-48, 10-30, 10-20, 20-1000, 20-900, 20-8 00, 20-700, 20-600, 20-500, 20-480, 20-400, 20-300, 20-200, 20-100, 20-50, 20-50, 20-66, 20-48, 20-30, 30-1000, 30-900, 30-800, 30-700, 30-600, 30-500, 30-480, 30-400, 30-300, 30-200, 30-100, 30-50, 30-50, 30-66, 30-48, 30-40, 40-1000, 40-900, 40-800, 40-700, 40-600, 40-5 00, 40-480, 40-400, 40-300, 40-200, 40-100, 40-60, 40-50, 40-66, 40-48, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-480, 50-400, 50-300, 50-200, 50-100, 50-60, 50-66, 60-1000, 60-900, 60-800, 60-700, 60-600, 60-500, 60-480, 60-400, 60-300, 60-200, 60-100, 70-100 0, 70-900, 70-800, 70-700, 70-600, 70-500, 70-480, 70-400, 70-300, 70-200, 70-100, 70-80, 70-90, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500, 80-480, 80-400, 80-300, 80-200, 80-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 particles.In some embodiments, the library includes the following particle sets: 2-19, 48-480, 48-66, 66-480, 220-240, 40-60, 48-66, 50-70, or 60-80. In some embodiments, the library comprises at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 48, 50, 55, 60, 65, 66, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 600, 562, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000 particle sets. In some embodiments, the library comprises 2, 10, 15, 20, 24, 48, 66, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 particle sets.

[0326] In certain embodiments, the particle set library of the present disclosure may include one, two, three, four, five, or more particle sets. In certain embodiments, each particle set may include one, two, three, four, five, or more types of particles. In certain embodiments, a particle set may include a single type of particle. In certain embodiments, a particle set may include multiple types of particles. For example, without limitation, a particle set may include particles bound to the same barcode or particles bound to different barcodes, or a combination thereof. In certain embodiments, a particle set may include particles bound to a comPACT polypeptide. In certain embodiments, a particle set may include particles bound to the same comPACT polypeptide, different compact polypeptides, or a combination thereof.

[0327] [Dextramers and tetramers] The comPACT polypeptide can be attached to a dextran, such as a biotinylated dextran or a streptavidin-coated dextran. Modified dextrans are described in more detail in Bethune et al., BioTechniques 62:123-130 Mar. 2017 and U.S. Publication No. 2015 / 0329617, which is incorporated herein by reference in its entirety. The biotinylated comPACT polypeptide can be attached to a streptavidin-coated dextran. In certain embodiments, the dextran is coated with streptavidin. In certain embodiments, the streptavidin is covalently bound to the dextran. In certain embodiments, the streptavidin is non-covalently bound to the biotin-dextran.

[0328] comPACT can also be constructed into tetramers that contain one, two, three, or four biotinylated comPACT proteins bound to a streptavidin core. The tetramer can also contain a fluorophore, such as phycoerythrin (PE) or allophycocyanin (APC), bound to the streptavidin core. In certain embodiments, the fluorophore is selected from the group including PerCP, Cy3, Cy5, and Alexa488. In certain embodiments, the fluorophore is a quantum dot (a non-limiting example is Qdot800). In certain embodiments, any fluorophore with a high extinction coefficient can be used. MHC class I and II tetramers are well known in the art. MHC class I tetramers are described in further detail in Burrow SR et al., J Immunol December 1, 2000, 165 (11) 6229-6234, and MHC class II tetramers are described in further detail in Nepom GT, J Immunol March 15, 2012, 188 (6) 2477-2482, both of which are incorporated herein by reference in their entireties.

[0329] [Method of producing comPACT polypeptide] [Antigen prediction] To produce comPACT, one of the initial steps may include identifying the patient's tumor-specific antigens (e.g., neo-antigens). The compositions generated by this method may then be utilized in T cell-mediated immunization processes, for example, for patient-specific cancer immunotherapy. Identifying the patient's putative neo-antigens (tumor or pathogen) may utilize in silico prediction algorithm programs that analyze tumor, viral, or bacterial sequencing data, including whole genome, whole exome, or transcriptome sequencing data, to identify one or more mutations that correspond to the putatively expressed neo-antigens. In addition, human leukocyte antigen (HLA) typing may be determined from the patient's tumor or blood sample, and this HLA information may be utilized in predictive algorithms for MHC binding, along with the identified putative neo-antigen peptide sequences (see Fritsch et al., 2014, Cancer Immunol Res., 2:522-529, the entire contents of which are incorporated herein by reference). Commonly found HLAs in human populations can also be included in the neoantigen prediction algorithm, such as HLA-A*02,24,01;HLA-B*35,44,51;DRB1*11,13,07 in Caucasians, HLA-A*02,03,30;HLA-B*35,15,44;DRB1*13,11,03 in Afro-Brazilians, and HLA-A*24,02,26;HLA-B*40,51,52;DRB1*04,15,09 in Asians. Specific pairings of HLA alleles can also be used. Common alleles found in human populations are further described in Bardi et al. (Rev Bras Hematol Hemoter. 2012; 34(1): 25-30.).

[0330] Additional examples of methods for identifying neo-antigens include sequencing combined with mass spectrometry and MHC presentation prediction (e.g., U.S. Publication No. 2017 / 0199961), and sequencing combined with MHC binding affinity prediction (e.g., issued U.S. Patent No. 9,115,402). In addition, methods useful for identifying whether neo-antigen-specific T cells are present in a patient sample can be used in combination with the methods described herein, for example, as described in U.S. Publication No. 2017 / 0003288 and PCT / US17 / 59598, which are incorporated herein by reference in their entireties. These analyses provide a ranked list of candidate neo-antigen peptides for the patient that can be readily synthesized using routine methods for screening cognate antigen-specific T cells.

[0331] [Restriction digest assembly] In general, preparation of comPACT polynucleotides can be accomplished by procedures disclosed herein and by recognized recombinant DNA techniques, such as preparation of plasmid DNA, cleavage of DNA with restriction enzymes, ligation of DNA, transformation or transfection of the host, culture of the host, and isolation and purification of the expressed fusion complexes. Such procedures are generally known and are disclosed in standard references such as Sambrook et al., supra.

[0332] In some aspects, DNA encoding an MHC class I heavy chain can be obtained from a suitable cell line, such as, for example, human lymphoblastoid cells. In various configurations, the gene or cDNA encoding the class I heavy chain can be amplified by polymerase chain reaction (PCR) or other means known in the art. In some aspects, the PCR product can also contain a sequence encoding a linker, and / or one or more restriction enzyme sites for ligation of such a sequence.

[0333] In some embodiments, a vector encoding a comPACT polynucleotide can be prepared by ligating sequences encoding an MHC class I heavy chain and β2-microglobulin to sequences encoding an antigenic peptide.

[0334] DNA encoding the antigenic peptide can be obtained by isolating DNA from a natural source or by known synthetic methods, such as the phosphotriester method. See, for example, Oligonucleotide Synthesis, IRL Press (ed. M. Gait, 1984). Synthetic oligonucleotides can also be prepared using commercially available automated oligonucleotide synthesizers. A DNA sequence encoding a universal target sequence discussed herein can be inserted between the sequence encoding the signal sequence and the sequence encoding the antigenic peptide, and a second universal target sequence can be inserted between the sequence encoding the antigenic peptide segment and the sequence encoding the β2-microglobulin segment. In some embodiments, the segments can be joined using a ligase.

[0335] [PCR assembly] In some embodiments, comPACT can be constructed via polymerase chain reaction (PCR) amplification. As with the restriction digestion method, DNA encoding the MHC heavy chain and β2-microglobulin can be obtained from a suitable source. A second DNA fragment encoding the selected signal sequence can also be obtained from a suitable source. Both DNA fragments may have different universal target sequences such that the primer of one universal sequence does not anneal to the second universal sequence. Two sequences encoding the selected antigen peptide can be synthesized; one forward primer with the antigen sequence at its 5' end and the complement of the universal primer sequence on the MHC DNA fragment at its 3' end; and one reverse primer with the reverse complement of the selected antigen sequence at its 5' end and the reverse complement of the universal primer from the signal sequence fragment at its 3' end. A PCR reaction using all four DNA fragments and primers at the 5' end of the signal sequence fragment and the 3' end of the MHC allele fragment amplifies two DNA fragments, one with the signal sequence at its 3' end and the antigen sequence at its 5' end, and one with the antigen sequence at its 3' end and the MHC allele at its 3' end. Additional PCR amplification cycles anneal the overlapping antigen peptide sequences to yield a single full-length DNA fragment. In some embodiments, the signal peptide fragment further comprises a promoter sequence. In some embodiments, the MHC fragment further comprises a purification cluster and / or a polyA tail.

[0336] [Transfection, transduction, and genetic modification of host cells] The comPACT polynucleotide may be inserted into the host cell via any known suitable method, including, but not limited to, transfection, transduction, electroporation, lipofection, sonoporation, mechanical disruption, or viral vectors. Exemplary transfection reagents include, but are not limited to, FectorPro, Expifectamine, lipofectamine, polyethylenimine (PEI), Fugene, or any other transfection reagent that provides optimal transfection rates based on the cell type, transfection system, transfection type, transfection conditions, and construct to be transfected. Transfected. In some examples, Expifectamine is used to transfect mammalian cells with comPACT polynucleotides. In some examples, polyethylenimine is used to transfect mammalian cells with comPACT polynucleotides. In some examples, FectorPro is used to transfect mammalian cells with comPACT polynucleotides.

[0337] The comPACT polynucleotide can be expressed transiently or stably in the host cell. In some embodiments, the comPACT polynucleotide is integrated into the host genome. In other embodiments, the comPACT polynucleotide remains extrachromosomal. Any suitable gene editing technique known in the art can also be used to modify the host cell with the comPACT polynucleotide, including CRISPR / Cas9, zinc finger nucleases, or TALEN nucleases.

[0338] [Expression] Many strategies can be used to express comPACT polypeptides. For example, comPACT polynucleotides can be incorporated into a suitable vector by known methods, such as by using restriction enzymes and ligases (see, e.g., Sambrook et al., supra). A vector can be selected based on factors related to the cloning protocol. For example, the vector can be compatible with and have an appropriate replicon for the host being used. Suitable host cells include eukaryotic and prokaryotic cells and can be cells that can be easily transformed and exhibit rapid growth in culture. Examples of host cells include prokaryotes such as E. coli and Bacillus subtilis, and eukaryotes such as animal cells and yeast, e.g., mammalian cells and human cells. Non-limiting examples of mammalian cells that can be used as hosts to express comPACT include J558, NSO, SP2-O, 293T, Expi293, and CHO (and any derivatives or modifications of J558, NSO, SP2-O, 293T, Expi293, and CHO cell lines). Other examples of possible hosts include insect cells such as Sf9, which can be grown using common culture conditions. See Sambrook et al., supra. In various embodiments, cells expressing comPACT polypeptides can be identified using known methods. For example, expression of comPACT polypeptides can be determined by ELISA, FACS, or Western blot. In certain embodiments, expression of comPACT polypeptides can be determined by ELISA, FACS, or Western blot using an antibody probe against the MHC heavy chain portion of comPACT, or an antibody against an affinity tag such as His6 (SEQ ID NO:34), or streptavidin reagent if comPACT is biotinylated.

[0339] In some embodiments, comPACT is expressed in mammalian cells. There are several advantages to expressing proteins in mammalian cells rather than E. coli cells. Proteins expressed in E. coli cells must be carefully purified from lipopolysaccharide (LPS). When proteins are expressed in mammalian cells, there is no LPS contamination of the purified protein. In addition, mammalian cells are more likely to properly fold mammalian proteins because they produce proteins with the correct post-translational modifications required for proper folding, including proper formation of disulfide bonds. In addition, mammalian cells provide the correct chaperone proteins to assist protein folding in the endoplasmic reticulum or Golgi apparatus. This increases the purification of homogeneously folded proteins compared to proteins expressed in E. coli cells.

[0340] In some aspects, comPACT is expressed in prokaryotic cells. In certain embodiments, prokaryotic cells genetically engineered to post-translationally modify comPACT. In certain embodiments, comPACT expressed in prokaryotic cells is substantially free of LPS or has no detectable LPS as measured using LPS detection methods known in the art.

[0341] comPACT may be substantially free of LPS. comPACT may be free of LPS, e.g., comPACT may have no detectable LPS as measured using LPS detection methods known in the art. comPACT may be glycosylated. comPACT may have one or more post-translational modifications. comPACT may be modified via one or more post-translational modifications, such as, for example, glycosylation, upon expression in a eukaryotic or, in certain embodiments, mammalian cell. comPACT may include one or more post-translational modifications. comPACT may be (1) substantially free of LPS or free of LPS, and (2) glycosylated.

[0342] [Exemplary comPACT workflow process] FIG. 23 shows an exemplary schematic diagram of comPACT protein construction and expression. Sense and antisense oligos encoding the desired neo-antigen peptide sequence can be synthesized and annealed to form double-stranded oligos with overhangs at the 5' and 3' ends, which can be ligated to a plasmid containing the β2M gene and MHC alleles. The complete comPACT oligo can be amplified into a double-stranded amplicon and transfected into cells for protein expression and optional biotinylation. comPACT protein can be assessed by SDS-PAGE. comPACT polynucleotides can then be selected to scale up plasmid production in E. coli. Protein producer cells are transfected with the selected plasmid, and comPACT is purified from the producer cells for use in functional assays.

[0343] [Purification (chromatography)] The expressed comPACT polypeptides can be isolated and purified by known methods. For example, comPACT containing a His6 affinity tag (SEQ ID NO:34) can be purified via affinity chromatography on a metal affinity chromatography column (e.g., a Ni-NTA column or any other metal affinity resin column described herein, such as Co2+, Ca2+, Zn2+, Cu2+ resin or any combination thereof (including Ni2+)) by procedures generally known and disclosed. In addition, comPACT containing human HLA sequences can be purified by affinity chromatography on a monoclonal antibody-Sepharose column by procedures generally known and disclosed.

[0344] [Methods for isolating antigen-specific T cells] The comPACT libraries described herein have been used to isolate antigen-specific T cells and can be used to isolate any cell that presents a neo-antigen. A diagram of the T cell isolation process, according to one embodiment, is shown in Figure 26. This process may also be referred to herein as the "imPACT" or "imPACT isolation technology" method.

[0345] The steps and components of the imPACT isolation technology method include, but are not limited to, steps (1)-(5) shown in FIG. 26: (1) Creating a comPACT element library generated for patient-specific neoantigen T cell isolation (2) Adding unique DNA oligonucleotides, neoIDs, or barcodes to the comPACT element library (3) Each individual comPACT polypeptide and its corresponding neoID barcode, DNA oligonucleotide, neoID, or barcode, is bound to two separate fluorescent streptavidin proteins (phycoerythrin (PE) and allophycocyanin (APC) in the example provided in Figure 26). (4) This assembly process yielded two paired barcode fluorescent tetramers for each comPACT polypeptide and barcode element. (5) All comPACT polypeptides and neoID barcodes, DNA oligonucleotides, neoIDs, or tetramer libraries constructed with barcodes targeting predicted neoantigen candidates for each patient are pooled together to isolate neoantigen-specific T cells from the subject's peripheral blood.

[0346] The use of the comPACT library to identify and characterize neoantigen-specific T cells is also shown in panels 6-8 of FIG. 26. Incubation of the comPACT-neoID library with patient samples (6) is followed by fluorescence activated cell sorting (FACS) (7). A fixed number of T cells engineered to express the tool's neoTCR can be added to the patient sample as an internal positive control to calibrate each analysis. Dual fluorescently labeled (PE and APC) tetramer-bound neoantigen-specific T cells, as well as internal positive control cells and potential non-specific T cells, are sorted as single cells into individual wells of a plate for subsequent RT-PCR analysis, including barcoding and neo-TCR sequencing (8).

[0347] [Barcode signal-to-noise (S / N) analysis] True positive neo-antigen-specific double-labeled T cells can be separated from false positive T cells identified by flow cytometry by sequence analysis of the neo-ID barcodes bound to isolated T cells. The presence of multiple copies of the same neo-ID barcode results in a higher ratio of specific neo-ID barcode species compared to non-specifically bound barcodes. This results in a higher signal-to-noise barcode ratio (S / N). Non-specific T cells bind a relatively equal number of different tetramer species, resulting in a lower ratio of distinct neo-ID barcodes. A schematic of non-specific vs. specific T cell binding is shown in Figure 27A (non-specific) and Figure 27B (specific). The numbers indicate the various neo-ID barcodes. In Figure 27A, the ratio of the unique DNA copy number of the most predominant neo-ID divided by the second most predominant neo-ID is 1, indicating a cell with non-specific binding of comPACT elements. In Figure 27B, the ratio of unique DNA copies of the most predominant neoID divided by the second most predominant neoID is 5, indicating that this T cell is bound by the predominant comPACT element and represents a true positive neoantigen-specific CD8 T cell. This can be further confirmed by functional characterization of neoTCR engineered T cells cloned from that individual cell.

[0348] [S / N1 and S / N2 analysis] In some embodiments, one TCR may recognize two different neo-antigens. In such cases, even if the T cells are specific, the S / N ratio may be less than 10. In such cases, two different S / N calculations may be used: S / N1 and S / N2. S / N1 is the highest signal divided by the second highest signal, and S / N2 is the highest signal from one mutation divided by the highest signal from a different mutation. In the S / N2 analysis, the highest signal from a different mutation may not be the second highest signal in the sample.

[0349] In an illustrative example, eight different TCRs can be identified in one sample. Six of them can have S / N1 ratios greater than 10 and can be confirmed as specific neo-antigen T cells. The other two T cells can have S / N1 ratios less than 10. However, S / N2 can be greater than 10. Cloning two TCRs shows that they can recognize two different neo-antigens that share the same mutation, explaining the reason for the low S / N1 ratio. In some embodiments, S / N2 analysis can be useful to distinguish non-specific from specific cells when there are multiple neo-antigens derived from the same mutation.

[0350] In some embodiments, a higher signal to noise ratio indicates higher TCR binding specificity.

[0351] [Threshold] In some embodiments, if the barcode signal to noise S / N1 or S / N2 ratio exceeds a threshold, the isolated T cell is identified as the antigen-specific T cell.

[0352] In some embodiments, the threshold is at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 or more. In some embodiments, the threshold is at least 2, 5, 10, or 20 or more. In some embodiments, the ratio corresponds to the specificity of the isolated antigen-specific T cells. In some embodiments, the signal to noise ratio is 10 or more.

[0353] [Sign] As used herein, "identifying label" or "identifying labels" refers to a molecule or compound used to label a particle set. In some embodiments, the identifying label is a fluorophore. In some embodiments, the identifying label is a metal, a lanthanide, a quantum dot, a radioisotope, a nanoparticle, or a dye. Any suitable fluorophore can be used, including, but not limited to, allophycocyanin (APC), phycoerythrin (PE), fluorescein (FITC), rhodamine, Texas Red, DAPI, C2, Cy3, Cy5, Cy7, AlexaFluor fluorophores, BODIPY fluorophores, DyLight fluorophores, FluoProbes fluorophores, or any combination thereof.

[0354] [Barcode] As used herein, "barcode" or "barcode" refers to a nucleotide sequence used to tag and identify a particular peptide, including but not limited to an antigenic peptide. In certain embodiments, the barcode is selected from the group consisting of a 3-mer, a 4-mer, a 5-mer, a 6-mer, a 7-mer, an 8-mer, a 9-mer, a 10-mer, an 11-mer, a 12-mer, a 13-mer, a 14-mer, a 15-mer, a 16-mer, a 17-mer, an 18-mer, a 19-mer, and a 20-mer. In certain embodiments, the barcode is an 8-mer.

[0355] In some embodiments, the distinct particle pairs comprise a unique antigenic peptide and a defined barcode operably associated with the identity of the antigenic peptide.

[0356] In some embodiments, a first particle comprises a first barcode and the second particle comprises a second barcode that is different from the second barcode, and the first and second barcodes are associated with an identity of an antigen.

[0357] In some embodiments, the particle pair includes a third particle that includes a third barcode that is different from the first and second barcodes, and the first, second, and third barcodes are associated with an identity of the antigen.

[0358] Exemplary barcodes and barcode structures are provided below in Table A. Barcodes are sometimes referred to as "NeoIDs." TIFF0007611836000001.tif39170

[0359] In some embodiments, the barcode signal to noise ratio is based on at least one barcode. In such embodiments, each of the paired particles comprises the same antigen, a different label, and at least one barcode, where at least one barcode is associated with a neo-antigen. In some embodiments, the paired particles have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 barcodes.

[0360] In some cases, aggregates of particles with fluorophore labels can result in high average fluorescence intensity of a single fluorophore of stained cells during isolation. This is not due to specific binding of fluorescent particles but due to non-specific binding of comPACT particle aggregates, resulting in high neo-antigen barcode S / N since there can be many identical barcodes non-specifically bound to T cells. To address this issue, a dual barcode system can be used.

[0361] In some embodiments, each particle pair of comPACT library elements contains at least two barcodes. Dual barcoding conjugates two different DNA barcodes per antigen to each comPACT tetramer, respectively. An illustration of a comparison of single and dual barcodes for one antigen is shown in FIG. 28. In the top panel, the same neo-antigen is associated with two different particles, two different fluorophores, and a single barcode labeled "1". In the bottom panel, the same antigen is associated with two different particles, two different fluorophores, and two different barcodes labeled "1" and "2". This increases the discrimination of false positives with high signal-to-noise ratios caused by tetramer aggregation. The signal-to-noise ratios of each fluorescent particle and each DNA barcode assigned to the same antigen can be analyzed separately.

[0362] In some embodiments, each particle pair of comPACT library elements contains at least two barcodes. Dual barcoding conjugates two different DNA barcodes per antigenic peptide to each comPACT tetramer, respectively. An illustration of a comparison of single and dual barcodes for one antigenic peptide is shown in FIG. 28. In the top panel, the same antigenic peptide is associated with two different particles, two different fluorophores, and a single barcode labeled "1". In the bottom panel, the same antigenic peptide is associated with two different particles, two different fluorophores, and two different barcodes labeled "1" and "2". This increases the discrimination of false positives with high signal-to-noise ratios caused by tetramer aggregation. The signal-to-noise ratios of each fluorescent particle and each DNA barcode assigned to the same antigenic peptide can be analyzed separately.

[0363] [Cell samples] The imPACT method (i.e., the imPACT isolation technique) can be used to isolate immune cells, such as T cells and B cells, from any suitable patient-derived sample that contains immune cells, including, but not limited to, blood, plasma, peripheral blood mononuclear cell (PBMC) samples, bone marrow, tumor infiltrating lymphocyte (TIL) samples, tissues, solid tumors, hematological cancers, liquid tumors, or any combination thereof. For example, CD4+ and CD8+ T cells can be labeled and sorted from PBMCs or TILs using anti-CD4 and anti-CD8 fluorescent antibodies, and the live population of CD4+ and CD8+ single positive cells is sorted using fluorescence activated cell sorting (FACS) to isolate only CD4+ or CD8+ cells. In some embodiments, T cells that are positive for both CD4 and CD8 can be isolated using anti-CD3 fluorescent antibodies followed by FACS. In addition, the imPACT method can be used for antibody discovery of B cells. One skilled in the art can determine the type of immune cells that are isolated for the type or types of comPACT proteins used. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a PBMC sample. In some embodiments, the sample is a solid tumor sample. In some embodiments, the sample is a hematological tumor sample. In some embodiments, the sample is a bone marrow sample. In some embodiments, the sample is a tumor sample containing tumor infiltrating lymphocytes. The T cells can be CD8+ T cells or CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are human T cells. In some embodiments, the T cells are human CD8+ T cells.

[0364] [Isolation of T cells] In another aspect, provided herein is a method of isolating an antigen-specific T cell, the method comprising the steps of: (a) providing a polypeptide comprising, in an amino-terminal to carboxyl-terminal direction, (i) a first universal target peptide, (ii) an antigenic peptide, (iii) a second universal target peptide that is different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide, wherein the polypeptide is bound to a particle; (b) providing a sample known or suspected to contain one or more T cells; (c) contacting the polypeptide with the sample, wherein the contacting provides conditions sufficient for a single T cell to bind to the polypeptide attached to the particle; and (d) isolating a single T cell associated with the particle.

[0365] Isolation and identification of patient-derived and antigen-specific T cells using comPACT as described herein can include incubating comPACT protein with patient-derived T cells or a sample containing patient-derived T cells. In some embodiments, a library containing at least two comPACTs can be incubated with patient-derived T cells. T cells can be prepared using standard methods starting from tissue such as blood, lymph nodes, or tumors.

[0366] Incubation of comPACT or a comPACT library with the T cell suspension allows complete and thorough exposure of the particle-bound antigenic peptides to the various T cell receptors. This method may include rocking or rolling the cells. In some embodiments, comPACT is bound to particles.

[0367] Following incubation of comPACT or comPACT library (both bound to particles) and T cells, bound coMPACT-T cell complexes are selectively separated or selectively collected. T cells are likely to bind many identical copies of the same comPACT library elements (i.e., individual comPACT polypeptides and comPACT polypeptides bound to particles) and can be separated based on these interactions. For example, if the comPACT or comPACT associated with the particles contains a fluorophore or is bound to the particles with a fluorophore, fluorescence activated cell sorting (FACS), including single cell sorting, can be used to selectively separate T cells. If comPACT or its associated particles are attached to magnetic particles, applying a magnet to the suspension allows separation of particles complexed with antigen-paired T cells and removal of unpaired T cells. Alternatively, if the comPACT associated particles are polystyrene particles, unpaired T cells can be separated by gravity (e.g., centrifugation). After removal of unpaired T cells, in some embodiments, the separated bound particles are washed at least once to remove non-specifically associated T cells.

[0368] The comPACT-bound T cells can also be separated by FACS into individual collection vessels, such as multi-well plates. The individual collection vessels can be single cell reaction vessels. For example, components used for downstream processing and analysis can be added to each single cell reaction vessel. The comPACT-bound T cells can be separated by FACS into bulk collection vessels (e.g., all separated T cells are collected in the same vessel).

[0369] comPACT-bound T cells can also be individually isolated in droplets using a droplet-generating microfluidic device (i.e., a "droplet generator"). Droplet generators used to encapsulate single cells are known to those of skill in the art, for example, as described in U.S. Publication No. 2006 / 0079583, U.S. Publication No. 2006 / 0079584, U.S. Publication No. 2010 / 0021984, U.S. Publication No. 2015 / 0376609, U.S. Publication No. 2009 / 0235990, and U.S. Publication No. 2004 / 0180346.

[0370] After comPACT-binding T cells are isolated into single cell reaction vessels (e.g., isolated in individual wells or droplets), the nucleic acids of the comPACT-binding T cells can be further processed for downstream analysis. Specifically, expressed TCRα and TCRβ mRNA transcripts can first be converted to cDNA by reverse transcription, and the cDNA can be amplified for next generation sequencing (NGS) methods known to those of skill in the art, including, but not limited to, sequencing by synthesis techniques (e.g., Illumina or any other NGS sequencing machine).

[0371] [Methods for identifying T cell antigen specificity] In certain embodiments, the presently disclosed subject matter provides methods for identifying the antigen specificity of T cells. In certain embodiments, the T cell isolation methods described herein provide information regarding the antigen specificity of the isolated T cells. For example, but not by way of limitation, the information regarding the antigen specificity can be obtained by nucleic acid analysis of the isolated T cells. In certain embodiments, the nucleic acid of the isolated T cells can be analyzed to determine the sequence of the T cell receptor gene sequences (e.g., TCR alpha and TCR beta sequences). In certain embodiments, the information regarding the antigen specificity of the isolated T cells can be used for downstream applications. Non-limiting examples of downstream applications include analysis of immune repertoires, manufacturing processes, and clinical follow-up of patients undergoing immunotherapy. In certain embodiments, the information regarding the antigen specificity of the isolated T cells can be used to prepare reagents and compositions for producing cells useful for adoptive cell transfer therapy.

[0372] In non-limiting embodiments, immune repertoire monitoring is performed. In certain embodiments, immune repertoire monitoring is performed before, during, or after treatment. In certain embodiments, the treatment is immunotherapy. Non-limiting examples of immunotherapy include administration of vaccines, oncolytic viruses, antibodies, T cells expressing chimeric antigen receptors, T cells expressing recombinant T cell receptors, and tumor infiltrating lymphocytes.

[0373] [Treatment method] In certain embodiments, the subject matter of the present disclosure provides methods of treatment, including but not limited to, inducing and / or increasing an immune response in a subject in need of treatment. In certain embodiments of the present disclosure, the methods of treatment disclosed herein include the isolation and / or administration of cells. For example, but not limited to, the cells used in the methods described herein can be obtained from the subject in certain embodiments. In certain embodiments, the cells are tumor cells, non-cancer cells, T cells, or any combination thereof. In certain embodiments, nucleic acid can be extracted from the cells as outlined herein. In certain embodiments, the nucleic acid of the cells can be sequenced as outlined herein. In certain embodiments, the information obtained from the subject, e.g., nucleic acid sequence information, provides information regarding antigen-specific T cells. In certain embodiments, the information relates to the identity (e.g., amino acid sequence) of the antigenic peptide. In certain embodiments, the antigenic peptide is a tumor neo-antigen. In certain embodiments, the information relates to the identity of the MHC sequence.

[0374] In certain embodiments, the methods described herein relate to the treatment of cancer. In certain embodiments, the cancer is a solid cancer. Non-limiting examples of tumors treatable by the methods described herein include, for example, carcinoma, lymphoma, sarcoma, blastoma, and leukemia. Non-limiting specific examples include, for example, breast cancer, pancreatic cancer, liver cancer, lung cancer, prostate cancer, colon cancer, renal cancer, bladder cancer, head and neck cancer, thyroid carcinoma, soft tissue sarcoma, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancer of all histopathological types, angiosarcoma, hemangiosarcoma, osteosarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, s ... Cancers of the genital tract, rectum, rectum, rectum-sarcoma, sarcoma, sarcoma-associated tumors, such as sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovium, testicular cancer, uterine cancer, cervical cancer, gastrointestinal cancer, mesothelioma, and cancers associated with viral infections, including but not limited to human papilloma virus (HPV)-associated tumors (e.g., cervical, vaginal, vulvar, head and neck, anal, and penile cancer).

[0375] In certain embodiments, a comPACT minigene comprising the candidate antigenic peptide is generated according to the methods disclosed herein. In certain embodiments, a comPACT polypeptide comprising the antigenic peptide is generated. In certain embodiments, a particle comprising a comPACT polypeptide is generated according to the methods disclosed herein. In certain embodiments, at least one set of particles comprising a comPACT polypeptide is generated according to the methods disclosed herein.

[0376] In certain embodiments of the therapeutic methods disclosed herein, T cells are isolated by binding to particles. In certain embodiments, the T cells so isolated are obtained from a subject. In certain embodiments, the T cells are CD8 + T cells.

[0377] In certain embodiments, the isolated T cells are selected and their genomes are analyzed. In certain embodiments, the TCR sequences of the isolated T cells are obtained. In certain embodiments, the TCR gene sequences or portions thereof are inserted into a homologous recombination template. In certain embodiments, the homologous recombination template comprises features described in International Patent Application No. PCT / US2018 / 058230, the contents of which are incorporated herein by reference in their entirety.

[0378] In certain embodiments, the T cells are modified by inserting a homologous recombination template comprising the TCR gene sequences, or a portion thereof, of the isolated T cell.

[0379] In certain embodiments, the modified T cells are adoptively transferred into the patient. Adoptive cell transfer (ACT) is an effective form of immunotherapy that involves the transfer of immune cells with anti-tumor activity into cancer patients. The lymphocytes used for adoptive transfer can be derived from the blood or stroma of resected tumors, although other sources of such cells are known in the art. In certain embodiments, the lymphocytes used in ACT can be administered in a single dose. Such administration can be by injection, e.g., intravenous injection. In certain embodiments, the lymphocytes can be administered in multiple doses. Dosing can be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing can be once a month, once every two weeks, once a week, or once every other day. Administration of the cytotoxic lymphocytes can be continued for as long as necessary. In certain embodiments, the methods described herein can be used to determine the immune repertoire of a subject. In certain embodiments, the immune repertoire is analyzed before, during, and / or after treatment. In certain embodiments, the treatment is cancer treatment. In certain embodiments, the cancer treatment is immunotherapy. In certain embodiments, the immunotherapy comprises administration of an antibody. In certain embodiments, the immunotherapy comprises adoptive cell transfer of T cells. In certain embodiments, the T cells comprise a recombinant TCR or a chimeric antigen receptor. In certain embodiments, the immune repertoire provides information for providing targeted therapy.

[0380] [Methods for modifying cells] In certain embodiments, the presently disclosed subject matter provides methods for modifying cells. For example, but not by way of limitation, modified cells can be obtained using the methods and compositions described herein.

[0381] In certain embodiments, the presently disclosed subject matter provides a method of modifying a cell by introducing and recombining a homologous recombination (HR) template nucleic acid sequence into an endogenous locus of the cell. In certain embodiments, the cell is modified by a non-viral method. In certain embodiments, the HR template nucleic acid sequence is circular. In certain embodiments, the HR template nucleic acid sequence is linear. In certain embodiments, the HR template nucleic acid sequence comprises a first and a second homology arm. In certain embodiments, the homology arms can be from about 300 bases to about 2000 bases. For example, each homology arm can be 1000 bases. In certain embodiments, the homology arms can be homologous to a first and a second endogenous sequence of the cell. In certain embodiments, the endogenous locus is a TCR locus. For example, the first and second endogenous sequences are within a TCR alpha locus or a TCR beta locus. In certain embodiments, the HR template comprises a TCR gene sequence. In non-limiting embodiments, the TCR gene sequence is a patient-specific TCR gene sequence. In non-limiting embodiments, the TCR gene sequence is identified and obtained using the methods described herein. For example, a method for identifying the antigen specificity of a T cell can be used to obtain a TCR gene sequence from a patient, and the TCR sequence can be incorporated into the HR template. In certain embodiments, the HR template includes a TCR alpha gene sequence and a TCR beta gene sequence. Further information regarding HR template nucleic acids and methods for modifying the cells can be found in International Patent Application No. PCT / US2018 / 058230, the contents of which are incorporated herein by reference.

[0382] In certain embodiments, constructs containing a gene of interest can be inserted into endogenous loci using non-viral gene editing methods. In certain embodiments, this can be achieved by using a homology repair template that contains the coding sequence of the gene of interest flanked by left and right HR arms. In certain embodiments, in addition to the HR arms, the gene of interest is sandwiched between a 2A peptide, a protease cleavage site upstream of the 2A peptide for removing the 2A tag from the upstream translated gene of interest, and a signal sequence; once integrated into the genome, the gene of interest in the expression gene cassette is transcribed as a single messenger RNA. In certain embodiments, during translation of the gene of interest messenger RNA, the flanking regions are separated from the gene of interest by the self-cleaving 2A peptide, and the protease cleavage site is cleaved for removal of the 2A sequence upstream of the translated gene of interest. In certain embodiments, in addition to the 2A and protease cleavage sites, a gly-ser-gly (GSG) linker can be inserted before each 2A peptide to further enhance separation of the gene of interest from other elements in the expression cassette. In certain embodiments, P2A peptides are used because of their superiority over other 2A peptides due to their efficient cleavage. In certain embodiments, two (2) P2A peptides and codon divergence are used to express the gene of interest without introducing exogenous epitopes from the remaining amino acids on either end of the gene of interest from the P2A peptide.

[0383] In certain embodiments, the neoTCR is integrated into the TCR alpha locus of a T cell, as described in PCT / US / 2018 / 058230. In certain embodiments, a homology repair template is used that includes neoTCR coding sequences flanked by left and right HR arms. In certain embodiments, the endogenous TCR beta locus is disrupted, resulting in expression of only the TCR sequences encoded by the neoTCR construct. In certain embodiments, the general strategy is applied using a circular HR template. In certain embodiments, the general strategy is applied using a linear template.

[0384] In certain embodiments, the target TCR alpha locus (Cα) is shown with the plasmid HR template and the resulting edited sequence and downstream mRNA / protein product are shown in Figures 67A and 67B. In certain embodiments, additional elements in the neoTCR cassette include: 2A = P2A ribosomal skip element; F = furin cleavage site upstream of 2A that removes the 2A tag from the upstream TCR β protein; HGH = human growth hormone signal sequence. The HR template of the neoTCR expression gene cassette includes two adjacent homology arms for direct insertion into the TCR alpha genomic locus targeted by the CRISPR Cas9 nuclease RNP containing the TCR alpha guide RNA. In certain embodiments, the homology arms (LHA and RHA) flank the neoE-specific TCR sequence of the neoTCR expression gene cassette. In certain embodiments, the protease cleavage site is any suitable protease cleavage site known to be used by one of skill in the art. In certain embodiments, any signal sequence known to one of skill in the art can be selected based on the desired transport and use.

[0385] In certain embodiments, once integrated into the genome, the neoTCR expression gene cassette is transcribed as a single messenger RNA from the endogenous TCR alpha promoter, which still contains a portion of the endogenous TCR alpha polypeptide from that individual T cell. In certain embodiments, during ribosomal polypeptide translation of the single neoTCR messenger RNA, the neoTCR sequence is separated from the endogenous CRISPR-disrupted TCR alpha polypeptide by self-cleavage at the P2A peptide. In certain embodiments, the encoded neoTCR alpha and neoTCR beta polypeptides are also separated from each other by cleavage by the endogenous cellular human furin protease, and a second self-cleaving P2A sequence motif is included in the neoTCR expression gene cassette (Figure 67B). In certain embodiments, the neoTCR alpha and neoTCR beta polypeptides are separately targeted by a signal leader sequence (e.g., from human growth hormone, HGH) to the endoplasmic reticulum for multimer assembly and transport of the neoTCR protein complex to the T cell surface. In certain embodiments, the inclusion of a furin protease cleavage site facilitates removal of the 2A sequence from the upstream TCR β chain to reduce potential interference with TCR β function, In certain embodiments, the inclusion of a gly-ser-gly linker before each 2A (not shown) further enhances the separation of the three polypeptides.

[0386] In certain embodiments, the three repeat protein sequences are codon diverged in the HR template to promote genomic stability. In certain embodiments, the two P2As are codon diverged with respect to each other and with respect to the two HGH signal sequences in the TCR gene cassette to promote stability of the neoTCR cassette sequence introduced into the genome of ex vivo modified T cells. In certain embodiments, the reintroduced 5' end of TRAC exon 1 (Figures 67A and 67B, vertical stripes) reduces the chance of the entire cassette being lost over time through removal of the intervening sequence of the two direct repeats.

[0387] The presently disclosed subject matter further provides compositions comprising cells modified by the methods disclosed herein.

[0388] Exemplary embodiments A. In certain non-limiting embodiments, the presently disclosed subject matter provides methods comprising: (a) contacting a sample with a plurality of distinct sets of particles, each particle comprising a unique antigenic peptide, an operably associated barcode, and at least one identifying label, wherein the sample comprises T cells, and the contacting comprises providing conditions suitable for a single T cell to bind to the unique antigenic peptide of at least one of the set of particles; (b) isolating one or more T cells bound to the particles; (c) identifying the barcodes of the particles bound to the isolated T cells; and (d) determining a ratio of each barcode.

[0389] A1. The method of A, wherein the ratio is calculated by identifying the copy number of the first barcode and the copy number of the second barcode and dividing the copy number of the first barcode by the copy number of the second barcode.

[0390] A2. The method of A or A1, wherein the unique antigenic peptide is the same for each distinct set of particles.

[0391] A3. Any one of the methods A-A2, wherein each distinct particle set comprises at least one or more barcodes, each barcode being associated with the identity of an antigenic peptide.

[0392] A4. Any one of the methods A-A3, wherein the ratio of each barcode corresponds to the antigen specificity of the isolated T cells.

[0393] A5. Any one of the methods A-A4, wherein the ratio of the first barcode exceeds a threshold value, the isolated T cell is identified as an antigen-specific T cell.

[0394] A6. The method of A5, wherein the threshold is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-5, 3-6, 4-7, 5-8, 5-10, 7-10 or more, or greater than 10.

[0395] A7. Any one of the methods A-A6, wherein the step of identifying the barcode comprises a nucleotide-based assay.

[0396] A8. The method of A7, wherein the nucleotide-based assay is a PCR, RT-PCR, sequencing, or hybridization assay.

[0397] A9. The method of A7 or 8, wherein the nucleotide-based assay determines (a) the sequence of each barcode and / or (b) the copy number of each barcode.

[0398] A10. Any of the methods A-A9, further comprising the step of obtaining a T cell receptor (TCR) CDR sequence.

[0399] A11. Any of the methods A-A10, further comprising the step of obtaining a TCR gene sequence.

[0400] A12. The method of A11, wherein the TCR sequence is a TCR alpha or TCR beta chain sequence.

[0401] A13. Any of the methods of A-A12 for identifying the antigen specificity of T cells.

[0402] A14. The method of A13, wherein the antigen specificity of the T cell comprises a TCR sequence of the T cell bound to a sequence of an antigen peptide.

[0403] A15. Any of the methods of A-A14, wherein at least one identifying label is the same in each distinct set of particles.

[0404] A16. Any of the methods A-A15, comprising at least two different identification marks.

[0405] A17. Any of the methods A-A16, wherein at least one identifying label is a fluorophore.

[0406] A18. The method of A17, wherein the fluorophore is selected from the group including allophycocyanin (APC) and phycoerythrin (PE).

[0407] A19. The method of A18, wherein the at least two different identifying labels are fluorophores, and the fluorophores are selected from the group including allophycocyanin (APC) and phycoerythrin (PE).

[0408] A20. Any of the methods A-A19, wherein the antigen peptide is selected from the group consisting of a tumor antigen, a neoantigen, a tumor neoantigen, a viral antigen, a bacterial antigen, a phosphoantigen, and a microbial antigen.

[0409] A21. The method of A20, wherein the neoantigen is identified from tumor sequencing data from the subject.

[0410] A22. The method of A21, wherein neoantigens are determined using an in silico prediction algorithm.

[0411] A23. The method of A22, wherein the predictive algorithm further comprises an MHC binding algorithm for predicting binding between a neoantigen and an MHC peptide.

[0412] A24. Any of the methods A-A23, wherein the sample is selected from a blood sample, a bone marrow sample, a tissue sample, a tumor sample, or a peripheral blood mononuclear cell (PBMC) sample.

[0413] A25. Any of the methods A-A24, wherein the T cells are human T cells.

[0414] A26. The method of A25, wherein the T cells are CD8+ T cells.

[0415] A27. Any of the methods of A-A24, comprising a library of distinct particle sets.

[0416] A28. The method of A27, wherein the library comprises between 2 and 500 distinct particle sets.

[0417] A29. Any of the methods A-A28, wherein each particle comprises an MHC peptide.

[0418] A30. The method of A29, wherein the MHC peptide is a human MHC peptide.

[0419] A31. The method of A29, wherein the MHC peptide is a class I HLA peptide.

[0420] A32. The method of A29, wherein the HLA peptide comprises an HLA-A, HLA-B, or HLA-C peptide.

[0421] A33. HLA-A*01:01、HLA-A*02:01、HLA-A*03:01、HLA-A *24:02、HLA-A*30:02、HLA-A*31:01、HLA-A*32:01、HLA-A*33 :01、HLA-A*68:01、HLA-A*11:01、HLA-A*23:01、HLA-A*30:01、HLA-A*25:01、HLA-A*25:01、HLA-A*26:02-A*HL、HL A-A*68:02、HLA-B*07:02、HLA-B*14:02、HLA-B*18:01、HLA-B*27:02、HLA-B*39:01、HLA-B*40:01、6:HLA-4B4 4 :01、HLA-B*50:01、HLA-B*57:01、HLA-B*58:01、HLA-B*08:01、HLA-B*15:01、HLA-B*15:03、HLA-B*35:02-B、HL、HL A-B*42:01、HLA-B*44:03、HLA-B*51:01、HLA-B*53:01、HLA-B*13:02、HLA-B*15:07、HLA-B*27:05、7:HLA-B 3 :01、HLA-B*38:01、HLA-B*41:02、HLA-B*44:05、HLA-B*49:01、HLA-B*52:01、HLA-B*55:01、HLA-C*02:04-C、HL、HL A-C*05:01、HLA-C*07:01、HLA-C*01:02、HLA-C*04:01、HLA-C*06:02、HLA-C*07:02、HLA-C*16:01、7:HLA-3C*0 :04、HLA-C*08:01、HLA-C*08:02、HLA-C*12:02、HLA-C*12:03 HLA-C*14:02 and HLA-C*15:02 were also expressed as HLA-C*17:01.

[0422] A34. In the case of HLA β2M, the A-A33 is also activated.

[0423] A35. The β2M proteins in the β2M proteins in A34 remained stable.

[0424] A36. The method of A35, wherein the β2M peptide comprises a mutation.

[0425] A37. The method of A36, wherein the mutation is S88C.

[0426] A38. Any of the methods A-A37, wherein each particle comprises a polypeptide comprising, in amino-terminal to carboxyl-terminal direction, (i) an antigenic peptide, (ii) a β2M peptide, and (iii) an MHC peptide.

[0427] A39. Any of the methods A-A38, wherein the antigenic peptide is 7-15 amino acids, 7-10, 8-9, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

[0428] A40. The method of A38 or A39, wherein the polypeptide is biotinylated.

[0429] A41. Any of the methods A-A40, wherein the particles are selected from the group consisting of magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles.

[0430] A42. Any of the methods of A-A41, wherein the particles are coated with streptavidin.

[0431] A43. Any of the methods of A-A42 for monitoring immune repertoire in a subject.

[0432] A44. The method of A43, further comprising monitoring changes in antigen-specific T cells in the subject.

[0433] A45. The method of A43 or A44, comprising administering immunotherapy to the subject.

[0434] A46. The method of A45, wherein the immunotherapy is adoptive cell transfer or a checkpoint inhibitor.

[0435] A47. Any of the methods A-A46 for identifying at least one TCR sequence.

[0436] A48. The method of A47, wherein at least one TCR sequence is a TCR alpha sequence, a TCR beta sequence, or a combination thereof.

[0437] A49. The method of A47 or A48, further comprising producing a soluble TCR polypeptide.

[0438] B. In certain non-limiting embodiments, the presently disclosed subject matter provides a library comprising at least two sets of particles, each set of particles comprising an antigenic peptide, a barcode operably associated with the identity of the antigenic peptide, and at least one identification label.

[0439] B1. The library of B, wherein at least one identifying label is the same in each set of particles.

[0440] B2. The library of B or B1, wherein each distinct particle set comprises at least two different identifying labels.

[0441] B3. Any of the libraries of B-B2, wherein at least one identifying label is a fluorophore.

[0442] B4. The library of B3, wherein the fluorophore is selected from the group consisting of allophycocyanin (APC) and phycoerythrin (PE).

[0443] B5. The library of B2, wherein the at least two different identifying labels are fluorophores, and the fluorophores are selected from the group consisting of allophycocyanin (APC) and phycoerythrin (PE).

[0444] C. In certain non-limiting embodiments, the presently disclosed subject matter provides a particle comprising at least one polypeptide, a barcode, and an identification label, wherein the polypeptide comprises an antigenic peptide, a β2M peptide, and an MHC peptide, and the barcode is operably associated with the identity of the antigenic peptide.

[0445] C1. The particles of C selected from the group consisting of magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles.

[0446] C2. The particle of C or C1, wherein the identifying label is a fluorophore.

[0447] C2. Any of the particles C-C2, coated with streptavidin.

[0448] C2. Any of the particles C-C3, in which the polypeptide is labeled.

[0449] D. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of treating cancer in a subject comprising the steps of: (a) preparing a plurality of particles, each comprising a plurality of labeled polypeptides, the polypeptides comprising an antigenic peptide, a β2M sequence, an HLA sequence, and a detectable label; (b) contacting the plurality of particles with a plurality of T cells from a subject under conditions suitable for antigen-specific binding of the T cells to the particles; (c) isolating the T cells bound to the particles and identifying TCR gene sequences of the isolated T cells; (d) preparing a polynucleotide comprising homology arms and at least one TCR gene sequence, the TCR gene sequence being located between the homology arms; (e) recombining the polynucleotide into an endogenous locus of a T cell of the subject; (f) culturing the modified T cells to produce a population of T cells; and (g) administering a therapeutically effective number of modified T cells to the subject, thereby treating the cancer.

[0450] E. In certain non-limiting embodiments, the presently disclosed subject matter provides methods of modifying a cell comprising: (a) introducing into a cell a homologous recombination (HR) template nucleic acid sequence comprising: (i) first and second homology arms that are homologous to a first and second endogenous sequence of the cell; (ii) a T cell receptor (TCR) gene sequence obtained by any of methods A-A49, wherein the TCR gene sequence is located between the first HR arm and the second HR arm; and (iii) a HR template nucleic acid sequence comprising a first 2A coding sequence located upstream of the TCR gene sequence and a second 2A coding sequence located downstream of the TCR gene sequence, wherein the first and second 2A coding sequences encode the same amino acid sequence but differ by codons from each other; and (b) recombining the HR template nucleic acid into an endogenous locus of the cell comprising a first and second endogenous sequence that are homologous to the first and second homology arms of the HR template nucleic acid.

[0451] F. In certain non-limiting embodiments, the presently disclosed subject matter provides compositions comprising modified cells, the modified cells comprising an exogenous nucleic acid sequence integrated into an endogenous locus, the exogenous nucleic acid sequence comprising: (a) a TCR gene sequence identified by any of the methods described in A-A49; and (b) a first 2A coding sequence located upstream of the TCR gene sequence and a second 2A coding sequence located downstream of the TCR gene sequence, the first and second 2A coding sequences encoding the same amino acid sequence but differing from each other by codons. EXAMPLES

[0452] The following examples are merely illustrative of the presently disclosed subject matter and should not be construed as limiting in any way.

[0453] Example 1: Design and cloning of comPACT minigenes by restriction digest cloning [Construction of comPACT minigene for restriction digestion] The basic components of a comPACT minigene include a signal sequence to direct secretion of the encoded protein, a universal target sequence such as a restriction site or primer binding site, the encoded antigenic peptide (or neoantigen, NeoE), a second universal target site, β2m, the extracellular domain of an MHC allele, and a purification cluster that allows for enzymatic modification (e.g., biotinylation) and purification of comPACT, for example, via an affinity tag. The cluster may also include protease cleavage sites and linker sequences between components, as formed and shown in the figures and examples. The minigene may also encode cysteine ​​mutations that may act as disulfide traps. Certain comPACT minigenes formed and disclosed herein encoded cysteine ​​mutations that act as disulfide traps. Such minigenes were formed to include disulfide traps to increase the success of production of comPACT polypeptides by stabilizing the protein. A diagram of a comPACT minigene is shown in FIG. 1. Additional restriction sites upstream and downstream of the MHC heavy chain sequence can be used to insert other MHC alleles to construct different MHC templates and to construct a library of MHC templates (Figure 2). The DNA encoding the signal sequence, universal targeting sequence, β2m, and the extracellular domain of the MHC allele is the base MHC template.

[0454] In the restriction digestion cloning method, each comPACT DNA construct is a base MHC template that contains three in-frame dummy antigen sequence inserts with stop codons and unique restriction sites for destruction of the uncut or religated template (Figure 3), and can be used as part of a ready-made platform for the rapid construction of libraries of antigen peptides complexed with their MHC alleles. The MHC alleles can also be modified or mutated (e.g., Y84A or Y84C), for example, to improve folding or increase binding of the antigen peptide to the MHC protein. In addition, the β2m protein can also be mutated (e.g., S88C) to conjugate it to a thiol dye.

[0455] In this example, the comPACT minigene is depicted with the following structure: a NotI restriction site at the 5' end; a signal sequence from human growth hormone hGH as shown in Table 1; a restriction site Blp1 upstream of the antigenic peptide region and a BamHI restriction site downstream of the antigenic peptide region as shown in Table 2; a coding linker sequence primarily of glycine and serine residues (i.e., a Gly-Ser linker); a β2m sequence; a second coding Gly-Ser linker sequence containing a BspI restriction site; an MHC heavy chain; a third coding Gly-Ser linker sequence containing a BstBI restriction site; and a coding purification cluster containing an AviTag (or any avidin / streptavidin) sequence, a TEV cleavage site, and a concatenated histidine tag. TIFF0007611836000002.tif45170TIFF0007611836000003.tif39170

[0456] [Restriction digest cloning and assembly of comPACT minigenes] Three different methods of inserting the encoded neo-antigen via restriction digestion are described here. In the first, as illustrated in Figure 4, an antigenic peptide (NeoE) encoding primer spans a first restriction site (BlpI in this example) at the 5' end and a second restriction site (BamHI in this example) at the 3' end. This primer amplifies a universal reverse primer that encodes the second restriction site, resulting in a primer dimer of approximately 70 bp.

[0457] In the second method, a primer encoding the antigenic peptide spans a second restriction site as its 5' end and is the reverse complement of the antigen-encoding sequence. This primer primes in the reverse direction of the template DNA encoding the signal sequence. Paired with a forward primer spanning the first restriction site sequence, the reaction produces a 70 bp product, or approximately a 140 bp product if a forward primer spanning a restriction site further upstream of the antigenic site is used.

[0458] In both the first and second methods, the insert is typically washed on a commercially available column, digested with the appropriate restriction enzyme, washed again on a commercially available column, and then ligated into the vector with the pre-digested MHC template. The ligation reaction is transformed into E. coli, and the plasmid prepared from the transformed E. coli is used in mammalian producer cell transfection reactions.

[0459] Example 2: Design and cloning of comPACT minigenes by primer annealing In a third variation of the MHC template vector ligation, PCR and restriction digestion were bypassed by annealing two reverse-complementary neoantigen-encoding primers. These primers were designed to have 5' and 3' ends that begin and end with complementary sequences that simulate overhangs from restriction digestion (Figure 5). The sense and antisense primers were incubated with T4 polynucleotide kinase and ATP to phosphorylate the 5' ends (Figure 22A). When these primers were annealed to each other, they formed a double-stranded oligonucleotide sequence with overhanging nucleotides as if they had been digested with a restriction enzyme.

[0460] The phosphorylated neoantigen insert (alternatively called neoepitope) was ligated to the precut MHC template in the vector. The comPACT minigene had the same structure as described in Example 1. The ligation product was then used for PCR amplification of the linear comPACT amplicon using bookend universal primers to amplify and sequence the complete comPACT minigene. 824 comPACT minigenes with unique neoantigen sequences (alternatively called neoepitope sequences) were generated using this method, with over 99% of the generated comPACT minigenes having the correct neoantigen sequence (alternatively called neoepitope sequence) (Figure 22B). The neoantigen sequences (alternatively called neoepitope sequences) cloned into the comPACT polypeptide and expressed as a polypeptide were based on tumor neoantigens identified from patient samples (e.g., tumor samples or other patient samples expressing tumor antigens). Based on the identified neoantigens, a set of predicted neoantigen sequences (alternatively called neoepitope sequences) was generated for each identified neoantigen.

[0461] E. coli was then transformed with the ligation product plasmid and plated onto selective agar plates containing ampicillin. Individual colonies were picked, grown overnight for plasmid purification, and sequenced for complete gene validation. After sequencing validation, plasmid lots were archived and grown in larger quantities.

[0462] Alternatively, if the precut MHC template vector retains a 5' phosphate on its overhanging end, T4 kinase is not used. The neo-antigen sequence (alternatively called the neo-epitope insert) of the annealed antigen insert can then be ligated with the cut MHC vector and the ligation product transformed into E. coli for plasmid production.

[0463] Example 3: Design and cloning of comPACT minigenes via PCR assembly [Construction of comPACT minigenes for PCR assembly] A fourth method of inserting an antigen, e.g., a neo-antigen (both of which, as used in this example for clarity, refer to the neoE insert in FIG. 6 and described as a neo-epitope in Example 2), may also be used. In this method, an antigen-encoding sequence (as used in this example for clarity, antigen-encoding sequence refers to the neo-epitope sequence described in Example 2) is inserted via polymerase chain reaction into an MHC template flanked by an upstream promoter and downstream polyadenylation signal to form a 2.5 kb minigene. A schematic of the PCR assembly reaction is shown in FIG.

[0464] In this example, the comPACT minigene is depicted with the following structure: a promoter at the 5' end; a signal sequence with a first universal target sequence; an encoded antigenic peptide; a second universal target sequence with an encoded linker sequence of primarily glycine and serine residues (i.e., a GlySer linker); a β2M sequence; a second encoded Gly-Ser linker sequence; an MHC heavy chain allele; a third Gly-Ser linker sequence; a purification cluster; and a polyA sequence. The universal target sequences are not the same in this exemplary method, i.e., they are different from each other.

[0465] [PCR assembly of comPACT minigenes] In this method, two primers (<60 nt) are synthesized with a selected antigen sequence (antigen sequence refers to neo-antigen sequence as used in this example for clarity). The first primer has the neo-antigen sequence (neo-antigen sequence refers to neo-epitope sequence as used in this example for clarity) at the 5' end, followed by a stretch of the second universal target sequence at the 3' end. The second primer has the reverse complement of the neo-antigen sequence (neo-antigen sequence refers to neo-epitope sequence as used in this example for clarity) at the 5' end and the reverse complement of the first universal sequence at the 3' end. These primers are mixed with a DNA fragment encoding the promoter region, signal sequence and first universal target sequence, and another DNA fragment encoding the second universal target sequence, β2m sequence, MHC allele, purification cluster and polyA sequence. Each antigen peptide primer anneals to its complementary sequence, and a PCR reaction is performed to amplify the neo-antigen sequence (as used in this example for clarity, neo-antigen sequence refers to the neo-epitope sequence described in Example 2) on either the promoter fragment or the MHC allele fragment. These two newly synthesized fragments each have a neo-antigen sequence (as used in this example for clarity, neo-antigen sequence refers to the neo-epitope sequence described in Example 2). A further PCR reaction, together with primers at the 5' end of the promoter sequence and the 3' end of the polyA sequence, allows the neo-antigen sequences (as used in this example for clarity, neo-antigen sequence refers to the neo-epitope sequence described in Example 2) to anneal to each other and prime the assembly of a full-length linear comPACT amplicon.

[0466] The fully assembled linear comPACT polynucleotide is then cleaned up for direct transfection into mammalian producer cells, bypassing the steps involved in E. coli and plasmid production altogether.

[0467] Example 4: Expression and purification of comPACT protein from plasmids [Protein expression] Neoantigen 12 (neo12) was ligated to the HLA-A2 template sequence via restriction digestion and ligation of NotI and BamHI restriction sites and inserted into an expression plasmid (pPACT0010) as previously described in Example 1.

[0468] Expi293 mammalian producer cells in a 30 mL shake flask volume were transfected on day 1 with pPACT0010 incubated with Expifcetamin transfection reagent. The enhancer included in the Expifectamine transfection kit was added on day 0. Samples were collected from cell supernatants on days 1 to 7 and secreted protein was assessed by SDS-PAGE and total protein staining using Safestain (ThermoFisher). The levels of secreted comPACT protein increased until day 3, at which point protein secretion plateaued (Figure 7). Secreted comPACT protein was initially identified by its apparent molecular weight (=53 kDa) and confirmed by Western blot using NTA-HRP to detect the His6 affinity tag (SEQ ID NO:34).

[0469] [Protein purification] Neol2 comPACT protein harvested on day 7 was purified by Ni-NTA affinity chromatography via binding of the His6 affinity tag (SEQ ID NO:34). Samples were assayed for total protein via SDS-PAGE and Safestain. Absence of comPACT protein in the affinity column flow-through (FT) fraction confirmed that the His6 tag (SEQ ID NO:34) was not cleaved during expression and purification (Figure 8). Purification yields were >400 mg per L of culture volume.

[0470] The neo12comPACT protein was biotinylated (discussed below in Example 5) and further purified by size-exclusion chromatography. A single major peak was observed, suggesting that the protein was properly folded, monomeric, and had little aggregation (FIG. 9). The second peak was ATP, which was added for the BirA-catalyzed biotinylation reaction.

[0471] Although Ni-NTA chromatography was used in Example 4, any HA affinity chromatography (including but not limited to metal affinity chromatography as described herein) can be used to purify HA-tagged comPACT.

[0472] [Production volume optimization and parallel production] Production of comPACT was scaled down from a 30 mL culture volume in shake flasks to 0.7 mL in a 96 deep-well shake block. Expi293 mammalian producer cells were transfected with plasmid DNA containing the pPACT0010 plasmid and secreted neo12comPACT protein was purified as previously described. 437 mg / L of purified neo12comPACT protein was collected from a 0.7 mL well volume (Figure 10), compared to the previously reported yield of >400 mg / L from a 30 mL purification experiment. The protein yield from the 0.7 mL experiment corresponds to >300 micrograms of protein, or approximately 1000 times the amount required for a typical flow cytometry experiment.

[0473] Next, parallel expression of multiple comPACT constructs was evaluated. Eight different comPACT constructs containing different neo-antigens (neo-antigens 10, 15, 64, 65, 66, 67, 80, and 83) were expressed in 30 mL shake flasks as a mid-throughput assay (Figure 11). Each comPACT construct was transfected into cells as previously described, and comPACT proteins were expressed and secreted into the cell supernatant. Expressed proteins were purified, concentrated, and normalized as previously described. Samples of crude supernatant and concentrated protein were assayed for total protein as previously described. comPACT proteins were purified by size exclusion chromatography (Figure 12). A single peak containing 2-20 mg of protein was seen for each protein, again suggesting that comPACT proteins are properly folded and monomeric.

[0474] Example 5: Expression and purification of comPACT proteins from linear amplicons In the previous examples, comPACT proteins were expressed from plasmids transfected into mammalian producer cells. As an alternative approach, a linear amplicon of the neo12 comPACT minigene (neoantigen 12 assembled into a minigene containing the HLA-A2 template sequence) flanked by promoter and polyA sequences was transfected into 0.7 mL of producer cells in a 96 deep-well plate. As a control, the pPACT0010 plasmid was also transfected into separate producer cells. Proteins from both samples were expressed, purified, and assayed for total protein as described above. Similar levels of expressed protein were produced by both the linear amplicon and the plasmid (Figure 13A), suggesting that proteins encoded by comPACT minigenes can be produced without the need for a plasmid intermediate. For direct transfection of producer cells, multiple different comPACT minigenes with different neoepitope sequences were produced (Figure 13B).

[0475] Additional comPACTs with different HLA alleles were generated using the annealing and phosphorylation workflow described in Example 2. Linear amplicons were derived from expression vectors using bookend PCR and universal primers and transfected into Expi293F cells for comPACT protein production (data not shown).

[0476] Example 6: Biotinylation of comPACT Protein [In vitro biotinylation of comPACT by isolated BirA enzyme] comPACT purified clusters contained a BirA recognition sequence (Avitag) for biotinylation. Purified comPACT proteins were either not biotinylated (no BirA treatment) or biotinylated with commercial BirA protein according to the manufacturer's instructions (with BirA treatment). After overnight BirA enzyme treatment, samples were bound to two different types of magnetic streptavidin beads (C1 and T1) and incubated to allow binding of biotinylated proteins to the streptavidin beads. Supernatants (SN) and beads ("pellet" P) were separated by SDS-PAGE. Samples were analyzed for total protein using Safestain and for the presence of comPACT protein by Western blot using NTA-HRP (Figure 14). In untreated samples, comPACT protein was found mainly in the SN fraction, confirming that it was not biotinylated. In the biotinylated samples, comPACT protein was found in both the C1 and T1 streptavidin bead pellet samples, but the interaction between the biotinylated protein and the C1 streptavidin beads was the most complete. No biotinylated comPACT protein was detected by Western blot in the C1 streptavidin bead-depleted supernatant, suggesting that approximately 100% of the comPACT protein was biotinylated.

[0477] comPACT proteins can also be biotinylated in the clarified supernatant prior to purification. As described above, multiple comPACT proteins were expressed in producer cells. Cell culture supernatants were collected and clarified by centrifugation. The clarified supernatants were treated with commercially available BirA protein according to the manufacturer's instructions, then purified by Ni-NTA affinity chromatography and biotinylation was assessed by Western blot (Figure 15). All comPACT proteins tested were biotinylated using this method, indicating that biotinylation of comPACT proteins in clarified cell supernatants is effective. Although Ni-NTA chromatography was used in Example 6, any HA affinity chromatography (including but not limited to metal affinity chromatography as described herein) can be used to purify HA-tagged comPACT.

[0478] To produce sufficient BirA for high-throughput biotinylation of comPACT proteins, a BirA protein containing a His6 tag (SEQ ID NO:34) was expressed in E. coli cells. This His6-tagged (SEQ ID NO:34) BirA was purified by Ni-NTA affinity chromatography (Figure 16B) and can be used to biotinylate comPACT proteins. A second version of BirA-His6 ("His6" disclosed as SEQ ID NO:34) containing a TEV-cleavable His6 tag (SEQ ID NO:34) was also expressed and purified by Ni-NTA affinity chromatography (Figure 16C). This BirA-TEV-His6 protein ("His6" disclosed as SEQ ID NO:34) can be purified via Ni-NTA, the His6 tag (SEQ ID NO:34) removed by TEV cleavage, and then tag-free BirA can be used to biotinylate comPACT proteins. After biotinylation of comPACT protein, the untagged BirA protein can then be purified by Ni-NTA affinity chromatography. In addition, TEV protease was heterologously expressed in E. coli for use in producing biotinylated comPACT protein, BirA-TEV-His6 ("His6" disclosed as SEQ ID NO:34) (FIG. 16A). Although Ni-NTA chromatography was used in Example 6, any HA affinity chromatography (including but not limited to metal affinity chromatography described herein) can be used to purify HA-tagged comPACT.

[0479] Cleavage of the His6 tag (SEQ ID NO:34) on the comPACT protein after biotinylation was also evaluated and the results are shown in FIG. 17. comPACT proteins were treated with BirA or left untreated and they were biotinylated as described above (lanes 1 and 2 in FIG. 17). A third sample of comPACT protein was treated with BirA and then with TEV to cleave the His6 tag (SEQ ID NO:34) present on the protein (lane 3). Samples were separated by SDS-PAGE and total protein was assessed with Safestain. All three samples contained equal amounts of comPACT protein. Biotinylation of comPACT protein and cleavage of the His6 tag (SEQ ID NO:34) were assessed by Western blot using SA-HRP reagent for the biotin signal and NTA-HRP reagent for the His6 tag (SEQ ID NO:34). The non-biotinylated sample showed no biotin signal but had a His6 signal (SEQ ID NO:34), and the biotinylated uncleaved sample had both signals. The biotinylated TEV cleaved sample had only a biotin signal, indicating that the His6 tag (SEQ ID NO:34) was successfully cleaved from the comPACT protein.

[0480] A third approach to biotinylating comPACT in vitro is to express BirA in Expi293 producer cells. Expi293 cells were generated that co-express BirA and a V5-tagged cell surface transduction marker. V5+ sorted transduced cells also express BirA (Figure 18). These cells can be used to produce biotinylated comPACT in vivo prior to comPACT protein purification.

[0481] Example 7: Antigen-specific T cell staining and affinity assessment using comPACT protein To compare antigen-specific T cell staining using comPACT proteins and generic peptide-MHC, comPACT dextramers were prepared according to published protocols (Bethune, MT et al. BioTechniques 62, 123-130, doi:10.2144 / 000114525 (2017)). T cells were engineered to express an A2 / neo12-specific TCR and stained with either HLA-A2 / neo12 peptide-MHC dextramer or HLA-A2 / neo12 peptide comPACT dextramer. Staining with comPACT dextramer was at least as efficient as staining with peptide-MHC dextramer (Figure 19). This data suggests that comPACT dextramer can be used to sort antigen-specific T cells for TCR sequencing.

[0482] Example 8: Functional T cell assay Beyond antigen-specific capture of T cells, the modular design and ease of manufacture of comPACT facilitates their use in functional T cell assays. For example, incorporation of a mutant form of β2m (S88C) allows comPACT to be labeled with maleimide-dye conjugates, assembled as NTAmers, and used to measure kinetic parameters of TCR-comPACT binding. S88C mutant comPACT proteins have been constructed and expressed at approximately 150 mg / L. These mutant comPACTs exhibit similar purity and elution profiles as unmutated comPACT (Figure 20). Other dyes, such as Cy5, can also be conjugated to S88C comPACT (Figure 21).

[0483] Example 9: comPACT library generation HLA allele diversity across the US human population was analyzed by bioinformatics from the Allele Frequency Net database (www.allelefrequencies.net) to identify the optimal number of alleles to include in the HLA repertoire to provide high coverage of the HLA frequency of interest. 9736 alleles were analyzed. Figure 24A shows an analysis of the proportion of patients in which one or both alleles from each of the HLA A, B, and C loci are covered by a library of 66 HLA alleles. A solid line indicates that one allele is covered, and a dashed line indicates that both alleles are covered. 66 alleles allow for coverage of at least 4 of the 6 HLA alleles per patient in >95% of the total population, and 6 / 6 alleles in >80% of the total population (Figure 24B). The most frequent HLA-I allele is HLA-A02:01, with a prevalence in the US of approximately 50%. Thus, the HLA library presented here for the first time maximizes the potential for broad implementation of personalized neoTCR-T cell therapy for global, diverse populations.

[0484] Next, a library of comPACT proteins with different neoepitopes and selected HLA alleles was generated. Neoepitope candidates were selected from the Immune Epitope Database (www.iedb.org). The complete sequence of each of the 66 HLA-I alleles in the repertoire was obtained from the IMGT database and modified to include the Y84C mutation. All clones were sequence verified and deposited in the database and reagent inventory. Ten neoepitope peptides were selected from the IEDB database and inserted into a panel of 36 HLA alleles. comPACT polypeptides of selected neoepitopes and HLA alleles were expressed and purified by a size-exclusion chromatography column (Agilent Sec Bio 300) coupled to an Agilent Infinity II HPLC system (SEC-HPLC) according to the manufacturer's instructions. The results are shown in Figures 25A-C. comPACT polypeptides were purified as monodisperse polypeptides, as assessed by SEC-HPLC by measuring the area under the curve of the monomer peak divided by the area under the entire chromatogram (Figures 25A and 25B). Most comPACT polypeptides were expressed at high titers (Figure 25C). At least one comPACT protein for each listed HLA allele was purified and characterized by HPLC, demonstrating that the comPACT platform is robust and suitable for many alleles.

[0485] Example 10: imPACT T cell isolation method Materials and Methods

[0486] [comPACT library preparation]

[0487] Paired PE and APC tetramer particles containing the three comPACT library elements and barcodes were prepared prior to the experiment. Biotinylated comPACT (1 μM, produced in-house) and DNA barcodes (1 μM, IDT) were mixed in a 3:1 molar ratio. PE-streptavidin (3.33 μM, Life Technologies) or APC-streptavidin (6.26 μM, Life Technologies) were added and reacted with biotin in a 1:4 ratio. After incubation, additional biotin was introduced to occupy free streptavidin sites.

[0488] [CD8 T cell staining]

[0489] Cells were incubated with 40 nM fluorescent comPACT tetramer for neoantigen-specific T cell staining. Fc receptor blocking solution was then added to minimize non-specific antibody staining. Samples were also incubated with an antibody cocktail containing FITC CD4, CD14, CD19, CD20, CD40, PerCp-Cy5.5 CD8, BV711 CD45RA, BV786 CD95, and BV510 IP26 (Biolegend) to identify T cell phenotype.

[0490] [Single cell sorting]

[0491] Fluorescently labeled cells were sorted into single cells using a FACSAria III (BD Biosciences). Cells were first sorted for T cell phenotype based on IP26 and CD3 staining, and then sorted for dual p-HLA binding based on APC and PE staining. comPACT positive cells were sorted into 96-well plates containing lysis buffer of 10 mM Tris and RNAse inhibitor (Promega).

[0492] [TCR cloning]

[0493] An RT-PCR master mix was prepared containing the following reagents: nuclease-free water (Invitrogen), 5x buffer (Qiagen), 10 mM dNTPs (Qiagen), alpha multi-primer mix, beta multi-primer mix, alpha antisense primer, beta antisense primer, DNA barcode sense primer, DNA barcode antisense primer (all primers were ordered through IDT), Onestep RT-PCR enzyme (Qiagen), and KOD polymerase (Millipore). The RT-PCR master mix was then added to each well to initiate reverse transcription and polymerase chain amplification of the TCR and DNA barcode sequences.

[0494] [Sensitivity and S / N Assay]

[0495] CD8 T cells expressing TCRs against the MART1 antigen (F5) or the neoantigen neo12 were incubated with fluorescent comPACT particles containing the corresponding comPACT neoantigen element (MART1 or neo12). Cells were stained and sorted via FACS as described above (Figure 29A-C).

[0496] CD8 T cells expressing TCR against the neo-antigen neo12 were doped into control PBMC samples at a ratio of 1 to 300000. The doped samples were incubated with a library of 33 tetramers containing neo12comPACT and 32 additional neo-antigen comPACT elements. Single cells were sorted based on the gating strategy described above for APC and PE double-labeled CD8 T cells (Figure 30).

[0497] [Specificity and S / N Assay]

[0498] CD8 T cells expressing TCR against neo-antigen neo12 were doped into control PBMC samples at a ratio of 1 to 300000 or at a ratio of 1 to 30000. PBMC alone was used as a negative control. Doped samples were incubated with a library containing neo12 comPACT and 28 irrelevant control comPACT elements. Single cells were sorted based on the gating strategy described above for APC and PE double-labeled CD8 T cells. The barcode and signal-to-noise ratio for each barcode associated with a given cell were determined.

[0499] [result] [sensitivity] Figure 29A provides a diagram of the endogenous TCR gating strategy for isolating PACT neoantigen CD8 T cells (top panel) and tetramer positive double labeled T cells (bottom panel). T cells expressing neoantigen F5 (Figure 29B) or neo12 (Figure 29C) were labeled and gated according to the gating strategy. Corresponding tetramer staining resulted in greater than 99% accuracy for F5 and neo12 comPACT neoantigen T cells. Replicates of this assay yielded greater than 99% tetramer staining for both gene edited F5 and neo12 T cells (data not shown). Thus, the imPACT method has high staining and sorting sensitivity of greater than 98% or 99%.

[0500] To test the sensitivity of the imPACT tetramer methodology, a cell doping experiment was performed. T cells expressing the neoantigen neo12 were doped into control PBMC samples at a ratio of 1 to 300000 (one neo12 T cell per 300000 PBMCs). imPACT tetramer analysis was performed using tetramers generated from neo12 comPACT and 32 irrelevant control comPACT. Cells positive for 33 comPACT tetramers were sorted from CD8 T cell gating as described above. Cells were sequenced for relevant TCR and neoID sequences. After sequencing, the signal-to-noise ratio (S / N) was used to determine the specificity of tetramer binding. The S / N calculation in this example was the DNA copy number of the most predominant neoID divided by the second most predominant neoID. In this example, an S / N of greater than 10 was considered to be specific binding of comPACT to T cells. The index flow results of IP26 staining of each cell indicate whether a given cell is gene edited or naive.

[0501] Table 4 summarizes the cells sorted from this experiment. All 1686717 cells were analyzed by flow cytometry, and 11 cells were sorted from the positive gate. TIFF0007611836000004.tif38170

[0502] Five of the 11 positive cells have a S / N higher than 10 (average S / N=83.1), while the other six have a S / N lower than 10 (average=1.1). Based on the ratio of neo12 doped cells (1:3 million) and the number of cells processed (1,686,717), there should be about 5-6 neo12 cells in the analyzed sample. The sequencing results show that 5 neo12 cells were isolated using the method. Thus, the imPACT tetramer method is sensitive enough to isolate antigen-specific cells at a low frequency of 1 / 300,000. Figure 30 shows the gating FACS cells of the 1:300,000 doped sample. TCR- indicates neo12 positive T cells, and TCR+ indicates nonspecifically bound T cells. The average S / N ratio of specific neo12 cells was 83, while the average S / N ratio of nonspecific cells was 1.1.

[0503] [Specificity] Next, the imPACT isolation method was evaluated for neo-antigen specificity. The neo12 doping assay was repeated with a second library of neo12 comPACT and 28 irrelevant control comPACT spiked PBMC samples. PBMCs alone were used as a negative control. Double positive cells were isolated, barcodes sequenced, and the S / N ratio of each barcode associated with a given cell was determined. Figure 31 shows the PE and ACP FACS gating data of neo12 antigen doped and undoped PBMC samples. A table summarizing the barcode S / N sequencing results is shown below. In the specificity test, the S / N average was 162, indicating 162 copies of neo12 neo-ID barcode for each non-neo12 neo-ID barcode. This indicates high specificity of the neo12 barcode for neo12 and cells sorted from PBMC samples. In contrast, the S / N average for the non-specific test was 1.7, indicating only 1.7 copies of the neo12 neoID barcode for each non-neo12 neoID barcode, indicating low specificity of the neo12 barcode in sorted cells in PMBC-only samples.

[0504] The assay was repeated with a comPACT library in which the 33 elements were added to a PBMC sample at a ratio of 1 neo12 T cell to 30,000 PBMCs. Figure 32A shows the gated double positive cells, and Figure 32B shows the barcode S / N average of specific and non-specific T cells. The signal-to-noise ratio of the 33 tetramer positive cells was determined. The S / N average of T cells with specific binding was 124.9 and the non-specific S / N ratio was 1.2, confirming the high specificity of the imPACT method for isolating neo-antigen-specific T cells. Table 3 summarizes the information of the sorted cells and whether the isolated cells were genetically modified (gene edited). TIFF0007611836000005.tif88170

[0505] Example 11: Isolation of neoantigen T cells from patient samples Materials and Methods [Preparation of tetramer]

[0506] Tetramers were prepared as previously discussed above.

[0507] [CD8 selection and cell staining]

[0508] Cryopreserved patient PBMCs were thawed and CD8 T cells were selected using a CD8+ T cell isolation kit (Miltenyi) following the manufacturer's recommended protocol. Isolated CD8 T cells were used for subsequent staining. Cells were incubated with 40 nM of fluorescent comPACT tetramer library for neoE-specific T cell staining. Fc receptor blocking solution was then added to minimize nonspecific antibody staining. Samples were incubated with an antibody cocktail including FITC CD4, CD14, CD19, CD20, CD40, PerCp-Cy5.5 CD8, BV711 CD45RA, BV786 CD95, and BV510 IP26 (Biolegend) to identify T cell phenotype. Live / dead near-infrared cell stain (Invitrogen) was used to distinguish between live and non-live cells. BV605 Annexin-V (Biolegend) was used to further distinguish between live and apoptotic cells.

[0509] [Single cell sorting and TCR cloning]

[0510] Fluorescently labeled cells were sorted into single cells using a FACSAria III (BD Biosciences). Viable CD8+ tetramer-positive cells were sorted into 96-well plates containing lysis buffer of 10 mM Tris and RNAse inhibitor (Promega). Cells were then frozen for subsequent TCR cloning. An RT-PCR master mix was prepared containing the following reagents: nuclease-free water (Invitrogen), 5x buffer (Qiagen), 10 mM dNTPs (Qiagen), alpha multi-primer mix, beta multi-primer mix, alpha antisense primer, beta antisense primer, DNA barcode sense primer, DNA barcode antisense primer (all primers were ordered through IDT), Onestep RT-PCR enzyme (Qiagen), and KOD polymerase (Millipore). The RT-PCR master mix was then added to each well to initiate reverse transcription and polymerase chain amplification of the TCR and DNA barcode sequences. Two additional rounds of PCR were performed to further amplify the TCR and DNA barcode sequences and add adapter sequences for next-generation sequencing (NGS).

[0511] [Next-generation sequencing]

[0512] Next generation sequencing was performed on a Miniseq (Illumina) using the recommended reagents. Library preparation was performed according to the protocol recommended by Illumina. Target species and PhiX were mixed in equal amounts to provide diversity.

[0513] [result] First, a stage IIIA melanoma patient sample (PACT032) was analyzed using a 26-element comPACT library containing HLA A02:01 allele types. 6Of the PBMCs, 231 were double positive for APC and PE. Cells were analyzed for neoID barcodes and the signal-to-noise ratio of all double positive cells was determined. Figure 33A shows a FACS dot plot of double positive T cells. After neoID sequencing, one T cell showed specificity for one mutation with a signal-to-noise ratio (S / N1) >10. This neo-antigen TCR was cloned and screened against the predicted neo-antigen. The remaining double positive cells all had a signal-to-noise ratio of 1 and were not specific for the neo-antigen associated with bound comPACT. A secondary screen (Figure 33B) confirmed the specificity of the neo-antigen TCR isolated by impact analysis. Table 5 below shows a summary of the signal-to-noise ratios of specific and non-specific T cells.

[0514] TIFF0007611836000006.tif28158

[0515] Example 12: S / N1 and S / N2 analysis to identify TCRs Next, a stage III melanoma patient sample (PACT077) was analyzed using a 138-element comPACT library containing HLA A02:01, A24:02, B18:01, and C07:01 types. 6Of the PBMCs, 250 were double positive for APC and PE. Cells were analyzed for neoID barcodes and the signal-to-noise ratio of all double positive cells was determined. Figure 34A shows a FACS dot plot of double positive T cells. Figure 34D shows neoantigen-specific T cells identified in peripheral blood. The asterisk indicates that the same TCR clonotype is found in tumor infiltrating lymphocytes (TILs) from tumor sequencing. After neoID sequencing, 25 T cells showed specificity for one mutation with a signal-to-noise ratio (S / N1) greater than 10 (Figure 34C). Figure 34B shows that all candidate cells were derived from antigen-experienced CD95+ cells. Neoantigen TCRs were cloned and screened against the predicted neoantigens. Figure 34E shows the percentage of neoTCR gene-edited lymphocytes capable of recognizing the cognate antigen. The remaining double positive cells all had a signal-to-noise ratio of 1 and were not specific for the neoantigen associated with bound comPACT. Table 6 below provides a summary of the T cell signal-to-noise ratios for selected neo-antigens.

[0516] TIFF0007611836000007.tif54156

[0517] Interestingly, analysis of TCRs from PACT077 identified eight distinct TCRs. Six of them had S / N1 ratios greater than 10, confirming that they were neoantigen-specific T cells. In the other two T cells, the S / N1 ratio was less than 10, but the S / N2 ratio was higher than 10 (Figure 34C). Cloning of two TCRs (TCR143 and TCR164) revealed that they could recognize two different neoantigens that share the same mutation, further explaining why the S / N1 was low (Figure 35A). These results indicate that the S / N2 ratio can be used to distinguish nonspecific from specific cells when multiple neoantigens derived from the same mutation are present.

[0518] A secondary screen (Figure 35B) confirmed the specificity of the neo-antigen TCRs isolated via imPACT analysis: TCR135, TCR136, TCR139, TCR142, TCR144, and TCR145.

[0519] FIG. 36 shows that the isolated TCRs varied among variants with different levels of clonality, truncality, and in situ neoantigen expression levels.

[0520] Example 13: Isolation of neoantigen T cells with dual neoID barcodes Materials and Methods [Preparation of tetramer]

[0521] Paired fluorescent tetramer particles were prepared as previously discussed above, except that each particle pair had a different unique neoID barcode associated with a neo-antigen (see Figure 28 for an illustration of dual-barcoded paired particles).

[0522] [CD8 selection and cell staining]

[0523] Cryopreserved patient PBMCs were thawed and CD8 T cells were selected using a CD8+ T cell isolation kit (Miltenyi) according to the manufacturer's recommended protocol. Isolated CD8 T cells were used for subsequent staining as previously described.

[0524] [Single cell sorting and TCR cloning]

[0525] Fluorescently labeled cells were sorted into single cells using a FACSAria III (BD Biosciences) as previously described.

[0526] [Next-generation sequencing]

[0527] Next generation sequencing was performed on a Miniseq (Illumina) using recommended reagents as previously described.

[0528] [result] PACT049 (stage 4 CRC, naive) PBMCs were screened using the imPACT process and dual barcoding. A 6-element dual barcoded comPACT library (HLA-B57:01, A01:01, and C06:02) was generated and used to investigate neo-antigen TCRs. 352 single cells were sorted. After sequencing analysis, three neo-antigen TCR candidates were identified against one HLA-B57:01 neo-antigen RCSPEQLKKAW (SEQ ID NO:208) (Figure 37A). These three candidates were sorted based on tetramer MFI (mean fluorescence intensity) and were all considered antigen-experienced CD95+ (Figure 33B). After cloning the PACT049 neo-antigen T cells, these TCRs were confirmed by dextramer staining with the relevant predicted neo-antigen (Figure 37C and Figure 37D).

[0529] Example 14: Additional isolation of neo-antigen T cells from patient samples Additional patient samples were incubated with the comPACT library and isolated according to the imPACT methodology described above. Patient samples were analyzed using the imPACT signal-to-noise method and single or dual barcode methods. A graph of the number of neoantigens and HLA types identified from each sample and cancer type is provided in Figure 38A, and the HLA types are tabulated in Figure 38B. Thirty-two neoantigen-specific TCRs were identified across five cancer types, with 13 HLA types identified in the vicinity. Neoantigens were identified in colorectal cancer (11), melanoma (7), bladder cancer (3), endometrial adenocarcinoma (1), and head and neck cancer (1) samples. Two patient samples (PACT056 and 095) did not yield a neoantigen-specific TCR. Four patient samples (PACT032, 052, 053, and 078) have one neoantigen-specific TCR. Multiple neoantigen-specific TCRs were isolated in each of the following seven samples: PACT035, 036, 037, 049, 077, 131, and 133. Multiple neoantigen-specific TCRs allow selection of the best TCR for the patient. Two samples (PACT077 and 078) contained peripheral TCRs found in situ by deep sequencing of TILs. These results indicate that the success rate of neoantigen-specific TCR identification was 100% in drug-treated patients and over 80% in untreated patients.

[0530] The results show that the imPACT technology can successfully isolate antigen-paired neoantigen-specific TCRs from patient samples with high accuracy and specificity. Because the imPACT technology targets neoantigens and the antigen presentation pathway is universal, the imPACT platform technology can be applied to a variety of cancer types, enabling the development of personalized neoTCR-T cell therapies to eradicate solid tumors.

[0531] Example 15: Reproducibility of T cell isolation method Next, the comPACT element library was used to analyze PBMCs from healthy donors. Fifteen pairs of fluorescent comPACT (HLA A02:01) containing neoantigens for cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza were generated as previously described. The comPACT library was incubated with PBMCs, and double-positive T cells were sorted and isolated. Neo-ID barcodes were sequenced, and TCRs were cloned and sequenced, as previously described. Experiments were performed in triplicate.

[0532] Figure 39A shows the percent antigen specificity for isolated T cells with neoantigens for CMV and EBV in each of the three replicates. Figure 39B shows the number of TCR alpha chains isolated from each experiment. Table 7 shows an overview of the identified TCR alpha chains. Fourteen unique TCR alpha chains were identified, with ten of the fourteen shared across all three experiments. This reproducibility experiment demonstrates that the imPACT method can consistently isolate similar levels of antigen-specific T cells from the same sample in independent experiments, demonstrating that the method of isolating double-positive T cells by incubating cells with paired comPACT tetramers with different fluorophores is highly reproducible in multiple settings.

[0533] TIFF0007611836000008.tif103170

[0534] Example 16: Comparison of T cell isolation with comPACT tetramer, dextramer, and trimer The isolation efficiency of tetramers, dextramers, and trimers of comPACT library elements in the dual staining method was evaluated. Streptavidin core tetramers with four copies of each comPACT element (tetramers), streptavidin core trimers with three copies of each comPACT element and a nucleic acid barcode (trimers + DNA), and dextramers of dextran polymers with multiple copies of comPACT elements with and without nucleic acid barcodes (dextramers and dextramers + DNA) were incubated with T cells gene-edited to overexpress F5 (MART1) or neo12 neoantigens. T cells were isolated based on the gating strategy described in Example 10 above, and the percentage of dual stained T cells isolated by each staining method was quantified. As shown in Figure 40A (F5 T cells) and Figure 40B (neol2 antigen-specific T cells), more than 98% of the gene-edited cells were stained with the corresponding comPACT element. The data show that similar staining efficiencies were achieved for DNA-containing tetramers, dextramers, trimer-tetramers, and DNA-containing dextramers on T cells containing a common TCR (F5 Mart-1) and a neoantigen TCR (neo12).

[0535] Example 17: Signal-to-noise analysis comparison PACTneo12 T cells, PACT M1W T cells, and viral donor PBMCs were incubated with trimer comPACT particles containing nucleic acid barcodes (trimer+DNA) and dextramer containing multiple copies of comPACT elements containing nucleic acid barcodes (dextramer+DNA). Cells were sorted into single cells by FACS. TCR alpha / beta and neoID barcodes were cloned and sequenced for each sample. All TCRs were confirmed to be correct for neo12, CMV, or M1W. S / N analysis was performed on each cell as previously described. The S / N ratio of each method (trimer, T; or dextramer, D) for each sample is shown in Figure 41A. The average S / N ratio of each method is provided in Figure 41B. Notably, analysis of the signal-to-noise ratio of neoID barcode DNA shows a higher signal-to-noise ratio for cells isolated with trimer+DNA particles compared to cells isolated with dextramer+DNA. The data show that trimer+DNA particles have a significantly better signal to noise ratio compared to dextramer+DNA.

[0536] Example 18: Isolation and characterization of neoantigen T cells from patient samples following cancer immunotherapy Subjects with pMMR colorectal cancer (not generally considered to respond to anti-PD1 antibody therapy) or endometrial adenocarcinoma were treated with AB122 (anti-PD-1 antibody). Pre-treatment blood samples were incubated with comPACT libraries and isolated according to the imPACT method described above to identify the baseline repertoire of neo-antigen-specific T cells. PBMCs were then collected at various time points and analyzed with the imPACT signal-to-noise ratio method to monitor the evolution during treatment of the mutation-targeted T cell repertoire. Changes in the neo-antigen-specific T cell repertoire during AB122 treatment were monitored to allow correlation of immune phenotype with clinical outcome.

[0537] [result] The top panels of Figures 42A-C show the longitudinal evolution of neoantigen-specific T cells in peripheral blood during treatment of patients with colorectal cancer PACT157 (Figure 42A) and PACT132 (Figure 42B) or endometrial cancer PACT131 (Figure 42C).

[0538] The bottom panels of Figures 42A-C show the neoantigen clonality and predicted neoantigen-HLA binding affinity for each sample, with the top dots indicating clonal variation and the bottom dots indicating subclonal variation.

[0539] The gene, HLA type, and neoantigen sequence for each TCR identified by the imPact method in each subject are also shown in Figures 42A-C.

[0540] Longitudinal monitoring of patients during treatment will allow analysis of neoantigen-targeting T cells to identify driver mutations that correlate with treatment efficacy. Additionally, monitoring changes in neoantigen-specific T cell repertoires in response to immunotherapy can inform next steps in treatment.

[0541] Example 19: Phenotypic and functional characterization of neoantigen-specific T cells from PACT131 T cells isolated by imPACT isolation technology from patient sample PACT311 were characterized for cell surface markers CD45RA, CD95, CD39, and CD103 via flow cytometry. Flow cytometry results of T cells isolated from the patient on days 1, 15, and 57 are shown in Figure 43. Black dots indicate neo-antigen specific T cells. CD45RA+CD95+ T cells have experienced antigen, while CD39+CD103+ positivity suggests that the T cells have transited the tumor compartment.

[0542] Next, three TCR clones (TCR200, TCR202, and TCR205) against the same PIK3CA neo-antigen target captured from patient samples were characterized. T cells were edited to express the selected TCR. The percentage of live, CD8+, and CD4+ T cells is shown in Figure 44A. Activation of neo-antigen-specific T cells was determined by incubating edited T cells with increasing amounts of HLA cognate peptide and measuring secretion of IFNγIL2 and TNFα. Cytokine release of each TCR clone is shown in Figure 44B. All T cells were activated by cognate neo-antigen. No cytokine release could be detected against non-cognate neo-antigens (data not shown).

[0543] Example 20: Validation of neoTCRs isolated from melanoma patient samples using the imPACT method Materials and Methods

[0544] [Preparation of comPACT libraries]

[0545] Somatic nonsynonymous mutations were identified in patient PACT135 using whole exome sequencing of tumor biopsies and the patient's normal PBMCs, and RNA-Seq transcriptome sequencing of the tumor biopsies. The patient had stage IV metastatic melanoma and was receiving anti-PD1 antibody therapy with nivolumab. 2566 coding mutations were identified. 632 neoepitopes were predicted from the tumor mutation count, and a library of 243 comPACT (neoepitope-HLA complexes) was generated across HLA-A*03:01, A*24:02, and C*12:03, as described in Examples 10 and 11. HLA typing was predicted by the OptiType program based on the patient's normal PBMC whole exome sequencing. Three of the six HLAs were covered by the library.

[0546] [T cell isolation]

[0547] PBMC and TIL samples were collected from subject PACT135 at various time points before or during anti-PD-1 antibody therapy. PBMC samples were collected on days 14, 43, and 84 after initiation of treatment. TIL samples were collected on day -37 before initiation of anti-PD1 treatment and on day 82 after initiation of treatment. T cells were incubated with comPACT libraries and neo-antigen-specific T cells were isolated using the imPACT method as described in Examples 10 and 11.

[0548] Fourteen TCRs specific for five neoantigen HLA were isolated; one neoTCR recognizing PUM1, one neoTCR recognizing TTP2, two neoTCRs recognizing IL8-HLA-A*24:01, and ten neoTCRs recognizing IL8-HLA-A*03:01. T cells expressing neoTCRs were expanded for 14 days in medium containing IL2, IL7, IL15, or combinations thereof. At the end of expansion, T cells maintained a "younger" T cell phenotype and gave rise to neoTCR-P1 T cells that exhibited T memory stem cell and T central memory cell phenotypes.

[0549] [neoTCR gene editing]

[0550] CD4 and CD8 T cells from healthy donors were engineered to express each identified neo-antigen-specific TCR using a CRISPR-based non-viral method, as described in International Patent Application WO2019089610, published May 9, 2019, which is incorporated herein by reference in its entirety.

[0551] [neoTCR expression]

[0552] Expression of neoTCR in gene-edited CD4 and CD8 T cells was analyzed using fluorophore-comPACT trimer dextran conjugates. Biotinylated comPACT proteins were bound to streptavidin dextramers and incubated with neoTCR CD4 and CD8 T cells. Binding of comPACT-dextramers to each neoTCR-expressing T cell was determined by methods described in more detail in Bethune et al. (BioTechniques 62:123-130 Mar. 2017) and Bethune et al. (eLife 5: 2016), each of which is incorporated herein by reference for all that it teaches. Dextramers were prepared using fluorescently labeled streptavidin (Life Technologies, Carlsbad, CA).

[0553] [Generation of matched autologous melanoma cell lines]

[0554] A matched autologous melanoma cell line was established from a patient biopsy (M489). As a negative control, a second cell line from a mismatched melanoma was established from another patient biopsy (M202). T cells expressing the neoTCR were assessed for functionality (expression of activation markers, cytokine secretion, antigen-specific target cell killing, and T cell proliferation) using matched and mismatched autologous melanoma cell lines.

[0555] [T cell activation]

[0556] neoTCR-expressing T cells were incubated with or without IFNγ and co-cultured with M489 matched melanoma tumor cell line and neoantigen-matched comPACT-dextramer. As a negative control, neoTCR T cells incubated with or without IFNγ were co-cultured with M202 mismatched melanoma tumor cell line and neoantigen-matched comPACT-dextramer. Stimulation of T cells with anti-CD3 antibody OKT3 was used as a positive control. Internalization of neoTCR after comPACT-dextramer binding was assessed via FACS.

[0557] Expression of activation markers 4-1BB and OX-40 was also determined in CD4 and CD8 neoTCR T cells co-cultured with matched and mismatched melanoma cell lines. Expression of activation markers was determined via FACS. Anti-OX-40 antibody (clone Ber-ACT35, catalog number 350012) and anti-4-1BB antibody (clone 4B4-1, catalog number 309810) were purchased from Biolegend.

[0558] [T cell cytotoxicity assay]

[0559] T cell-induced killing of tumor cells over time was monitored via immunofluorescence using an IncuCyte imaging system (Essen BioSciences). Each of the 14 neoTCR-expressing T cells was co-cultured with patient-matched M489 tumor cells. The neoTCR T cells were also co-cultured with the M202 mismatched cell line as a negative control. M489 and M202 tumor cells were labeled using NucLight Red lentivirus (Essen BioSciences).

[0560] M489 or M202 tumor cells were seeded at 25,000 cells / well in 96-well plates and incubated overnight in an incubator. The next day, neoTCR T cells were added at the following concentrations: 25,000 T cells / well (1:1 T cell:tumor cell ratio) or 100,000 T cells / well (5:1 T cell:tumor cell ratio). The co-culture preparations were then monitored by collecting time-lapse images at 2-h intervals for 12 days using an IncuCyte imaging system with a 10x objective.

[0561] [Cytokine secretion assay]

[0562] Cytokine production was assessed in the supernatants of co-cultured T cells and melanoma cell lines using a cytokine bead assay (CBA bead-based immunoassay, BD BioSciences). CBA is a multiplex bead-based immunoassay application of flow cytometry that allows simultaneous quantification of multiple proteins by efficiently capturing analytes using antibody-coated beads. After co-culture of T cells with target cells for 24 or 48 h, supernatants were collected and analyzed for IFNγ, IL-2, and TNFα secretion.

[0563] [result]

[0564] [Identification of neoTCR in PACT135 over time]

[0565] imPACT analysis led to the isolation of 14 TCRs specific for 5 neoantigen HLAs: one neoTCR recognizing PUM1, one neoTCR recognizing TTP2, two neoTCRs recognizing IL8-HLA-A24:02, and 10 neoTCRs recognizing IL8-HLA-A03:01. The neoantigen peptide sequences, alpha and beta TCR CDR3 sequences, and HLA alleles isolated from patient PACT135 are shown in Table 8 below. JPEG0007611836000009.jpg193170

[0566] Figure 45 provides a summary of the number of neo-antigen-specific T cells per CD8 T cell in each sample collected over the course of anti-PD-1 antibody treatment. Each box represents one T cell and each cross represents 10 T cells. Each row of boxes or crosses represents a unique neoTCR clonotype.

[0567] TCR219, TCR220, TCR223, TCR224, TCR225, TCR228, TCR229, TCR232, TCR240, and TCR241 recognize the IL8-KTYFKPFHPK neoantigen (sequence number: 256).

[0568] TCR221 and TCR227 recognize the IL8-YFKPFHPKF neoantigen (SEQ ID NO: 227).

[0569] TCR218 recognizes the TPP2-CFSEVSAKF neoantigen (SEQ ID NO:223).

[0570] TCR222 recognizes the PUM1-AMMDYFFQR neoantigen (SEQ ID NO:226).

[0571] [neoTCR expression]

[0572] Figure 46 shows that T cell gene editing efficiency was strong for 14 neoTCRs in both CD4 and CD8 T cells. In 13 neoTCR T cells, both CD4 and CD8 T cells bound to the cognate comPACT-dextramer complex. However, only CD8 T cells expressing neoTCR against PUM1 (TCR222) bound to the cognate comPACT-dextramer, and no binding of comPACT-dextramer was observed in CD4neoTCR T cells.

[0573] [T cell activation]

[0574] The neoTCR was internalized upon co-culture of neoTCR T cell cognate comPACT-Dextramer and melanoma matched cell line M489 with or without IFNγ pre-incubation (FIG. 47). The decrease in the percentage of comPACT-Dextramer positive T cells indicates neoTCR internalization, a surrogate marker of T cell activation. Cells incubated with RPMI medium alone did not internalize the neoTCR-bound Dextramer complex, whereas T cells incubated with anti-CD3 antibody OKT3 did internalize the neoTCR-bound Dextramer complex. The neo12 antigen was also used as a negative control for each sample.

[0575] NeoTCR T cells from patient PACT135 also expressed the activation markers 4-1BB (Figure 48) and OX40 (Figure 49) after incubation with the M489 cell line with or without IFNγ preincubation. No expression of 4-1BB or OX40 was observed in TCR222 CD4 T cells, as the neoTCR did not bind to its cognate neoantigen.

[0576] Pretreatment of tumor cells with IFNγ to activate the immunoproteasome and enhance HLA expression increased 4-1BB expression in IL8-HLA-A03 TCR (Figure 48). Pretreatment of tumor cells with IFNγ to activate the immunoproteasome and enhance HLA expression increased OX40 expression in IL8-HLA-A03 TCR (Figure 49).

[0577] [T cell cytotoxicity assay]

[0578] All 14 T cell preparations expressing the identified neoTCRs showed specific cytotoxicity against the matched autologous melanoma cell line M489 as determined by cytotoxicity assays. Figure 50 provides a graph of the percentage of tumor cell confluency after co-culture with all neoTCR T cells identified from PACT135 compared to the percentage of tumor cell confluency after mock treatment or incubation with RPMI medium or neo12 TCR T cells. Figures 51A and 51B provide individual graphs of the percentage of tumor cell confluency after co-culture with each neoTCR T cell.

[0579] T cells expressing neoTCR showed potent killing of matched tumor cells at both T cell:tumor cell ratios tested, 1:1 and 5:1 (data not shown). No cytotoxic activity was observed against the mismatched tumor cell line M202 (data not shown). Control samples had 42% nuclear confluence 96 hours after incubation compared to less than 20% nuclear confluence for each sample incubated with a 1:1 ratio of neoTCR T cells (p<0.000001 for each neoTCR T cell sample; Figure 50 and Figure 51A-B). Importantly, while tumor cell numbers decreased, T cell numbers increased, indicating that neoTCR T cells proliferated in response to cognate antigens endogenously expressed by the patient's matched tumor cells (data not shown). After 2 days of co-culture with cognate tumor cells, neoTCR T cells were activated, as indicated by the formation of clusters. The T cells proliferated and covered the entire surface of the well by day 5, with no detectable tumor cells.

[0580] Even neoTCRs with low frequency in PBMC or TIL samples, such as neoTCRs expressed in only one T cell, had strong activity against the patient's matched tumor cell line, resulting in high accuracy and sensitivity of the imPACT technology.

[0581] [T cell cytokine secretion]

[0582] The neoTCR expressing T cells were evaluated for antigen-specific cytokine production. The neoTCR T cells secreted IFNγ, IL-2 and TNFα cytokines after co-culture in the presence of the patient's matched melanoma cell line M489. No cytokine secretion was measured when the neoTCR T cells were co-cultured with the unmatched melanoma cell line M202. Figure 52 shows the secretion of IFNγ, IL2 and TNFα by TCR218 T cells after co-culture in the presence of M489. Figure 53 shows the secretion of IFNγ, IL2 and TNFα by TCR221 T cells and IFNγ by TCR227 T cells after co-culture in the presence of M489. Figure 54 shows the secretion of IFNγ and TNFα by TCR222 T cells after co-culture in the presence of M489. No IL2 was detected at 48 hours. Figure 55 shows IFNγ secretion by TCR219, TCR223, TCR224, TCR225, TCR229, TCR240 and TCR241 T cells after co-culture in the presence of M489 alone (black bars) and after 1 hour pre-treatment of M489 cells with IFNγ (gray bars). TCR220, TCR228 and TCR232 T cells secreted IFNγ after co-culture with M489 cells pre-treated with IFNγ. No IL2 or TNFα was detected at 48 hours.

[0583] Example 21: Validation of neoTCRs isolated from colorectal patient samples using the imPACT method Materials and Methods

[0584] [Preparation of comPACT libraries]

[0585] 144 neoepitopes were predicted in treatment-naive patients with colorectal cancer. As described in Examples 10 and 11, a library of 61 comPACT (neoepitope-HLA complexes) was generated across HLA-A*03:01, A*02:01 and B*07:03.

[0586] [T cell isolation]

[0587] PBMCs collected from a subject (PACT035) were incubated with the comPACT library. Neoantigen-specific T cells were isolated using the imPACT method as described in Examples 10 and 11. Seven neoTCR clonotypes against the COX6C protein were identified.

[0588] [neoTCR gene editing]

[0589] CD4 and CD8 T cells from healthy donors were engineered to express seven COX6C neoantigen-specific TCRs using a CRISPR-based non-viral method as described in International Patent Application WO2019089610, published May 9, 2019, which is incorporated herein by reference in its entirety.

[0590] [COX6C R20Q stable expressing cell line]

[0591] Using PACT precision genome engineering expertise, we generated stable tumor cell lines expressing the COX6C R20Q neo-antigen under the control of endogenous regulatory elements. The colon cancer cell line SW620, which expresses high levels of cell surface HLA-A02, was used to express the neo-antigen. SW620 cells were nucleofected with gRNA / Cas9 and HDR template to generate COX6C R20Q knock-in cell lines. Edited cells were single sorted and expanded. The COX6C locus was sequenced. Sequencing analysis showed that approximately 80% of single cells were highly edited. Four cell lines expressing COX6C R20Q at the endogenous locus were selected: SW620 cells expressing the wild-type COX6C gene, SW620 cells heterozygous for the COX6C-R20Q mutation, and two lines (one shown) of SW620 cells homozygous for the COX6C-R20Q mutation.

[0592] Expression of HLA-A02 in the modified SW620 cell line was confirmed by flow cytometry using BB7.2 anti-HLA*A2 antibody. K562 cell lines, which constitutively express HLA-A*02 and HLA-C*02, were used as positive and negative controls, respectively. SW620 cells were nucleofected with the GFP construct to confirm transfection efficiency.

[0593] [T cell activation]

[0594] Expression of the activation marker Nur77 was also determined in CD4 and CD8 TCR089neoTCR T cells co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation. As a negative control, TCR089neoTCR T cells were also co-cultured with wild-type SW620 cells or cultured alone. Cells were stained for Nur77 using anti-Nur77 mAb (eBiosciences) and Nur77 expression was assessed by flow cytometry.

[0595] [T cell cytotoxicity assay, IncuCyte]

[0596] neoTCR T cell-induced killing of tumor cells over time was also determined via immunofluorescence using an IncuCyte imaging system (Essen BioSciences). T cells expressing each of the seven identified COX6C neoTCR clonotypes were used in this assay. SW620 cells homozygous for the COX6C-R20Q mutation or wild-type SW620 cells were labeled red using NucLight Red lentivirus (Essen). Labeling the tumor cells red allows them to be distinguished from T cells in co-culture and allows tumor cell killing to be monitored over time. 40,000 tumor cells / well were seeded in a 96-well plate and left in the incubator overnight. The next day, neoTCR T cells were added at a 1:1 T cell:tumor cell ratio. Each neoTCR T cell was added to an individual tumor cell sample. RNP (T cells electroporated with ribonucleoprotein (RNP) complexes alone), neo12 TCR T cells, and medium alone were used as negative controls. Co-culture samples were monitored by collecting time-lapse images at 2-h intervals for 5 days using an IncuCyte imaging system with a 10x objective.

[0597] [T cell cytotoxicity assay, flow cytometry]

[0598] TCR089neoTCR T cells were assessed for antigen-specific T cell-mediated killing. 100,000 TCR089neoTCR T cells were co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation (+ / +) or wild-type SW620 at different T cell:tumor cell ratios.

[0599] After 24 h of co-culture of T cells and target cells, cells were stained for 20 min at 4 °C in the dark using a Live / Dead Cell Stain Kit (Live / Dead Near-Infrared Viability Stain for Flow, Cat. No. NC0584313, ThermoFisher). In cells with compromised membranes, the dye reacts with free amines both inside and on the cell surface, resulting in strong fluorescent staining. In live cells, the fluorescence intensity is lower because the reactivity of the dye is restricted to cell surface amines. The difference in intensity is typically more than 50-fold between live and dead cells, allowing easy distinction. After incubation, cells were washed and fixed with eBioscience IC Fixation Buffer (ThermoFisher, Cat. No. 00-8222-49) and analyzed by flow cytometry.

[0600] [Cytokine secretion assay]

[0601] TCR089neoTCR T cells were co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation or heterozygous for the COX6C-R20Q mutation at a T cell:tumor cell ratio of 5:1. As a positive control, T cells expressing TCR089 were co-cultured with SW620WT and pulsed with 1 μM for 1 h. As a negative control, T cells expressing TCR089 were co-cultured with SW620 wild-type cells. After 24 h, supernatants were collected and cytokine production was assessed using a cytokine bead assay (CBA, a bead-based immunoassay from BD BioSciences). CBA is a multiplex bead-based immunoassay application of flow cytometry that allows for simultaneous quantification of multiple proteins by efficiently capturing analytes using antibody-coated beads.

[0602] [result]

[0603] [Identification of neoTCR in PACT035]

[0604] Seven neoTCR clonotypes against the COX6C protein were identified in patient samples using the imPACT T cell isolation method (Figure 56, indicated by arrows). When engineered into CD4 T cells, two of the seven bound neoantigen-HLA complexes and are therefore CD8-independent neoTCRs. COX6C is a subunit of the mitochondrial enzyme cytochrome C oxidase, which is expressed at moderate levels in essentially all tissues. The neoantigen target peptide was residues 18-20 with the R20Q mutation. The neoTCRs that bound to the R20Q COX6C peptide (residues 18-26) were named TCR089, TCR091, TCR092, TCR094, TCR097, TCR098, and TCR099. The neoantigen peptide sequences, alpha and beta TCR CDR3 sequences, and HLA alleles isolated from patient PACT035 are shown in Table 9 below. JPEG0007611836000010.jpg92170

[0605] [Transfection of SW620 cell line]

[0606] Figure 57A shows no HLA*A2 expression in KV1858 cell line (expressing HLA*C2 but not HLA*A2). Figure 57B shows HLA*A2 expression in KV1832 cell line (expressing HLA*A2). Figure 57C shows HLA*A2 expression in SW620 cell line (expressing HLA*A2). Figure 57D shows GFP quantification in nucleofected SW620 cells. Isotype control antibody was used as a negative control.

[0607] [T cell activation]

[0608] Nur77 is an immediate early gene whose expression is rapidly upregulated by TCR signaling. Expression of Nur77 is rapidly upregulated by antigen-TCR signaling. Expression of Nur77 was detected in TCR089neoTCR T cells co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation (Figure 58). No induction of Nur77 was observed when TCR089neoTCR T cells were co-cultured alone or with SW620 cells expressing the WT COX6C protein.

[0609] [T cell cytotoxicity assay, IncuCyte]

[0610] Target cell killing was also measured for each COX6CneoTCR T cell. All neoTCR expressing T cells showed potent killing of SW620 homozygous tumor cells (Figure 59A). No cytotoxic activity was observed against SW620 cells expressing wild-type COX6C protein (Figure 59B). The amount of killing induced by the T cells was dose-dependent and increased with increasing T cell:tumor cell ratios.

[0611] IncuCyte images collected during killing assays using TCR089neoTCR T cells also showed that the number of tumor cells decreased while the number of T cells increased (data not shown), indicating that TCR089 T cells proliferated in response to cognate antigens endogenously expressed by SW620 cells homozygous for the COX6C-R20Q mutation.

[0612] [TCR089 T cell cytotoxicity assay, flow cytometry]

[0613] TCR089 killed SW620 cells homozygous for the COX6C-R20Q mutation, but not wild-type SW620 cells. No cytotoxic activity was observed against SW620 cells expressing wild-type COX6C protein (Figure 60). The amount of T cell-induced killing was dose-dependent and increased with increasing TCR089 T cell:tumor cell ratios.

[0614] [Cytokine secretion assay]

[0615] Strong IFN-secretion was measured in TCR089 T cell samples after co-culture with SW620 cells homozygous for the COX6C-R20Q mutation (Figure 61). Half the amount of IFNγ was measured when TCR089 T cells were co-cultured with SW620 cells heterozygous for the COX6C-R20Q mutation. Similar IFNγ was detected in TCR089 T cells alone, in the absence of tumor cells, or when T cells were co-cultured with wild-type SW620 cells.

[0616] Example 22: Method of treating cancer patients with neoT CRT cells Patients with cancer or other proliferative diseases may require interventional therapy to slow or stop cell proliferation and kill existing cells that may or do harm to the patient (e.g., cause pain, discomfort, or illness). Specifically, the neoTCR T cells disclosed herein may be used to treat cancer.

[0617] As shown in Figure 62, neo-E specific TCRs were isolated from patients treated with PD-1 therapy. RNA and DNA were collected from biopsies and tumor cell lines, and RNAseq and WES (whole exome sequencing) were performed on biopsies and cells. DNA was also collected from the patient's PBMCs for WES using controls. Once tumor antigens were identified using RNAseq and WES, an algorithm was used to select neoepitope candidates for screening using comPACT polypeptides (on which predicted neoepitopes are expressed) and imPACT isolation technology. A barcoded comPACT particle library was constructed and combined with the patient's sample. The comPACT particles were able to bind and capture neoepitope specific T cells.

[0618] Example 23: Example of imPACT isolation technology Based on computational prediction of patient-specific neoEs, we generated hundreds of capture reagents consisting of the patient's HLA class I subtypes loaded with the corresponding predicted neoEs (Peng et al. AACR 2019); we then isolated neoE-specific T cells and sequenced TCR alpha and beta. The isolated neoTCRs were functionally studied by generating primary human T cells expressing the neoTCR using non-viral precision genomic engineering to replace the endogenous TCR (Jacoby et al. AACR 2019, Sennino et al. AACR 2019).

[0619] T cell responses were analyzed in two patients with metastatic melanoma receiving anti-PD-1 therapy. NeoE-specific T cells were isolated at various time points from peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs). Patient PT476 showed a durable response; tumor mutation count (TMB) was 2556; 243 neoE-HLA complexes were generated across three HLA types, HLA-A03:01, A24:01, and C12:03. This resulted in the isolation of 17 TCRs specific for five neoE-HLAs. NeoE-specific T cells were present at baseline in TILs and expanded during treatment in TILs and PBMCs. Patient PT461 showed rapid disease progression with anti-PD-1; TMB was 61; 78 neo-E-HLA complexes were generated covering HLA-A02:01, A03:01, B07:02, C05:01 and C07:02, and 2 TCRs were isolated against 1 neo-E-HLA.

[0620] To further characterize the T cell response, T cells were genetically edited to express 14 different TCRs isolated from patient PT476 specific for neoE of mutated IL8, PUM1 and TPP2 genes. All 14 T cell preparations showed specific cytotoxicity against matched autologous melanoma cell lines established from biopsies of patient PT476 (50-75% tumor growth inhibition compared to growth of melanoma cell lines in coculture with unmatched control TCR in 96-hour assays using P:T 1:1, p<0.000001 for each comparison) and no cytotoxic effect against unmatched control human melanoma cell lines. When cocultured with matched autologous melanoma cell lines, neoE TCR T cells upregulated 4-1BB and OX-40, secreted IFNγ, IL-2 and TNFα, and induced T cell proliferation and degranulation. No response was seen when T cells were cocultured with unmatched targets.

[0621] These results indicate that anti-PD-1 therapy induces focused neoE-specific T cell responses against a limited number of neoEs and that non-viral precision genomic modification can successfully redirect T cells to neoE-expressing tumors, which could be used as an approach for personalized ACT therapy.

[0622] Similarly, in addition to anti-PD-1 therapy, other checkpoint therapies and additional combinations can be used; for example, anti-PD-L1 or anti-CTLA4 therapy can be used.

[0623] Biopsies and PBMCs were collected at multiple time points after anti-PD-1 treatment (Figure 45: PT476 responder, and Figure 63: PT461 non-responder), as partially described in Example 20. TILs and cell lines were established from patient biopsies. Using imPACT isolation technology, neoE-specific T cells were isolated and their evolution was monitored over time. NeoEs derived from non-synonymous mutations were predicted from baseline cell lines using whole exome analysis (WES) and RNAseq, and ranked according to predicted HLA binding affinity, truncality of mutations, and expression levels. NeoE-specific T cells were isolated using HLA-neoE capture reagents for the top-ranked neoEs. Figure 45 shows PT476: 243 neoE-HLA complexes were generated with three HLA types, HLA-A03:01, A24:01, and C12:03, and 17 TCRs specific for five neoE-HLAs were isolated. Figure 63 shows patient PT461: 78 neoE-HLA complexes were generated covering HLA-A02:01, A03:01, B07:02, C05:01, and C07:02, and 2 TCRs were isolated against 1 neoE-HLA.

[0624] Example 24: imPACT isolation technology method example Neo-E specific T cells can be isolated from patient samples as described in Example 22. In this example, the imPACT isolation technique identified 14 neoTCR-T candidates, including 12 IL-8 (HLA-A24:02 and HLA-A03:01) neoTCR-T candidates, 1 PUM1 (HLA-A3:01) neoTCR-T candidate, and 1 TPP2 (HLA-A24:02) neoTCR-T candidate.

[0625] As shown in Figure 62, the imPACT isolation technology method includes three work streams: gene editing, co-culture assays, and cell-based assays.

[0626] Genetic editing: CD8 and CD4 T cells from healthy donors were precisely engineered to express neoTCR. Briefly, neoE-specific TCR sequences were cloned into homologous recombination (HR) DNA templates. These HR templates were used with site-specific nucleases to engineer primary human T cells. The single-step (non-viral) precise genomic engineering seamlessly replaced the endogenous TCR with the patient's neoE-specific TCR (native sequence), whose expression is under endogenous control.

[0627] Co-culture assay: neoTCR-P1 T cells were co-cultured with a melanoma cell line (M489) or a mismatched melanoma tumor cell line derived from a baseline biopsy of the same patient at a 1:1 or 5:1 end product to target (P:T) ratio. Target cell killing was assessed over 6 days using the IncuCyte system. Expression of the proliferation marker Ki67 was assessed by flow cytometry at 48 hours. Expression of activation markers was assessed by flow cytometry at 24 hours. Cytokine secretion was measured in cell supernatants at 48 hours using the BD Cytokine Bead Array (CBA) Human Th1 / Th2 Cytokine Kit II.

[0628] Co-culture assays: Peptide-HLA: recognition / stimulation, target cell killing, proliferation, activation markers, and cytokine secretion assays were performed.

[0629] Example 25: Engineered neoTCR-T cells kill autologous tumor cells As shown in Figure 64, neoTCR-T cells kill autologous melanoma tumor cells. Using time-lapse microscopy of tumor cell death and T cell proliferation, neoTCR-T cells were co-cultured with an autologous melanoma cell line stably expressing red fluorescent protein (nuclear RFP). More specifically, neoTCR was targeted to the IL-8-HLA-A*03:01 neo-antigen on the melanoma cell line and the neoTCR was cultured with the IL-8-HLA-A*03:01 neo-antigen melanoma cell line (top three images in Figure 64) and compared to a negative control (bottom three images in Figure 64). Images shown here were collected at time 0 (left panel), day 2 (middle panel), and day 5 (right panel). Thus, neoTCR is specific for tumor neo-antigen and can effectively kill autologous tumor cells.

[0630] The ability of neoTCR-T cells to kill autologous tumor cells can also be seen in Figure 65A. neoTCR-T cells were co-cultured with autologous (black bars) or mismatched melanoma tumor cells (white bars) and the percentage of Ki67 (proliferation marker) expressing CD8neoTCR T cells was assessed by flow cytometry after 48 hours. *p<0.05 compared to mismatched melanoma tumor cells (t-test with Holm-Sidak method for correction of multiple comparisons). T cells expressing neoTCRneo12 were used as negative control.

[0631] Similarly, neoTCR-T cells were shown to express activation markers upon co-culture with autologous tumor cells. This can be seen in Figure 65B. neoTCR-T cells were co-cultured with autologous (black bars) or mismatched tumor cell lines (white bars) and the percentage of CD8neoTCR T cells expressing the activation marker 4-1BB (top) or CD4neoTCR T cells expressing the activation marker OX40 (bottom bar graph) was assessed by flow cytometry after 24 hours. *p<0.05 compared to mismatched melanoma tumor cells (t-test with Holm-Sidak method for multiple comparison correction). T cells expressing neoTCRneo12 were used as a negative control. To measure upregulation of OX-40 in CD4neoTCR T cells, melanoma cells were pretreated with IFNγ for 24 hours before co-culture with T cells.

[0632] Finally, neoTCR-T cells were shown to secrete interferon-gamma when co-cultured with autologous tumor cells. This can be seen in Figure 65C. neoTCR-T cells were co-cultured with autologous melanoma tumor cells and IFNγ secretion was assessed after 48 hours by flow cytometry (CBA). Mock T cells were used as a negative control. *p<0.05 (t-test using the Holm-Sidak method for correction of multiple comparisons).

[0633] Together, this shows that newly generated neoTCR-T cells expressing TCRs isolated using the imPACT isolation technique upregulated markers of activation and proliferation upon co-culture with autologous tumor cells, and more importantly, all neoTCR-T cells specifically killed patient-derived autologous melanoma cells.

[0634] Example 26: Administration of neoTCR-T cells eradicates tumors in mice and in vitro engineered cell lines Neoantigen-expressing tumors were implanted into the flanks of 15 NOD scid gamma (NSG) mice. Tumors were 95 mm in size. 3When the mice reached the target number, they were divided into two groups: a control group that received PBS (7 mice) and a group that received neoTCR T cells (5*10 6 The treatment group (8 mice) received 100 total T cells / mouse; gene editing efficiency 50%). As shown in Figure 66A, after injection of neoTCR-T cells (see arrows in figure), the tumor size decreased and by day 19 the tumor was completely eradicated. Figure 66B shows the number of human CD8 T cells / mL present in mouse blood 4 days after neoTCR-T cell injection and 35 days after neoTCR-T cell injection. Although NSG mice lack human cytokines, neoTCR-T cells are present in the circulation after tumor eradication, indicating that neoTCR-T cells not only kill target cells but also proliferate and persist.

[0635] Example 27: Design of neoTCR-T cells specific for somatic tumor mutations The subclonal mutational nature of tumor progression in primary tumors and metastases creates problems in the field of oncology because oncology drugs are "individualized" in the sense that they are designed to target proteins, chemicals, or cells that harbor specific mutations (e.g., many small molecule drugs are designed to target tumors based on point mutations). Because tumors often mutate (mutations accumulate during the length of cancer growth and disease) in later stages of primary tumors and metastases, drugs that worked well to shrink tumors and slow tumor progression when the tumor was first detected and treated may lose their effectiveness over time.

[0636] Disclosed herein is the ability to design and generate neoTCR-T cells specific for somatic tumor mutations. Using algorithmic and bioinformatics approaches, we identified tumor-exclusive somatic mutations expressed by all cancer cells in a patient.

[0637] Example 28: neoTCR-T cells address all HLA types of the world's population There are 13,000 HLAs in the human population. Each person has a set of 6 HLAs. As a result, less than 1% of any neo-E-HLA tumor targets are the same between patients (data generated from analyses of 60,000 patients (Hartmaier et al. (2017) Genome Medicine) and 20,000 patients (Schumacher and Schrieber (2015) Science)). Analyses performed predict that the HLA allele catalogs of PBMC matches will have >99% coverage of at least 1 HLA allele, >90% coverage of 4-6 alleles, and >60% coverage of all 6 alleles in potential US clinical trial subjects.

[0638] Example 29: neoTCR therapy can be used against tumors with low, medium and high mutational load The imPACT isolation technique is highly sensitive and can detect neoantigens on tumors with any level of mutational load. For example, neoTCR-T cells can be used to treat tumors with low tumor mutational load, such as prostate and breast cancer, tumors with medium tumor mutational load, such as ovarian and colorectal cancer, and tumors with high tumor mutational load, such as bladder and melanoma cancer.

[0639] Thus, the imPACT isolation technology described herein can be used to design, engineer, and generate neoTCRs for tumors with low, medium, and high tumor mutational load. In certain embodiments, the imPACT isolation technology method can be used to detect neoantigens in tumors with low, medium, and high tumor mutational load. In certain embodiments, the imPACT isolation technology method has been used to detect neoantigens in tumors with low, medium, and high tumor mutational load. In certain embodiments, the imPACT isolation technology method can be used to generate compositions comprising neoTCRs for treating tumors with low, medium, and high tumor mutational load in patients afflicted with such tumors. In certain embodiments, the imPACT isolation technology method can be used to cause a population of neoTCRs to treat tumors with low, medium, and high tumor mutational load in patients afflicted with such tumors.

[0640] Example 30: Methods of treating patients with neoTCR-T cell therapy The first step in treating a patient with neoTCR-T cell therapy is to screen the patient. Once screening and biopsy are performed, the patient can be enrolled and leukapheresis is performed. During the time of production of neoTCR T cells (patient-specific) described herein, including the creation of the comPACT library, screening imPACT isolation technology, and editing of T cells expressing neoTCR, the patient can optionally be enrolled in a bridging therapy (e.g., standard of care including first-line, second-line, third-line, and subsequent treatments for a particular cancer indication). Such a bridging therapy can be prescribed and administered for between 0 and 60 days, with an average of 21 to 42 days. Following such optional bridging therapy, the patient can be prescribed a conditioning chemotherapy. This conditioning chemotherapy can be given 5, 4, and 3 days prior to administration of the neoTCR T cell therapy. On the day of administration, the patient receives an infusion of neoTCR. The tumor is then evaluated.

[0641] Example 31: Compositions and methods for treating non-cancer diseases and disorders using neoTCR T cells Diseases other than cancer, including but not limited to, can be treated with neoTCR T cell therapy. In particular, any disease or disorder that causes the affected cell population to produce disease / disorder-specific neoantigens can be treated with neoTCR T cell therapy. Such cells include viral, fungal, or bacterial infected cells that, as a result of the infection, present infection-specific neoantigens detectable by neoTCR T cells. Cells associated with inflammatory or autoimmune diseases may also present disease-specific neoantigens for which neoTCR T cells are produced. For example, if a patient suffers from an allergy or inflammatory disease, neoTCRs can be generated against neoantigens specific to ligands of inflammatory cytokines presented by inflammatory cells.

[0642] Example 32: Imaging methods using neoTCR T cells Once the comPACT and imPACT isolation technology methods have been applied to patient tumor samples, the neoTCR T cells can be used to treat disease as described herein, and can also be used to image, detect, and / or monitor tumor burden, progression, remission, and eradication by labeling the neoTCR T cells with a detectable label (e.g., any label that can be imaged, such as a dye or zirconium label; see, e.g., U.S. Patent No. 8,771,966, incorporated herein by reference in its entirety).

[0643] For example, neoTCR T cells can be genetically modified to express a dye or fluorescent protein. Such labeled neoTCR T cells can be used to determine the efficacy of neoTCR T cell therapy in eradicating tumors; here, if labeled neoTCR T cells can be imaged proliferating and spreading, it can be inferred that the neoTCR T cell therapy is effective because the cells differentiate into T effector cells upon encountering the target antigen.

[0644] For example, neoTCR T cells can be labeled with an agent such as zirconium 89. Such labeled neoTCR T cells can be used to determine the presence of tumors (before, during, or after) neoTCR T cell therapy based on the interaction, or lack thereof, between the neoTCR T cells and tumor cells (if present).

[0645] Cells other than tumor cells that present neo-antigens that allow for the generation of neoTCR T cells can also be imaged, including, but not limited to, those described in Example 30. For example, if an inflammatory disease is treated by designing and administering neoTCR T cell therapy using the methods described herein such that an inflammatory cell specific neoepitope (e.g., a neoepitope on a ligand of an inflammatory cytokine that causes inflammation) is found and neoTCR T cells are designed and generated therefrom, the same neoTCR T cells can be labeled with an imaging agent to later determine whether the ligand-presenting cells are still present or whether the neoTCR has effectively eradicated or sufficiently reduced such cell populations to improve the patient's condition.

[0646] Example 33: Methods for determining the efficacy of neoTCR-T cell therapy and for adjusting the dosing of neoTCR-T cell therapy When a neoTCR-T cell therapy is administered to a patient, efficacy can be monitored using imaging methods known in the art. For example, a patient can be infused with a neoTCR-T cell therapy as described herein, followed by administration of a tumor tracer, and imaged 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after administration of such tracer. In certain embodiments, the tracer can be administered the same week as the neoTCR-T cell therapy, the week following neoTCR-T cell therapy, two weeks after neoTCR-T cell therapy, three weeks after neoTCR-T cell therapy, or one or more months after neoTCR-T cell therapy.

[0647] In certain embodiments, if the tumor does not progress but does not shrink (visualized using imaging) after neoTCR-T cell therapy, an additional dose of neoTCR-T cell therapy may be given. In certain embodiments, if the tumor does not shrink and possibly increases in size (visualized using imaging) after neoTCR-T cell therapy, an additional dose of neoTCR-T cell therapy may be given.

[0648] In certain embodiments, tracers and imaging are used every 3-6 months after neoTCR-T cell therapy to monitor the status of the disease. In certain embodiments, tracers and imaging are used every 6-12 months after neoTCR-T cell therapy to monitor the status of the disease.

[0649] Example 34: Methods of Treating Patients Using neoT CRT Cells Candidate neoepitopes are synthesized and cloned into comPACT polynucleotides as described in Examples 1-9. Briefly, polynucleotide sequences encoding candidate antigenic peptides are inserted into the MHC template described in Figures 1-5. Mammalian cells are seeded and transfected with comPACT polynucleotides containing the candidate antigenic peptide sequences. The transfected cells express and secrete comPACT polypeptides into the cell culture medium. Conditioned medium is collected from the cells, and the comPACT polypeptides are purified by size-exclusion chromatography. The purified comPACT polypeptides are then assembled into multimeric particles (e.g., tetramers, dextramers, NTAmers) that contain multiple copies of the comPACT polypeptide, a DNA barcode, and a fluorophore (e.g., APC or PE). One advantage of this approach is that multiple candidate neoepitopes can be produced and screened simultaneously in a high-throughput manner. Furthermore, this process can be automated. Two different types of particles, the first containing APC and the second containing PE as fluorophore, are combined to obtain a particle set capable of recognizing patient-specific antigenic peptides.

[0650] To identify neoTCR T cells and their TCR sequences, freshly isolated or cryopreserved T cells from patients are stained with multimer particles and a set of antibodies for phenotypic characterization. Viable and barcoded T cells are single-cell sorted, and their DNA and RNA are extracted and analyzed by next-generation sequencing. Sequencing data obtained from comPACT-positive T cells are analyzed to identify and validate predicted antigen peptides, neoTCR T cells, and validated neoepitope TCR candidates and their sequences, as described in Examples 11-13.

[0651] The identified neoepitope TCR sequence is cloned into a homology-directed recombination (HDR) template for genome editing in T cells. Further details of the template sequence and structure can be found in International Patent Application No. PCT / US2018 / 058230, the contents of which are incorporated herein by reference. Freshly collected or previously cryopreserved T cells of the patient are engineered for TCR gene disruption and HDR template incorporation using non-viral methods. Endogenous TCR loci can be disrupted using a CRISPR / Cas9 approach involving gRNAs of the endogenous loci of the TCR-α and TCR-β gene sequences. The HDR template is recombined with one of the endogenous disrupted TCR gene sequences to introduce the identified neoepitope TCR. Thus, the modified T cells lack expression of the endogenous TCR and express the neoepitope TCR. These neoTCR T cells are then adoptively transferred into patients to specifically target tumor cells expressing the neoantigen.

[0652] Compared to other adoptive cell transfer methods, this process has important advantages, including but not limited to: i) it is flexible, as it allows personalized targeting of tumor-exclusive mutations presented in the context of patient-specific HLA; ii) it provides a clinical tool to attack cancer cells expressing neo-antigens that are not expressed on the cell surface; iii) it works regardless of patient ethnicity or cancer type; and iv) multiple steps can be automated, resulting in a small manufacturing footprint.

[0653] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[0654] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. 1. An in vitro method for identifying antigen specificity of a T cell, comprising: a. contacting a sample with a plurality of distinct sets of particles, i. each particle comprises a polypeptide comprising a unique antigen peptide, a β2M peptide, and an MHC peptide, an operably associated barcode, and at least one identification label; ii. the sample contains T cells; and iii. The contacting step includes providing conditions suitable for a single T cell to bind to a unique antigenic peptide of at least one of the particle sets; b. isolating one or more T cells bound to the particles; c. identifying the barcode of the particle bound to the isolated T cell; d. determining the ratio of the most predominant barcode identified in c. to the second most predominant barcode; and e. Determining the antigen specificity of the T cells based on the ratio of the most predominant barcode to the second most predominant barcode. A method comprising:

2. 2. The method of claim 1, wherein the ratio is calculated by dividing the copy number of the most predominant barcode by the copy number of the second most predominant barcode.

3. The method of claim 1 or 2, wherein the unique antigenic peptide is the same for each distinct set of particles.

4. 4. The method of claim 1, wherein each distinct particle set comprises at least one or more barcodes, each barcode being associated with the identity of an antigenic peptide.

5. 5. The method of claim 1, wherein the ratio of each barcode corresponds to the antigen specificity of the isolated T cells.

6. A method described in any one of claims 1 to 5, wherein the isolated T cells are identified as antigen-specific T cells when the ratio of the most predominant barcode to the second most predominant barcode exceeds a threshold value.

7. The method of claim 6 , wherein the threshold value is at least two or greater than two.

8. The method of claim 1 , wherein identifying the barcode comprises a nucleotide-based assay.

9. The method of claim 8, wherein the nucleotide-based assay is a PCR, RT-PCR, sequencing, or hybridization assay.

10. 10. The method of claim 8 or 9, wherein the nucleotide-based assay determines (a) the sequence of each barcode and / or (b) the copy number of each barcode.

11. 11. The method of claim 1, further comprising obtaining T cell receptor (TCR) CDR sequences.

12. 12. The method of claim 1, further comprising obtaining a TCR gene sequence.

13. The method of claim 12, wherein the TCR sequence is a TCR alpha chain sequence.

14. 13. The method of claim 12, wherein the TCR sequence is a TCR beta chain sequence.

15. 15. The method of claim 1, wherein the antigen specificity of the T cell comprises the sequence of the antigenic peptide and the TCR sequence of the bound T cell.

16. 16. The method of claim 1, wherein at least one identification label is the same in each distinct set of particles.

17. 17. The method of claim 1, comprising at least two different identification marks.

18. 18. The method of claim 1, wherein at least one identifying label is a fluorophore.

19. 19. The method of claim 18, wherein the fluorophore is selected from the group comprising allophycocyanin (APC) and phycoerythrin (PE).

20. 18. The method of claim 17, wherein the at least two different identifying labels are fluorophores, and the fluorophores are selected from the group comprising allophycocyanin (APC) and phycoerythrin (PE).

21. 21. The method of any one of claims 1 to 20, wherein the antigenic peptide is selected from the group consisting of tumor antigens, neoantigens, tumor neoantigens, viral antigens, bacterial antigens, phosphoantigens, and microbial antigens.

22. 22. The method of claim 21, wherein the neoantigen is identified from tumor sequencing data obtained using a sample from the subject.

23. 23. The method of claim 22, wherein neoantigens are determined using an in silico prediction algorithm.

24. 24. The method of claim 23, wherein the predictive algorithm further comprises an MHC binding algorithm for predicting binding between a neo-antigen and an MHC peptide.

25. 25. The method of any one of claims 1 to 24, wherein the sample is selected from a blood sample, a bone marrow sample, a tissue sample, a tumor sample, or a peripheral blood mononuclear cell (PBMC) sample.

26. 26. The method of any one of claims 1 to 25, wherein the T cell is a human T cell.

27. 27. The method of claim 26, wherein the T cells are CD8+ T cells.

28. 28. The method of claim 1 , comprising a library of distinct particle sets.

29. 30. The method of claim 28, wherein the library comprises between 2 and 500 distinct particle sets.

30. 30. The method of claim 1, wherein each particle comprises an MHC peptide.

31. 31. The method of claim 30, wherein the MHC peptide is a human MHC peptide.

32. 31. The method of claim 30, wherein the MHC peptide is a class I HLA peptide.

33. The method of claim 32, wherein the class I HLA peptide comprises an HLA-A, HLA-B, or HLA-C peptide.

34. The HLA peptides are HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33: 01, HLA-A*68:01, HLA-A*11:01, HLA-A*23:01, HLA-A*30:01, HLA-A*33:03, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA -A*68:02, HLA-B*07:02, HLA-B*14:02, HLA-B*18:01, HLA-B*27:02, HLA-B*39:01, HLA-B*40:01, HLA-B*44:02, HLA-B*46: 01, HLA-B*50:01, HLA-B*57:01, HLA-B*58:01, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*35:01, HLA-B*40:02, HLA- B*42:01, HLA-B*44:03, HLA-B*51:01, HLA-B*53:01, HLA-B*13:02, HLA-B*15:07, HLA-B*27:05, HLA-B*35:03, HLA-B*37:0 1, HLA-B*38:01, HLA-B*41:02, HLA-B*44:05, HLA-B*49:01, HLA-B*52:01, HLA-B*55:01, HLA-C*02:02, HLA-C*03:04, HLA- C*05:01, HLA-C*07:01, HLA-C*01:02, HLA-C*04:01, HLA-C*06:02, HLA-C*07:02, HLA-C*16:01, HLA-C*03:03, HLA-C*07:0 34. The method of claim 33, comprising 4. HLA-C*08:01, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, or HLA-C*17:

01.

35. 35. The method of any one of claims 1 to 34, wherein each particle comprises an HLA peptide and a β2M peptide.

36. The method of claim 35, wherein the β2M peptide is a human β2M peptide.

37. The method of claim 36 , wherein the β2M peptide comprises a mutation.

38. 38. The method of claim 37, wherein the mutation is S88C.

39. 39. The method of any one of claims 1 to 38, wherein each particle comprises a polypeptide comprising, from the amino terminus to the carboxyl terminus, (i) an antigenic peptide, (ii) a β2M peptide, and (iii) an MHC peptide.

40. 40. The method of any one of claims 1 to 39, wherein the antigenic peptide is 7-15 amino acids, 7-10, 8-9, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

41. 41. The method of claim 39 or 40, wherein the polypeptide is biotinylated.

42. 42. The method of any one of claims 1 to 41, wherein the particles are selected from the group consisting of magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles.

43. 43. The method of any one of claims 1 to 42, wherein the particles are coated with streptavidin.

44. 44. A method according to any one of claims 1 to 43 for monitoring the immune repertoire in a sample obtained from a subject.

45. 45. The method of claim 44, further comprising monitoring changes in antigen-specific T cells in a sample obtained from the subject.

46. The method described in claim 44 or 45, wherein the monitoring is performed prior to administering immunotherapy to the subject.

47. 47. The method of claim 46, wherein the immunotherapy is adoptive cell transfer or a checkpoint inhibitor.

48. 48. A method according to any one of claims 1 to 47 for identifying at least one TCR sequence.

49. 49. The method of claim 48, wherein at least one TCR sequence is a TCR alpha sequence, a TCR beta sequence, or a combination thereof.

50. 50. The method of claim 48 or 49, further comprising producing a soluble TCR polypeptide.

51. 1. A method of producing an altered T cell population for treating cancer in a subject, comprising: a. preparing a plurality of particles each comprising a plurality of labeled polypeptides, the polypeptides comprising an antigenic peptide, a β2M sequence, an HLA sequence, and a detectable label; b. contacting the plurality of particles with a plurality of T cells of a sample obtained from the subject under conditions suitable for antigen-specific binding of the T cells to the particles; c. isolating the particle-bound T cells and identifying the TCR gene sequences of the isolated T cells by the method of any one of claims 1 to 50; d. Preparing a polynucleotide comprising homology arms and at least one TCR gene sequence, wherein the TCR gene sequence is located between the homology arms; e. Recombining the polynucleotide into an endogenous locus of a T cell of the subject; and f. Culturing the modified T cells to produce a population of T cells. A method comprising:

52. An in vitro method for modifying an isolated cell, comprising: a. In isolated cells i. first and second homologous recombination (HR) arms that are homologous to first and second endogenous sequences of the isolated cell; ii. a T cell receptor (TCR) gene sequence obtained by the method of any one of claims 1 to 50, the TCR gene sequence being located between a first HR arm and a second HR arm; and iii. a first 2A coding sequence located upstream of the TCR gene sequence and a second 2A coding sequence located downstream of the TCR gene sequence, the first and second 2A coding sequences encoding the same amino acid sequence but with different codons; introducing a HR template nucleic acid sequence comprising: b. Recombining the HR template nucleic acid into an endogenous locus of the isolated cell that contains first and second endogenous sequences that are homologous to the first and second HR arms of the HR template nucleic acid. A method comprising:

53. 53. A method of producing a composition comprising a modified cell, comprising obtaining a modified cell by the method of claim 52, wherein the modified cell comprises an exogenous nucleic acid sequence integrated into an endogenous locus, the exogenous nucleic acid sequence comprising: a. a TCR gene sequence identified by the method of any one of claims 1 to 50, and b. a first 2A coding sequence located upstream of the TCR gene sequence and a second 2A coding sequence located downstream of the TCR gene sequence, the first and second 2A coding sequences coding for the same amino acid sequence but with different codons; A method comprising:

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