Compositions and methods for identifying antigen-specific T cells

Abstract

Disclosed herein are antigenic peptide-MHC complexes, referred to as compPACT polypeptides and compPACT polynucleotides, and methods of producing such complexes. Also disclosed herein are methods of producing libraries of compPACT polynucleotides and polypeptides, and exemplary uses thereof in capturing cancer neo-epitope reactive T cells with high accuracy. Dual particle detection methods for detecting neo-antigen-specific T cells with improved sensitivity and specificity are provided. Also provided are signal-to-noise ratio analysis of isolated T cells for detecting neo-antigen-specific T cells with improved T cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 804,649 filed on February 12, 2019, U.S. Provisional Application No. 62 / 826,823 filed on March 29, 2019, U.S. Provisional Application No. 62 / 876,380 filed on July 19, 2019, and U.S. Provisional Application No. 62 / 867,165 filed on June 26, 2019, the contents of which are incorporated by reference in their entireties and claim priority.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, generated on January 21, 2020, is named 087520_0125_SL.txt and is 292,725 bytes in size. Background Art

[0005] T cells are the primary mediators of adaptive immunity. Guided by the specificity of each T cell's unique T cell receptor (TCR), T cells regulate autoimmunity, help activate 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. The identification of relevant peptide-MHC complex ligands plays a role in understanding immune responses to tumors and pathogens. MHC complex ligands are also valuable for understanding responses to self and dietary antigens. This understanding enables clinically beneficial immunotherapies (e.g., TCR gene transfer and vaccines) that initiate, amplify, or attenuate immune responses to target antigens.

[0006] Mutated “neo-epitopes” are important targets for both endogenous and engineered immune responses against cancer. Neo-epitope-reactive tumor-infiltrating leukocytes (TILs) are present in the endogenous pool and regress tumors following adoptive transfer. Similarly, tumor mutational burden predicts the clinical effectiveness of CTLA-4 or PD-1 blockade, suggesting that these checkpoint inhibition strategies affect tumor regression by unleashing neo-epitope-reactive T cells. Because neo-epitopes arise from somatic mutations in tumor cells, they are not typically presented by thymic epithelial cells to induce central tolerance. Therefore, T cell responses against these neo-epitopes are tumor-specific, can be of high affinity, and are patient-specific (i.e., private). From a clinical perspective, this presents both an opportunity and a challenge: neo-epitopes are excellent targets for immunotherapy, but TCR isolation methods should have sufficiently high throughput to enable therapeutic applications at a clinically useful scale.

[0007] There is an unmet need for rapid and robust TCR ligand discovery technologies for basic and translational research. Peptide-MHC multimers enable the sorting of T cells based on the antigen specificity of the TCR, an important step in isolating tumor-specific TCRs for gene therapy. The current typical peptide-MHC production protocol begins with the solid-phase synthesis of the peptide ligand of interest. Simultaneously, generic β2-microglobulin and related MHC class I molecules are heterologously expressed in Escherichia coli, generating misfolded inclusion bodies. Each peptide is added to a refolding reaction containing β2-microglobulin and related MHC class I molecules. Finally, the correctly refolded ternary complex fraction can be purified and formulated for peptide-MHC multimer production. To facilitate the parallel production of a specific MHC molecule with many different peptide ligands, Schumacher and colleagues designed a photocleavable peptide that binds a specific MHC molecule as a conditional ligand. A single refolding reaction is performed to generate an MHC molecule bound to its conditional ligand. Upon exposure to UV light, the conditional ligand is cleaved and exchanged for the desired peptide present in excess. Many such exchange reactions can be performed in parallel, enabling the construction of a pMHC library for that specific MHC allele. Even so, this state-of-the-art technology has challenging limitations. First, the production, purification, and refolding of MHC molecules expressed in E. coli inclusion bodies are laborious, and the yield of correctly folded peptide-MHC complexes is low. Second, the turnaround time (weeks) for commercial peptide synthesis is inconsistent with the optimal timescale for personalized on-demand TCR gene therapy targeting patient-specific new epitopes. Third, many predicted ligands cannot be used to screen T cells by this method because the biophysical properties of the peptide (e.g., hydrophobicity) prevent its synthesis or exchange. Fourth, the exchange efficiency is often poor (<50% for most predicted HLA-binding peptides). The resulting mixture of correctly folded exchanged MHC and misfolded unliganded MHC results in multimeric staining with a low signal-to-noise ratio, a problem that is exacerbated when T cells are screened with multiplexed pools of peptide-MHC reagents. Fifth, designing and validating conditional ligands for each new MHC allele is a laborious and unreliable task. Since the MHC locus is the most multi-allelic locus in the human genome, this is a major obstacle to implementing new epitope-targeted gene therapy in patients with different MHC haplotypes. Taken together, these limitations emphasize the need for new technologies in this field. Various compositions and methods for generating peptide-MHC multimers that address these limitations are disclosed herein. SUMMARY OF THE INVENTION

[0009] The present disclosure provides compositions and methods for identifying new epitopes, identifying and isolating T cell receptors, engineering primary cells to express specific T cell receptors, expanding engineered T cells, and treating disorders using cell therapy. In various embodiments, the present invention provides improved cell therapies and compositions for identifying new epitopes, identifying and isolating T cell receptors, engineering primary cells to express specific T cell receptors, expanding engineered T cells, and treating proliferative diseases, disorders, and conditions.

[0010] In one aspect, the present invention provides a method for identifying the antigen specificity of a T cell, comprising providing two or more different particle collections, each different particle collection comprising a unique antigenic peptide and at least one defined barcode operably associated with the identity of the antigenic peptide, and each collection comprising a first particle comprising a first identification marker and a second particle comprising a second identification marker different from the first identification marker; providing a sample known or suspected to contain one or more T cells; contacting the sample with the two or more particle collections, wherein the contacting comprises providing conditions sufficient for a single T cell to bind to a unique antigen of at least one particle collection; separating the one or more T cells bound to the particle collection by their associated first and second identification markers; performing an assay to identify the one or more barcodes associated with the particle collection bound to the separated T cells; determining a ratio of the barcodes bound to the separated T cells, wherein the ratio is calculated by identifying the first copy number of the primary barcode and the second copy number of the different 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.

[0011] In one aspect, provided herein is a method for identifying the antigen specificity of a T cell, comprising: obtaining or having obtained at least one antigen-specific T cell that is bound to two or more different sets of particles, each different set of particles comprising a unique antigenic peptide and at least one defined barcode operably associated with the identity of the antigenic peptide, wherein each set comprises a first particle comprising a first identification marker and a second particle comprising a second identification marker different from the first identification marker; performing or having performed at least one assay to identify one or more barcodes detectably bound to the set of particles bound to the T cell; and determining or having determined the ratio of barcodes bound to the T cell that identifies the antigen specificity of the T cell, wherein the ratio is calculated by identifying the first copy number of a primary barcode and the second copy number of a different barcode and dividing the first copy number by the second copy number.

[0012] In one aspect, provided herein is a method for identifying the antigen specificity of a T cell, comprising: obtaining or having obtained a dataset comprising data associated with one or more barcodes detectably bound to a collection of particles directly or indirectly bound to a T cell, wherein the one or more barcodes are each operably associated with a unique antigenic peptide; and determining or having determined a ratio of barcodes bound to a T cell identifying the antigen specificity of the T cell, wherein the ratio is calculated by identifying a first copy number of a primary barcode and a second copy number of a different barcode and dividing the first copy number by the second copy number.

[0013] In some embodiments, a data set includes one or more barcodes and one or more barcode copy numbers.

[0014] In some embodiments, the unique antigenic peptide is the same for each different collection of particles.

[0015] In some embodiments, the first particle comprises a first barcode and the second particle comprises a second barcode different from the first barcode, wherein the first and second barcodes are associated with the identity of the antigen.

[0016] In some embodiments, the ratio of the barcodes corresponds to the antigen specificity of the isolated T cells.

[0017] In some embodiments, if the ratio of the barcodes is above a threshold, the isolated T cell is identified as an antigen-specific T cell.

[0018] In some embodiments, the threshold is at least 2 or greater.

[0019] In some embodiments, the threshold is at least 5 or greater.

[0020] In some embodiments, the threshold value is at least 10 or greater.

[0021] In some embodiments, the threshold is between 2 and 5.

[0022] In some embodiments, the threshold is between 5 and 10.

[0023] In some embodiments, the threshold value is at least or greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-5, 3-6, 4-7, 5-8, 5-10, 7-10, or greater than 10.

[0024] In some embodiments, the assay is a nucleotide-based assay.

[0025] In some embodiments, the nucleotide-based assay is a PCR assay, a RT-PCR assay, a sequencing assay, or a hybridization assay.

[0026] In some embodiments, the assay determines the sequence of one or more barcodes.

[0027] In some embodiments, the assay determines the sequence and copy number of one or more barcodes.

[0028] In some embodiments, the method further comprises obtaining a T cell receptor (TCR) CDR sequence.

[0029] In some embodiments, the method further comprises obtaining TCR alpha and beta chain sequences.

[0030] In some embodiments, the antigen specificity of a T cell comprises each of (a) the sequence of the antigenic peptide and (b) the TCR sequence of the bound T cell.

[0031] In some embodiments, the first identification label of each first particle is the same in each set.

[0032] In some embodiments, the second identification label of each second particle is the same in each set.

[0033] In some embodiments, the first identification label of each first particle is the same in each set, and wherein the second identification label of each second particle is the same in each set.

[0034] In some embodiments, the first and second identification labels are fluorophores.

[0035] In some embodiments, the first fluorophore is allophycocyanin (APC).

[0036] In some embodiments, the second fluorophore is phycoerythrin (PE).

[0037] In some embodiments, the collection of particles comprises a third particle comprising a third barcode that is different from the first and second barcodes, wherein the first, second, and third barcodes are associated with the identity of the antigen.

[0038] In some embodiments, the unique antigenic peptide is selected from the group consisting of a tumor antigenic peptide, a neoantigenic peptide, a tumor neoantigenic peptide, a viral antigenic peptide, a bacterial antigenic peptide, a phosphoantigenic peptide, and a microbial antigenic peptide.

[0039] In some embodiments, the unique antigenic peptide is a neoantigenic peptide.

[0040] In some embodiments, where the neoantigens are derived from tumor sequencing data of a subject, the tumor sequencing data is used to identify one or more somatic mutations relative to wild-type present in the data.

[0041] In some embodiments, neoantigens are identified using computer prediction algorithms.

[0042] In some embodiments, the prediction algorithm further comprises an MHC binding algorithm to predict binding between neoantigens and MHC peptides.

[0043] 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.

[0044] In some embodiments, the sample is a PBMC sample.

[0045] In some embodiments, the sample is a tumor sample.

[0046] In some embodiments, the sample is a bone marrow sample.

[0047] In some embodiments, the T cells are human T cells.

[0048] In some embodiments, the T cells are CD8+ T cells.

[0049] In some embodiments, the CD8+ T cells are human CD8+ T cells.

[0050] In some embodiments, the method comprises a library of different particle collections.

[0051] In some embodiments, the library includes between 2 and 500 different collections of particles.

[0052] In some embodiments, the library includes at least 60 different collections of particles.

[0053] In some embodiments, each particle comprises an MHC peptide.

[0054] In some embodiments, the MHC peptide is a mammalian MHC peptide.

[0055] In some embodiments, the MHC peptide is a human MHC peptide.

[0056] In some embodiments, the MHC peptide is a class I HLA peptide.

[0057] In some embodiments, the HLA peptide comprises an HLA-A, HLA-B, or HLA-C peptide.

[0058] In some embodiments, the HLA peptides comprise 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, HL A-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*2 9:02,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*35:0 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 :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.

[0059] In some embodiments, the HLA peptide comprises a Y84A or Y84C mutation.

[0060] In some embodiments, each particle comprises a HLA I peptide and a β2M peptide.

[0061] In some embodiments, the β2M peptide is a mammalian β2M peptide.

[0062] In some embodiments, the β2M peptide is a human β2M peptide.

[0063] In some embodiments, the β2M peptide comprises a mutation that allows or increases binding to a thiol dye.

[0064] In some embodiments, the mutation is S88C.

[0065] In some embodiments, each particle comprises a polypeptide comprising, in the amino to carboxyl terminal direction, (i) an antigenic peptide, (ii) a β2M peptide, and (iii) an MHC peptide.

[0066] In some embodiments, the polypeptide further comprises a first universal target peptide preceding the antigenic peptide, and a second universal target peptide different from the first universal target peptide between the antigenic peptide and the β2M peptide.

[0067] In some embodiments, each particle comprises a polypeptide comprising, in the amino to carboxyl terminal direction, (i) a first universal target peptide, (ii) an antigenic peptide, (iii) a second universal target peptide different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide.

[0068] 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.

[0069] In some embodiments, the polypeptide comprising the unique antigenic peptide is biotinylated.

[0070] In some embodiments, each particle in the distinct collection of particles comprises a streptavidin core and at least one copy of a unique antigenic peptide.

[0071] In some embodiments, the particle comprises one, two, three, or four copies of a unique antigenic peptide.

[0072] In one aspect, provided herein is a library comprising two or more different particle collections, each particle collection comprising a unique antigenic peptide and a defined barcode operably associated with the identity of the antigenic peptide, wherein each collection comprises a first particle comprising a first identification marker and a second particle comprising a second identification marker different from the first identification marker.

[0073] In some embodiments, the identifying label is a fluorophore.

[0074] In some embodiments, the fluorophore is APC or PE.

[0075] In one aspect, provided herein is a particle comprising a tetrameric solid support bound to a unique barcode and three or fewer attached polypeptide molecules comprising, in the amino 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.

[0076] In some embodiments, the polypeptide further comprises a first universal target peptide preceding the antigenic peptide, and a second universal target peptide different from the first universal target peptide between the antigenic peptide and the β2M peptide.

[0077] In some embodiments, the solid support is a streptavidin core.

[0078] In some embodiments, the polypeptide molecule is biotinylated.

[0079] In some embodiments, the polypeptide molecule is bound to the streptavidin core via a biotin-streptavidin interaction.

[0080] In some embodiments, the particle further comprises an identification label.

[0081] In some embodiments, the identifying label is a fluorophore.

[0082] In some embodiments, the fluorophore is APC or PE.

[0083] In one aspect, provided herein are libraries comprising particles, wherein the library comprises two or more different particles, wherein each different particle comprises a unique antigenic peptide.

[0084] In one aspect, the present invention provides a method for monitoring the immune repertoire of a subject, comprising: providing two or more different particle collections, each different particle collection comprising a unique antigenic peptide and at least one defined barcode operably associated with the identity of the antigenic peptide, and each collection comprising a first particle comprising a first identification marker and a second particle comprising a second identification marker different from the first identification marker; providing a sample known or suspected to contain one or more T cells, wherein the sample is obtained from the subject over time; contacting the sample with the two or more particle collections, wherein the contacting comprises providing conditions sufficient for a single T cell to bind to a unique antigen of at least one particle collection; isolating one or more T cells associated with the first and second identification markers; 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 identifying a first copy number of the primary barcode from step (e) and a second copy number of a different barcode and dividing the first copy number by the second copy number; identifying the 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.

[0085] In some embodiments, the first and second identification labels are fluorophores.

[0086] In some embodiments, the fluorophore is APC or PE.

[0087] In one aspect, the present invention provides a method for monitoring the immune repertoire in a subject, comprising obtaining or having obtained a dataset comprising data associated with one or more barcodes that are detectably bound directly or indirectly to T cells, wherein the one or more barcodes are each operably associated with a unique antigenic peptide; determining or having determined a ratio of barcodes that bind to T cells that identify T cell antigen specificity, wherein the ratio is calculated by identifying a first copy number of the primary barcode from step (a) and a second copy number of a different 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 cells; and monitoring changes in the antigen-specific T cells identified by the method in the subject.

[0088] In some embodiments, a data set includes one or more barcodes and one or more barcode copy numbers.

[0089] In some embodiments, the unique antigenic peptide is the same for each different collection of particles.

[0090] In some embodiments, monitoring changes in T cells comprises administering a soluble, labeled antigen-specific TCR to the subject.

[0091] In some embodiments, immunotherapy is administered to the subject based on the identified changes in antigen-specific T cells.

[0092] In some embodiments, the immunotherapy is a checkpoint inhibitor.

[0093] 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).

[0094] In one aspect, the present invention provides a method for identifying an antigen, comprising providing two or more different sets of particles, each different set of particles comprising a unique antigenic peptide and at least one defined barcode operably associated with the identity of the antigenic peptide, and each set comprising a first particle comprising a first identification marker and a second particle comprising a second identification marker different from the first identification marker; providing a sample known or suspected to contain one or more T cells; contacting the sample with the two or more sets of particles, wherein the contacting comprises providing conditions sufficient for a single T cell to bind to a unique antigen of at least one set of particles; isolating one or more T cells associated with the first and second identification markers; 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 identifying a first copy number of the primary barcode and a second copy number of the different barcode from step (e) 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 cell.

[0095] In some embodiments, the first and second identification labels are fluorophores.

[0096] In some embodiments, the fluorophore is APC or PE.

[0097] In one aspect, provided herein is a method of identifying an antigen, comprising obtaining or having obtained a dataset comprising data associated with one or more barcodes that are detectably bound directly or indirectly to T cells, wherein each of the one or more barcodes is operably associated with a unique antigenic peptide; determining or having determined a ratio of barcodes bound to T cells, wherein the ratio is calculated by identifying a first copy number of a primary barcode and a second copy number of a different barcode from step (a) and dividing the first copy number by the second copy number; and identifying the sequence of a unique antigenic peptide bound to an antigen-specific T cell.

[0098] In some embodiments, step (a) comprises obtaining a T cell-based sample and performing an assay thereon to obtain a dataset.

[0099] In some embodiments, a data set includes one or more barcodes and one or more barcode copy numbers.

[0100] In some embodiments, the unique antigenic peptide is the same for each different collection of particles.

[0101] In one aspect, the present invention provides a method for identifying HLA and peptide complexes, comprising providing two or more different sets of particles, each different set of particles comprising a unique antigenic peptide and at least one defined barcode operably associated with the identity of the antigenic peptide, and each set comprising a first particle comprising a first identification marker and a second particle comprising a second identification marker different from the first identification marker; providing a sample known or suspected to contain one or more T cells; contacting the sample with the two or more sets of particles, wherein the contacting comprises providing conditions sufficient for a single T cell to bind to a unique antigen of at least one set of particles; isolating one or more T cells associated with the first and second identification markers; 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 identifying a first copy number of the primary barcode and a second copy number of the different barcode from step (e) and dividing the first copy number by the second copy number; and identifying HLA and peptide complexes bound to antigen-specific T cells.

[0102] In some embodiments, the first and second identification labels are fluorophores.

[0103] In some embodiments, the fluorophore is APC or PE.

[0104] In one aspect, provided herein is a method for identifying HLA and peptide complexes, comprising obtaining or having obtained a dataset comprising data associated with one or more barcodes that are detectably bound directly or indirectly to T cells, wherein the one or more barcodes are each operably associated with a unique antigenic peptide; determining or having determined a ratio of barcodes bound to T cells, wherein the ratio is calculated by identifying a first copy number of a primary barcode from step (a) and a second copy number of a different barcode and dividing the first copy number by the second copy number; and identifying HLA and peptide complexes bound to antigen-specific T cells.

[0105] In some embodiments, step (a) comprises obtaining a T cell-based sample and performing an assay thereon to obtain a dataset.

[0106] In some embodiments, a data set includes one or more barcodes and one or more barcode copy numbers.

[0107] In some embodiments, the unique antigenic peptide is the same for each different collection of particles.

[0108] In one aspect, the present invention provides a method for identifying a subject for treatment with immunotherapy, comprising providing two or more different sets of particles, each different set of particles comprising a unique antigenic peptide and at least one defined barcode operably associated with the identity of the antigenic peptide, and each set comprising a first particle comprising a first identification marker and a second particle comprising a second identification marker different from the first identification marker; providing a sample known or suspected to contain one or more T cells; contacting the sample with the two or more sets of particles, wherein the contacting comprises providing conditions sufficient for a single T cell to bind to a unique antigen of at least one set of particles; isolating one or more T cells associated with the first and second identification markers; 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 identifying a first copy number of the primary barcode from step (e) and a second copy number of the different barcode and dividing the first copy number by the second copy number.

[0109] In some embodiments, the first and second identification labels are fluorophores.

[0110] In some embodiments, the fluorophore is APC or PE.

[0111] In one aspect, provided herein is a method of identifying a subject for treatment with an immunotherapy, comprising obtaining or having obtained a dataset comprising data associated with one or more barcodes that detectably bind directly or indirectly to T cells, wherein the one or more barcodes are each operably associated with a unique antigenic peptide; and determining or having determined a ratio of barcodes bound to T cells, wherein the ratio is calculated by identifying a first copy number of a primary barcode from step (a) and a second copy number of a different barcode and dividing the first copy number by the second copy number.

[0112] In some embodiments, step (a) comprises obtaining a T cell-based sample and performing an assay thereon to obtain a dataset.

[0113] In some embodiments, a data set includes one or more barcodes and one or more barcode copy numbers.

[0114] In some embodiments, the unique antigenic peptide is the same for each different collection of particles.

[0115] 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.

[0116] In some embodiments, the antibody comprises a monoclonal antibody.

[0117] In one aspect, the present invention provides a method for identifying a unique TCR sequence, comprising providing two or more different particle collections, each different particle collection comprising a unique antigenic peptide and at least one defined barcode operably associated with the antigenic peptide identity, and each collection comprising a first particle comprising a first identification marker and a second particle comprising a second identification marker different from the first identification marker; providing a sample known or suspected to contain one or more T cells; contacting the sample with the two or more particle collections, wherein the contacting comprises providing conditions sufficient for a single T cell to bind to a unique antigen of at least one particle collection; isolating one or more T cells associated with the first and second identification markers; 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 identifying a first copy number of the primary barcode from step (e) and a second copy number of the different barcode and dividing the first copy number by the second copy number; and identifying a unique TCR sequence.

[0118] In some embodiments, the first and second identification labels are fluorophores.

[0119] In some embodiments, the fluorophore is APC or PE.

[0120] In one aspect, provided herein is a method of identifying a unique TCR sequence, comprising obtaining or having obtained a dataset comprising data associated with one or more barcodes that are detectably bound directly or indirectly to a T cell, wherein the one or more barcodes are each operably associated with a unique antigenic peptide; determining or having determined a ratio of barcodes bound to T cells, wherein the ratio is calculated by identifying a first copy number of a primary barcode from step (a) and a second copy number of a different barcode and dividing the first copy number by the second copy number; and identifying a unique TCR sequence.

[0121] In some embodiments, step (a) comprises obtaining a T cell-based sample and performing an assay thereon to obtain a dataset.

[0122] In some embodiments, a data set includes one or more barcode sequences and one or more barcode copy numbers.

[0123] In some embodiments, the unique antigenic peptide is the same for each different collection of particles.

[0124] In some embodiments, the method further comprises producing a soluble TCR polypeptide comprising the identified unique TCR sequence.

[0125] In some embodiments, the soluble TCR polypeptide is linked to a label or a drug.

[0126] In some embodiments, the method is repeated to identify at least two unique TCR sequences.

[0127] In some embodiments, the method further comprises producing a library comprising at least two unique TCR sequences.

[0128] In one aspect, provided herein is a method of treating cancer in a subject, comprising obtaining or having obtained a dataset comprising data associated with one or more barcodes detectably bound directly or indirectly to T cells, wherein each of the one or more barcodes is operably associated with a unique antigenic peptide; determining or having determined a ratio of barcodes bound to T cells that identify T cell antigen specificity, wherein the ratio is calculated by identifying a first copy number of a primary barcode from step (a) and a second copy number of a different barcode and dividing the first copy number by the second copy number; identifying a TCR sequence of at least one or both T cells, and generating an engineered T cell comprising at least one or both TCR sequences; and administering the engineered T cell to the subject.

[0129] In some embodiments, the method further comprises administering immunotherapy.

[0130] In some embodiments, the immunotherapy is a checkpoint inhibitor.

[0131] In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0132] In some embodiments, the T cells are autologous.

[0133] In some embodiments, the engineered T cells are autologous.

[0134] In some embodiments, the unique antigenic peptide is presented by HLA class I on the surface of cells of the subject's cancer.

[0135] In certain embodiments, the subject matter disclosed herein provides a method for treating T cells. In certain embodiments, the method includes: (a) contacting a sample with a plurality of different particle sets; (b) isolating one or more T cells bound to particles; (c) identifying the barcodes of particles bound to separated T cells; and (d) determining the ratio of each barcode. In certain embodiments, each particle comprises a unique antigenic peptide, an operably associated barcode, and at least one identification marker. In certain embodiments, the sample comprises T cells. In certain embodiments, contacting includes providing conditions suitable for a single T cell to bind to a unique antigenic peptide of at least one particle set.

[0136] 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 different set of particles. In certain embodiments, each different set of particles comprises at least one or more barcodes, wherein each barcode is associated with the 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, if the ratio of the first barcode is above a threshold, the isolated T cell is identified as an antigen-specific T cell. In certain embodiments, the threshold is at least or greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-5, 3-6, 4-7, 5-8, 5-10, 7-10 or greater than 10.

[0137] In certain embodiments, identifying a barcode 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 number of copies of each barcode. In certain embodiments, the nucleotide-based assay determines (a) the sequence of each barcode and / or (b) the number of copies of each barcode.

[0138] 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.

[0139] In certain embodiments, the method includes identifying the antigen specificity of the T cell. In certain embodiments, the antigen specificity of the T cell includes the sequence of the antigenic peptide and the TCR sequence of the bound T cell.

[0140] In certain embodiments, at least one identification label is the same in each different particle set. In certain embodiments, the method includes at least two different identification labels. In certain embodiments, at least one identification label is a fluorophore. In certain embodiments, the fluorophore is selected from the group including allophycocyanin (APC) and phycoerythrin (PE). In certain embodiments, at least two different identification labels are fluorophores, wherein the fluorophore is selected from the group including allophycocyanin (APC) and phycoerythrin (PE).

[0141] In certain embodiments, the antigenic peptide is selected from the group consisting of tumor antigens, neoantigens, tumor neoantigens, viral antigens, bacterial antigens, phosphoantigens, and microbial antigens. In certain embodiments, the neoantigens are identified from tumor sequencing data of the subject. In certain embodiments, a computer prediction algorithm is used to determine the neoantigens. In certain embodiments, the prediction algorithm further includes an MHC binding algorithm to predict the binding between the neoantigen and the MHC peptide.

[0142] 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, wherein the T cell is a human T cell. In certain embodiments, the T cell is a CD8 + T cells.

[0143] In certain embodiments, the method comprises a library of different particle sets. In certain embodiments, the library comprises 2 to 500 different 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 comprise 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, H LA-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,HL A-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*0 3:03, 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.

[0144] 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.

[0145] In certain embodiments, each particle comprises a polypeptide comprising (i) an antigenic peptide, (ii) a β2M peptide, and (iii) an MHC peptide in the amino to carboxyl terminal direction. In certain embodiments, the antigenic peptide is 7-15 amino acids in length, 7-10, 8-9, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. 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 particles are coated with streptavidin.

[0146] In certain embodiments, the subject matter disclosed herein provides a method for monitoring the immune repertoire in a subject. In certain embodiments, the method includes monitoring changes in antigen-specific T cells in a subject. In certain embodiments, the method includes administering immunotherapy to the subject. In certain embodiments, immunotherapy is adoptive cell transfer or checkpoint inhibitors. In certain embodiments, any method disclosed herein is used to monitor the immune repertoire in a subject.

[0147] In certain embodiments, the currently disclosed subject matter provides a method for identifying at least one TCR sequence. In certain embodiments, 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 manufacturing a soluble TCR polypeptide. In certain embodiments, any method disclosed herein is used to identify at least one TCR sequence.

[0148] In certain embodiments, the currently disclosed subject matter provides a library of particles. In certain embodiments, the library includes 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 identification tag. In certain embodiments, at least one identification tag is the same in each particle set. In certain embodiments, there are at least two different identification tags in each different particle set. In certain embodiments, at least one identification tag is a fluorophore. In certain embodiments, the fluorophore is selected from the group including allophycocyanin (APC) and phycoerythrin (PE). In certain embodiments, at least two different identification tags are fluorophores, wherein the fluorophore is selected from the group including allophycocyanin (APC) and phycoerythrin (PE).

[0149] In certain embodiments, the currently disclosed subject matter further provides particles. In certain embodiments, the particles comprise at least one polypeptide, a barcode, and an identification tag. In certain embodiments, the polypeptide comprises an antigenic peptide, a β2M peptide, and an MHC peptide. In certain embodiments, the barcode is operably associated with the identity of the antigenic peptide. In certain embodiments, the particles are selected from the group consisting of magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles. In certain embodiments, the identification tag is a fluorophore. In certain embodiments, the particles are coated with streptavidin. In certain embodiments, the polypeptide is labeled.

[0150] In certain embodiments, the currently disclosed subject further discloses a method for treating cancer in a subject. In certain embodiments, the method includes: (a) preparing multiple particles, each particle comprising multiple labeled polypeptides; (b) contacting multiple particles with multiple T cells from a subject under conditions suitable for T cells to specifically bind to particle antigens; (c) separating T cells bound to particles and identifying TCR gene sequences of separated T cells; (d) preparing polynucleotides comprising homology arms and at least one TCR gene sequence; (e) recombining polynucleotides into endogenous loci of subject T cells; (f) culturing modified T cells to produce T cell groups; and (g) administering a therapeutically effective number of modified T cells to a subject, thereby treating cancer. In certain embodiments, the polypeptide comprises an antigenic peptide, a β2M sequence, an HLA sequence, and a detectable marker. In certain embodiments, the TCR gene sequence is patient-specific. In certain embodiments, the TCR gene sequence is located between homology arms.

[0151] In certain embodiments, the currently disclosed subject matter further discloses a method for modifying a cell. In certain embodiments, the method includes: (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 homologous to the first and second endogenous sequences of the cell; (b) a T cell receptor (TCR) gene sequence obtained by any method 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, wherein the first and second 2A coding sequences encode the same amino acid sequence that is codon-diverged from each other. 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 and second HR arms. 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 joint. In certain embodiments, the sequence encoding a flexible joint 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 the second 2A coding sequence.

[0152] In certain embodiments, the currently disclosed subject matter further discloses a composition comprising a modified cell. In certain embodiments, the modified cell comprises 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 method disclosed herein. In certain embodiments, the first and second 2A coding sequences encode the same amino acid sequence with codon divergence 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 joint. In certain embodiments, the sequence encoding the flexible joint is located immediately upstream of the 2A coding sequence. In certain embodiments, the flexible joint 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

[0154] These and other features, aspects and advantages of the disclosed compositions and methods will be better understood from the following description and accompanying drawings, in which:

[0155] Figure 1 The design of an exemplary compACT minigene is shown. SS refers to an optional signal sequence; US1 refers to the first universal target site; NeoE refers to a neo-epitope, i.e., an antigenic peptide sequence site; US2 refers to a second universal target site; L1 refers to an optional first linker sequence; Beta2m refers to a beta-2-microglobulin domain sequence; L2 refers to an optional second linker sequence; MHC heavy chain refers to an MHC heavy chain allele; L3 refers to an optional third linker sequence; and purification cluster refers to an optional purification cluster having a biotinylation sequence, a protease cleavage site, and an affinity tag sequence. Although Figure 1His6 (SEQ ID NO:34) (6-His tag (SEQ ID NO:34)) is shown as an affinity tag, but any other suitable affinity tag may be used, including but not limited to histidine tags of varying lengths (poly-His 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, poly-Asp tags, poly-Phe tags, protein C, streptavidin / biotin tags, Strep-tags, protein G, or any other protein purification tag capable of purifying compPACT polypeptides.

[0156] In addition, although Figure 1 The use of nickel resin (see "Ni" in the figure) to purify the His6-tagged (SEQ ID NO: 34) compPACT polypeptide is shown, but other His6 (SEQ ID NO: 34) affinity resins have been used. Specifically, zinc resin has been used to successfully purify the His6-tagged (SEQ ID NO: 34) compPACT polypeptide from solution. Cobalt and calcium resins are two other exemplary His6 (SEQ ID NO: 34) affinity resins that can be used.

[0157] Figure 2 Shown is a schematic diagram of an exemplary modular off-the-shelf platform for rapid assembly of libraries of antigenic peptide ligands complexed with selected MHC alleles. Figure 2 SEQ ID NOs: 9, 11 and 13 are disclosed respectively in order of appearance.

[0158] Figure 3 Schematic diagram of an exemplary restriction digestion cloning reaction to replace a virtual insert in an MHC template with a selected new epitope sequence. The virtual insert (underlined, bold; top sequence in the figure) contains four stop codons in different frames and unique restriction sites for destroying uncut or re-ligated templates. The restriction sites on either side of the insert are shown in boxes. The bottom sequence group shows the NeoE insert after the virtual insert has been cut and ligated to the correct NeoE sequence. Figure 3 SEQ ID NOs: 270-274 are disclosed respectively in order of appearance.

[0159] Figure 4Schematic diagram of an exemplary restriction digestion cloning reaction for inserting a selected neo-epitope sequence into an MHC template. The neo-epitope sequence (underlined, bold) is synthesized as a primer, flanked by two different restriction sites (boxed). A universal primer with a reverse complementary sequence to the 3' restriction site is used in a PCR reaction to form a double-stranded primer dimer of the neo-epitope sequence. Restriction digestion of both the neo-epitope and the MCH template vector allows a ligation reaction to insert the selected neo-epitope sequence into the MHC template sequence. The ligation reaction is transformed into E. coli, and the plasmid prepared from the transformed E. coli is used for a mammalian production cell transfection reaction. Figure 4 SEQ ID NOs: 275-276, 271-274 and 277 are disclosed in order of appearance, respectively.

[0160] Figure 5 Figure 1 is an exemplary alternative format for restriction digestion cloning reactions to insert selected neo-epitope sequences into MHC templates. Two complementary NeoE encoding primers are synthesized with partial 5' and 3' restriction sites. These primers are annealed and mimic the overhangs of restriction digestion. The pre-cut vector (whose overhangs retain primarily 5' phosphates) is then ligated to the annealed NeoE insert and the ligation product is transformed into E. coli for plasmid production. Figure 5 SEQ ID NOs: 278-279, 271-274 and 277 are disclosed in order of appearance, respectively.

[0161] Figure 6 is a diagram of an exemplary PCR-based method for inserting a selected neo-epitope sequence in an MHC template. Two complementary NeoE encoding primers are synthesized, the forward primer has a 3' sequence to the second universal site in the MHC template; and the reverse primer has a 3' sequence to the complementary sequence of the first universal site in the MHC template. These primers are mixed with a 5' fragment of the MHC template with the first universal sequence site and a second fragment of the MHC template with the second universal site and the remainder of the compPACT minigene. The first PCR amplification cycle generates two nucleotide fragments, one fragment encoding the first universal site region with the downstream neo-epitope and the other encoding the neo-epitope followed by the remainder of the compPACT gene. The two fragments that overlap in the unique neo-epitope sequence are then assembled, and the complete assembly is amplified and cleaned up for transfection.

[0162] Figure 7 Shown are total protein expression in 30 mL of mammalian cells transfected with the compACT gene (Neo12) over a 7 day time course and Western blot using NTA-HRP reagent to detect the His-tag.

[0163] Figure 8A gel showing the Ni-NTA affinity chromatography purification of Neo12 comPACT protein is shown. Pre stands for crude lysate, FT stands for flow through, W stands for wash, and E stands for eluted.

[0164] Fig. 9 A size exclusion chromatogram of purified Neo12 protein is shown. The major peak is Neo12 protein and the minor peak is ATP added during the biotinylation step.

[0165] Fig.10 Shows something like Figure 8 For the purification experiments shown, 0.7 cell culture volume was used.

[0166] Fig.11 Crude and purified proteins of eight different NeoE comPACT proteins, each with a different antigenic sequence, are shown. Fig.11 SEQ ID NOs: 11 and 13 are disclosed respectively in order of appearance.

[0167] Fig.12 Shows Fig.11 Size exclusion chromatograms of eight NeoE comPACT proteins.

[0168] Fig.13A Compared with NeoE comPACT proteins (particularly Neo12 comPACT proteins) produced from plasmids, the use of Figure 6 NeoE comPACT proteins (particularly Neo12 comPACT proteins) were generated using the PCR assembly method (linear amplicon) described in . Fig. 13B A DNA gel of linear amplicons generated by the PCR assembly method is shown. Each lane contains a comPACT minigene (specifically the Neo12 comPACT minigene) with a different neo-epitope sequence.

[0169] Fig.14 A streptavidin bead pull-down assay is shown to test the complete biotinylation of the compPACT protein. Fig.14 "(His6)" is disclosed as SEQ ID NO:34.

[0170] Fig.15 Biotinylation of different compACT proteins in crude cell lysates, visualized by western blotting using streptavidin-HRP, is shown. Fig.15 "His6" is disclosed as SEQ ID NO:34. Fig.15 Also disclosed are SEQ ID NOs: 280, 280-281 and 281, respectively, in order of appearance.

[0171] Fig.16A , 16B and 16C show the BirA enzyme in E. coli ( Fig. 16B and 16C ) and TEV protease ( Fig.16A ) production and purification.

[0172] Fig.17 Biotinylation of the compPACT protein using BirA (lane 2) and cleavage of the His6 tag (SEQ ID NO: 34) using TEV protease (lane 3) are shown.

[0173] Fig.18 Cell sorting of cells transduced with BirA and V5 based on V5 expression is shown.

[0174] Fig.19 Antigen-specific capture of T cells using multimeric compPACT protein is shown.

[0175] Fig. 20 Shown is the production of compACT NTAmers using the S88Cβ2M compPACT protein.

[0176] Fig.21 The coupling of Cy5 to the S88C comPACT protein monomer is shown.

[0177] Fig.22A An example diagram of the cloning strategy for making compACT polynucleotides is shown. Fig. 22B Sequence validation statistics obtained from 824 individual compPACT polynucleotides are presented.

[0178] Fig.23 An example diagram of the workflow for making compACT polynucleotides and proteins is provided.

[0179] Fig.24A and 24B The percentage of patients covered by the highest HLA I allele relative to the size of the compPACT HLA repertoire in the United States is shown.

[0180] Fig.25A The monodispersity of compPACT proteins is shown for a representative selection of compPACT proteins. Fig.25B The compPACT protein yields of a representative selection of compPACT proteins are shown. Fig.25C The expression of a representative selection of compPACT proteins is shown.

[0181] Fig.26A schematic diagram of the imPACT signal-to-noise neoantigen T cell isolation process is provided.

[0182] Fig.27A A graphical representation of the nonspecific barcode signal intensity is provided to identify signal and noise. Fig.27B A graphical representation of specific barcode signal intensity is provided to identify signal and noise.

[0183] Fig.28 Illustrations of single and dual barcodes are provided.

[0184] Fig.29A A schematic diagram of the double-tetramer staining procedure is provided. Fig.29B Data showing imPACT tetramer staining of MART5 antigen-specific T cells are presented. Fig.29C Data showing imPACT tetramer staining of neo12 antigen-specific T cells are presented.

[0185] Fig.30 An example of isolated neoantigen T cell sensitivity by neoID signal-to-noise ratio is provided.

[0186] Fig.31 An example of the specificity of the impact neoantigen isolation process (i.e., imPACT isolation technology) is provided.

[0187] Fig.32A Provided are FACS plots of gene-edited specific T cells (solid squares) that exhibited a NeoID signal-to-noise ratio greater than 10. Nonspecific T cells (circles) exhibited a S / N<10. Fig.32B Quantification of signal-to-noise ratios and mean values ​​for specific and nonspecific T cells are shown.

[0188] Fig.33A Examples of imPACT analysis of PBMC samples and validation of imPACT-isolated TCRs using a single barcoding approach are provided. Fig.33A SEQ ID NOs: 282-284 are disclosed respectively in order of appearance. Fig.33B Examples of imPACT analysis of PBMC samples and validation of imPACT-isolated TCRs using a single barcoding approach are provided.

[0189] Fig.34A Provided are FACS plots of double-stained T cells from imPACT analysis of PBMC samples using a single barcoding approach. Fig.34A SEQ ID NOs: 285-292 are disclosed respectively in order of appearance. Fig.34B Provided are FACS plots of CD45RA and CD95 stained T cells after double tetramer staining. Fig.34B SEQ ID NOs: 285-292 are disclosed respectively in order of appearance. Fig.34C A table is provided summarizing the TRA (SEQ ID NOs: 293-300, respectively, in order of appearance), TRB (SEQ ID NOs: 285-292, respectively, in order of appearance), genes, and neoantigenic peptides (SEQ ID NOs: 204, 203, 203, 203, 205, 203, and 206-207, in order of appearance) sequences of T cells isolated after imPACT analysis. Fig.34D A summary of isolated neoantigen-specific T cells is provided. Fig.34E A summary of the number of isolated lymphocytes identified for each TCR using the imPACT approach is provided.

[0190] Fig.35A An example of a validation screen of the imPACT analysis of PBMC samples used in Figure 34 is provided. Fig.35A SEQ ID NOs: 207, 301, 206 and 302 are disclosed respectively in order of appearance. Fig.35B An example of a validation screen of the imPACT analysis of PBMC samples used in Figure 34 is provided.

[0191] Fig.36 A graphical representation of the mutation-targeted T cells of the PBMC samples used in Figure 34 is provided.

[0192] Fig.37A Provided are FACS plots of double-stained T cells from imPACT analysis of PBMC samples using the dual barcoding approach. Fig.37B Provided are FACS plots of CD45RA and CD95 stained T cells after double tetramer staining. Fig.37B SEQ ID NOs: 303-305 are disclosed respectively in order of appearance. Fig.37C A table is provided that summarizes the TRA, TRB, gene, and neoantigen peptide sequences of T cells isolated after imPACT analysis. In order of appearance, Fig.37CAll disclose "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 and 208, respectively. Fig.37D An example of a validation screen of the imPACT assay using the compPACT9 dextramer is provided.

[0193] Fig.38A A summary of neoantigen-specific TCRs isolated from patient samples is provided. Fig.38B A table summarizing the HAL type, cancer, number of targets, and number of TCRs found in TILs is provided.

[0194] Fig.39A Antigenic specificity of HCMV and EBV T cells is shown. Fig.39B The number of TCR hits is shown.

[0195] Fig.40A A comparison of tetramer, trimer and dextramer separation methods using F5T cells is shown. Fig.40B A comparison of tetramer, trimer, and dextramer isolation methods using neo12 T cells is shown.

[0196] Fig.41A A comparison of trimer and dextramer isolation methods using PBMC samples and neo12 T cells, CMV T cells, and M1WT cells is shown. Fig.41B The average barcode signal-to-noise ratio when trimers or dextramers were used is shown.

[0197] Fig.42A Shown are the changes in neoantigen-specific T cells in the peripheral blood of patient PACT157 over time. Fig.42A SEQ ID NOs: 315-317 are disclosed respectively in order of appearance. Fig.42B Shown are the changes in neoantigen-specific T cells in the peripheral blood of patient PACT132 over time. Fig.42BSEQ ID NOs: 318-319 are disclosed respectively in order of appearance. Fig.42C Shown are the changes in neoantigen-specific T cells in the peripheral blood of patient PACT131 over time. Fig.42C SEQ ID NOs: 320-323 are disclosed respectively in order of appearance.

[0198] Fig.43 Phenotypic characterization of neoantigen-specific T cells from one patient is shown.

[0199] Fig.44A Functional characterization of TCR clones isolated against the PIK3CA neoantigen target is shown. Fig.44B Functional characterization of TCR clones isolated against the PIK3CA neoantigen target is shown.

[0200] Fig.45 A summary of the number of neoantigen-specific T cells per CD8 T cell in each T cell sample collected during anti-PD-1 antibody treatment of patient PACT135 is provided. Fig.45 "KTYFKPFHPK" is disclosed as SEQ ID NO: 256 and "YFKPFHPKF" is disclosed as SEQ ID NO: 227.

[0201] Fig.46 Robust T cell gene editing efficiency of 14 neo TCRs in both CD4 and CD8 T cells was demonstrated.

[0202] Fig.47 Shown is internalization of the neo TCR upon co-culture with the neoTCR T cell cognate comPACT-dextramer and the melanoma-matched cell line M489, with and without IFNγ pre-incubation.

[0203] Fig.48 NeoTCR T cells derived from patient PACT135 were shown to express the activation marker 4-1BB.

[0204] Fig.49 NeoTCR T cells derived from patient PACT135 were shown to express the activation marker OX40.

[0205] Fig.50 A graph of the percent confluence of tumor cells after co-culture with all neoTCR T cells identified from PACT135 is provided.

[0206] Fig.51A Separate graphs of the percent confluence of tumor cells after coculture with each neoTCR T cell are provided. Fig.51BSeparate graphs of the percent confluence of tumor cells after coculture with each neoTCR T cell are provided.

[0207] Fig.52 Shown is the secretion of IFNγ, IL2, and TNFα by TCR218 T cells after co-culture with M489 cells.

[0208] Fig.53 Shown are the secretion of IFNγ, IL2, and TNFα by TCR221 and TCR227 T cells after co-culture with M489 cells.

[0209] Fig.54 Shown is the secretion of IFNγ and TNFα by TCR222 T cells after co-culture with M489 cells.

[0210] Fig.55 Shown is IFNγ secretion by TCR219, TCR220, TCR223, TCR224, TCR225, TCR228, TCR229, TCR232, TCR240, and TCR241 T cells after co-culture with M489 cells with or without IFNγ pretreatment.

[0211] Fig.56 A summary of the numbers of neoantigen-specific T cells isolated from patient PACT035 is provided.

[0212] Fig.57A HLA-A2 expression in the KV1858 cell line is shown. Fig.57B HLA-A2 expression in the KV1832 cell line is shown. Fig.57C HLA-A2 expression in the SW620 cell line is shown. Fig.57D GFP expression in transfected SW620 cells is shown.

[0213] Fig.58 Shown is the expression of Nur77 in TCR089 neoTCR T cells co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation.

[0214] Fig.59A T cells expressing neoTCR were shown to kill SW620 homozygous tumor cells. Fig.59B Showed no killing of wild-type SW620 cells.

[0215] Fig.60 It was shown that TCR089 killed SW620 cells homozygous for the COX6C-R20Q mutation, but not wild-type SW620 cells.

[0216] Fig.61Shown is IFNγ secretion in TCR089 T cells after co-culture with SW620 cells homozygous for the COX6C-R20Q mutation.

[0217] Fig.62 Methods for the imPACT separation technology are shown: NeoE-specific TCRs are isolated from patients treated with checkpoint inhibitors, sequenced, tumor antigens identified, algorithms used to select neoepitopes for screening using compPACT peptides and the imPACT separation technology, and neoepitope-specific T cells captured.

[0218] Fig.63 Shown are patient samples from patients who did not respond to anti-PD-1 therapy, patient neoE-HLA complex resolution and resulting identified TCRs. Fig.63 SEQ ID NO: 324 is disclosed.

[0219] Fig.64 neoTCR-T cells were shown to kill autologous melanoma tumor cells.

[0220] Fig.65A The ability of neoTCR-T cells to kill autologous tumor cells was demonstrated. Fig.65B showed that neoTCR-T cells expressed activation markers when co-cultured with autologous tumor cells. Fig.65C showed that neoTCR-T cells secreted interferon-γ when co-cultured with autologous tumor cells.

[0221] Fig.66A NeoTCR T cell therapy was shown to eradicate tumors implanted in mice. Fig.66B Shown are the number of human CD8 T cells / mL present in the blood of mice on day 4 after neoTCR T cell infusion and on day 35 after infusion.

[0222] Fig.67A and 67B A neoantigen-specific TCR construct design for integration of a neoantigen-specific TCR construct (neoTCR) into the TCRα locus is described. Fig.67A Illustrated are the target TCRα locus (endogenous TRAC, upper panel) and its CRISPR Cas9 target site (horizontal stripes, cleavage site designated by arrow), as well as a circular plasmid homologous recombination (HR) template (lower panel) carrying the polynucleotide encoding the neoTCR, which is located between the left and right homology arms (“LHA” and “RHA”, respectively) before integration of the RNP:CRISPR / Cas9 complex. Fig.67BIllustrated are the integrated neoTCR in the TCRα locus (upper panel), the transcribed and spliced ​​neoTCR mRNA (middle panel), and the translation and processing of the expressed neoTCR (lower panel).

[0223] Detailed description

[0224] definition

[0225] Unless otherwise stated, the terms used in the claims and specification are defined as follows.

[0226] As used herein, the term "antigen-specific T cells" refers to cells that are distinguished from each other by their T cell receptors (TCRs), which confer their antigen specificity.

[0227] 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 having a peptide in the antigen binding groove.

[0228] As used herein, "antigen" includes any antigen, including patient-specific antigens.

[0229] "Antigenic peptide" and "antigenic peptide" as well as "neo-epitope" and "NeoE" are used interchangeably and refer to a peptide derived from an antigen identified on a cell of interest (e.g., if it is a tumor cell, an antigen expressed by a tumor cell) that is incorporated into a compPACT polypeptide using the molecular biology techniques described herein. In addition, as explicitly stated in the Examples, the terms "neo-antigenic sequence" and "neo-antigenic insert" may have the same meaning as "antigenic peptide" and "antigenic peptide" as well as "neo-epitope" and "NeoE". The term also refers to a peptide or peptide fragment that is capable of binding to an MHC molecule.

[0230] “Antigen-MHC complex” and “antigen-MHC” and “recombinant antigen-MHC complex” and “peptide MHC” and “p / MHC” and “neoantigen-MHC complex” are all used interchangeably to refer to the ternary complex consisting of HLA / MHC heavy chain, β2M chain and antigen peptide.

[0231] "Anti-CTLA4 antibodies" Antibodies attach to CTLA-4 and stop it from working. This can boost the body's immune response against cancer cells. Includes ipilimumab. Includes AB154 (Arcus), tiragolumab (Genentech / Roche), BMS-986297 (BMS), MK-7684 (Merck), and etigilimab (OncoMed). In addition to anti-CTLA4 antibodies, CTLA4 inhibitors (large and small molecules) can be used in combination with any neoTCR product.

[0232] "Anti-PD-1 antibody" and "antibody that binds to PD-1" and "anti-PD-1 therapy" refer to antibodies that bind and are capable of binding to PD-1 with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting PD-1. In certain embodiments, antibodies that bind or are capable of binding to PD-1 are capable of blocking the interaction of PD-1 and PD-L1 and enhancing 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 (large and small molecules) can be used in combination with any neoTCR product.

[0233] "Anti-PD-L1 antibodies" and "antibodies that bind to PD-L1" and "anti-PD-L1 therapy" refer to antibodies that bind and are capable of binding to PD-L1 with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting PD-L1. In certain embodiments, antibodies that bind or are capable of binding to PD-L1 are capable of blocking the interaction of PD-1 and PD-L1 and enhancing 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 (large and small molecules) may be used in combination with any neoTCR product.

[0234] "Attachment moiety" refers to any chemical or biological moiety that can be used to attach a polynucleotide or polypeptide to a chemical or biological matrix. As used herein, an attachment moiety is used to attach a polynucleotide or polypeptide to a particle.

[0235] "Checkpoint inhibitors" refer to a type of drug that blocks certain proteins produced by some types of immune system cells (such as T cells) and some cancer cells. These proteins help keep immune responses in check and can prevent T cells from killing cancer cells. When these proteins are blocked, the "brakes" of the immune system are released, and T cells are better able to 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.

[0236] "Barcode" and "barcode sequence" and "nucleotide barcode" and "barcoded polynucleotide" and "neoID" and "neoID barcode" are used interchangeably and refer to a nucleotide sequence used to label and identify a specific peptide.

[0237] "Barcoded particles" refer to particles to which a barcode is attached.

[0238] "Beta-2-microglobulin", "β-2-microglobulin", "β2M" are used interchangeably and have the same meaning.

[0239] "comPACT" and "comPACT construct" are used interchangeably and refer to a polynucleotide or polypeptide, including a neoantigen and an MCH complex, depending on the context in which the term is used. A comPACT may also include a signal sequence, a universal target site, a linker, and a purification cluster. Figure 1 A non-limiting representation of compPACT is shown.

[0240] "comPACT library" and "comPACT-neoID library" are used interchangeably and refer to one or more compPACTs.

[0241] "comPACT minigene" or "comPACT polynucleotide" or "comPACT gene" or "comPACT polynucleotide molecule" are used interchangeably and refer to a nucleic acid sequence encoding a compPACT protein.

[0242] "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 forming the MHC display portion. The compPACT polypeptides described herein may also optionally comprise linker sequences between any or all of the individual components of the compPACT polypeptide. Figure 1 An example of placing an alternative linker sequence in a compACT polypeptide is shown in the compPACT minigene.

[0243] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0244] "Epitope" or "epitope tag" refers to an affinity tag in which a peptide sequence is genetically engineered into a polypeptide and in which 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 by a continuous amino acid sequence (linear epitope) or contain non-continuous amino acids (conformational epitope), for example, due to the folding of the antigen, which are in close spatial proximity. "Linker" refers to any amino acid sequence (or nucleic acid sequence encoding such an amino acid sequence) used to connect components in a fusion protein. When applied to compPACT proteins (fusion proteins), linkers can be used, for example, to connect NeoE to β2M or β2M to MHC heavy chain or MHC heavy chain to purification clusters.

[0245] "Host cell" and "production cell" both refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the initially transformed cell are included herein.

[0246] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, 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 aspects, an individual or subject is a human.

[0247] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0248] "MHC complex" refers to a complex comprising β2-microglobulin and MHC heavy chain. The MHC complex can be a polypeptide or a polynucleotide encoding such a polypeptide. The MHC complex is contained in all compPACT proteins, and the polynucleotide encoding such β2-microglobulin and MHC heavy chain is contained in all compPACT minigenes. Figure 1 and Figure 2 Two examples of inclusion of MHC complexes in the compPACT minigene are shown. Fig.11Western blot of compACT protein containing MHC class I heavy chain complex is shown.

[0249] "MHC display portion" refers to an MHC class I heavy chain comprising the α1, α2 and α3 domains.

[0250] "MHC" refers to the major histocompatibility complex, which is a group of genes that encode cell surface proteins essential to the acquired immune system or the recognition of foreign molecules. The main function of MHC molecules is to bind to foreign antigens (including antigens presented on endogenous cells that are harmful to the 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.

[0251] "MHC class I" refers to the subgroup of the MHC family that contains the Beta-2-microglobulin subunit.

[0252] "Neoantigen" refers to an antigen with at least one change that makes the presentation of the neoantigen or neoantigen different from its corresponding wild-type antigen, for example, the change is a mutation in the polypeptide sequence, and the difference is a difference in post-translational modification or expression level. "Neoantigen" and "tumor neoantigen" refer to specific antigens on cells that can be used to identify targets for killing. When applied to cancer and tumors, neoantigens are antigens specific to tumors or cancers. When applied to pathogens and pathogen-infected cells, neoantigens are antigens specific to pathogens or pathogen-infected cells. "Tumor neoantigen" refers to neoantigens derived from tumors or cancers, such as from patient tumors.

[0253] “neoTCR product” and “neoTCR T cell therapy” and “neoTCR T cell treatment” and “neoTCR T cell” are used interchangeably and refer to genetically engineered T cells that express a TCR that recognizes a neoepitope that has been identified and designed using compPACT polypeptide and polynucleotide and imPACT separation technologies.

[0254] "Neo 12" and "Neo 12 protein" refer to exemplary neo-epitopes.

[0255] "NTAmer" refers to a complex comprising a compACT polypeptide.

[0256] By "ready-made" is meant, with respect to the design of compPACT polynucleotides and compPACT polypeptides made therefrom, a compPACT minigene comprising a Beta-2-microglobulin, an MHC heavy chain allele, and a location to insert a neo-epitope in such a construct. In certain embodiments, the order of the construct from 5' to 3' is 1) neo-epitope, 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 with additional elements is Figure 2 The construct disclosed in .

[0257] "Operably associated" means that, with respect to particle construction, each particle constructed using a given compPACT (in which a specific neoantigen is expressed) is associated with one or more barcodes that are unique to that particle. In this way, downstream sequencing to determine which barcodes bind to specific cells can be used to determine which compPACT (and therefore which neoantigen) is responsible for that binding.

[0258] "Particle", "assembly of particles", "pair of particles" and "different assembly of particles" all mean that, with respect to the term "particle", a core of a compACT comprising a matrix capable of being specifically sorted or separated and to which components of the compPACT (as well as additional polypeptides, polynucleotides and chemicals) can be attached. In certain embodiments, a "assembly of particles" refers to a plurality of particles.

[0259] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0260] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition or formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0261] As used herein, "polynucleotide" or "nucleic acid" are used interchangeably and include any compound and / or substance containing a nucleotide polymer. Each nucleotide is composed of a base, particularly 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. Typically, nucleic acid molecules are described by a base sequence, whereby the base represents the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically expressed as 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 also includes synthetic forms of DNA and RNA and two or more mixed polymers comprising these molecules. Those skilled in the art will understand which form is being referred to, for example, based on the context in which the polynucleotide is 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 may comprise naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugar or phosphate backbone linkages or chemically modified residues. Polynucleotides encompass DNA and RNA molecules suitable as vectors for direct expression of polypeptides of the invention in vitro and / or in vivo.

[0262] "Proliferative disorder" refers to excessive cell proliferation of one or more subpopulations of cells in a multicellular organism, resulting in damage (i.e., discomfort or reduced life expectancy) to the multicellular organism. Cell proliferative disorders can occur in different types of animals and humans. As used herein, proliferative disorders include neoplastic disorders.

[0263] "Protein" and "polypeptide" are used interchangeably herein.

[0264] "Purification cluster" refers to an optional portion of compPACT that includes genetically encoded elements that allow purification of compPACT.

[0265] "Signal sequence" refers to a short peptide present at the N-terminus of a newly synthesized protein that is destined for the secretory pathway. A signal sequence may be included in compACT design and production.

[0266] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the disease in the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some aspects, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0267] "Universal target site," "universal target sequence," and "universal sequence" are used interchangeably and refer to a polynucleotide sequence that can be cleaved by a restriction endonuclease, or a primer binding site that can be used for primer binding and amplification of a desired sequence.

[0268] "Vector", "expression vector" and "expression construct" are used interchangeably and refer to discrete elements used to introduce heterologous DNA into cells for expression or replication thereof. As used herein, a vector can be engineered and used to express in vivo or in vitro a polypeptide gene product encoded by a coding sequence inserted into the vector.

[0269] "Young" or "youthful" when referring to T cells refers to memory stem cells (T MSC ) and central memory cells (T CM ). These cells have T cell proliferation upon specific activation and are capable of multiple cell divisions. They also have the ability to engraft after re-infusion, rapidly differentiate into effector T cells upon exposure to their cognate antigens and target and kill tumor cells, as well as ongoing cancer monitoring and control.

[0270] As used herein, the terms "barcoded T cells," "paired T cells," "T cell-bound nanoparticles," and "T cell-paired antigen-MHC complexes" refer to complexes of T cells with T cell receptors that bind to antigenic peptides presented by MHC molecules on the barcoded NP-antigen-MHC complex (i.e., particle-comPACT complex).

[0271] As used herein, "antibody" or "antibodies" is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. "Antibody fragment" refers to a molecule that is different from an intact antibody and contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. 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 (dAb); and multispecific antibodies formed from antibody fragments.

[0272] The term "in vivo" refers to processes that occur in living organisms, including cells.

[0273] As used herein, the term "mammal" includes humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0274] In the context of two or more nucleic acids or polypeptide sequences, the term "percent sequence identity" refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, have a specific percentage of identical nucleotides or amino acid residues, as measured using one of the following sequence comparison algorithms (e.g., BLASTP and BLASTN or other algorithms available to the skilled person) or by visual inspection. Depending on the application, "percent sequence identity" can exist over a region of the compared sequences, such as over a functional domain, or alternatively, over the full length of the two sequences to be compared.

[0275] To perform sequence comparison, one sequence is usually used as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Then, based on the established program parameters, the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence.

[0276] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized 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 Ausubel et al., below).

[0277] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0278] It must be noted 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.

[0279] Other interpretative conventions

[0280] The ranges described herein are understood to be shorthand for all values ​​within the range, including the endpoints. For example, a range of 1 to 50 is understood to include any number or fraction thereof, a combination of numbers or fractions thereof, or a subrange 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 fraction from the group).

[0281] introduction

[0282] T cell-mediated immunity is characterized by the activation of antigen-specific cytotoxic T cells that are able to induce the death of cells displaying antigens in major histocompatibility complexes (MHC) on their surfaces. These cells displaying antigen-loaded MHC complexes include virus-infected cells, cells with intracellular bacteria, cells with internalized or phagocytosed extracellular protein sources, and cancer cells displaying tumor antigens.

[0283] The natural class I MHC heavy chain contains about 350 amino acids; the natural β2-microglobulin contains about 100 amino acids, and the class I antigenic peptides are generally about 7 to about 15 amino acids in length. The class I heavy chain is encoded by the genes of the major histocompatibility complex, which are called HLA-A, -B and -C in humans and H-2K, D and L in mice. The class I heavy chain and β2-microglobulin are encoded on different chromosomes, respectively. The antigenic peptides are generally processed by cells from protein sources, such as viruses, bacteria or cancer cells. Different variants of the polypeptides encoded by the human HLA-A, -B and -CMHC genes and the mouse H-2K, D and LMHC genes have been identified.

[0284] Embodiments of the methods disclosed herein relate to methods for making single molecules, wherein a selected neoantigen is linked to an MHC complex comprising β2-microglobulin (β2M) and an MHC heavy chain. Different MHC heavy chains can be linked to β2M molecules to form different numbers of MHC templates. The method disclosed herein for inserting a neoantigen into an MHC template by restriction digestion or PCR-based assembly using a universal target sequence flanking a neoepitope insertion site (also referred to as a neoantigen insertion site) results in a library of different neoantigen-MHC complexes that can be constructed in a high-throughput method, which can be personalized for a specific patient. These complexes, referred to as "comPACT proteins," can then be linked to, for example, particles, barcode particles, or surfaces for isolating and identifying patient-specific T cell populations targeting patient-specific neoantigens. Methods for linking antigen-MHC complexes and uses of such complexes are disclosed in PCT / US2018 / 21611, filed on March 8, 2018, which is incorporated herein by reference in its entirety.

[0285] Nucleotide and peptide compositions

[0286] MHC complex

[0287] Briefly, as used herein, a compPACT 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, β2-microglobulin, and an MHC class I heavy chain, comprising the α1, α2, and α3 domains forming the MHC display portion. The compPACT polypeptides described herein may also optionally comprise a linker sequence between any or all of the individual components of the compPACT polypeptide. Figure 1 An example of placing an optional linker sequence in a compPACT polypeptide is shown in the compPACT minigene of . The MHC display portion may include a recombinant MHC molecule. The design and manufacture of a single compPACT polypeptide and a library of compPACT polypeptide molecules are described in International Application PCT / US2019 / 025415 filed on April 2, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, the compPACT polypeptide may comprise a disulfide trap, as described in U.S. Publication No. 2009 / 0117153 and U.S. Publication No. 2008 / 0219947; each of which is incorporated herein by reference. The antigen-MHC complex formed by the compPACT protein results in the display of antigens, enabling them to be recognized by cognate TCR molecules. In some embodiments, the MHC complex may be an MHC class I (MHCI) complex paired with a CD8-positive (CD8+) T "killer" cell. In some embodiments, the MHC complex may be an MHC class II (MHC II) complex paired with a CD4-positive (CD4+) T cell.

[0288] In some embodiments, the MHC class I heavy chain sequence of compPACT can include a single amino acid substitution, addition and / or deletion, such as a substitution of Tyr-84 with a non-aromatic amino acid other than proline. In these embodiments, the amino acid substitution can 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 can be a modified or uncommon amino acid. In one embodiment, the MHC class I heavy chain sequence of compPACT comprises a substitution of tyrosine 84 to alanine. In another embodiment, the MHC class I heavy chain sequence of compPACT comprises a substitution of Tyrosine-84 to cysteine.

[0289] β2-microglobulin (β2M) may include recombinant β2M molecules. In some embodiments, the β2M sequence may include single amino acid substitutions, additions and / or deletions as described above. In one embodiment, the substitution includes a substitution of 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, wherein the substitution does not negatively affect the function of β2M within the compPACT polypeptide, and the substitution allows the conjugation of a thiol-reactive portion. For example, such substitutions may be achieved by screening for cysteine ​​in proteins using mutagenesis techniques known to those skilled in the art. Such thiol-reactive portions may be used to detect β2M or the entire compPACT polypeptide. In certain embodiments, the thiol-reactive portion is a thiol-reactive dye (fluorophore) conjugate, which allows the use of compPACT to measure the kinetic parameters of TCR-comPACT binding (see, e.g., Example 8). In certain embodiments, the thiol-reactive moiety is a dye (fluorophore) that contains a thiol-reactive cross-linker reactive group, including but not limited to maleimide, iodoacetamide or its derivatives, haloacetyl, pyridyl disulfide, and all other thiol-reactive conjugation 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).

[0290] universal sequence

[0291] Antigenic peptides are usually flanked by universal sequences or portions thereof. These sequences allow for rapid, high-throughput methods for replacing or inserting antigenic peptide encoding nucleotides in polynucleotide MHC templates. Universal sequences may include restriction sites for cloning based on restriction digestion. Exemplary restriction sites include, but are not limited to, Nco1, BamHI, BlpI, BspEI, BstBI, Xbal, HindIII, EcoRI, ApaI, NotI, any restriction site not present in β2M, MHC heavy chain, NeoE, signal sequence (if present) purification cluster (if present) or any fusion of its components (including optional linker sequences), and any combination thereof. Alternatively, universal sequences may be primer binding sites. Universal primer sequences known in the art may be used in the compositions and methods disclosed herein, or the sequence may be different from the universal primer sequences previously described and may be designed to promote specific binding / amplification and eliminate non-specific binding / amplification. The length of the universal sequence may be between 4-50, 4-15, 15-40, 15-35, 15-30, 20-40, 25-40, or 30-40 nucleotides. The length of the universal sequence may 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 some embodiments, the length of the universal target sequence is 4-8 nucleotides. In other embodiments, the length of the universal target sequence is between 9-25 nucleotides. In other embodiments, the length of the universal target sequence is between 25-35 nucleotides. In other embodiments, the length of the universal target sequence is at least about 15 nucleotides. In certain aspects, one or more universal target sequences are not present in the genetic material being manipulated, for example, to reduce or eliminate off-target effects and / or increase specificity.

[0292] Connectors

[0293] In various embodiments, compPACT 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 and connect the carboxyl terminus of the antigenic peptide segment to the amino terminus of the β2-microglobulin segment. In a non-limiting example, when compPACT is expressed, the connected peptide ligand can fold into the binding groove, producing a functional compPACT 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.

[0294] In various embodiments, compPACT 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 and connect the carboxyl terminus of the β2-microglobulin segment to the amino terminus of the MHC heavy chain segment. In a non-limiting example, when compPACT is expressed, the β2-microglobulin and the MHC heavy chain may fold into the binding groove, producing a molecule that can play a role in promoting T cell expansion. In various embodiments, the linker may include at least about 15 amino acids, 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.

[0295] In various embodiments, compPACT may comprise 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 and connect it to the amino terminus of the purification cluster. 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 some embodiments, the linker is only 2 or 3 amino acids.

[0296] In certain embodiments, the same joint can be used for the first and second joints, and optionally the third joint (if present). In certain embodiments, the same joint is used for the first, second and third joints. In certain embodiments, all three joints are (G4S)4 joints (SEQ ID NO: 19). In certain embodiments, all three joints are (G3S)n joints (SEQ ID NO: 201). In certain embodiments, all three joints are (GSGGS)n joints (SEQID NO: 11).

[0297] In certain embodiments, all three linkers are (GCGGS)n linkers (SEQ ID NO: 13).

[0298] In certain embodiments, different sequences are used for each of the first and second linkers, and optionally the third linker if present.

[0299] In certain embodiments, two of the first, second, and optional third linkers are the same and one is different.

[0300] Any suitable flexible linker sequence known in the art can be used. Suitable linker sequences include, but are not limited to, glycine-serine sequences comprising repeating units of 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 can be used for any one of the first, second and third linkers. In certain embodiments, a cleavable linker is only used for the first, second or third linker. In certain embodiments, a cleavable linker is only used for the first linker. In certain embodiments, a cleavable linker is only used for the second linker. In certain embodiments, a cleavable linker is only used for the third linker.

[0301] In certain embodiments, the linkers (first, second and / or third) may be selected from the group consisting of linkers that are either rigid or less flexible.

[0302] signal sequence

[0303] In various embodiments, the compPACT polynucleotides and polypeptides comprise 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 may 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 compPACT protein to the secretory pathway (for secretion of compPACT from the cell) may be used.

[0304] 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.

[0305] The signal sequence may be between 70 and 80 nucleotides in length. The signal sequence may be between 40-90, 40-60, 45-70, 50-80, 60-90, 55-70, 60-80 or 70-80 nucleotides in length. The signal peptide may be between 10-30, 10-20, 15-30 or 20-30 amino acids in length.

[0306] Promoter

[0307] The comPACT polynucleotide composition may further include a promoter for transcribing the encoded polynucleotide into an mRNA transcript that can be translated by a host cell. The source of the promoter may be prokaryotic, viral or eukaryotic (e.g., but not limited to mammals). Any suitable promoter for gene transcription in cells 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 EF1a, 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 a specific polymerase (e.g., T7 or Sp6 RNA polymerase) to be present, a promoter that is constitutive in the absence of a repressor and is inducible in the presence of an inducer (e.g., but not limited to, a lac promoter that is constitutive in the absence of a lac repressor but can be induced by IPTG or lactose), an inducible promoter, a repressible promoter or a regulated promoter. In certain embodiments, the prokaryotic promoter is 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.

[0308] In some embodiments, the compACT polynucleotide comprises an SV40 or any viral promoter. In certain embodiments, depending on the cell line and reagents, a strong viral promoter may be beneficial.

[0309] In some embodiments, the compACT polynucleotide comprises a CMV promoter.

[0310] Affinity tags

[0311] The comPACT polynucleotide composition may further comprise at least one sequence encoding an affinity tag or epitope tag. In some embodiments, the compPACT polynucleotide comprises at least two affinity tag or epitope tag sequences. Any suitable affinity tag or epitope tag can be used in a compPACT polynucleotide or polypeptide. Such epitope tags include, but are not limited to, AviTag (or any avidin / streptavidin tag), strep tags, polyhistidine (His6)-tags (SEQ ID NO: 34), FLAG-tags, HA-tags, and Myc-tags. The sequences in the polynucleotide compPACT gene are translated into peptides in the compPACT polypeptide. These epitope tags can be used for affinity chromatography purification or quantification of expressed compPACT polypeptides. For example, the His6 tag (SEQ ID NO: 34) can be used to purify the compPACT protein by HA-tag binding affinity chromatography. In certain embodiments, metal ion resins 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 compPACT proteins. In certain embodiments, a mixture of Ni2+ and Zn2+ resins is used to purify HA-tagged compPACT proteins. In certain embodiments, the resin is an immobilized metal affinity chromatography resin (IMAC). In certain embodiments, the metal ions are coupled to the resin matrix via a chelating ligand. In certain embodiments, the metal ions are coupled to the resin matrix using nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA).

[0312] In addition, AviTag encodes a known biotinylation site that is recognized by the BirA enzyme. Inclusion of this peptide sequence in a protein allows the sequence to be biotinylated by enzymatic modification by BirA. Thus, a compPACT polypeptide comprising an AviTag (or any avidin / streptavidin tag) sequence and a His6 tag (SEQ ID NO: 34) can be biotinylated, purified via a 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 assessed by biotin visualization using streptavidin or other avidin reagents. In some embodiments, a compPACT polynucleotide comprises an AviTag (or any avidin / streptavidin tag) sequence. In some embodiments, a compPACT polypeptide comprises an AviTag (or any avidin / streptavidin) epitope. In some embodiments, a compPACT polynucleotide comprises a His6 sequence (SEQ ID NO: 34). In some embodiments, the compPACT polypeptide comprises a His6 epitope (SEQ ID NO: 34). In some embodiments, the compPACT polynucleotide comprises an AviTag (or any avidin / streptavidin) sequence and a His6 sequence (SEQ ID NO: 34). In some embodiments, the compPACT polypeptide comprises an AviTag (or any avidin / streptavidin) epitope and a His6 epitope (SEQ ID NO: 34).

[0313] Protease cleavage site

[0314] The comPACT polynucleotide composition may also include a sequence encoding a protease cleavage site in the purification cluster. The cleavage site may be encoded between the first and second affinity tag sequences and allows for cleavage of the second affinity tag from the comPACT protein once the comPACT has been expressed and subjected to a round of purification. 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.

[0315] PolyA tail

[0316] The compPACT 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 compPACT polynucleotide is assembled by PCR for direct transfection into a host cell (e.g., not in an expression construct or vector). Any suitable polyA tail and sequence motif may be used in the compPACT 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 compPACT polynucleotide comprises a BHG polyA tail sequence.

[0317] Antigen sequence

[0318] The length of the antigen sequence (i.e., the sequence of the neoantigen to which the neoepitope portion of the compPACT polypeptide is designed to bind) can be between 20-60, 20-30, 25-35, 20-45, 30-45, 40-60, or 45-60 nucleotides. 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 originated from an exogenous antigen and then subsequently became an endogenous antigen (e.g., an intracellular virus). The antigenic peptide can be or be derived from a tumor antigen, a neoantigen, a tumor neoantigen, a viral antigen, a bacterial antigen, a phosphoantigen, or a microbial antigen. In one embodiment, the antigenic peptide is a neoantigen. The antigenic peptide can be selected from patient data and can contain one or more somatic mutations.

[0319] In order to make an inclusive compPACT library with multiple neo-epitopes and thus multiple compPACT polypeptides, it is necessary to predict and identify antigenic sequences. Prediction of antigenic peptides can include prediction algorithms and they can be designed to predict the binding of antigenic peptides or neo-antigens to MHC alleles. Prediction of antigenic peptides is discussed further below.

[0320] In some embodiments, the length of the nucleotide sequence encoding the antigenic peptide is between 20-60, 20-30, 25-35, 20-45, 30-45, 40-60, or 45-60 nucleotides. In other embodiments, the length of the nucleotide sequence encoding the antigenic peptide is between 20-30 nucleotides. In some embodiments, the length of the antigenic peptide is 7-15 amino acids, 7-10, 8-9, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0321] Biotinylation

[0322] The compPACT proteins described herein can be further biotinylated by any suitable method. One such method utilizes BirA biotin-protein ligase and is commercially available. A specific amino acid sequence called the AviTag sequence (GLNDIFEAQKIEWHE (SEQ ID NO: 30)) is encoded in the target protein. BirA ligase, d-biotin and ATP are added to the reaction mixture containing the target protein. BirA covalently links biotin to the lysine in the AviTag sequence, thereby biotinylating the target protein. The new biotinylated protein can then be purified and used for downstream applications. Other methods known in the art for biotinylating proteins can also be used. For clarity, any applicable avidin / streptavidin sequence can be used for compPACT protein preparation.

[0323] Expression constructs and vectors

[0324] The compPACT polynucleotide molecule can be inserted into an expression construct or expression vector, such as for plasmids (to increase the number of expression constructs or expression vectors encoding the compPACT polynucleotide for protein production) and 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 Escherichia coli or Bacillus subtilis plasmids; eukaryotic expression vectors, such as mammalian expression vectors or yeast expression vectors; or viral vectors, such as adenoviral expression vectors, lentiviral expression vectors, vaccinia expression vectors or baculovirus expression vectors. The mammalian expression construct or expression vector 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 modified cell line of CHO, HEK293, Expi293 or HeLa cell lines, and any other suitable mammalian cell line. Mammalian expression constructs or expression vectors can be used for primary mammalian cell lines, such as immune cells or tumor cells obtained directly from an organism (e.g., human) or collected (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 for (e.g., transfection) insect cell lines such as Sf9 or Sf12 (or any derivatives or modifications thereof) or yeast cell lines such as Pichia pastoris (or any derivatives or modifications thereof), as appropriate. In addition, the expression construct or expression vector may contain a nucleotide barcode. The nucleotide barcode of each expression construct or vector can be unique. In some embodiments, the nucleotide sequence encoding the signal sequence, β-2-microglobulin, and MHC allele can be connected to an expression construct or expression vector with a non-coding or simulated antigen insertion sequence. The non-coding antigen insertion sequence can then be removed by appropriate cloning techniques (e.g., restriction digestion), and the desired antigen sequence (in this case, for clarity, the antigen sequence refers to the new antigen sequence) can be inserted by ligation or any other appropriate cloning technique.

[0325] In some aspects, provided herein are compPACT libraries comprising two or more compPACT polypeptides. Such libraries are created by encoding two or more compPACT polypeptides in an expression construct or expression vector. In certain embodiments, each expression construct or expression vector comprises a single compPACT polynucleotide. In certain embodiments, the number of expression constructs or expression vectors (each expression construct or expression vector may be the same expression construct or expression vector) is the same as the number of different compPACT polynucleotides. In certain embodiments, compPACT polynucleotides are inserted into expression constructs or expression vectors using the same or different universal target sites. In other aspects, provided herein are MHC libraries comprising two or more MHCs. Such libraries are created 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 created by encoding two or more HLA polypeptides in an expression construct or expression vector.

[0326] Host cells

[0327] On the other hand, provided herein is a host cell comprising a polynucleotide molecule or expression construct as described herein. The host cell can be any suitable host cell known in the art, including but not limited to bacterial cells such as Escherichia coli or Bacillus subtilis, or eukaryotic host cells such as Chinese hamster ovary (CHO), J558, NSO, SP2-0, HEK293, HEK293T, Expi293, HeLa, insect cell lines (e.g., Sf9 or Sf12), yeast cells such as Pichia pastoris, based on constructs and vectors, select other suitable eukaryotic or prokaryotic cell lines that are scientifically reasonable, or any derivative or modification of any such cell line. The host cell can also stably express the biotinylase BirA. The host cell can be a primary cell or an immortalized cell line.

[0328] 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 stem cells, tumor cells, immortalized cells and fetal cells. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the cell is an Escherichia coli cell. In some embodiments, the cell expresses the BirA protein or a fragment thereof.

[0329] In certain embodiments, any expression construct or expression vector described herein can be inserted into a host cell (e.g., by transfection, transformation, or a similar process based on the host cell type) for polypeptide production. In certain embodiments, an expression construct or expression vector encoding a compPACT polypeptide library, MHC, or HLA as described above can be inserted into a host cell (e.g., by transfection, transformation, or a similar process based on the host cell type) for polypeptide production and purification. In certain embodiments, an expression construct or expression vector encoding a compPACT polypeptide library as described below can be inserted into a host cell (e.g., by transfection, transformation, or a similar process based on the host cell type) for polypeptide production and purification.

[0330] library

[0331] In certain embodiments, the library comprises two or more different compPACT polynucleotide molecules. In certain embodiments, the library comprises two or more different polypeptide molecules. In certain embodiments, the library comprises two or more different compPACT polypeptide molecules attached to a particle.

[0332] In certain embodiments, any of 1) a compPACT polynucleotide library, 2) a compPACT polypeptide library, or 3) a compPACT polypeptide attached to a particle library comprises more than two respective molecules in such respective libraries. In certain embodiments, any of 1) a compPACT polynucleotide library, 2) a compPACT polypeptide library, or 3) a compPACT polypeptide attached to a particle library has no upper limit on the number of respective molecules in such respective libraries, and in turn comprises as many respective compPACT polypeptides, compPACT polypeptides attached to particles, or compPACT polynucleotides as possible. In certain embodiments, the upper limit is determined by the number of tumor neoantigens detected. In certain embodiments, the upper limit is determined by the number of potential neoepitopes identified based on the tumor neoantigens detected. In certain embodiments, the upper limit is determined by an algorithm.

[0333] The library may comprise from 2 to 1000 compPACT polypeptides, compPACT polypeptides attached to particles, or compPACT 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, 20-800,20-700,20-600,20-500,20-480,20-400,20-300,20-200,20-100,20-50,20-50,20-66,20-48,20-30,30-1000,30-900,30-800,30-700,30-600,30-500,30-480,30-400,30-300,30-200,30-100,30-50,30-50,30-66,30-48,30-40,40-1000,40-900,40-800,40-7 00,40-600,40-500,40-480,40-400,40-300,40-200,40-100,40-60,40-50,40-66,40-48,50-1000,50-900,50-800,50-700,50-600,50-500,50-480,50-400,50-300,50-200,50-100,50-60,50-66,60-1000,60-900,60-800,60-700,60-600,60-500,60-480,60-400,60- 70-600, 80-500, 80-480, 80-400, 80-300, 80-200, 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, 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.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 compPACT polypeptides, compPACT polypeptides attached to particles, or compPACT 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, 330 In some embodiments, the library comprises 2, 10, 15, 20, 24, 48, 66, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 compPACT polypeptides, compPACT polypeptides, or compPACT polynucleotides attached to particles. In some embodiments, two or more compPACT polypeptides in the library, compPACT polypeptides attached to a particle, or compPACT polynucleotides have different neo-epitope sequences and different MHC sequences.

[0334] In certain embodiments, the library comprises two or more compPACT polynucleotides, wherein each compPACT polynucleotide in the library comprises a neo-epitope sequence and an MHC heavy chain sequence corresponding to a neo-antigen detected from a patient sample.

[0335] In some embodiments, the library comprises greater than or equal to two different polynucleotide molecules, wherein each different polynucleotide molecule comprises (i) a first universal sequence, (ii) a nucleotide sequence encoding an antigenic peptide, wherein the nucleotide sequence is different for each of the greater than or equal to two 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 different for each of the greater than or equal to two polynucleotide molecules.

[0336] Likewise, HLA-A*01:01,HLA-A*02:01,HLA-A*03:01,HL A*24:02,A*A*30:02,A*31:01,A*32:01,A.A *33:01,A*68:01,A*11:01,A*23:01,A*30:01,A*33:03,A*25:01,A*26:01,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,HL-B*50:01,HL-B*57:01,HL-B*58:01,HL-B*08:01,HL-B*15:01,HL-B*15:03,HL-B*35:01,HL-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,H LA-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*0 7:04,HLA-C*08:01,HLA-C*08:02,HLA-C*12:02,HLA-C*12:0 3, HLA-C*14:02, HLA-C*15:02 and HLA-C*17:01 have not yet been expressed.One of the most popular songs is HLA-A*01:01,HLA-A*02:01,HLA-A*03:0 ,SONG-A*24:02,SONG-A*30:02,SONG-A*31:01,SONG-A*32:01,HL A-A*33:01,SONG-A*68:01,SONG-A*11:01,SONG-A*23:01,SONG-A*30:01,SONG-A*33:03,SONG-A*25:01,SONG-A*26:01,SONG-A*2 9:02,SONG-A*68:02,SONG-B*07:02,SONG-B*14:02,SONG-B*18:01,SONG-B*27:02,SONG-B*39:01,SONG-B*40:01,SONG-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 ,SONG-B*37:01,SONG-B*38:01,SONG-B*41:02,SONG-B*44:05,SONG-B*49:01,SONG-B*52:01,SONG-B*55:01,SONG-C*02:02,SONG-C *03:04,SONG-C*05:01,SONG-C*07:01,SONG-C*01:02,SONG-C*04:01,SONG-C*06:02,SONG-C*07:02,SONG-C*16:01,SONG-C*03:0 3,SONG-C*07:04,SONG-C*08:01,SONG-C*08:02,SONG-C*12:02,SONG-C*12:03,SONG-C*14:02,SONG-C*15:02 and SONG-C*17:01 continued.

[0337] In the new version,HLA sounds like HLA-A*01:01,HLA-A*02:01,HLA-A*03:0 1,SONG-A*11:01,SONG-A*23:01,SONG-A*24:02,SONG-A*25:01,H LA-A*26:01,SONG-A*29:02,SONG-A*30:01,SONG-A*30:02,SONG-A*31:01,SONG-A*32:01,SONG-A*33:01,SONG-A*33:03,SONG-A* 68:01,SONG-A*68:02,SONG-B*07:02,SONG-B*08:01,SONG-B*13:02,SONG-B*14:02,SONG-B*15:01,SONG-B*15:03,SONG-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,SONG-B*40:02,SONG-B*41:02,SONG-B*42:01,SONG-B*44:02,SONG-B*44:03,SONG-B*44:05,SONG-B*46:01,SONG-B*49: 01,NOT-B*50:01,NOT-B*51:01,NOT-B*52:01,NOT-B*53:01,NOT-B*55:01,NOT-B*57:01,NOT-B*58:01,NOT-C*01:02,HL A-C*02:02,SONG-C*03:03,SONG-C*03:04,SONG-C*04:01,SONG-C*05:01,SONG-C*06:02,SONG-C*07:01,SONG-C*07:02,SONG-C*0 7:04,SONG-C*08:01,SONG-C*08:02,SONG-C*12:02,SONG-C*12:03,SONG-C*14:02,SONG-C*15:02,SONG-C*16:01,SONG-C*17:01。In certain embodiments, the HLA library consists of 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*33:02, -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*38:0 1,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*4 6:01,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,HL A-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.

[0338] In some embodiments, the library comprises greater than or equal to two different polypeptide molecules, wherein for each of the greater than or equal to two polypeptide molecules, the antigenic peptide is not identical, and wherein each different polypeptide is attached to a particle. In some embodiments, the library further comprises a uniquely defined barcode sequence operably associated with the identity of each different polypeptide.

[0339] Embodiments include barcoded polynucleotides 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 compPACT is attached to a particle with a uniquely defined barcode sequence. This allows for efficient association between a given antigen and a given barcode, where the barcode is unique to the pair.

[0340] The barcoded polynucleotide can be ssDNA or dsDNA. The polynucleotide comprising the barcode can be modified at its 5' end to include an attachment portion for attachment to a particle. For example, a polynucleotide comprising a barcode sequence is conjugated to a biotin molecule to bind to a streptavidin core attached to a particle (e.g., dextran). However, any suitable attachment portion can be used to attach the polynucleotide to a particle. As described herein and understood by those skilled in the art, suitable attachment portions are known in the art. Non-limiting examples of attachment portions include thiols, maleimides, adamantane, cyclodextrins, amines, carboxyls, azides, and alkynes.

[0341] Particles

[0342] As used herein, "nanoparticles" or alternatively "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 compPACT and barcodes. In certain embodiments, in addition to compPACT and barcodes, additional entities (e.g., fluorophores or other imaging agents) can be attached to the particles. In certain embodiments, in addition to compPACT and barcodes, 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 compPACT. In certain embodiments, compPACT proteins, barcodes, imaging agents, and additional proteins can be attached to the particles. In certain embodiments, multiple compPACT proteins are attached to the particles.

[0343] In some embodiments, the particle is magnetic, such as for separation using a magnet. In some embodiments, the magnetic particle includes magnetic iron oxide. The example of magnetic particles includes but is not limited to Dynabead (Thermo Fisher). In some embodiments, the particle is a polystyrene particle, such as for separation by gravity. In other embodiments, the particle can be a surface, a bead or a polymer. The example of a bead includes but is not limited to agarose (agarose) beads and agarose (sepharose) beads. In a specific embodiment, the particle can be fluorescent or directly or indirectly attached to a fluorophore.

[0344] According to certain embodiments, the particle is modified with an attachment portion for attaching additional molecules. The modification of the particle includes an attachment portion that can be paired (e.g., covalently bound) with a corresponding homologous (e.g., complementary) attachment portion attached to a polynucleotide. Any suitable attachment portion can be used to modify the particle and the polynucleotide detection label for attachment. Non-limiting examples of attachment portions include streptavidin / biotin systems, thiol groups (e.g., cysteine) and cysteine ​​reactive moieties (e.g., maleimide, adamantane and cyclodextrin), amino and carboxyl groups, as well as azido and alkynyl groups. In some embodiments, the attachment portion may include a cleavage portion. In other embodiments, the attachment portion combined with a complementary homologous attachment portion 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 (eg, Dynabeads MyOne Streptavidin T1 beads from ThermoFisher Scientific), and the polynucleotides can be biotinylated for attachment to the modified particles.

[0345] The particles can be dextran, such as biotinylated dextran or streptavidin-coated dextran. Modified dextran is further described in detail in Bethune et al., BioTechniques 62: 123-130 Mar. 2017 and U.S. Publication No. 2015 / 0329617, which are incorporated herein by reference in their entirety. Biotinylated compPACTs can be attached to streptavidin-coated dextran.

[0346] The comPACT protein can also be assembled into a tetramer, including 1, 2, 3 or 4 biotinylated comPACT proteins bound to a streptavidin core. The tetramer can also contain a fluorophore, such as phycoerythrin (PE) or allophycocyanin (APC) bound to a streptavidin core. MHC class I and class II tetramers are well known in the art. MHC class I tetramers are further described in detail in Burrow SR et al., J Immunol December 1, 2000, 165 (11) 6229-6234, and MHC class II tetramers are further described in detail in Nepom GT, J Immunol March 15, 2012, 188 (6) 2477-2482, both of which are incorporated herein by reference in their entirety.

[0347] The comPACT proteins can also be assembled into multimers. In some embodiments, the comPACT protein multimers can be dimers, trimers, tetramers, pentamers, hexamers, or higher order multimers. In some embodiments, the multimers can include at least two or more comPACT proteins. In some embodiments, the multimers can include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 comPACT proteins.

[0348] Particle collections and libraries

[0349] Also contemplated are different sets of particles, each different set of particles comprising a unique antigenic peptide (as used herein, referred to as compPACT) and at least one defined barcode operably associated with the identity of the antigenic peptide. The set of particles 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 different sets of particles include at least two particles. In some embodiments, the different sets of particles include at least three particles. In some embodiments, the different sets of particles include at least four particles. In some embodiments, the unique antigenic peptide (as used herein, referred to as compPACT) comprises a compPACT polynucleotide molecule or a polypeptide molecule.

[0350] Libraries of different particle collections are also contemplated. Libraries of different particle collections may include 2 to 1000 particle collections. 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, 2 0-700,20-600,20-500,20-480,20-400,20-300,20-200,20-100,20-50,20-50,20-66,20-48,20-30,30-1000,30-900,30-800,30-700,30-600,30-500,30-480,30-400,30-300,30-200,30-100,30-50,30-50,30-66,30-48,30-40,40-1000,40-900,40-800,40-700,40-600,40-50 0,40-480,40-400,40-300,40-200,40-100,40-60,40-50,40-66,40-48,50-1000,50-900,50-800,50-700,50-600,50-500,50-480,50-400,50-300,50-200,50-100,50-60,50-66,60-1000,60-900,60-800,60-700,60-600,60-500,60-480,60-400,60-300,60-200,60-100,70-10 00,70-900,70-800,70-700,70-600,70-500,70-480,70-400,70-300,70-200,70-100,70-80,70-90,80-1000,80-900,80-800,80-700,80-600,80-500,80-480,80-400,80-300,80-200,80-100,100-150,150-200,200-250,250-300,300-350,350-400,400-450 or 450-500 particles collection.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 particle collections. 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 collections of particles. In some embodiments, the library comprises 2, 10, 15, 20, 24, 48, 66, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 collections of particles.

[0351] In certain embodiments, a library of particle assemblies of the present disclosure may comprise one, two, three, four, five or more particle assemblies. In certain embodiments, each particle assembly may comprise one, two, three, four, five or more types of particles. In certain embodiments, a particle assembly may comprise a single type of particle. In certain embodiments, a particle assembly may comprise multiple types of particles. For example, but not by way of limitation, a particle assembly may comprise particles bound to the same barcode or particles bound to different barcodes, or a combination thereof. In certain embodiments, a particle assembly may comprise particles bound to a compPACT polypeptide. In certain embodiments, a particle assembly may comprise particles bound to the same compPACT polypeptide, different compPACT polypeptides, or a combination thereof.

[0352] Dextramer and Tetramer

[0353] The compPACT polypeptide can be attached to a dextran, such as a biotinylated dextran or a streptavidin-coated dextran. Modified dextran is further described in detail in Bethune et al., BioTechniques 62: 123-130 Mar. 2017 and U.S. Publication No. 2015 / 0329617, which are incorporated herein by reference in their entirety. The biotinylated compPACT polypeptide can be attached to a streptavidin-coated dextran. In certain embodiments, the dextran is coated with streptavidin. In certain embodiments, streptavidin is covalently conjugated to the dextran. In certain embodiments, streptavidin is non-covalently coupled to biotin-dextran.

[0354] comPACT can also be assembled into tetramers, including 1, 2, 3 or 4 biotinylated comPACT proteins bound to a streptavidin core. The tetramer can also contain a fluorophore, such as phycoerythrin (PE) or allophycocyanin (APC) bound to a 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 class 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 entirety.

[0355] Method for preparing compACT polypeptide

[0356] Antigen prediction

[0357] To make compACT, one of the initial steps can include identifying the patient's tumor-specific antigens (e.g., neoantigens). The composition produced by this method can then be used for T cell-mediated immune processes, for example, for patient-specific cancer immunotherapy. In order to identify the patient's putative neoantigens (tumors or pathogens), a computer prediction algorithm program can be used to analyze tumor, virus or bacterial sequencing data (including whole genome, whole exome or transcriptome sequencing data) to identify one or more mutations corresponding to the putative expressed neoantigens. In addition, human leukocyte antigen (HLA) typing can be determined from the patient's tumor or blood sample, and the HLA information can be used together with the identified putative neoantigen peptide sequence for MHC binding prediction algorithms, (see, Fritsch et al., 2014, Cancer Immunol Res., 2: 522-529, the entire contents of which are incorporated herein by reference). Common HLAs in the population can also be included in the new antigen prediction algorithm, such as HLA-A*02,24,01 in Caucasians; HLA-B*35,44,51; DRB1*11,13,07; HLA-A*02,03,30 in Afro-Brazilians; HLA-B*35,15,44; DRB1*13,11,03; and HLA-A*24,02,26 in Asians; HLA-B*40,51,52; DRB1*04,15,09. Specific pairs of HLA alleles can also be used. Common alleles found in the population are further described in Bardi et al. (Rev Bras Hematol Hemoter. 2012; 34(1):25–30.)

[0358] Other examples of methods for identifying new antigens include combining sequencing with mass spectrometry and MHC presentation predictions (e.g., U.S. Publication No. 2017 / 0199961), and combining sequencing with MHC binding affinity predictions (e.g., published U.S. Patent No. 9,115,402). In addition, methods that can be used to identify the presence or absence of new antigen-specific T cells in patient samples 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, all of which are incorporated herein by reference. These analyses produce a ranked list of patient candidate new antigen peptides (which can be easily synthesized using conventional methods) for screening cognate antigen-specific T cells.

[0359] Restriction digestion assembly

[0360] In general, the preparation of compACT polynucleotides can be accomplished by the procedures disclosed herein and recognized recombinant DNA techniques, such as preparation of plasmid DNA, cleavage of DNA with restriction enzymes, DNA ligation, transformation or transfection of hosts, culturing of hosts, and isolation and purification of expressed fusion complexes. Such procedures are generally known and disclosed in standard references, such as Sambrook et al., supra.

[0361] In some aspects, DNA encoding MHC class I heavy chains can be obtained from a suitable cell line, such as human lymphoblastoid cells. In various configurations, genes or cDNA encoding class I heavy chains can be amplified by polymerase chain reaction (PCR) or other means known in the art. In some aspects, the PCR product may also include a sequence encoding a joint, and / or one or more restriction enzyme sites for connecting such sequences.

[0362] In some embodiments, a vector encoding a compACT polynucleotide can be prepared by linking sequences encoding MHC class I heavy chain and β2-microglobulin to a sequence encoding an antigenic peptide.

[0363] The DNA encoding the antigenic peptide can be obtained by isolating DNA from a natural source or by a known synthetic method such as the phosphotriester method. See, for example, Oligonucleotide Synthesis, IRL Press (M. Gait, ed., 1984). Synthetic oligonucleotides can also be prepared using a commercially available automated oligonucleotide synthesizer. The DNA sequence encoding the 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, a ligase can be used to connect the segments.

[0364] PCR assembly

[0365] In some aspects, compACT can be assembled by polymerase chain reaction (PCR) amplification. Similar to the restriction digestion method, DNA encoding MHC heavy chain and β2-microglobulin can be obtained from a suitable source. A second DNA fragment encoding a selected signal sequence can also be obtained from a suitable source. The two fragments of DNA may have different universal target sequences, so that a primer for one universal sequence will not anneal to the second universal sequence. Two sequences encoding the selected antigenic peptide can be synthesized; a forward primer having an antigen sequence at its 5' end and a complementary sequence to the universal primer sequence on the MHC DNA fragment at its 3' end; a reverse primer having a reverse complementary sequence to the selected antigen sequence at its 5' end and a reverse complementary sequence to the universal primer from the signal sequence fragment at its 3' end. A PCR reaction with primers for all four DNA fragments and a 5' end signal sequence fragment and a 3' end MHC allele fragment will result in the amplification of two DNA fragments, one having a 3' end signal sequence and a 5' end antigen sequence, and the other having a 3' end antigen sequence and a 3' end MHC allele. Further PCR amplification cycles will anneal the overlapping antigen peptide sequences and produce 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.

[0366] Transfection, transduction and genetic modification of host cells

[0367] The compPACT polynucleotide can be inserted into the host cell by a known appropriate 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, polyethyleneimine (PEI), Fugene or any other transfection reagent that provides the best transfection rate based on cell type, transfection system, transfection type, transfection conditions and construct to be transfected. In some instances, Expifectamine is used to transfect mammalian cells with compPACT polynucleotides. In some instances, polyethyleneimine is used to transfect mammalian cells with compPACT polynucleotides. In some instances, FectorPro is used to transfect mammalian cells with compPACT polynucleotides.

[0368] The compPACT polynucleotide can be expressed transiently or stably in a host cell. In some embodiments, the compPACT polynucleotide is integrated into the host genome. In other embodiments, the compPACT polynucleotide remains extrachromosomal. Any suitable gene editing technology known in the art can also be used to modify host cells with compPACT polynucleotides, including CRISPR / Cas9, zinc finger nucleases, or TALEN nucleases.

[0369] Express

[0370] A variety of strategies can be used to express compPACT polypeptides. For example, compPACT polynucleotides can be incorporated into suitable vectors by known methods, such as by using restriction enzymes and ligases (see, e.g., Sambrook et al., supra). The vector can be selected based on factors related to the cloning scheme. For example, the vector can be compatible with the host being used and have a suitable replicon. Suitable host cells include eukaryotic cells and prokaryotic cells, and can be cells that are easy to transform and show rapid growth in culture medium. Examples of host cells include prokaryotes such as Escherichia coli and Bacillus subtilis, and eukaryotic organisms such as animal cells and yeast, such as mammalian cells and human cells. Non-limiting examples of mammalian cells that can be used as hosts to express compPACT include J558, NSO, SP2-O, 293T, Expi293 and CHO (and any derivatives or modifications of any 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 conventional culture conditions. See Sambrook et al., supra. In various embodiments, cells expressing compPACT polypeptides can be identified using known methods. For example, expression of the compPACT polypeptide can be determined by ELISA, FACS, or Western blotting. In certain embodiments, expression of the compPACT polypeptide can be determined by ELISA, FACS, or Western blotting using an antibody probe to the MHC heavy chain portion of compPACT or an antibody to an affinity tag such as His6 (SEQ ID NO: 34), or a streptavidin reagent (if compPACT has been biotinylated).

[0371] In some aspects, compACT is expressed in mammalian cells. The benefits of expressing proteins in mammalian cells rather than in E. coli cells are many. Proteins expressed in E. coli cells must be carefully purified from lipopolysaccharides (LPS). Expression of proteins in mammalian cells does not result in contamination of the purified protein with LPS. In addition, mammalian cells are more likely to correctly fold mammalian proteins because mammalian cells produce proteins with the correct post-translational modifications required for correct folding (including the correct formation of disulfide bonds). In addition, mammalian cells provide the correct chaperone proteins to assist in protein folding in the endoplasmic reticulum or Golgi apparatus. Compared to proteins expressed in E. coli cells, this results in increased purification of uniformly well-folded proteins.

[0372] In some aspects, compPACT is expressed in a prokaryotic cell. In certain embodiments, the prokaryotic cell has been genetically modified to post-translationally modify compPACT. In certain embodiments, compPACT expressed in a prokaryotic cell is substantially free of LPS or has no detectable LPS as measured using an LPS detection method known in the art.

[0373] comPACT can be substantially free of LPS. comPACT can be free of LPS, for example, comPACT can have no detectable LPS as measured using an LPS detection method known in the art. comPACT can be glycosylated. comPACT can have one or more post-translational modifications. comPACT can be modified by expression in a eukaryotic cell or in a specific embodiment in a mammalian cell, for example, by one or more post-translational modifications such as glycosylation. comPACT can include one or more post-translational modifications. comPACT can be (1) substantially free of LPS or free of LPS, and (2) glycosylated.

[0374] Example compPACT workflow

[0375] Fig.23An exemplary schematic diagram of the assembly and expression of compPACT protein is shown. Sense and antisense oligonucleotides encoding the desired neoantigenic peptide sequence are synthesized and annealed to form double-stranded oligonucleotides with overhangs at the 5' and 3' ends, which can then be ligated into a plasmid containing the β2M gene and MHC alleles. The complete compPACT oligo can be amplified into a double-stranded amplicon and transfected into cells for protein expression and optional biotinylation. The compPACT protein can be evaluated by SDS-PAGE. The compPACT polynucleotide can then be selected for amplification of plasmid production in E. coli. Protein production cells are transfected with the selected plasmid, and compPACT is then purified from the production cells for functional assays.

[0376] Purification (Chromatography)

[0377] The expressed compPACT polypeptide can be isolated and purified by known methods. For example, compPACT comprising a His6 affinity tag (SEQ ID NO: 34) can be purified by 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+)) and by known and published procedures. In addition, compPACT comprising human HLA sequences can be purified by affinity chromatography on a monoclonal antibody-Sepharose column by known and published procedures.

[0378] Method for isolating antigen-specific T cells

[0379] The compACT library described herein has been used to isolate antigen-specific T cells and can be used to isolate any cell that presents a new antigen. According to one embodiment, a schematic diagram of the T cell isolation process is shown in Fig.26 This process may also be referred to herein as 'imPACT' or the 'imPACT Separation Technology' method.

[0380] The steps and components of the imPACT separation technology method include but are not limited to Fig.26 Steps (1)-(5) shown in the figure:

[0381] (1) Creation of a compACT component library for patient-specific neoantigen T cell isolation

[0382] (2) Add unique DNA oligonucleotides, neoIDs, or barcodes to the compACT component library

[0383] (3) Each individual compACT peptide and its corresponding NeoID barcode, DNA oligonucleotide, neoID or barcode is linked to two separate fluorescent streptavidin proteins (in Fig.26 In the example provided, phycoerythrin (PE) and allophycocyanin (APC) are combined

[0384] (4) This assembly process results in two paired barcoded fluorescent tetramers for each compACT polypeptide and barcode element.

[0385] (5) Tetramer libraries assembled with all compACT peptides and NeoID barcodes, DNA oligonucleotides, neoID, or barcodes targeting each patient's predicted neoantigen candidates are combined and used to isolate neoantigen-specific T cells from the subject's peripheral blood.

[0386] The use of the compPACT library to identify and characterize neoantigen-specific T cells is also shown Fig.26 Small Figure 6-8 The compACT-neoID library is incubated with patient samples (6) followed by fluorescence-activated cell sorting (FACS) (7). A fixed number of T cells engineered to express the tool 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 nonspecific 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).

[0387] Barcode signal-to-noise ratio (S / N) analysis

[0388] True positive neoantigen-specific dual-labeled T cells can be resolved from identified false positive T cells by flow cytometry through sequence analysis of the neoID barcodes bound to isolated T cells. The presence of multiple copies of the same neoID barcode produces a high proportion of specific neoID 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 relatively equal numbers of different tetramer species, resulting in a lower proportion of different neoID barcodes. A schematic diagram of non-specific versus specific T cell binding is shown in Fig.27A (nonspecific) and Fig.27B (specificity). Numbers indicate different neoID barcodes. Fig.27A The ratio of the number of unique DNA copies of the most dominant neoID to the second most dominant neoID was 1, indicating cells that were nonspecifically bound by the compPACT element. Fig.27BThe ratio of the number of unique DNA copies of the most dominant neoID divided by the second most dominant neoID was 5, indicating that this T cell was engaged by the dominant compACT element and represented a true positive neoantigen-specific CD8 T cell. This could be further confirmed by functional characterization of T cells engineered with neoTCRs cloned from single cells.

[0389] S / N1 and S / N2 analysis

[0390] In some embodiments, one TCR may recognize two different neoantigens. In this case, even if the T cell is specific, the S / N ratio may be lower than 10. In this case, two different S / N calculations can be used, S / N1 and S / N2. S / N1 is the highest signal divided by the second highest signal, while 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.

[0391] In an illustrative example, 8 different TCRs can be identified in one sample. 6 of them may have an S / N1 ratio greater than 10 and can be confirmed as specific neoantigen T cells. For the other 2 T cells, the S / N1 ratio may be lower than 10. However, the S / N2 may be higher than 10. Cloning these 2 TCRs shows that they can recognize two different neoantigens that share the same mutation, explaining the reason for the low S / N1 ratio. In some embodiments, when there are multiple neoantigens derived from the same mutation, S / N2 analysis can be used to call nonspecific from a specific cell.

[0392] In some embodiments, a higher S / N ratio indicates higher TCR binding specificity.

[0393] Threshold

[0394] In some embodiments, if the barcode signal-to-noise ratio S / N1 or S / N2 ratio is above a threshold, the isolated T cell is identified as the antigen-specific T cell.

[0395] In some embodiments, the threshold value is at least or greater than 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. In some embodiments, the threshold value is at least or greater than 2, 5, 10 or 20. In some embodiments, the ratio corresponds to the specificity of the isolated antigen-specific T cells. In some embodiments, the S / N ratio is 10 or greater.

[0396] mark

[0397] As used herein, "identification marker" or "identification label" refers to a molecule or compound used to label a collection of particles. In some embodiments, the identification label is a fluorophore. In some embodiments, the identification 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 fluorophore, BODIPY fluorophore, DyLight fluorophore, FluoProbes fluorophore, or any combination thereof.

[0398] Barcode

[0399] As used herein, "barcode" or "barcodes" refers to a nucleotide sequence used to tag and identify specific peptides (including but not limited to antigenic peptides). In certain embodiments, the barcode is selected from the group consisting of 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer and 20-mer. In certain embodiments, the barcode is an 8-mer.

[0400] In some embodiments, different pairs of particles contain unique antigenic peptides and defined barcodes operably associated with the identity of the antigenic peptides.

[0401] In some embodiments, the first particle comprises a first barcode and the second particle comprises a second barcode that is different from the second barcode, wherein the first and second barcodes are associated with the identity of the antigen.

[0402] In some embodiments, a particle pair comprises a third particle comprising a third barcode that is different from the first and second barcodes, wherein the first, second, and third barcodes are associated with the identity of the antigen.

[0403] Exemplary barcodes and barcode structures are provided below in Table A. The barcode may also be referred to as a "neoID."

[0404]

[0405] In some embodiments, the barcode signal-to-noise ratio is based on at least one barcode. In such embodiments, each paired particle comprises the same antigen, a different label, and at least one barcode, wherein at least one barcode is associated with a new 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.

[0406] In some cases, aggregates of particles labeled with fluorophores can result in high mean fluorescence intensity of a single fluorophore staining cells during separation. This is not due to specific binding of the fluorescent particles, but rather to nonspecific binding of compACT particle aggregates, resulting in high neoantigen barcode S / N, as there may be a large number of identical barcodes nonspecifically binding to T cells. A dual barcode system can be used to address this issue.

[0407] In some embodiments, each particle pair of the comPACT library element comprises at least two barcodes.Dual barcoding Two different DNA barcodes for each antigen are conjugated to each comPACT tetramer, respectively. Fig.28 Comparison plots of single and dual barcoding for one antigen are shown. In the top plot, the same neoantigen is associated with two different particles, two different fluorophores, and a single barcode, labeled "1". In the bottom plot, the same antigen is associated with two different particles, two different fluorophores, and two different barcodes, labeled "1" and "2". This results in increased recognition of false positives with high signal-to-noise ratios caused by tetramer aggregation. The signal-to-noise ratio assigned to each fluorescent particle and each DNA barcode for the same antigen can be analyzed individually.

[0408] In some embodiments, each particle pair of the comPACT library element comprises at least two barcodes.Dual barcoding Two different DNA barcodes for each antigenic peptide are conjugated to each comPACT tetramer, respectively. Fig.28Comparison graphs of single and dual barcoding for one antigenic peptide are shown. In the top graph, the same antigenic peptide is associated with two different particles, two different fluorophores, and a single barcode, labeled "1". In the bottom graph, the same antigenic peptide is associated with two different particles, two different fluorophores, and two different barcodes, labeled "1" and "2". This results in an increased recognition of false positives with high signal-to-noise ratios caused by tetramer aggregation. The signal-to-noise ratio assigned to each fluorescent particle and each DNA barcode for the same antigenic peptide can be analyzed individually.

[0409] Cell samples

[0410] The imPACT method (i.e., the imPACT separation technique) can be used to separate immune cells, such as T cells and B cells, from any suitable patient-derived sample containing 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, blood cancers and liquid tumors, or any combination thereof. For example, both CD4+ and CD8+ T cells can be labeled and sorted from PBMCs or TILs using anti-CD4 and anti-CD8 fluorescent antibodies, and live cell populations of CD4+ and CD8+ single positive cells can be sorted using fluorescence activated cell sorting (FACS) to separate only CD4+ or CD8+ cells. In some embodiments, anti-CD3 fluorescent antibodies can be used followed by FACS to separate T cells that are positive for both CD4 and CD8. In addition, the imPACT method can also be used for antibody discovery of B cells. Those skilled in the art can determine the type of immune cells to be separated based on the type of compPACT protein 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 blood tumor sample. In some embodiments, the sample is a bone marrow sample. In some embodiments, the sample is a tumor sample comprising tumor infiltrating lymphocytes. The T cell can be a CD8+T cell or a CD4+T cell. In some embodiments, the T cell is a CD8+T cell. In some embodiments, the T cell is a CD4+T cell. In some embodiments, the T cell is a human T cell. In some embodiments, the T cell is a human CD8+T cell.

[0411] T cell isolation

[0412] In another aspect, provided herein is a method for isolating antigen-specific T cells, the method comprising the following steps: (a) providing a polypeptide comprising, in the amino-terminal to carboxyl-terminal direction, (i) a first universal target peptide, (ii) an antigenic peptide, (iii) a second universal target peptide different from the first universal target peptide, (iv) a β2M peptide, and (v) an MHC peptide, wherein the polypeptide is attached 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 comprises providing conditions sufficient for a single T cell to bind to the polypeptide attached to the particle, and (d) isolating the single T cell associated with the particle.

[0413] Isolating and identifying patient-derived and antigen-specific T cells using compPACT as described herein can include incubating the compPACT protein with patient-derived T cells or a sample containing patient-derived T cells. In some embodiments, a library comprising at least two compPACTs can be incubated with patient-derived T cells. T cells can be prepared starting from tissues such as blood, lymph nodes, or tumors using standard methods.

[0414] Incubation of comPACT or a comPACT library with a T cell suspension allows complete and thorough exposure of the particle-bound antigenic peptides to various T cell receptors. The method may include shaking or rotating the cells. In some embodiments, comPACT is associated with the particle.

[0415] After incubation of compPACT or a compPACT library (both bound to the particle) and T cells, the bound compPACT-T cell complexes are selectively isolated or selectively collected. T cells may bind to many identical copies of the same compPACT library element (i.e., a single compPACT polypeptide and a compPACT polypeptide associated with the particle) and can be separated based on these interactions. For example, if compPACT or a compPACT associated with a particle contains a fluorophore, or is attached to a particle with a fluorophore, fluorescence-associated cell sorting (FACS), including single cell sorting, can be used to selectively separate T cells. If compPACT or particles associated therewith are attached to magnetic particles, applying a magnet to the suspension can separate particles complexed with antigen-paired T cells and remove unpaired T cells. Alternatively, if the particles associated with compPACT are polystyrene particles, unpaired T cells can be separated by gravity (e.g., centrifugation). After removing unpaired T cells, in some embodiments, the separated bound particles are washed at least once to remove any non-specifically bound T cells.

[0416] The compPACT-bound T cells can also be separated by FACS into individual collection containers, such as multiwell plates. The individual collection containers can be single cell reaction containers. For example, components for downstream processing and analysis can be added to each single cell reaction container. The compPACT-bound T cells can be separated by FACS into a large volume collection container (e.g., each separated T cell is collected in the same container).

[0417] Compact-bound T cells can also be individually isolated in droplets using a droplet-generating microfluidic device (i.e., a "droplet generator"). Droplet-generating devices for encapsulating 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.

[0418] After the compPACT-binding T cells are separated into single-cell reaction vessels (e.g., separated in a single well or droplet), the nucleic acid of the compPACT-binding T cells can be further processed for downstream analysis. Specifically, the expressed TCRα and TCRβ mRNA transcripts can first be converted into cDNA by reverse transcription, and the amplified cDNA is used for next-generation sequencing (NGS) methods known to those skilled in the art, including but not limited to synthetic sequencing technology (e.g., Illumina or any other NGS sequencer).

[0419] Methods for identifying T cell antigen specificity

[0420] In certain embodiments, the subject matter of the present disclosure provides a method for identifying the antigen specificity of T cells. In certain embodiments, the T cell separation method described herein provides information about the antigen specificity of the separated T cells. For example, but not limited to, information about antigen specificity can be obtained by nucleic acid analysis of separated T cells. In certain embodiments, the nucleic acid of the separated T cells can be analyzed to determine the sequence of T cell receptor gene sequences (e.g., TCRα and TCRβ sequences). In certain embodiments, information about the antigen specificity of the separated T cells can be used for downstream applications. Non-limiting examples of downstream applications include immune repertoire analysis, manufacturing methods, and clinical follow-up of patients receiving immunotherapy. In certain embodiments, information about the antigen specificity of the separated T cells can be used to prepare reagents and compositions for manufacturing cells that can be used for adoptive cell transfer therapy.

[0421] In non-limiting embodiments, monitoring of the immune repertoire is performed. In certain embodiments, monitoring of the immune repertoire is performed before, during, or after treatment. In certain embodiments, 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, tumor infiltrating lymphocytes.

[0422] Treatment

[0423] In certain embodiments, the currently disclosed subject matter provides treatment methods, including but not limited to inducing and / or increasing an immune response in a subject in need thereof. In certain embodiments of the present disclosure, the treatment methods disclosed herein relate to the separation and / or administration of cells. For example, but not limited to, in certain embodiments, the cells used in the methods described herein can be obtained from a subject. In certain embodiments, the cells are tumor cells, non-cancerous cells, T cells, or any combination thereof. In certain embodiments, nucleic acids can be extracted from cells as described herein. In certain embodiments, the nucleic acids of the cells can be sequenced as outlined herein. In certain embodiments, information obtained from the subject, such as nucleic acid sequence information, provides information about antigen-specific T cells. In certain embodiments, the information relates to the identity (e.g., amino acid sequence) of an antigenic peptide. In certain embodiments, the antigenic peptide is a tumor neoantigen. In certain embodiments, the information relates to the identity of an MHC sequence.

[0424] 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 that can be treated by the methods described herein include, for example, carcinomas, lymphomas, sarcomas, blastomas, and leukemias. Non-limiting specific examples include, e.g., breast cancer, pancreatic cancer, liver cancer, lung cancer, prostate cancer, colon cancer, kidney cancer, bladder cancer, head and neck cancer, thyroid cancer, 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, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, testicular cancer, uterine cancer, cervical cancer, gastrointestinal cancer, mesothelioma, cancers associated with viral infection such as, but not limited to, human papillomavirus (HPV)-associated tumors (e.g., cervical cancer, vaginal cancer, vulvar cancer, head and neck cancer, anal cancer, and penile cancer).

[0425] In certain embodiments, a compPACT minigene comprising a candidate antigen-peptide is generated according to the methods disclosed herein. In certain embodiments, a compPACT polypeptide comprising an antigenic peptide is generated.

[0426] In certain embodiments, particles comprising a compPACT polypeptide are produced according to the methods disclosed herein. In certain embodiments, at least one collection of particles comprising a compPACT polypeptide are produced according to the methods disclosed herein.

[0427] In certain embodiments of the methods of treatment disclosed herein, T cells are isolated by binding to particles. In certain embodiments, T cells isolated in this manner are obtained from a subject. In certain embodiments, the T cells are CD8 + T cells.

[0428] In certain embodiments, the isolated T cells are sorted and their genomes are analyzed. In certain embodiments, the TCR sequence of the isolated T cells is obtained. In certain embodiments, the TCR gene sequence or a portion thereof is inserted into a homologous recombination template. In certain embodiments, the homologous recombination template includes the features described in International Patent Application No. PCT / US2018 / 058230, the contents of which are incorporated herein by reference in their entirety.

[0429] In certain embodiments, the T cell is modified by inserting a homologous recombination template comprising the TCR gene sequence of the isolated T cell or a portion thereof.

[0430] In certain embodiments, the modified T cells are adoptively transferred to the patient. Adoptive cell transfer (ACT) is an effective form of immunotherapy and involves transferring immune cells with anti-tumor activity into cancer patients. Lymphocytes used for adoptive transfer can be derived from blood or the 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. This administration can be by injection, such as intravenous injection. In certain embodiments, lymphocytes can be administered in multiple doses. Administration can be once, twice, three times, four times, five times, six times or more than six times a year. Administration can be once a month, once every two weeks, once a week or once every other day. Cytotoxic lymphocytes can continue to be administered 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, treatment is cancer treatment. In certain embodiments, cancer treatment is immunotherapy. In certain embodiments, immunotherapy includes administration of antibodies. In certain embodiments, immunotherapy includes 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 to provide targeted therapies.

[0431] Methods of modifying cells

[0432] 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.

[0433] In certain embodiments, the subject matter disclosed herein provides a method for modifying cells by introducing and recombining homologous recombination (HR) template nucleic acid sequences into the endogenous loci of cells. In certain embodiments, cells are modified by non-viral methods. 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 arm may be about 300 bases to about 2,000 bases. For example, each homology arm may be 1,000 bases. In certain embodiments, the homology arm may be homologous to the first and second endogenous sequences of the cell. In certain embodiments, the endogenous locus is a TCR locus. For example, the first and second endogenous sequences are in the TCR α locus or the TCR β 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, methods for identifying T cell antigen specificity can be used to obtain the sequence of a TCR gene from a patient, and the TCR sequence can be incorporated into a HR template. In certain embodiments, the HR template comprises a TCR alpha gene sequence and a TCR beta gene sequence. Additional information about HR template nucleic acids and methods for modifying cells thereof can be found in International Patent Application No. PCT / US2018 / 058230, the contents of which are incorporated herein by reference.

[0434] In certain embodiments, a construct comprising a target gene can be inserted into an endogenous locus using a non-viral gene editing method. In certain embodiments, this can be achieved by using a homologous repair template comprising a coding sequence of a target gene flanking the left and right HR arms. In certain embodiments, in addition to the HR arms, the target gene is sandwiched between a 2A peptide, a protease cleavage site located upstream of the 2A peptide to remove the 2A tag from the target gene translated upstream, and a signal sequence; wherein once integrated into the genome, the gene of the target expression gene cassette is transcribed into a single messenger RNA. In certain embodiments, during the translation of the target gene messenger RNA, the flanking region is disconnected from the target gene by self-cleaving the 2A peptide, and the protease cleavage site is cut to remove the upstream 2A sequence from the target gene of translation. In certain embodiments, in addition to 2A and protease cleavage sites, a glycine-serine-glycine (GSG) linker can be inserted before each 2A peptide to further enhance the separation of the target gene from other elements in the expression cassette. In certain embodiments, P2A peptides are used because they are superior to other 2A peptides due to their effective cleavage. In certain embodiments, two (2) P2A peptides and codon divergence are used to express a target gene without introducing any foreign epitopes from the remaining amino acids at either end of the target gene from the P2A peptides.

[0435] In certain embodiments and as described in PCT / US / 2018 / 058230, the neoTCR is integrated into the TCR alpha locus of a T cell. In certain embodiments, a homologous repair template containing a neoTCR coding sequence flanked by left and right HR arms is used. In certain embodiments, the endogenous TCR beta locus is destroyed, resulting in expression of only the TCR sequence 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.

[0436] In certain embodiments, the target TCRα locus (Cα) is shown together with the plasmid HR template, and the resulting edited sequence and downstream mRNA / protein products are shown in Fig.67A and 67BIn certain embodiments, the additional elements in the neoTCR cassette include: 2A = P2A ribosomal skipping element; F = furin cleavage site upstream of 2A, which removes the 2A tag from the upstream TCRβ protein; HGH = human growth hormone signal sequence. The HR template of the neoTCR expression gene cassette includes two flanking homology arms for direct insertion into the TCRα genomic locus targeted by the CRISPR Cas9 nuclease RNP with the TCRα 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 those skilled in the art that can be used. In certain embodiments, any signal sequence known to those skilled in the art can be selected based on desired trafficking and use.

[0437] In certain embodiments, once integrated into the genome, the neoTCR expression gene cassette is transcribed into a single messenger RNA from an endogenous TCRα promoter that still includes a portion of an endogenous TCRα polypeptide from the individual's T cells. In certain embodiments, during ribosomal polypeptide translation of a single neoTCR messenger RNA, the neoTCR sequence is disconnected from the endogenous CRISPR-destroyed TCRα polypeptide by self-cleavage at the P2A peptide. In certain embodiments, the encoded neoTCRα and neoTCRβ polypeptides are also disconnected from each other by cleavage by endogenous cellular human furin and a second self-cleavage P2A sequence motif contained in the neoTCR expression gene cassette ( Fig.67B ). In certain embodiments, the neoTCRα and neoTCRβ polypeptides are targeted to the endoplasmic reticulum by signal leader sequences (e.g., derived from human growth hormone, HGH) for multimeric assembly and transport of the neoTCR protein complex to the surface of T cells. In certain embodiments, the inclusion of a furin cleavage site helps to remove the 2A sequence from the upstream TCRβ chain to reduce potential interference with TCRβ function. In certain embodiments, the inclusion of a gly-ser-gly linker (not shown) before each 2A further enhances the separation of the three polypeptides.

[0438] In certain embodiments, the three repeated protein sequences are codon diverged within the HR template to promote genomic stability. In certain embodiments, within the TCR gene cassette, the two P2A codons diverge relative to each other, and the two HGH signal sequences are codon diverged relative to each other to promote the stability of the introduced new TCR cassette sequence within the genome of the ex vivo engineered T cell. In certain embodiments, the 5' end ( Fig.67A and 67B, vertical bars) reduces the likelihood that the entire cassette will be lost over time by removing the intervening sequence between the two direct repeats.

[0439] The presently disclosed subject matter further provides compositions comprising cells modified by the methods disclosed herein.

[0440] Exemplary embodiments

[0441] A. In certain non-limiting embodiments, the presently disclosed subject matter provides a method comprising: (a) contacting a sample with a plurality of different particle collections, wherein each particle comprises a unique antigenic peptide, an operably associated barcode, and at least one identification marker, wherein the sample comprises T cells, and wherein the contacting comprises providing conditions suitable for binding of a single T cell to a unique antigenic peptide of at least one particle collection; (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.

[0442] A1. The method of A above, wherein 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.

[0443] A2. The method of A or A1 above, wherein the unique antigenic peptide is the same for each different set of particles.

[0444] A3. The method of any of A-A2 above, wherein each different set of particles comprises at least one or more barcodes, wherein each barcode is associated with the identity of the antigenic peptide.

[0445] A4. The method of any of A-A3 above, wherein the ratio of each barcode corresponds to the antigen specificity of the isolated T cells.

[0446] A5. The method of any one of A-A4 above, wherein if the ratio of the first barcode is above a threshold, the isolated T cell is identified as an antigen-specific T cell.

[0447] A6. The method of A5 above, wherein the threshold is at least or greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-5, 3-6, 4-7, 5-8, 5-10, 7-10 or greater than 10.

[0448] A7. The method of any of A-A6 above, wherein identifying the barcode comprises a nucleotide-based assay.

[0449] A8. The method of A7 above, wherein the nucleotide-based assay is a PCR, RT-PCR, sequencing or hybridization assay.

[0450] A9. The method of A7 or A8 above, wherein the nucleotide-based assay determines (a) the sequence of each barcode and / or (b) the copy number of each barcode.

[0451] A10. The method of any of A-A9 above, further comprising obtaining a T cell receptor (TCR) CDR sequence.

[0452] A11. The method of any one of items A-A10 above, further comprising obtaining a TCR gene sequence.

[0453] A12. The method of A11 described above, wherein the TCR sequence is a TCRα or TCRβ chain sequence.

[0454] A13. The method according to any one of A-A12 above, which is used to identify the antigen specificity of T cells.

[0455] A14. The method of A13 above, wherein the antigen specificity of the T cell includes the sequence of the antigen peptide and the TCR sequence of the bound T cell.

[0456] A15. The method of any of A-A14 above, wherein the at least one identification label is the same in each different set of particles.

[0457] A16. The method of any of A-A15 above, comprising at least two different identification markers.

[0458] A17. The method of any of the preceding A-A16, wherein the at least one identification label is a fluorophore.

[0459] A18. The method of the aforementioned A17, wherein the fluorophore is selected from the group consisting of allophycocyanin (APC) and phycoerythrin (PE).

[0460] A19. The method of A18 above, wherein the at least two different recognition labels are fluorophores, wherein the fluorophores are selected from the group consisting of allophycocyanin (APC) and phycoerythrin (PE).

[0461] A20. The method of any one of the preceding A-A19, wherein the antigenic 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.

[0462] A21. The method of A20 described above, wherein the neoantigen is identified from tumor sequencing data of the subject.

[0463] A22. The method of A21 above, wherein a computer prediction algorithm is used to determine the new antigens.

[0464] A23. The method of A22 above, wherein the prediction algorithm further comprises an MHC binding algorithm to predict the binding between the new antigen and the MHC peptide.

[0465] A24. The method of any of A-A23 above, 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.

[0466] A25. The method of any one of the preceding A-A24, wherein the T cells are human T cells.

[0467] A26. The method of A25 above, wherein the T cells are CD8+ T cells.

[0468] A27. The method of any of the preceding claims A-A26, wherein the method comprises a library of different particle collections.

[0469] A28. The method of A27 described above, wherein the library comprises 2 to 500 different particle collections.

[0470] A29. The method of any of A-A28 above, wherein each particle comprises an MHC peptide.

[0471] A30. The method of A29 described above, wherein the MHC peptide is a human MHC peptide.

[0472] A31. The method of A29 described above, wherein the MHC peptide is a class I HLA peptide.

[0473] A32. The method of A29 described above, wherein the HLA peptide comprises an HLA-A, HLA-B or HLA-C peptide.

[0474] A33. The method of the aforementioned A32, wherein the HLA peptide comprises 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.

[0475] A34. The method of any of A-A33 above, wherein each particle comprises an HLA peptide and a β2M peptide.

[0476] A35. The method of A34 described above, wherein the β2M peptide is human β2M peptide.

[0477] A36. The method of A35 described above, wherein the β2M peptide comprises a mutation.

[0478] A37. The method of A36 described above, wherein the mutation is S88C.

[0479] A38. The method of any of A-A37 above, wherein each particle comprises a polypeptide comprising, in the amino to carboxyl terminal direction, (i) an antigenic peptide, (ii) a β2M peptide, and (iii) an MHC peptide.

[0480] A39. The method of any one of A-A38 above, 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.

[0481] A40. The method of A38 or A39 described above, wherein the polypeptide is biotinylated.

[0482] A41. The method of any of the preceding A-A40, wherein the particles are selected from the group consisting of magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles.

[0483] A42. The method of any of the preceding A-A41, wherein the particles are coated with streptavidin.

[0484] A43. The method of any of A-A42 above, which is used to monitor the immune repertoire in a subject.

[0485] A44. The method of A43 described above, further comprising monitoring changes in antigen-specific T cells in the subject.

[0486] A45. The method of A43 or A44 described above, comprising administering immunotherapy to the subject.

[0487] A46. The method of A45 described above, wherein the immunotherapy is adoptive cell transfer or a checkpoint inhibitor.

[0488] A47. The method of any one of A-A46 above, which is used to identify at least one TCR sequence.

[0489] A48. The method of A47 described above, wherein the at least one TCR sequence is a TCRα sequence, a TCRβ sequence, or a combination thereof.

[0490] A49. The method of A47 or A48 described above, further comprising producing a soluble TCR polypeptide.

[0491] B. In certain non-limiting embodiments, the presently disclosed subject matter provides a library comprising at least two collections of particles, each collection of particles comprising an antigenic peptide, a barcode operably associated with the identity of the antigenic peptide, and at least one identification tag.

[0492] B1. The library of B above, wherein the at least one identification tag is the same in each particle collection.

[0493] B2. The library of B or B1 above, comprising at least two different identification tags in each different particle set.

[0494] B3. The library of any of the aforementioned B-B2, wherein the at least one recognition label is a fluorophore.

[0495] B4. The library of the aforementioned B3, wherein the fluorophore is selected from the group consisting of allophycocyanin (APC) and phycoerythrin (PE).

[0496] B5. The library of the aforementioned B2, wherein the at least two different recognition labels are fluorophores, wherein the fluorophores are selected from the group consisting of allophycocyanin (APC) and phycoerythrin (PE).

[0497] C. In certain non-limiting embodiments, the presently disclosed subject matter provides a particle comprising at least one polypeptide, a barcode, and an identification tag, wherein the polypeptide comprises an antigenic peptide, a β2M peptide, and an MHC peptide, and wherein the barcode is operably associated with the identity of the antigenic peptide.

[0498] C1. The particles of the aforementioned C, which are selected from the group consisting of magnetic beads, agarose beads, styrene polymer particles and dextran polymer particles.

[0499] C2. The particle of C or C1 above, wherein the identification label is a fluorophore.

[0500] C3. The particle of any of C-C2 above, which is coated with streptavidin.

[0501] C4. The particle of any of the preceding C-C3, wherein the polypeptide is labeled.

[0502] D. In certain non-limiting embodiments, the subject matter of the present disclosure provides a method for treating cancer in a subject, comprising: (a) preparing a plurality of particles, each particle comprising a plurality of labeled polypeptides, wherein the polypeptides comprise 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 specific binding of the T cells to the particle antigen; (c) isolating T cells bound to the particles and identifying the TCR gene sequences of the isolated T cells; (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; (f) culturing the modified T cells of the step to produce a T cell population; and (g) administering a therapeutically effective number of the modified T cells to the subject, thereby treating cancer.

[0503] E. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for modifying a cell, comprising: (a) introducing a homologous recombination (HR) template nucleic acid sequence into the cell, wherein the HR template nucleic acid sequence comprises (i) first and second homology arms homologous to first and second endogenous sequences of the cell, (ii) a T cell receptor (TCR) gene sequence obtained by a method according to any one of A-A49, wherein the TCR gene sequence is located between the first and second HR arms, 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, wherein the first and second 2A coding sequences encode the same amino acid sequence that are codon divergent from each other; and (b) recombining the HR template nucleic acid into an endogenous locus of the cell, the locus comprising first and second endogenous sequences homologous to the first and second homology arms of the HR template nucleic acid.

[0504] F. In certain non-limiting embodiments, the presently disclosed subject matter provides a composition comprising a modified cell, 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 a method according to any one of 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, wherein the first and second 2A coding sequences encode the same amino acid sequence that are codon divergent from each other. Example

[0505] The following examples are merely illustrative of the presently disclosed subject matter and should not be considered limiting in any way.

[0506] Example 1 Design and cloning of the compACT minigene by restriction digestion cloning

[0507] Construction of the compACT minigene for restriction digestion:

[0508] The basic components of the comPACT minigene include a signal sequence that directs secretion of the encoded protein, a universal target sequence such as a restriction site or primer binding site, an encoded antigenic peptide (or neoantigen, NeoE), a second universal target site, β2m, an extracellular domain of an MHC allele, and a purification cluster, for example, to achieve enzymatic modification (e.g., biotinylation) and purification of comPACT via an affinity tag. The cluster may also contain a protease cleavage site and a linker sequence between the components shown in the figures and in the examples. The minigene may also encode a cysteine ​​mutation that can act as a disulfide trap. Certain comPACT minigenes prepared and disclosed herein encode cysteine ​​mutations that act as disulfide traps. In order to increase the success rate of making comPACT polypeptides by stabilizing the protein, such minigenes are made to include a disulfide trap. Figure 1 A schematic representation of the compACT minigene is shown. 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 MHC template libraries ( Figure 2 ). DNA encoding the signal sequence, universal target sequence, β2m and the extracellular domain of the MHC allele is the basic MHC template.

[0509] For the restriction digestion cloning approach, each compACT DNA construct is a basic MHC template with a virtual antigen sequence insert containing a stop codon in three frames and a unique restriction site for destruction of uncut or religated template ( Figure 3 ), and can be used as part of a ready-made platform for rapid assembly of a library of antigenic peptides complexed with that MHC allele. The MHC allele can also be modified or mutated (e.g., Y84A or Y84C), for example, to improve folding or increase binding of the antigenic peptide to the MHC protein. In addition, the β2m protein can also be mutated (e.g., S88C), for example, to allow it to bind to a thiol dye.

[0510] In this example, a compACT minigene having the following structure is shown: 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; an encoded linker sequence (i.e., a Gly-Ser linker) consisting primarily of glycine and serine residues; a β2m sequence; a second encoded Gly-Ser linker sequence with a BspI restriction site; an MHC heavy chain; a third encoded Gly-Ser linker sequence with a BstBI restriction site; and an encoded purification cluster with an AviTag (or any avidin / streptavidin) sequence, a TEV cleavage site, and a tandem histidine tag.

[0511]

[0512]

[0513]

[0514] Restriction digestion cloning and assembly of comPACT minigenes

[0515] This article describes three different methods for inserting encoded neoantigens by restriction digestion. In the first method, e.g. Figure 4 As shown, the primer encoding the antigen peptide (NeoE) spans the first restriction site at the 5' end (in this example, BlpI) and the second restriction site at the 3' end (in this example, BamHI). This primer amplifies the universal reverse primer encoding the second restriction site, generating a primer dimer of about 70 bp.

[0516] In the second method, the primer encoding the antigen peptide spans the second restriction site as the 5' end, and is the reverse complementary sequence of the antigen coding sequence. This primer is initiated in the reverse direction of the template DNA encoding the signal sequence. Paired with the forward primer that spans the first restriction site sequence, if a forward primer that spans the restriction site farther upstream of the antigen site is used, the reaction produces a product of 70bp, or a product of about 140bp.

[0517] In the first and second methods, the insert is typically cleaned on a commercial column, digested with an appropriate restriction endonuclease, cleaned again on a commercial 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.

[0518] Example 2 Design and cloning of the compACT minigene by primer annealing

[0519] 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 mimic the overhangs generated by restriction digestion ( Figure 5 ). The sense and antisense primers were incubated with T4 polynucleotide kinase and ATP to phosphorylate the 5' end ( Fig.22A ). When these primers anneal to each other, they form a double-stranded oligonucleotide sequence with overhanging nucleotides, as if it had been digested with a restriction enzyme.

[0520] The phosphorylated neoantigen insert (or neoepitope) was ligated to the pre-cut 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, and the complete comPACT minigene was amplified and sequenced using bookend universal primers. 824 comPACT minigenes with unique neoantigen sequences (or neoepitope sequences) were prepared using this method, of which more than 99% of the generated comPACT minigenes had the correct neoantigen sequence (or neoepitope sequence) ( Fig. 22B ). The neoantigen sequences (or alternatively referred to as neoepitope sequences) cloned into compPACT polynucleotides and expressed as polypeptides are 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 series of predicted neoantigen sequences (alternatively referred to as neoepitope sequences) are made for each identified neoantigen.

[0521] Next, E. coli is transformed with the ligation product plasmid and plated onto selective agar plates containing ampicillin. Single colonies are picked and grown overnight for plasmid purification and sequencing for complete genetic verification. After sequencing verification, plasmid batches are archived and multiplied into larger quantities.

[0522] Alternatively, if the pre-cut MHC template vector retains a 5' phosphate at its overhang, T4 kinase is not used. The annealed antigen insert neoantigen sequence (also called neoepitope insert) can then be ligated to the cut MHC vector and the ligation product transformed into E. coli for plasmid production.

[0523] Example 3 Design and cloning of compPACT minigenes by PCR assembly

[0524] Structure of the compPACT minigene for PCR assembly:

[0525] A fourth approach of inserting antigens, such as neoantigens, can also be used (for clarity, as used in this example, both refer to Figure 6 In this method, the antigen coding sequence (for clarity, as used in this example, the antigen coding sequence refers to the neo-epitope sequence described in Example 2) is inserted into the MHC template, which is flanked by an upstream promoter and a downstream polyadenylation signal to form a 2.5 kb minigene by polymerase chain reaction. Figure 6 shown.

[0526] In this example, the compACT minigene is shown to have 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 consisting primarily of 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. In this exemplary method, the universal target sequences are not identical, i.e., they are different from each other.

[0527] PCR assembly of compPACT minigenes:

[0528] In this method, two primers (<60nt) with a selected antigen sequence (for clarity, as used in this example, the antigen sequence refers to the neoantigen sequence) are synthesized. The first primer has a neoantigen sequence at the 5' end (for clarity, as used in this example, the neoantigen sequence refers to the neoepitope sequence described in Example 2), followed by a second universal target sequence segment at the 3' end. The second primer has the reverse complement of the neoantigen sequence at the 5' end (for clarity, as used in this example, the neoantigen sequence refers to the neoepitope sequence described in Example 2), and the reverse complement of the first universal sequence at the 3' end. These primers are mixed with a DNA fragment encoding a promoter region, a signal sequence, and a first universal target sequence, and another DNA fragment encoding a second universal target sequence, a β2m sequence, an MHC allele, a purification cluster, and a polyA sequence. Each antigen peptide primer anneals with its complementary sequence, and a PCR reaction is performed to amplify the neoantigen sequence (for clarity, as used in this example, the neoantigen sequence refers to the neoepitope sequence described in Example 2) to the promoter fragment or the MHC allele fragment. The two newly synthesized fragments now each have a neoantigen sequence (for clarity, as used in this example, the neoantigen sequence refers to the neoepitope 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 neoantigen sequences (for clarity, as used in this example, the neoantigen sequence refers to the neoepitope sequence described in Example 2) to anneal to each other and initiate the assembly of the full-length linear compPACT amplicon.

[0529] The fully assembled linear compACT polynucleotide is then cleaned for direct transfection into mammalian production cells, bypassing the use of E. coli and plasmid production steps.

[0530] Example 4 Expression and purification of comPACT protein in plasmid

[0531] Protein expression

[0532] Neoantigen12 (neo12) was ligated to the HLA-A2 template sequence and inserted into the expression plasmid (pPACT0010) by restriction digestion and ligation at NotI and BamHI restriction sites as previously described in Example 1.

[0533] On day -1, Expi293 mammalian production cells in a 30 mL shake flask volume were transfected with pPACT0010 incubated with Expifectamine 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 proteins were assessed by SDS-PAGE and total protein staining using Safestain (ThermoFisher). Secreted compPACT protein levels increased until day 3, when protein secretion plateaued ( Figure 7 The secreted compACT protein was initially identified by its apparent molecular weight (=53 kDa) and confirmed by Western blotting using NTA-HRP to detect the His6 affinity tag (SEQ ID NO: 34).

[0534] Protein purification

[0535] Neo12 comPACT protein collected on day 7 was purified by Ni-NTA affinity chromatography via binding of the His6 affinity tag (SEQ ID NO: 34). The total protein of the samples was analyzed by SDS-PAGE and Safestain. The lack of comPACT protein in the flow-through (FT) fraction of the affinity column confirmed that the His6 tag (SEQ ID NO: 34) was not cleaved during expression and purification ( Figure 8 ). The purified yield per liter of culture volume is >400 mg.

[0536] Neo12 comPACT protein was biotinylated (discussed in Example 5 below) and further purified by size exclusion chromatography. A single major peak was observed, indicating that the protein was properly folded and monomeric with little aggregation ( Fig. 9 ). The second peak is ATP, which is added for the biotinylation reaction catalyzed by BirA.

[0537] Although Ni-NTA chromatography was used in Example 4, any HA-affinity chromatography (including but not limited to the metal affinity chromatography described herein) can be used to purify HA-tagged compPACT.

[0538] Optimization of production volumes and parallel production

[0539] The production of comPACTs was scaled down from a 30 mL culture volume in a shake flask to 0.7 mL in a 96-deep well shake block. Expi293 mammalian production cells were transfected with plasmid DNA containing the pPACT0010 plasmid, and the secreted Neo12 comPACT protein was purified as previously described. 437 mg / L of purified Neo12 comPACT protein was collected from the 0.7 mL well volume, compared to a yield of >400 mg / L from the 30 mL purification experiment described previously ( Fig.10 ). The protein yield of the 0.7 mL experiment corresponds to >300 μg of protein, or approximately 1000-fold higher than required for a typical flow cytometry experiment.

[0540] Next, the parallel expression of multiple comPACT constructs was evaluated. Eight different comPACT constructs with different neoantigens (neoantigens 10, 15, 64, 65, 66, 67, 80, and 83) were expressed in 30 mL shake flasks as a medium-throughput assay ( Fig.11 Each comPACT construct was transfected into cells as described above, where the comPACT protein was expressed and secreted into the cell supernatant. The expressed protein was purified, concentrated, and standardized as described above. Total protein was determined for crude supernatant samples and concentrated protein as described above. The comPACT protein was purified by size exclusion chromatography ( Fig.12 A single peak containing 2-20 mg of protein was observed for each protein, also indicating that the compACT proteins were correctly folded and monomeric.

[0541] Example 5 Expression and purification of compACT protein from linear amplicon

[0542] In the previous examples, compPACT proteins were expressed from plasmids transfected into mammalian production cells. As an alternative, a linear amplicon of the neo12compPACT minigene (neoantigen 12 assembled into a minigene with an HLA-A2 template sequence) flanked by a promoter sequence and a polyA sequence was transfected into 0.7 mL of production cells in a 96-deep well plate. As a control, the pPACT0010 plasmid was also transfected into separate production cells. As previously described, proteins from both samples were expressed, purified, and assayed for total protein. Both the linear amplicon and the plasmid produced similar levels of expressed protein ( Fig.13A ), demonstrating that the protein encoded by the compPACT minigene can be produced without the need for a plasmid intermediate. Several different compPACT minigenes with different neo-epitope sequences have been generated ( Fig. 13B ) for direct transfection of producer cells.

[0543] Additional compPACTs with different HLA alleles were prepared using the annealing and phosphorylation workflow described in Example 2. Linear amplicons were obtained from the expression vector using bookend PCR and universal primers and transfected into Expi293F cells for compPACT protein production (data not shown).

[0544] Example 6 Biotinylation of comPACT protein

[0545] In vitro biotinylation of compPACT with isolated BirA enzyme

[0546] The comPACT purification cluster includes a BirA recognition sequence (Avitag) for biotinylation. Purified comPACT protein was either not biotinylated (no BirA treatment) or biotinylated with commercial BirA protein according to the manufacturer's instructions (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 the biotinylated protein to bind to the streptavidin beads. The supernatant (SN) and beads ("pellet", P) were separated by SDS-PAGE. Samples were assayed for total protein with Safestain, and the presence of comPACT protein was determined by western blotting and NTA-HRP ( Fig.14). In the untreated samples, compPACT protein was found primarily in the SN fraction, confirming that it was not biotinylated. In the biotinylated samples, compPACT protein was found in both the C1 and T1 streptavidin beads pelleted samples, although the interaction between the biotinylated protein and the C1 streptavidin beads was most complete. No biotinylated compPACT protein was detected by western blotting in the C1 streptavidin beads depleted supernatant, indicating that approximately 100% of the compPACT protein was biotinylated.

[0547] The compPACT proteins can also be biotinylated in the clarified supernatant prior to purification. As described previously, multiple compPACT proteins were expressed in production cells. Cell culture supernatants were collected and clarified by centrifugation. The clarified supernatant was treated with commercial BirA protein according to the manufacturer's instructions, then purified by Ni-NTA affinity chromatography and assessed for biotinylation by western blotting ( Fig.15 ). All tested compPACT proteins were biotinylated using this method, indicating that biotinylation of compPACT 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 described herein) can be used to purify HA-tagged compPACT.

[0548] In order to produce enough BirA for high-throughput biotinylation of compACT protein, BirA protein with His6 tag (SEQ ID NO:34) was expressed in E. coli cells. This BirA with His6 tag (SEQ ID NO:34) was purified by Ni-NTA affinity chromatography ( Fig. 16B ) and can be used to biotinylate compPACT protein. A second version of BirA-His6 (disclosed as "His6" of SEQ ID NO: 34) with a TEV-cleavable His6 tag (SEQ ID NO: 34) was also expressed and purified by Ni-NTA affinity chromatography ( Fig. 16C ). This BirA-TEV-His6 protein (disclosed as "His6" of SEQ ID NO:34) can be purified by Ni-NTA, the His6 tag (SEQ ID NO:34) can be removed by TEV cleavage, and the untagged BirA can then be used for biotinylated compPACT protein. After biotinylation of the compPACT protein, the untagged BirA protein can be purified by Ni-NTA affinity chromatography. In addition, TEV protease was heterologously expressed in E. coli to bind to BirA-TEV-His6 (disclosed as "His6" of SEQ ID NO:34) ( Fig.16A) are used together for biotinylated compPACT protein production. Although Ni-NTA chromatography was used in Example 6, any HA-affinity chromatography (including but not limited to the metal affinity chromatography described herein) can be used to purify HA-tagged compPACT.

[0549] The cleavage of the His6 tag (SEQ ID NO: 34) on the compPACT protein after biotinylation was also evaluated, and the results were as follows Fig.17 The comPACT protein was treated with BirA or not for biotinylation as described previously ( Fig.17 Lanes 1 and 2 of the Figure 10). A third sample of the compPACT protein was treated with BirA and then TEV to cleave the His6 tag (SEQ ID NO: 34) present on the protein (lane 3). The samples were separated by SDS-PAGE and the total protein was assessed by Safestain. All three samples contained equal amounts of compPACT protein. Biotinylation of the comPACT protein and cleavage of the His6 tag (SEQ ID NO: 34) were assessed by Western blotting using SA-HRP reagent for the biotin signal and NTA-HRP reagent for the His6 tag (SEQ ID NO: 34). The non-biotinylated sample did not show a biotin signal, but did have a His6 signal (SEQ ID NO: 34), and the biotinylated and non-cleaved sample had both signals. The biotinylated and TEV-cleaved sample only had a biotin signal, indicating that the His6 tag (SEQ ID NO: 34) had been successfully cleaved from the compPACT protein.

[0550] A third approach to biotinylating compACT in vitro was to express BirA in Expi293 producer cells. Expi293 cells co-expressing BirA and cell surface transduction labeled with V5 were generated. Transduced cells sorted for V5+ also expressed BirA ( Fig.18 ). These cells can be used to produce biotinylated compPACT in vivo prior to compPACT protein purification.

[0551] Example 7 Antigen-specific T cell staining and affinity assessment using compACT protein

[0552] To compare antigen-specific T cell staining using compPACT proteins and conventional peptide-MHC, compPACT 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 A2 / neo12-specific TCRs and stained with either HLA-A2 / neo12 peptide-MHC dextramers or HLA-A2 / neo12 peptide compPACT dextramers. Staining with compPACT dextramers was at least as effective as staining with peptide-MHC dextramers ( Fig.19 ). This data suggests that compPACT dextramers can be used to sort antigen-specific T cells for TCR sequencing.

[0553] Example 8 Functional T cell assay

[0554] In addition to antigen-specific capture of T cells, the modular design and ease of production of compPACTs facilitate their use in functional T cell assays. For example, incorporation of a mutant version of β2m (S88C) enabled compPACT to be labeled with a maleimide dye conjugate, assembled as an NTAmer, and used to measure kinetic parameters of TCR-comPACT binding. S88C-mutated compPACT proteins were constructed and expressed at ~150 mg / L. These mutant compPACTs exhibited similar purity and elution profiles to unmutated compPACT ( Fig. 20 Other dyes such as Cy5 can also be conjugated to S88C comPACT ( Fig.21 ).

[0555] Example 9 comPACT library production

[0556] HLA allele diversity in the U.S. population was analyzed by bioinformatics from the Allele Frequencies Network database (www.allelefrequencies.net) to identify the optimal number of alleles included in the HLA repertoire to achieve high coverage of the subject's HLA frequencies. 9736 alleles were analyzed. Fig.24A An analysis of the percentage of patients in which one or two alleles from each of the HLA A, B, and C loci are covered by the 66 HLA allele library is shown. The solid line indicates that 1 allele is covered, while the dashed line indicates that two alleles are covered. The 66 alleles enable 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 population ( Fig. 24B ). The most common HLA-I allele is HLA-A02:01, with a prevalence of approximately 50% in the United States. Therefore, the HLA library presented here for the first time has the greatest potential to enable the widespread implementation of personalized NeoTCR-T cell therapy for different populations around the world.

[0557] Next, a compPACT protein library with different neo-epitopes and selected HLA alleles was prepared. The neo-epitope 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 list. Ten neo-epitope peptides were selected from the IEDB database and inserted into the group of 36 HLA alleles. The compPACT polypeptides expressing the selected neo-epitopes and HLA alleles were purified by a size exclusion chromatography column (Agilent Sec Bio 300) (SEC-HPLC) connected to an Agilent Infinity II HPLC system according to the manufacturer's instructions. The results are shown in Fig.25A The compACT polypeptide was purified as a monodisperse polypeptide ( Fig.25A and 25B Most compACT peptides are expressed at high titers ( Fig.25C ). At least one comPACT protein for each HLA allele described has been purified and characterized by HPLC, demonstrating that the comPACT platform is robust and applicable to many alleles.

[0558] Example 10 Impact T cell isolation method

[0559] Materials and methods

[0560] comPACT library preparation

[0561] Paired PE and APC tetramer particles with three compPACT library elements and barcodes were prepared before the experiment. Biotinylated compPACT (1 μM, generated in-house) and DNA barcodes (1 μM, IDT) were mixed at a molar ratio of 3:1. PE-streptavidin (3.33 μM, Life Technologies) or APC-streptavidin (6.26 μM, Life Technologies) were added to react with biotin at a ratio of 1:4. After incubation, additional biotin was introduced to occupy free streptavidin sites.

[0562] CD8 T cell staining

[0563] Cells were incubated with 40 nM fluorescent compACT tetramer for neoantigen-specific T cell staining. Fc receptor blocking solution was then added to minimize nonspecific 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 the phenotype of T cells.

[0564] Single cell sorting

[0565] 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 with 10 mM Tris and RNAse inhibitor (Promega).

[0566] TCR cloning

[0567] A RT-PCR master mix containing the following reagents was prepared: nuclease-free water (Invitrogen), 5x buffer (Qiagen), 10 mM dNTPs (Qiagen), alpha multiprimer mix, beta multiprimer 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.

[0568] Sensitivity and S / N determination

[0569] CD8 T cells expressing TCRs against the MART1 antigen (F5) or the neoantigen neo12 were incubated with fluorescent comPACT particles bearing the corresponding comPACT neoantigen element (MART1 or neo12). Cells were stained and sorted by FACS as described above ( Fig.29A -C).

[0570] CD8 T cells expressing TCRs against the neoantigen neo12 were spiked into control PBMC samples at a ratio of 1:300,000. The spiked samples were incubated with a 33-tetramer library that included neo12 comPACT and 32 additional neoantigen comPACT elements. Single cells were sorted based on the gating strategy described above for APC and PE dual-labeled CD8 T cells ( Fig.30 ).

[0571] Specificity and S / N determination

[0572] CD8 T cells expressing TCRs against the neoantigen neo12 were spiked into control PBMC samples at a ratio of 1 to 300,000 or 1 to 30,000. PBMCs alone were used as negative controls. The spiked 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 dual-labeled CD8 T cells. The barcodes and the signal-to-noise ratio of each barcode associated with a given cell were determined.

[0573] result

[0574] Sensitivity

[0575] Fig.29A A schematic diagram of the endogenous TCR gating strategy used to isolate PACT neoantigen CD8 T cells (top) and tetramer-positive double-labeled T cells (bottom) is provided. Fig.29B ) or Neo12( Fig.29C ) T cells were labeled and gated according to the gating strategy. The corresponding tetramer staining produced an accuracy of more than 99% for F5 and Neo12 compPACT neoantigen T cells. Repetition of this assay resulted in tetramer staining of gene-edited F5 and neo12 T cells exceeding 99% (data not shown). Therefore, the imPACT method has a high staining and sorting sensitivity of greater than 98% or 99%.

[0576] To test the sensitivity of the imPACT tetramer method, a cell doping experiment was performed. T cells expressing the new antigen neo12 were doped into the control PBMC sample at a ratio of 1 to 300,000 (1 neo12 T cell per 300,000 PBMCs). The imPACT tetramer analysis was performed using tetramers prepared from neo12 compPACT and 32 unrelated control compPACTs. Cells that were positive for 33 compPACT tetramers were sorted from the CD8 T cell gating as described above. The cells were sequenced for the 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 of this example is the DNA copy number of the most dominant neoID divided by the DNA copy number of the second most dominant neoID. In this example, an S / N greater than 10 is considered to be a specific binding of compPACT to T cells. The indexed flow results of IP26 staining for each cell indicate whether a given cell is gene-edited or naive.

[0577] The cells sorted from this experiment are summarized in Table 4. 1686,717 total cells were analyzed by flow cytometry and 11 cells were sorted from the positive gate.

[0578]

[0579] Five of the 11 positive cells had an S / N higher than 10 (average S / N = 83.1), while the other six had an S / N lower than 10 (average = 1.1). Based on the ratio of Neo12 doped cells (1:3000,000) and the number of cells treated (1,686,717), there should be approximately 5-6 neo12 cells in the analyzed sample. The sequencing results showed that 5 neo12 cells were isolated using this method. Therefore, the imPACT tetramer method is sensitive enough to isolate antigen-specific cells at a low frequency of 1 / 300,000. Fig.30 Gated FACS cells of a 1:300,000 spiked sample are shown. TCR- indicates neo12 positive T cells, while TCR+ indicates non-specific binding T cells. The average S / N ratio of specific neo12 cells was 83, while the average S / N ratio of non-specific cells was 1.1.

[0580] Specificity

[0581] Next, the neoantigen specificity of the imPACT isolation method was evaluated. The neo12 doping assay was repeated using a second library containing Neo12 comPACT spiked into PBMC samples and 28 unrelated control comPACTs. PBMCs alone were used as negative controls. Double-positive cells were isolated, barcodes were sequenced, and the S / N ratio for each barcode associated with a given cell was determined. Fig.31 PE and ACP FACS gating data for neo12 antigen doped PBMC samples and undoped PBMC samples are shown. Tables summarizing the barcode S / N sequencing results are shown below each test. In the specific test, the S / N average was 162, indicating that there were 162 copies of the neo12 neoID barcode for each non-neo12 neoID barcode. This indicates that the neo12 barcode is highly specific for sorting cells from neo12 and PBMC samples. In contrast, the S / N average in the non-specific test was 1.7, indicating that there were only 1.7 copies of the neo12 neoID barcode for each non-Neo12 neoID barcode. This indicates the low specificity of the neo12 barcode for sorting cells in PMBC-only samples.

[0582] The assay was repeated with a compACT library with 33 elements added to PBMC samples at a ratio of 1 neo12 T cell to 30,000 PBMCs. Fig.32A Gated double-positive cells are shown, while Fig.32B The barcode S / N averages for specific and nonspecific T cells are shown. The signal-to-noise ratios of 33 tetramer-positive cells were determined. The average S / N for T cells with specific binding was 124.9, while the nonspecific S / N ratio was 1.2, confirming the high specificity of the imPACT method for isolating neoantigen-specific T cells. Table 3 summarizes the information of the sorted cells and whether the isolated cells were genetically modified (gene edited).

[0583]

[0584]

[0585] Example 11 Isolation of neoantigen T cells from patient samples

[0586] Materials and methods

[0587] Tetramer preparation

[0588] Tetramers were prepared as described previously.

[0589] CD8 selection and cell staining

[0590] 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. Cells were incubated with 40nM fluorescent compACT tetramer library for NeoE-specific T cell staining. Fc receptor blocking solution was then added to minimize nonspecific antibody staining. The samples were incubated with an antibody cocktail containing FITC CD4, CD14, CD19, CD20, CD40, PerCp-Cy5.5 CD8, BV711CD45RA, BV786 CD95 and BV510 IP26 (Biolegend) to identify the phenotype of T cells. 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 live and apoptotic cells.

[0591] Single cell sorting and TCR cloning

[0592] Fluorescently labeled cells were sorted into single cells using a FACSAria III (BD Biosciences). Live CD8+ tetramer-positive cells were sorted into 96-well plates containing lysis buffer containing 10mM Tris and RNAse inhibitors (Promega). The cells were then frozen for subsequent TCR cloning. An RT-PCR master mix containing the following reagents was prepared: nuclease-free water (Invitrogen), 5x buffer (Qiagen), 10mM dNTP (Qiagen), α multi-primer mixture, β multi-primer mixture, α antisense primer, β 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 append adapter sequences for next-generation sequencing (NGS).

[0593] Next-generation sequencing

[0594] Next generation sequencing was performed on 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.

[0595] result

[0596] First, a stage IIIA melanoma patient sample (PACT032) was analyzed using a 26-element comPACT library with the HLA A02:01 allele type. 6 Of the 10 PBMCs, 231 were doubly positive for APC and PE. The cells were analyzed for neoID barcodes and the signal-to-noise ratio of all doubly positive cells was determined. Fig.33A FACS dot plots of double positive T cells are shown. After neoID sequencing, one T cell showed specificity for one mutation with a signal-to-noise ratio (S / N1) greater than 10. This neoantigen TCR was cloned and screened against the predicted neoantigen. The remaining double positive cells all had a signal-to-noise ratio of 1 and had no specificity for the neoantigen associated with the bound compACT. Secondary screening ( Fig.33B ) confirmed the specificity of the neoantigen TCRs isolated by impact analysis. Table 5 below provides a summary of the signal-to-noise ratios for specific and nonspecific T cells.

[0597] Table 5

[0598] TCR clonotype count S / N(UMI) TCR+ 230 1 PACT32-TCR75 1 13

[0599] Example 12 S / N1 and S / N2 analysis to identify TCRs

[0600] Next, a stage III melanoma patient sample (PACT077) was analyzed using a 138-element comPACT library with HLA A02:01, A24:02, B18:01, and C07:01 types. 6 Of the PBMCs, 250 were double positive for APC and PE. The cells were analyzed for neoID barcodes and the signal-to-noise ratio of all double positive cells was determined. Fig.34A FACS dot plots of double positive T cells are shown. Fig.34D Neoantigen-specific T cells identified in peripheral blood are shown. Asterisks indicate that the same TCR clonotype was found in tumor-infiltrating lymphocytes (TILs) by tumor sequencing. After neoID sequencing, 25 T cells showed specificity for a mutation with a signal-to-noise ratio (S / N1) greater than 10 ( Fig.34C ). Fig.34B All candidate cells were shown to be derived from antigen experienced CD95+ cells. Neoantigen TCRs were cloned and screened against predicted neoantigens. Fig.34E The percentage of neoTCR gene-edited lymphocytes that recognize the cognate antigen is shown. The remaining double-positive cells all have a signal-to-noise ratio of 1 and are not specific for the neoantigen associated with the bound compPACT. Table 6 below summarizes the signal-to-noise ratios of T cells against selected neoantigens.

[0601] Table 6

[0602] SEQ ID NO: Neoantigens Average S / N 203 EYIPGTTFL 25 204 IYNIIVTTL 43 205 KTSVALHLI 19 206 HLSLELLGVD 21 207 DEYIPGTTF 32

[0603] Interestingly, analysis of TCRs from PACT077 identified 8 different TCRs. 6 of them had an S / N1 ratio greater than 10 and were confirmed to be specific neoantigen T cells. For the other 2 T cells, the S / N1 ratio was lower than 10, but the S / N2 ratio was higher than 10 ( Fig.34C ). Cloning of two TCRs (TCR143 and TCR164) showed that they could recognize two different neoantigens that shared the same mutation, further explaining the low S / N1 ( Fig.35A ). These results suggest that the S / N2 ratio can be used to distinguish nonspecific cells from specific cells when multiple neoantigens derived from the same mutation are present.

[0604] Secondary screening ( Fig.35B ) confirmed the specificity of the neoantigen TCRs (i.e., TCR135, TCR136, TCR139, TCR142, TCR144, and TCR145) isolated by imPACT analysis.

[0605] Fig.36 Isolated TCRs were shown to vary between mutations, with different levels of clonality, truncation, and in situ neoantigen expression.

[0606] Example 13 Isolation of neoantigen T cells using dual NEOID barcodes

[0607] Materials and methods

[0608] Tetramer preparation

[0609] Paired fluorescent tetramer particles were prepared as described above, except that each particle pair had a different unique neoID barcode associated with a neoantigen (see Fig.28 Figure 3. Dual-barcoded paired particles.

[0610] CD8 selection and cell staining

[0611] 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 described previously.

[0612] Single cell sorting and TCR cloning

[0613] Fluorescently labeled cells were sorted into single cells using FACSAria III (BD Biosciences) as described previously.

[0614] Next-generation sequencing

[0615] Next generation sequencing was performed on a Miniseq (Illumina) using recommended reagents as described previously.

[0616] result

[0617] PACT049 (stage 4 CRC, naive) PBMCs were screened using the imPACT approach and dual barcoding. A six-element dual-barcoded compPACT library (HLA-B57:01, A01:01, and C06:02) was generated and used to interrogate neoantigen TCRs. 352 single cells were sorted. After sequencing analysis, three neoantigen TCR candidates were identified for one HLA-B57:01 neoantigen, RCSPEQLKKAW (SEQ ID NO:208) Fig.37A ). These three candidates were sorted based on tetramer MFI (mean fluorescence intensity) and were all considered to be antigen-experienced CD95+ ( Fig.33B After cloning PACT049 neoantigen T cells, these TCRs were confirmed by dextramer staining with the relevant predicted neoantigens ( Fig.37C and Fig.37D ).

[0618] Example 14 Additional Isolation of Neoantigen T Cells from Patient Samples

[0619] Additional patient samples were incubated with the comPACT library and separated according to the imPACT method described above. Patient samples were analyzed using the imPACT signal-to-noise method and single or dual barcode methods. Fig.38A A plot of the number of neoantigens and HLA types identified from each sample and cancer type is provided in Fig.38BHLA types are listed in . 32 neoantigen-specific TCRs were identified in five cancer types and 13 HLA types from the periphery. Neoantigens were identified in samples of colorectal cancer (11), melanoma (7), bladder cancer (3), endometrial adenocarcinoma (1), and head and neck cancer (1). Two patient samples (PACT056 and 095) did not produce any neoantigen-specific TCRs. Four patient samples (PACT032, 052, 053, and 078) had 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 enable selection of the best TCR for the patient. Two samples (PACT077 and 078) had peripheral TCRs that were also discovered in situ by deep sequencing of TILs. These results showed that the success rate of neoantigen-specific TCR identification was 100% in patients receiving drug treatment and greater than 80% in patients not receiving treatment.

[0620] The results showed that the imPACT technology can successfully isolate antigen-paired, neoantigen-specific TCRs from patient samples with high precision and specificity. Because the imPACT technology targets neoantigens and the antigen presentation pathway is universal, the imPACT platform technology can be applied to different cancer types, enabling the development of personalized NeoTCR-T cell therapies to eradicate solid tumors.

[0621] Example 15 Reproducibility of T cell isolation method

[0622] Next, the PBMCs of healthy donors were analyzed using the compPACT element library. 15 paired fluorescent compPACTs (HLA A02:01) with new antigens for cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza were prepared as described previously. The compPACT library was incubated with PBMCs, and double-positive T cells were sorted and isolated. As described previously, the neoID barcodes were sequenced, and the TCRs were cloned and sequenced. The experiment was performed in triplicate.

[0623] Fig.39A The percentage of antigen specificity of isolated T cells with neoantigens against CMV and EBV in each of three replicates is shown. Fig.39BThe number of TCRα chains isolated from each experiment is shown. Table 7 provides a summary of the identified TCRα chains. 14 unique TCRα chains were identified, and 10 of the 14 were shared across all three experiments. This reproducibility experiment shows that the imPACT method can consistently isolate antigen-specific T cells at similar levels from the same sample in independent experiments, indicating that the method of isolating double-positive T cells by incubating cells with paired compPACT tetramers with different fluorophores is highly reproducible in multiple settings.

[0624] Table 7

[0625]

[0626] Example 16 Comparison of T cell separation using compACT tetramers, dextramers, and trimers

[0627] The separation efficiency of tetramers, dextramers, and trimers of compPACT library elements in a dual staining approach was evaluated. Tetramers of streptavidin cores with four copies of each compPACT element (tetramers), trimers of streptavidin cores with three copies of each compPACT element and a nucleic acid barcode (trimers + DNA), and dextramers of dextran polymers with multiple copies of compPACT elements (with and without nucleic acid barcodes) (dextramers and dextramers + DNA) were incubated with T cells that had been 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 Fig.40A (F5 T cells) and Fig.40B (neo12 antigen-specific T cells) As shown, more than 98% of the gene-edited cells were stained with the corresponding compACT element. The data show that for T cells with a common TCR (F5 Mart-1) and a new antigen TCR (neo12), tetramers, dextramers, trimers containing DNA, and dextramers containing DNA achieved similar staining efficiencies.

[0628] Example 17 Comparison of signal-to-noise analysis

[0629] PACT Neo12 T cells, PACT M1W T cells, and virus donor PBMCs were incubated with trimer comPACT particles with nucleic acid barcodes (Trimer+DNA) and dextramers with nucleic acid barcodes that have multiple copies of the comPACT element (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 described previously. For each sample, the S / N ratio for each method (trimer, T; or dextramer, D) is shown in Fig.41A middle. Fig.41B The average S / N ratios for each method are provided in . Of note, analysis of the signal-to-noise ratio of neoID barcoded DNA showed that cells isolated with Trimer+DNA particles had a higher signal-to-noise ratio compared to cells isolated with Dextramer+DNA. The data suggest that Trimer+DNA particles have a better S / N ratio than Dextramer+DNA.

[0630] Example 18 Isolation and Characterization of Neoantigen T Cells from Patient Samples Following Cancer Immunotherapy

[0631] Subjects with pMMR colorectal cancer (generally considered unresponsive to anti-PD1 antibody therapy) or endometrial adenocarcinoma were treated with AB122 (anti-PD-1 antibody). Pre-treatment blood samples were incubated with the compPACT library and separated according to the imPACT method described above to identify the baseline repertoire of neoantigen-specific T cells. PBMCs were then collected at different time points and analyzed by the imPACT signal-to-noise method to monitor the therapeutic evolution of the mutation-targeted T cell repertoire. Changes in the neoantigen-specific T cell repertoire during AB122 treatment were monitored to achieve correlation of the immune phenotype with clinical outcomes.

[0632] result

[0633] Fig.42A -C shows the effect of PACT157 ( Fig.42A ) and PACT132( Fig.42B ); or endometrial cancer PACT131( Fig.42C ) Longitudinal evolution of neoantigen-specific T cells in peripheral blood during the course of the patients.

[0634] Fig.42A -C shows the neoantigen clonality and predicted neoantigen-HLA binding affinity for each sample. The top dots represent clonal mutations, while the bottom dots represent subclonal mutations.

[0635] Fig.42A -C also shows the gene, HLA type, and neoantigen sequence of each TCR identified in each subject by the imPact method.

[0636] Longitudinal monitoring of patients during treatment could enable analysis of T cells targeting neoantigens and identification of driver mutations associated with treatment benefit. In addition, monitoring changes in the repertoire of neoantigen-specific T cells in response to immunotherapy could inform next steps in treatment.

[0637] Example 19 Phenotypic and functional characterization of new antigen-specific T cells from PACT131

[0638] T cells isolated from patient sample PACT131 by the imPACT separation technology method were characterized by flow cytometry for cell surface markers CD45RA, CD95, CD39, and CD103. Flow cytometry results of T cells isolated from patients on days 1, 15, and 57 are shown in Figure 2. Fig.43 Black dots represent neoantigen-specific T cells. CD45RA+CD95+ T cells experienced antigen, while CD39+CD103+ positivity indicated that T cells had trafficked through the tumor compartment.

[0639] Next, three TCR clones (TCR200, TCR202, and TCR205) directed against the same PIK3CA neoantigen target captured from patient samples were characterized. T cells were edited to express the selected TCRs. Fig.44A The percentages of live, CD8+, and CD4+ T cells are shown in Figure 2. Activation of neoantigen-specific T cells was determined by incubating edited T cells with increasing amounts of HLA-cognate peptides and measuring the secretion of IFNγIL2 and TNFα. Cytokine release of each TCR clone is shown in Figure 2. Fig.44B All T cells were activated by cognate neoantigens. No cytokine release was detected against non-cognate neoantigens (data not shown).

[0640] Example 20 Validation of NEOTCR isolated from melanoma patient samples using the IMPACT method

[0641] Materials and methods

[0642] comPACT library preparation

[0643] Whole exome sequencing of tumor biopsy and patient normal PBMCs and RNA-Seq transcriptome sequencing of tumor biopsy were used to identify somatic non-synonymous mutations in patient PACT135. The patient had stage IV metastatic melanoma and was undergoing anti-PD1 antibody treatment with nivolumab. 2566 coding mutations were identified. 632 new epitopes were predicted from the tumor mutation load, and 243 compPACT (new epitope-HLA complex) libraries were generated in 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 whole exome sequencing of the patient's normal PBMCs. The library covers 3 of the 6 HLAs.

[0644] T cell isolation

[0645] PBMC and TIL samples were collected from subject PACT135 at different time points before or during anti-PD-1 antibody treatment. PBMC samples were collected on days 14, 43, and 84 after the start of treatment. TIL samples were collected on day -37 before the start of anti-PD1 treatment and on day 82 after the start of treatment. T cells were incubated with the compPACT library and neoantigen-specific T cells were isolated using the imPACT method described in Examples 10 and 11.

[0646] Fourteen TCRs specific for five neoantigen-HLAs were isolated; one neoTCR recognized PUM1, one neoTCR recognized TTP2, two neoTCRs recognized IL8-HLA-A*24:01, and 10 neoTCRs recognized IL8-HLA-A*03:01. T cells expressing neoTCRs were expanded for 14 days in medium containing IL2, IL7, IL15, or a combination thereof. At the end of expansion, T cells retained a "younger" T cell phenotype, generating NeoTCR-P1 T cells that exhibited both T memory stem cell and T central memory cell phenotypes.

[0647] NeoTCR gene editing

[0648] As described in International Patent Application No. WO2019089610 published on May 9, 2019 (herein incorporated by reference in its entirety), healthy donor-derived CD4 and CD8 T cells were engineered using a CRISPR-based non-viral approach to express a TCR specific for each identified neoantigen.

[0649] neoTCR expression

[0650] The expression of neoTCR in gene-edited CD4 and CD8 T cells was analyzed using a fluorophore-comPACT trimer dextran complex. Biotinylated compPACT protein was bound to streptavidin dextramer and incubated with neoTCR CD4 and CD8 T cells. The binding of compPACT-dextramer to corresponding T cells expressing neoTCR was determined by the method 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. Dextramer was prepared by using fluorescently labeled streptavidin (Life Technologies, Carlsbad, CA).

[0651] Matched autologous melanoma cell line production

[0652] A matched autologous melanoma cell line was established from a patient's biopsy (M489). As a negative control, a second cell line from a mismatched melanoma was established from a biopsy from a different patient (M202). The functionality of T cells expressing NeoTCRs (expression of activation markers, cytokine secretion, antigen-specific target cell killing, and T cell proliferation) was assessed using matched and mismatched autologous melanoma cell lines.

[0653] T cell activation

[0654] NeoTCR-expressing T cells were incubated with or without IFNγ and co-cultured with the M489-matched melanoma tumor cell line and neoantigen-matched comPACT-dextramer. As a negative control, neoTCR T cells incubated with or without IFNγ were also co-cultured with the M202-mismatched melanoma tumor cell line and neoantigen-matched comPACT-dextramer. T cells stimulated with the anti-CD3 antibody OKT3 were used as a positive control. Internalization of NeoTCR after comPACT-dextramer binding was assessed by FACS.

[0655] The 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. The expression of activation markers was determined by FACS. Anti-OX-40 antibody (clone Ber-ACT35, cat#350012) and anti-4-1BB antibody (clone 4B4-1, cat#309810) were purchased from Biolegend.

[0656] T cell cytotoxicity assay

[0657] T cell-induced killing of tumor cells over time was monitored by immunofluorescence using the IncuCyte imaging system (Essen BioSciences). Each of the 14 neoTCR-expressing T cells was co-cultured with patient-matched M489 tumor cells. NeoTCR T cells were also co-cultured with the M202 mismatched cell line as a negative control. M489 and M202 tumor cells were labeled with NucLight Red Lentivirus (Essen BioSciences).

[0658] M489 or M202 tumor cells were seeded in 96-well plates at 25,000 cells / well 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-hour intervals for 12 days using an IncuCyte imaging system with a 10X objective.

[0659] Cytokine secretion assay

[0660] Cytokine production in the supernatant of co-cultured T cells and melanoma cell lines was assessed using a cytokine bead assay (CBA BEAD-BASED IMMUNOASSAY, BD BioSciences). CBA is a flow cytometry multiplex bead-based immunoassay application that allows for simultaneous quantification of multiple proteins by efficiently capturing analytes using antibody-coated beads. After co-culturing T cells and target cells for 24 or 48 hours, supernatants were collected and analyzed for secretion of IFNγ, IL-2, and TNFα.

[0661] result

[0662] Identification of neoTCRs in PACT135 over time

[0663] imPACT analysis resulted in the isolation of 14 TCRs specific for five neoantigen-HLAs: one neoTCR recognized PUM1, one neoTCR recognized TTP2, two neoTCRs recognized IL8-HLA-A24:02, and ten neoTCRs recognized IL8-HLA-A03:01. The neoantigen peptide sequences, α and β TCR CDR3 sequences, and HLA alleles isolated from patient PACT135 are shown in Table 8 below.

[0664]

[0665]

[0666] Fig.45 A summary of the number of neoantigen-specific T cells per CD8 T cell in each sample collected during anti-PD-1 antibody treatment is provided. Each box represents one T cell and each cross represents ten T cells. Each column of boxes or crosses represents a unique neoTCR clonotype.

[0667] TCR219, TCR220, TCR223, TCR224, TCR225, TCR228, TCR229, TCR232, TCR240 and TCR241 recognize the IL8-KTYFKPFHPK neoantigen (SEQ ID NO:256).

[0668] TCR221 and TCR227 recognize the IL8-YFKPFHPKF neoantigen (SEQ ID NO: 227).

[0669] TCR218 recognizes the TPP2-CFSEVSAKF neoantigen (SEQ ID NO:223).

[0670] TCR222 recognizes the PUM1-AMMDYFFQR neoantigen (SEQ ID NO: 226).

[0671] neoTCR expression

[0672] Fig.46 Strong T cell gene editing efficiency was shown for 14 neo TCRs in both CD4 and CD8 T cells. For 13 neoTCR T cells, CD4 and CD8 T cells bound the homologous compPACT-dextramer complex. However, only CD8 T cells expressing a neoTCR (TCR222) against PUM1 bound to the homologous compPACT-dextramer, and no compPACT-dextramer binding was observed in CD4 neoTCR T cells.

[0673] T cell activation

[0674] When co-cultured with the neoTCR T cell-cognate comPACT-dextramer and the melanoma-matched cell line M489, neo TCR was internalized ( Fig.47). A decrease in the percentage of comPACT-dextramer-positive T cells indicates internalization of neoTCR, a surrogate marker of T cell activation. Cells cultured with RPMI medium alone did not internalize the dextramer complex bound to neoTCR, whereas T cells cultured with the anti-CD3 antibody OKT3 internalized the dextramer complex bound to neoTCR. Neo12 antigen was also used as a negative control for each sample.

[0675] NeoTCR T cells derived from patient PACT135 also expressed the activation marker 4-1BB ( Fig.48 ) and OX40( Fig.49 ). No expression of 4-1BB or OX40 was observed in TCR222 CD4 T cells because the neoTCR does not bind the cognate neoantigen.

[0676] When tumor cells were pretreated with IFNγ to activate the immunoproteasome and enhance HLA expression, 4-1BB expression in the IL8-HLA-A03 TCR was increased ( Fig.48 When tumor cells were pretreated with IFNγ to activate the immunoproteasome and enhance HLA expression, OX40 expression in the IL8-HLA-A03 TCR was increased ( Fig.49 ).

[0677] T cell cytotoxicity assay

[0678] 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. Fig.50 A graph is provided of the percent confluence of tumor cells after co-culture with all neoTCR T cells identified from PACT135 compared to the percent confluence of tumor cells after mock treatment or incubation with RPMI medium or neo12 TCR T cells. Fig.51A and 51B Separate graphs of the percent confluence of tumor cells after coculture with each neoTCR T cell are provided.

[0679] T cells expressing neoTCR showed strong killing of matched tumor cells at all tested T cell:tumor cell ratios (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 compared to less than 20% nuclear confluence at 96 hours post-incubation in each sample incubated with neoTCR T cells at a 1:1 ratio (p<0.000001 for each neoTCR T cell sample; Fig.50 and Fig.51A -B). Importantly, while the number of tumor cells decreased, the number of T cells increased, indicating that neoTCR T cells proliferated in response to the cognate antigen endogenously expressed by the patient-matched tumor cells (data not shown). After two days of coculture with syngeneic tumor cells, neoTCR T cells became activated, as evidenced by the formation of clusters. T cells proliferated and by day 5, they covered the entire surface of the well, and no tumor cells were detected.

[0680] Even neoTCRs with low frequencies in PBMC or TIL samples, such as those expressed in only one type of T cell, showed strong activity against patient-matched tumor cell lines, demonstrating the high accuracy and sensitivity of the imPACT technology.

[0681] T cell cytokine secretion

[0682] Antigen-specific cytokine production by NeoTCR-expressing T cells was assessed. NeoTCR T cells secreted IFNγ, IL-2, and TNFα cytokines after co-culture in the presence of the patient-matched melanoma cell line M489. No cytokine secretion was measured when neoTCR T cells were co-cultured with the mismatched melanoma cell line M202. Fig.52 Secretion of IFNγ, IL2, and TNFα by TCR228 T cells after co-culture in the presence of M489 is shown. Fig.53 Shown are 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. Fig.54 Shown is 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. Fig.55Shown are the secretion of IFNγ by TCR219, TCR223, TCR224, TCR225, TCR229, TCR240 and TCR241 T cells after co-culture in the presence of M489 alone (black bars) and after pre-treatment of M489 cells with IFNγ for one hour (grey bars). TCR220, TCR228 and TCR232 T cells secrete IFNγ after co-culture with IFNγ pre-treated M489 cells. No IL2 or TNFα was detected at 48 hours.

[0683] Example 21 Validation of NEOTCR isolated from colorectal patient samples using the IMPACT method

[0684] Materials and methods

[0685] comPACT library preparation

[0686] 144 new epitopes were predicted for the treatment of patients with colorectal cancer who had never received treatment As described in Examples 10 and 11, 61 compPACT (neo-epitope-HLA complex) libraries were generated in HLA-A*03:01, A*02:01 and B*07:03.

[0687] T cell isolation

[0688] PBMCs collected from a subject (PACT035) were incubated with the compPACT library. Neoantigen-specific T cells were isolated using the imPACT method described in Examples 10 and 11. Seven neoTCR clonotypes against the COX6C protein were identified.

[0689] NeoTCR gene editing

[0690] As described in International Patent Application No. WO2019089610 published on May 9, 2019 (herein incorporated by reference in its entirety), healthy donor-derived CD4 and CD8 T cells were engineered using a CRISPR-based non-viral approach to express seven COX6C neoantigen-specific TCRs.

[0691] COX6C R20Q stable expression cell line

[0692] PACT precision genome engineering expertise was used to generate stable tumor cell lines that express the COX6C R20Q neoantigen 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 neoantigen. SW620 cells were nucleofected with gRNA / Cas9 and HDR template to make COX6C R20Q knock-in cell lines. Edited cells were single-sorted and propagated. The COX6C locus was sequenced. Sequencing analysis showed that approximately 80% of single cells were highly edited. Four cell lines expressing COX6C R20Q in the endogenous locus were selected: SW620 cells expressing the wild-type COX6C gene, SW620 cells heterozygous for the COX6C-R20Q mutation, and two SW620 cell lines homozygous for the COX6C-R20Q mutation (one is shown).

[0693] The expression of HLA-A02 in the engineered SW620 cell line was confirmed by flow cytometry using the BB7.2 anti-HLA*A2 antibody. The K562 cell lines constitutively expressing 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.

[0694] T cell activation

[0695] Expression of the activation marker Nur77 was also determined in CD4 and CD8 TCR089 neoTCR T cells co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation. As a negative control, TCR089 neoTCR T cells were also co-cultured with wild-type SW620 cells, or cultured alone. Cells were stained for Nur77 using an anti-Nur77 mAb (eBiosciences), and Nur77 expression was assessed by flow cytometry.

[0696] T cell cytotoxicity assay, Incucyte

[0697] NeoTCR T cell-induced tumor cell killing over time was also determined by immunofluorescence using the 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 marked red using NucLight Red lentivirus (Essen). Red-labeling tumor cells can distinguish them from T cells in co-culture and monitor tumor cell killing over time. 40,000 tumor cells / well were seeded in 96-well plates and placed in an incubator overnight. The next day, neoTCR T cells were added at a 1:1 ratio of T cells: tumor cells. Each neoTCR T cell was added to a separate tumor cell sample. RNP (T cells electroporated only with ribonucleoprotein (RNP) complexes), neo12 TCR T cells, and culture medium alone were used as negative controls. The 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.

[0698] T cell cytotoxicity assay, flow cytometry

[0699] Evaluation of antigen-specific T cell-mediated killing by TCR089 neoTCR T cells. 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.

[0700] After co-culturing T cells and target cells for 24 hours, the cells were stained for 20 minutes in the dark at 4°C using a live / dead cell staining kit (Live / Dead NearIR viability stain for flow, cat# NC0584313, ThermoFisher). In cells with damaged cell membranes, the dye reacts with free amines inside and on the cell surface to produce strong fluorescent staining. In live cells, the reactivity of the dye is limited to cell surface amines, resulting in lower fluorescence intensity. The intensity difference between live and dead cells is typically greater than 50 times, making it easy to distinguish. After incubation, the cells were washed, fixed with eBioscience IC fixation buffer (ThermoFisher, catalog number 00-8222-49) and analyzed by flow cytometry.

[0701] Cytokine secretion assay

[0702] TCR089 neoTCR T cells were co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation or SW620 cells 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 SW620 WT and pulsed with 1 μM for 1 hour. As a negative control, T cells expressing TCR089 were co-cultured with SW620 wild-type cells. Supernatants were collected after 24 hours and cytokine production was assessed using a cytokine bead assay (CBA, BEAD-BASED IMMUNOASSAY from BD BioSciences). CBA is a multiplex bead-based immunoassay application for flow cytometry that effectively captures analytes using antibody-coated beads, allowing for simultaneous quantification of multiple proteins.

[0703] result

[0704] Identification of neoTCRs in PACT035

[0705] Seven neoTCR clonotypes targeting COX6C protein were identified in patient samples using the imPACT T cell isolation method. Fig.56 , indicated by arrows). When engineered into CD4 T cells, two of the seven bind to the neoantigen-HLA complex and are therefore neoTCRs that are independent of CD8. 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 is residues 18-20 with an R20Q mutation. The neoTCRs that bind to the R20QCOX6C peptide (residues 18-26) are referred to as TCR089, TCR091, TCR092, TCR094, TCR097, TCR098, and TCR099. The neoantigen peptide sequences, α and βTCR CDR3 sequences, and HLA alleles isolated from patient PACT035 are shown in Table 9 below.

[0706]

[0707] Transfection of SW620 cell line

[0708] Fig.57A The KV1858 cell line showed no HLA*A2 expression (expressed HLA*C2 but not HLA*A2). Fig.57B HLA*A2 expression in the KV1832 cell line is shown (Expressing HLA*A2). Fig.57C HLA*A2 expression in the SW620 cell line is shown (Expressing HLA*A2). Fig.57DQuantification of GFP in nucleofected SW620 cells is shown. An isotype control antibody was used as a negative control.

[0709] T cell activation

[0710] Nur77 is an immediate early gene whose expression is rapidly upregulated by TCR signaling. Nur77 expression is rapidly upregulated by antigen-TCR signaling. Nur77 expression was detected in TCR089 neoTCRT cells co-cultured with SW620 cells homozygous for the COX6C-R20Q mutation ( Fig.58 ). No Nur77 induction was observed when TCR089 neoTCR T cells were cultured alone or with SW620 cells expressing WT COX6C protein.

[0711] T cell cytotoxicity assay, IncuCyte

[0712] Target cell killing was also measured for each COX6C neoTCR T cell. All neoTCR-expressing T cells showed robust killing of SW620 homozygous tumor cells ( Fig.59A No cytotoxic activity was observed against SW620 cells expressing wild-type COX6C protein ( Fig.59B ). The amount of T cell-induced killing was dose-dependent and increased with increasing T cell:tumor cell ratios.

[0713] IncuCyte images collected during killing assays using TCR089 neoTCR T cells also showed an increase in T cell numbers while tumor cell numbers decreased (data not shown). This suggests that TCR089 T cells proliferate in response to cognate antigens endogenously expressed by SW620 cells homozygous for the COX6C-R20Q mutation.

[0714] TCR089 T cell cytotoxicity assay, flow cytometry

[0715] 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 ( Fig.60 The amount of T cell-induced killing was dose-dependent and increased with increasing TCR089 T cell:tumor cell ratios.

[0716] Cytokine secretion assay

[0717] Strong IFNγ secretion was measured in TCR089 T cell samples after co-culture with SW620 cells homozygous for the COX6C-R20Q mutation ( Fig.61 ). When TCR089 T cells were co-cultured with SW620 cells heterozygous for the COX6C-R20Q mutation, half the amount of IFNγ was measured. Similar IFNγ was detected for TCR089 T cells alone, or when T cells were co-cultured with wild-type SW620 cells, in the absence of tumor cells.

[0718] Example 22 Method for treating cancer patients using NEOTCR T cells

[0719] Patients with cancer or another proliferative disease may require interventional therapy to slow or stop cell proliferation and kill existing cells that may or do cause harm to the patient (e.g., cause pain, discomfort, or disease). Specifically, the neoTCR T cells disclosed herein can be used to treat cancer.

[0720] like Fig.62 As shown, 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 the biopsies and cells. DNA was also collected from the patient's PBMCs for WES to use as a control. Once tumor antigens are identified using RNAseq and WES, algorithms are used to select neo-epitope candidates for screening using compPACT peptides (where predicted neo-epitopes are expressed) and imPACT separation technology. A library of barcoded compPACT particles is assembled and bound to patient samples. The compPACT particles are able to associate and capture neo-epitope-specific T cells.

[0721] Example 23 imPACT separation technology example

[0722] Based on computational predictions of patient-specific neoE, hundreds of capture reagents were prepared consisting of patient HLA class I subtypes loaded with the corresponding predicted neoE (Peng et al., AACR 2019); neoE-specific T cells were then isolated and TCRα and β sequenced. Primary human T cells expressing neoTCRs were generated using non-viral precision genome engineering, and isolated neoTCRs were functionally studied to replace endogenous TCRs (Jacoby et al., AACR 2019, Sennino et al., AACR 2019).

[0723] T cell responses were analyzed in two patients with metastatic melanoma receiving anti-PD-1 therapy. NeoE-specific T cells were isolated from peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) at different time points. Patient PT476 had a durable response; tumor mutational burden (TMB) was 2556; 243 neoE-HLA complexes were generated spanning 3 HLA types: HLA-A03:01, A24:01, and C12:03. This resulted in the isolation of 17 TCRs specific for 5 neoE-HLAs. T cells specific for neoE were present in TILs at baseline and expanded in TILs and PBMCs during treatment. Patient PT461 had rapid disease progression after anti-PD-1; TMB was 61; 78 neoE-HLA complexes covering HLA-A02:01, A03:01, B07:02, C05:01, and C07:02 were generated, resulting in the separation of 2 TCRs to 1 neoE-HLA.

[0724] To further characterize the T cell response, T cells were gene-edited to express 14 different TCRs isolated from patient PT476, specific for neoE in the mutated IL8, PUM1, and TPP2 genes. All 14 T cell preparations showed specific cytotoxicity to the matching autologous melanoma cell lines established from patient PT476 biopsies (compared with the growth of melanoma cell lines in co-culture with mismatched control TCRs, 50-75% tumor growth inhibition, 96 hours assay using P:T1:1, each comparison p<0.000001), and there was no cytotoxic effect on the mismatched control human melanoma cell lines. After co-culture with matching 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. When T cells were co-cultured with mismatched targets, no response was observed.

[0725] These results suggest that anti-PD-1 therapy induces focused neoE-specific T-cell responses against limited amounts of neoE and that nonviral precision genome engineering can successfully redirect T cells to neoE-expressing tumors, which can be used as an approach for personalized ACT therapy.

[0726] Similarly, in addition to anti-PD-1 therapy, other checkpoint therapies and additional combinations may be used; for example, anti-PD-L1 or anti-CTLA4 therapy may be used.

[0727] As described in part in Example 20, biopsies and PBMCs were collected at various time points after anti-PD-1 treatment ( Fig.45 :PT476 responder, and Fig.63: PT461 non-responders). TILs and cell lines were established from biopsies of patients. imPACT isolation technology was used to isolate NeoE-specific T cells and monitor their evolution over time. NeoEs derived from non-synonymous mutations were predicted using whole exome sequencing (WES) and RNAseq from baseline cell lines and ranked based on predicted HLA binding affinity, truncation of mutations, and expression level. HLA-NeoE capture reagents for the top ranked NeoEs were used to isolate NeoE-specific T cells. Fig.45 PT476 was shown to generate 243 neoE-HLA complexes spanning three HLA types (HLA-A03:01, A24:01 and C12:03) and to isolate 17 TCRs specific for five NeoE-HLAs. Fig.63 Patient PT461 was shown to have generated 78 neoE-HLA-complexes covering HLA-A02:01, A03:01, B07:02, C05:01 and C07:02, resulting in the separation of 2 TCRs to 1 neoE-HLA.

[0728] Example 24 imPACT separation technology method example

[0729] NeoE-specific T cells can be isolated from patient samples as described in Example 22. In this example, the imPACT separation technology led to the identification of 14 candidate neoTCR-Ts, 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.

[0730] like Fig.62 As shown, the imPACT separation technology approach includes three workflows: gene editing, co-culture assays, and cell-based assays.

[0731] Gene Editing: CD8 and CD4 T cells from healthy donors were precisely genome 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. Single-step (non-viral) precise genome engineering resulted in seamless replacement of the endogenous TCR with the patient's NeoE-specific TCR (native sequence), whose expression is endogenously regulated.

[0732] Co-culture assay: NeoTCR-P1 T cells were co-cultured with a melanoma cell line (M489) derived from a baseline biopsy of the same patient or a mismatched melanoma tumor cell line at a final product-to-target (P:T) ratio of 1:1 or 5:1. 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 in cell supernatants was measured at 48 hours using the BD Cytokine Bead Array (CBA) Human Th1 / Th2 Cytokine Kit II.

[0733] Co-culture assays: Peptide-HLA: Recognition / stimulation, target cell killing, proliferation, activation markers, and cytokine secretion assays were performed.

[0734] Example 25: Engineered NEOTCR-T cells kill autologous tumor cells

[0735] like Fig.64 As shown, 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 that stably expresses red fluorescent protein (nuclear RFP). More specifically, NeoTCRs were made to target the IL-8-HLA-A*03:01 neoantigen on a melanoma cell line, and these NeoTCRs were cultured with the IL-8-HLA-A*03:01 neoantigen melanoma cell line ( Fig.64 ) and compared them to the negative control ( Fig.64 The images shown here were collected at time 0 (left), 2 days (middle), and 5 days (right). Thus, the NeoTCR is specific for tumor neoantigens and is able to effectively kill autologous tumor cells.

[0736] The ability of neoTCR-T cells to kill autologous tumor cells can also be seen in Fig.65A NeoTCR-T cells were co-cultured with autologous (black bars) or mismatched melanoma tumor cells (white bars), and after 48 h, the percentage of CD8 neoTCR T cells expressing Ki67 (proliferation marker) was assessed by flow cytometry. *p<0.05 compared to mismatched melanoma tumor cells (t-test corrected for multiple comparisons using the Holm-Sidak method). T cells expressing NeoTCR neo12 were used as negative control.

[0737] Similarly, NeoTCR-T cells were shown to express activation markers when co-cultured with autologous tumor cells. Fig.65BNeoTCR-T cells were co-cultured with autologous (black bars) or mismatched tumor cell lines (white bars), and the percentage of CD8 neoTCR T cells expressing the activation marker 4-1BB (top) or the percentage of CD4 neoTCR T cells expressing the activation marker OX40 (bottom bar graph) was assessed by flow cytometry after 24 hours. *p<0.05 compared with mismatched melanoma tumor cells (t-test corrected for multiple comparisons using the Holm-Sidak method). T cells expressing NeoTCRneo12 were used as negative control. To measure the upregulation of OX-40 in CD4 neoTCR T cells, melanoma cells were pretreated with IFNγ for 24 hours before co-culture with T cells.

[0738] Finally, we showed that NeoTCR-T cells secrete interferon-γ when co-cultured with autologous tumor cells. Fig.65C NeoTCR-T cells were co-cultured with autologous melanoma tumor cells, and IFNγ secretion was assessed by flow cytometry (CBA) after 48 h. Mock T cells were used as negative control. *p<0.05 (t-test corrected for multiple comparisons using the Holm-Sidak method).

[0739] Together, this showed that newly generated NeoTCR-T cells expressing TCRs isolated using imPACT isolation technology upregulated markers of activation and proliferation when co-cultured with autologous tumor cells. More importantly, all NeoTCR-T cells specifically killed patient-derived autologous melanoma cells.

[0740] Example 26 Tumor eradication in mice and in vitro in engineered cell lines using NEOTCR-T cell administration

[0741] Neoantigen-expressing tumors were implanted into the flanks of 15 NOD scid gamma (NSG) mice. 3 The mice were divided into two groups: a control group (7 mice), which received PBS; and a treatment group (8 mice), which was administered neoTCR T cells (5*10 6 Total T cells / mouse; gene editing efficiency 50%) Fig.66A As shown, after infusion of NeoTCR-T cells (see arrows in the figure), tumor size decreased and the tumor was completely eradicated by day 19. Fig.66BThe number of human CD8 T cells / mL present in the blood of mice at day 4 after neoTCRT cell infusion and at day 35 after neoTCR-T cell infusion is shown. Despite the lack of human cytokines in NSG mice, the presence of NeoTCR-T cells in the circulation after tumor eradication indicates that NeoTCR-T cells not only kill target cells but also proliferate and persist.

[0742] Example 27 Design of NEOTCR-T cells specific for trunk tumor mutations

[0743] The subclonal mutational nature of tumor progression in both primary tumors and metastases creates a problem in the field of oncology because oncology drugs are "personalized," meaning they are designed to target proteins, chemicals, or cells with specific mutations (e.g., many small molecule drugs are designed to target tumors based on point mutations). Because tumors often mutate in later stages of the primary tumor and in metastatic stages (mutations accumulate as cancer grows and the disease persists), drugs that work well to shrink or slow tumor progression when the tumor is first detected and treated may lose effectiveness over time.

[0744] Disclosed here is the ability to design and engineer neoTCR-T cells specific for trunk tumor mutations. Using algorithmic and bioinformatic approaches, tumor-specific trunk mutations expressed by all cancer cells in a patient were identified.

[0745] Example 28 NEOTCR-T cells address all HLA types in the global population

[0746] There are 13,000 HLAs in the human population. Each person has a set of 6 HLAs. Therefore, less than 1% of any NeoE-HLA tumor target is identical between patients (data generated after analyses of 60,000 patients (Hartmaier et al. (2017) Genome Medicine) and 20,000 patients (Schumacher & Schrieber (2015) Science). The analyses performed showed that the catalog of HLA alleles interrogated by PBMCs was: >99% had at least 1 HLA allele coverage, >90% had 4 to 6 alleles coverage, and >60% of potential US trial subjects were expected to have all 6 alleles covered.

[0747] Example 29 NEOTCR therapy can be used for tumors with low mutation load, medium mutation load and high mutation load

[0748] Because the imPACT separation technology is extremely sensitive, it is possible to detect neoantigens on tumors at all levels 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 intermediate tumor mutational load such as ovarian and colorectal cancer, and tumors with high tumor mutational load such as bladder and melanoma cancer.

[0749] Therefore, the imPACT separation technology described herein can be used to design, engineer and manufacture NeoTCRs for low, medium and high tumor mutation load tumors. In some aspects, the imPACT separation technology method can be used to detect new antigens on low, medium and high tumor mutation load tumors. In some aspects, the imPACT separation technology method has been used to detect new antigens on low, medium and high tumor mutation load tumors. In some aspects, the imPACT separation technology method can be used to prepare a composition comprising NeoTCR to treat low, medium and high tumor mutation load tumors in patients with such tumors. In some aspects, the imPACT separation technology method can be used to prepare NeoTCR populations to treat low, medium and high tumor mutation load tumors in patients with such tumors.

[0750] Example 30 Method for treating a patient using NEOTCR-T cell therapy

[0751] The initial step of treating patients with NeoTCR-T cell therapy is to screen patients. Once screening and biopsy are performed, patients can register and undergo leukapheresis. During the manufacture of NeoTCR T cells (patient-specific) as described herein, including compPACT library creation, imPACT separation technology screening and T cell editing to express NeoTCR, patients can optionally participate in bridging therapy (e.g., a standard of care therapy, including first-line, second-line, third-line and later-line therapy for specific cancer indications). Such bridging therapy can be prescribed and applied between 0 and 60 days (average between 21 and 42 days). After this optional bridging therapy, conditional chemotherapy can be prescribed to patients. This conditional chemotherapy can be performed 5, 4 and 3 days before applying NeoTCR T cell therapy. On the day of application, the patient will receive an infusion of NeoTCR. Thereafter, the tumor will be assessed.

[0752] Example 31 Compositions and methods for treating non-cancer diseases and conditions using NEOTCR T cells

[0753] Without limitation, NeoTCR T cell therapy can be used to treat diseases other than cancer. Specifically, NeoTCR T cell therapy can be used to treat any disease or condition that causes a diseased cell population to produce disease / disorder specific neoantigens. Such cells include cells infected with viruses, fungi, or bacteria, which present infection-specific neoantigens that can be detected by NeoTCR T cells due to infection. Cells associated with inflammatory or autoimmune diseases may also present disease-specific neoantigens from which NeoTCR T cells can be generated. For example, if a patient suffers from an allergy or inflammatory disease, a NeoTCR specific for a neoantigen of an inflammatory cytokine ligand presented on an inflammatory cell can be prepared.

[0754] Example 32 Method for Imaging Using NEOTCR T Cells

[0755] Once the compPACT and imPACT separation technology methods are applied to a patient tumor sample, the NeoTCR T cells can be used to treat the disease, as described herein, and can also be used to image, detect and / or monitor tumor burden, progression, remission and eradication. This can be accomplished by labeling the NeoTCR T cells with a detectable marker (e.g., any marker that can be imaged, such as with a dye or with a zirconium marker; see, e.g., U.S. Pat. No. 8,771,966, the entirety of which is incorporated herein by reference).

[0756] For example, NeoTCR T cells can be genetically modified to express dyes or fluorescent proteins. Such labeled NeoTCRT cells can be used to determine the efficacy of NeoTCR T cell therapy in eradicating tumors; if the labeled NeoTCR T cells can be imaged to proliferate and expand, it can be inferred that the NeoTCR T cell therapy is effective because the cells differentiate into T effector cells when they encounter the target antigen.

[0757] 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 any tumor prior to, during, or after NeoTCR T cell therapy (before, during, or after) based on the interaction or lack of interaction between the NeoTCR T cells and tumor cells (if present).

[0758] Without limitation, cells other than tumor cells can also be imaged that present new antigens that allow the manufacture of NeoTCR T cells. Such cells include, but are 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 new epitope (e.g., a new epitope on an inflammatory cytokine ligand that causes inflammation) is discovered and NeoTCR T cells are designed and manufactured thereby, 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 effectively eradicates or sufficiently reduces such cell populations to improve the patient's disease state.

[0759] Example 33 Methods for determining the efficacy of NEOTCR-T cell therapy and methods for adjusting the dose of NEOTCR-T cell therapy

[0760] After 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, and then a tumor tracer can be administered, which can be imaged 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after such tracer administration. In certain embodiments, the tracer can be administered in the same week as the neoTCR-T cell therapy, in a week after the neoTCR-T cell therapy, in two weeks after the neoTCR-T cell therapy, in three weeks after the neoTCR-T cell therapy, or one or more months after the neoTCR-T cells.

[0761] In certain embodiments, if the tumor does not progress but does not shrink following neoTCR-T cell therapy (as observed using imaging), additional neoTCR-T cell therapy administrations may be given. In certain embodiments, if the tumor does not shrink following neoTCR-T cell therapy and optionally increases in size (as observed using imaging), additional neoTCR-T cell therapy administrations may be given.

[0762] In certain embodiments, the disease state is monitored every 3-6 months using tracers and imaging after neoTCR-T cell therapy. In certain embodiments, the disease state is monitored every 6-12 months using tracers and imaging after neoTCR-T cell therapy.

[0763] Example 34 Method of using NEOTCR T cells to treat patients

[0764] As described in Examples 1-9, new epitope candidates were synthesized and cloned into compACT polynucleotides. Briefly, the polynucleotide sequence encoding the candidate antigenic peptide was inserted into Figure 1-5 The MHC template described in . Mammalian cells are inoculated and transfected with compPACT polynucleotides comprising candidate antigenic peptide sequences. The transfected cells express and secrete compPACT polypeptides in cell culture medium. Conditioned medium from the cells is collected and the compPACT polypeptides are purified by size exclusion chromatography. The purified compPACT polypeptides are then assembled with multimeric particles (e.g., tetramers, dextramers, NTAmers) that contain multiple copies of the compPACT polypeptide, a DNA barcode, and a fluorophore (e.g., APC or PE). One of the advantages of this approach is the simultaneous high-throughput production and screening of multiple new epitope candidates. In addition, the process can be automated. Two different types of particles (the first with APC and the second with PE as a fluorophore) are combined to obtain a collection of particles that can recognize patient-specific antigenic peptides.

[0765] To identify the sequences of neoTCR T cells and their TCRs, freshly isolated or cryopreserved T cells from patients were stained with multimeric particles and a panel of antibodies for phenotypic characterization. Live and barcoded T cells were sorted into single cells, and their DNA and RNA were extracted and analyzed by next generation sequencing. As described in Examples 11-13, sequencing data obtained from compACT positive T cells were analyzed to identify and validate predicted antigenic peptides, neoTCR T cells and validated neo-epitope TCR candidates and their sequences.

[0766] The identified new epitope TCR sequences are cloned into homology-directed recombination (HDR) templates for genome editing in T cells. Further details of the sequence and structure of the template can be found in International Patent Application No. PCT / US2018 / 058230, the contents of which are incorporated herein by reference. T cells from patients, whether freshly collected or previously cryopreserved, are engineered for disruption of TCR genes and integration of HDR templates using non-viral methods. CRISPR / Cas9 methods containing gRNAs for endogenous loci of TCR-α and TCR-β gene sequences can be used to disrupt endogenous TCR loci. The HDR template will be recombined with one of the endogenously disrupted TCR gene sequences to introduce the identified new epitope TCR. Therefore, the engineered T cells lack expression of endogenous TCRs and express new epitope TCRs. These neoTCR T cells are then adoptively transferred into patients and specifically target tumor cells expressing new antigens.

[0767] This process has significant advantages over other adoptive cell transfer approaches, including but not limited to: i) it is flexible as it allows for personalized targeting of tumor-specific mutations presented in the context of patient-specific HLA; ii) it provides a clinical tool to attack cancer cells expressing neoantigens that are not expressed on the cell surface; iii) it is effective regardless of the patient's ethnicity or cancer type; and iv) it can automate multiple steps and has a small manufacturing footprint.

[0768] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details may be made without departing from the spirit and scope of the invention.

[0769] All references, published patents, and patent applications cited throughout the text of this specification are incorporated herein by reference in their entirety for all purposes.

Claims

1. A method comprising: a. contacting the sample with multiple different particle collections, i. wherein each particle of each set consists of three polypeptides including an antigenic peptide, an operably associated barcode and at least one identification tag, ii. wherein the sample comprises T cells, and iii. Among them, The contacting comprises providing conditions suitable for a single T cell to bind to a unique antigenic peptide of at least one collection of particles; b. separating one or more T cells bound to the particles; c. identifying barcodes of particles bound to isolated T cells; d. determining the ratio of the most dominant barcode to the second most dominant barcode identified in (c); and e. Antigen specificity of T cells was determined based on the ratio of the most dominant barcode to the second most dominant barcode.

2. The method of claim 1, wherein the ratio is calculated by identifying the number of copies of the most dominant barcode and the number of copies of the second most dominant barcode and dividing the number of copies of the most dominant barcode by the number of copies of the second most dominant barcode.

3. The method of any one of claims 1-2, wherein the unique antigenic peptide is the same for each different collection of particles.

4. The method of any one of claims 1-2, wherein each distinct collection of particles comprises at least one or more barcodes, wherein each barcode is associated with the identity of the antigenic peptide.

5. The method of any one of claims 1-2, wherein the ratio corresponds to the antigen specificity of the isolated T cells.

6. The method of any one of claims 1-2, wherein the isolated T cells are identified as antigen-specific T cells if the ratio of the most dominant barcodes is above a threshold.

7. The method of claim 6, wherein the threshold value is at least or greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-5, 3-6, 4-7, 5-8, 5-10, 7-10 or greater than 10.

8. The method of any one of claims 1-2, 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. The method of claim 8, wherein the nucleotide-based assay determines (a) the sequence of each barcode and / or (b) the copy number of each barcode.

11. The method of any one of claims 1-2, further comprising obtaining a T cell receptor (TCR) CDR sequence.

12. The method of any one of claims 1-2, further comprising obtaining a TCR gene sequence.

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

14. The method of any one of claims 1-2, which is used to identify the antigen specificity of T cells.

15. The method of claim 14, 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. The method of any one of claims 1-2, wherein the at least one identification marker is the same in each different set of particles.

17. The method of any one of claims 1-2, comprising at least two different identification markers.

18. The method of any one of claims 1-2, wherein the at least one identifying label is a fluorophore.

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

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

21. The method of any one of claims 1-2, wherein the antigenic 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.

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

23. The method of claim 22, wherein a computer prediction algorithm is used to identify neoantigens.

24. The method of claim 23, wherein the prediction algorithm further comprises an MHC binding algorithm to predict binding between neoantigens and MHC peptides.

25. The method of any one of claims 1-2, 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. The method of any one of claims 1-2, wherein the T cells are human T cells.

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

28. The method of any one of claims 1-2, wherein the method comprises a library of different particle collections.

29. The method of claim 28, wherein the library comprises 2 to 500 different collections of particles.

30. The method of any one of claims 1-2, wherein each particle comprises an MHC peptide.

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

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

33. The method of claim 30, wherein the HLA peptide comprises an HLA-A, HLA-B, or HLA-C peptide.

34. The method of claim 33, wherein the HLA peptides comprise 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.

35. The method of any one of claims 1-2, wherein each particle comprises an HLA peptide and a β2M peptide.

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

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

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

39. The method of any one of claims 1-2, wherein each particle comprises a polypeptide comprising, in the amino to carboxyl terminal direction, (i) an antigenic peptide, (ii) a β2M peptide, and (iii) an MHC peptide.

40. The method of any one of claims 1-2, 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. The method of claim 39, wherein the polypeptide is biotinylated.

42. The method of any one of claims 1-2, wherein the particles are selected from the group consisting of magnetic beads, agarose beads, styrene polymer particles, and dextran polymer particles.

43. The method of any one of claims 1-2, wherein the particles are coated with streptavidin.

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