Methods of identifying antigen-specific t cell receptors

Abstract

The present disclosure provides methods of identifying antigen-specific T cell receptors and their cognate ligands, including high throughput methods of identifying and sequencing T cell receptors able to bind a specific antigen. Methods of identifying cancer cell-specific antigens are further provided.

Description

TITLE OF THE INVENTIONMETHODS OF IDENTIFYING ANTIGEN-SPECIFIC T CELL RECEPTORSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Appl. Ser. No. 63 / 715,053, filed November 1, 2024, the entire disclosure of which is incorporated herein by reference.INCORPORATION OF SEQUENCE LISTING

[0002] A sequence listing containing the file named “MDCC030WO_ST26.xml” which is 14,723 bytes (measured in MS-Windows®) and created on October 24, 2025, and comprises 16 sequences, is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to the field of immunotherapy, and more specifically to compositions and methods for identifying antigen-specific T cell receptors and their cognate ligands.BACKGROUND OF THE INVENTION

[0004] A T cell-mediated immune response is initiated when T cells interact with immune cells or antigen-presenting cells via T cell receptors (TCRs). In certain processes, this interaction is mediated by major histocompatibility complex (MHC) molecules, including human leukocyte antigens (HLA) that are specialized to present peptide antigens at the cell surface as peptide:MHC (pMHC) antigens. Understanding which pMHC antigens can be targeted by T cells, and which TCRs mediate interaction with specific pMHC antigens is an important objective for cancer immunology, infectious disease research, and autoimmunity research. However, there is an enormous number of possible TCR sequences and pMHC combinations, making the process of identifying antigen-specific T cells and the TCRs mediating their specificity extremely complex.SUMMARY OF THE INVENTION

[0005] In one aspect, the present disclosure provides methods for producing a nucleic acid- barcoded peptide / MHC tetramers, the methods comprising contacting a set of biotinylated1US_ACTIVE\131538682W-1peptide MHC monomers with a pool of streptavidin conjugated nucleic acids to produce a nucleic acid-barcoded peptide MHC tetramer, wherein the pool comprises a unique combination of at least two distinct streptavidin conjugated nucleic acids, each comprising a distinct barcode sequence. In certain embodiments, the methods further comprise producing a nucleic acid-barcoded peptide MHC tetramer library by performing said method using a plurality of sets of biotinylated peptide MHC monomers, wherein each set of biotinylated peptide MHC monomers is contacted with a unique pool of streptavidin conjugated nucleic acids. In other embodiments, the methods further comprise producing the set of biotinylated peptide MHC monomers by UV peptide exchange prior to contacting the set with the pool of streptavidin conjugated nucleic acids. In some embodiments, the methods comprise contacting the peptide MHC tetramer with free biotin. In certain embodiments, the peptide MHC tetramer is non-fluorescent. In certain embodiments, the streptavidin conjugated nucleic acids further comprise a capture sequence. The method of the present disclosure, in some embodiments, may further comprise contacting the pool of streptavidin conjugated nucleic acids with fluorophore-conjugated streptavidin. In one embodiment, the fluorophore-conjugated streptavidin is not conjugated to a nucleic acid molecule.

[0006] In another aspect, the present disclosure provides a nucleic acid-barcoded peptide MHC tetramer produced by the disclosed methods. In certain embodiments, the present disclosure provides a nucleic acid-barcoded peptide MHC tetramer library produced by the disclosed methods. In further embodiments, the present disclosure provides a nucleic acid-barcoded peptide MHC tetramer library produced by the disclosed method, wherein the peptide MHC tetramers of the library are non-fluorescent. The present disclosure, in further embodiments, provides a nucleic acid-barcoded peptide MHC tetramer or a nucleic acid-barcoded peptide MHC tetramer library, wherein the nucleic acid-barcoded peptide MHC tetramer or the plurality of nucleic acid-barcoded peptide MHC tetramers each further comprise at least one streptavidin conjugated fluorophore. In a further aspect, the present disclosure provides methods of determining a specificity of at least one T cell receptor, the method comprising: (a) contacting a plurality of T cells with a nucleic acid-barcoded peptide MHC tetramer library provided herein; (b) contacting the plurality of T cells with an anti-streptavidin antibody; (c) separating at least one peptide MHC tetramer bound T cell from unbound T cells; and (d) determining the barcode sequence of at least one peptide MHC tetramer and a sequence of a polynucleotide encoding a TCRa variable region or a polynucleotide encoding a TCRP variable region of the at least one peptide MHC tetramer bound T cell. In yet another aspect2US_ACTIVE\131538682W-1the present disclosure provides a method of determining a specificity of at least one T cell receptor, the method comprising: (a) contacting a plurality of T cells with a nucleic acid- barcoded peptide MHC tetramer library of the present disclosure, wherein the plurality of nucleic acid-barcoded peptide MHC tetramers each further comprise at least one streptavidin conjugated fluorophorc; (b) separating at least one peptide MHC tetramer bound T cell from unbound T cells; and (c) determining the barcode sequence of at least one peptide MHC tetramer and a sequence of a polynucleotide encoding a TCRa variable region or a polynucleotide encoding a TCRP variable region of the at least one peptide MHC tetramer bound T cell. In some embodiments, a method of the present disclosure comprises determining the barcode sequence of a plurality of peptide MHC tetramers. In further embodiments, a method of the present disclosure comprises determining the sequence of the polynucleotide encoding the TCRa variable region and the polynucleotide encoding the TCRP variable region of the at least one peptide MHC tetramer bound T cell. In some embodiments, the antistreptavidin antibody comprises a fluorescent label. In certain embodiments, separating comprises fluorescent activated cell sorting (FACS). In yet further embodiments, the streptavidin conjugated nucleic acids further comprise a capture sequence. In some embodiments, the capture sequence is complementary to a nucleic acid sequence of a polynucleotide molecule coupled to a substrate, and the method further comprises contacting the at least one peptide MHC tetramer bound T cell with the substrate prior to determining the barcode sequence of at least one peptide MHC tetramer and a sequence of a polynucleotide encoding a TCRa variable region or a polynucleotide encoding a TCRP variable region of the at least one peptide MHC tetramer bound T cell. In further embodiments, a method of the present disclosure comprises separating a plurality of peptide MHC tetramer bound T cells from unbound T cells. In yet further embodiments, a method of the present disclosure comprises determining the barcode sequence of a plurality of peptide MHC tetramers and the sequence of the polynucleotide encoding the TCRa variable region or the polynucleotide encoding a TCRP variable region of the plurality of peptide MHC tetramer bound T cells. In still further embodiments, a method of the present disclosure comprises determining the sequence of the polynucleotide encoding the TCRa variable region and the polynucleotide encoding the TCR variable region of the plurality of peptide MHC tetramer bound T cells.

[0007] In another aspect, the present disclosure provides a nucleic acid-barcoded peptide MHC tetramer library comprising a plurality of nucleic acid-barcoded peptide MHC tetramers each comprising four biotin molecules, at least one streptavidin conjugated nucleic acid comprising3US_ACTIVE\131538682W-1a barcode sequence, and four peptide MHC monomers, wherein: each peptide MHC monomer of each MHC peptide tetramer comprises an identical peptide, the library comprises a plurality of MHC tetramers comprising identical peptides, and at least two MHC tetramers of the plurality of MHC tetramers comprising identical peptides comprise distinct barcode sequences. In some embodiments, the library comprises a plurality' of MHC tetramers comprising a plurality of distinct peptides.

[0008] In a further aspect, the present disclosure provides methods of determining a specificity of at least one T cell receptor, the method comprising: (a) contacting a plurality of T cells with the nucleic acid-barcoded peptide MHC tetramer library as described herein; (b) contacting the plurality of T cells with an anti-streptavidin antibody; (c) separating at least one peptide MHC tetramer bound T cell from unbound T cells; and (d) determining the barcode sequence of at least one peptide MHC tetramer and a sequence of a polynucleotide encoding a TCRa variable region or a polynucleotide encoding a TCRP variable region of the at least one peptide MHC tetramer bound T cell. In some embodiments, step (d) comprises determining the barcode sequence of a plurality of peptide MHC tetramers. In another aspect, the present disclosure provides method of determining a specificity of at least one T cell receptor, the method comprising: (a) contacting a plurality of T cells with a nucleic acid-barcoded peptide MHC tetramer library of the present disclosure, wherein the plurality of nucleic acid-barcoded peptide MHC tetramers each further comprise at least one streptavidin conjugated fluorophore; (b) separating at least one peptide MHC tetramer bound T cell from unbound T cells; and (c) determining the barcode sequence of at least one peptide MHC tetramer and a sequence of a polynucleotide encoding a TCRa variable region or a polynucleotide encoding a TCRP variable region of the at least one peptide MHC tetramer bound T cell. In further embodiments, a method of the present disclosure comprises determining the sequence of the polynucleotide encoding the TCRa variable region and the polynucleotide encoding the TCRP variable region of the at least one peptide MHC tetramer bound T cell. In yet further embodiments, the anti- streptavidin antibody comprises a fluorescent label. In certain embodiments, separating comprises fluorescent activated cell sorting (FACS). In further embodiments, the streptavidin conjugated nucleic acid further comprises a capture sequence. In yet further embodiments, the capture sequence is complementary to a nucleic acid sequence of a polynucleotide molecule coupled to a substrate, and the method further comprises contacting the at least one peptide MHC tetramer bound T cell with the substrate prior to determining the barcode sequence of at least one peptide MHC tetramer and a sequence of a polynucleotide encoding a TCRa variable4US_ACTIVE\131538682W-1region or a polynucleotide encoding a TCRP variable region of the at least one peptide MHC tetramer bound T cell. In some embodiments, a method of the present disclosure comprises separating a plurality of peptide MHC tetramer bound T cells from unbound T cells. In further embodiments, a method of the present disclosure comprises determining the barcode sequence of a plurality of peptide MHC tetramers and the sequence of the polynucleotide encoding the TCRa variable region or the polynucleotide encoding a TCRP variable region of the plurality of peptide MHC tetramer bound T cells. In yet further embodiments, a method of the present disclosure comprises determining the sequence of the polynucleotide encoding the TCRa variable region and the polynucleotide encoding the TCRP variable region of the plurality of peptide MHC tetramer bound T cells.

[0009] In another aspect, the present disclosure provides kits comprising: (a) a set of biotinylated peptide MHC monomers; and (b) a pool of streptavidin conjugated nucleic acids, wherein the pool comprises a unique combination of at least two distinct streptavidin conjugated nucleic acids, each comprising a distinct barcode sequence. In some embodiments, the kits further comprise free biotin, fluorophore conjugated streptavidin, or an antistreptavidin antibody. In further embodiments, the streptavidin conjugated nucleic acids further comprise a capture sequence. In certain embodiments, the kits further comprise a substrate coupled to a polynucleotide molecule comprising a nucleic acid sequence that is complementary to said capture sequence. In yet further embodiments, the kits comprise a nucleic acid-barcoded peptide MHC tetramer library as described herein. In yet further embodiments, the kits further comprise an anti-streptavidin antibody. In some embodiments, the streptavidin conjugated nucleic acid further comprises a capture sequence. In yet further embodiments, the kits further comprise a substrate coupled to a polynucleotide molecule comprising a nucleic acid sequence that is complementary to said capture sequence.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0011] Fig. 1 shows aspects of the disclosed method for identifying antigens and antigenspecific TCRs. Panel A shows a conceptual workflow of an embodiment of the method. Custom DNA barcoded tetramers are made and used to stain T cells. Tetramer positive cells5US_ACTIVE\131538682W-1are sorted and single-cell sequenced, yielding the TCR sequences and identity of the bound pMHC. Panel B shows a cell sample stained individually with pMHC tetramers and analyzed via flow cytometry (left) or in a pooled fashion using the method disclosed herein (right). Each cell is a row, each DNA barcode is a column; pMHC were dual-barcoded. Labels on plot denote pMHC represented by the barcode pattern. All tetramer positive populations detected by flow cytometry were detected by the method disclosed herein.

[0012] Fig. 2 shows that the disclosed method can recover validated antigen-binding TCRs from a single cell. Panel A shows Experiment 1 in which antigen- specific TCRs were identified in patient samples using the disclosed method. TCR sequences were introduced into retroviral vectors and primary human CD8 T cells were transduced to create TCR-T cells. Cells were then stained with APC / PE fluorescent tetramers to validate the ability of the TCR to bind tetramer. The number in the bottom right of the plot signifies the clone size - i.e., the number of single cells with the identical TCR identified in disclosed method. A light gray number denotes that TCR was called a binder, dark gray denotes a non-validated binder via fluorescent tetramer staining. Panel B shows a plot of the number of times a cell was observed using the disclosed method for both validated and non-validated TCRs. Most TCRs observed two or more times validated. About 50% of cells observed a single time validated. One large clone (13 cells) did not validate. This may be due to the presence of a second TCR chain that the assay did not pick up. Panel C shows Experiment 2: TCR-T validation as in Panel A on another set of TCRs identified using the disclosed method. Panel D shows a plot of the number of times the TCRs in Panel C were observed. All TCRs shown were validated binders.

[0013] Fig. 3 demonstrates that 3000 tetramers or more can be used in a single stain. A population of cells containing known antigen-specific TCRs against 10 p:HLA was taken, and stained with those 10 p:HLA using a triple DNA barcoding scheme, in the absence (Panel A) or presence (Panel B) of up to 3000 tests of irrelevant tetramers which were encoded with other DNA barcodes. DNA barcode UMI heatmap plots show that antigen specific cells (signified by cells with 3 distinct dark gray bands) are readily identifiable in the presence of the decoy tetramers. A modest increase in “non-specific” signal is seen in the presence of decoy tetramers. Panel C and Panel D show binding patterns of two antigen-specific TCR clonotypes identified in panel A and panel B. Each line represents a specific DNA barcode. Binding (Log2 UMI) of the DNA barcodes associated with the tetramer known to bind the clone’s TCR (black triangles) remained constant with added decoy tetramer. Open circles show Log2 UMI of the barcodes associated with other tetramers contained within the 106US_ACTIVE\131538682W-1p:HLA pool. Black closed circles show the UMI counts of decoy tetramer associated barcodes. The data show that while non-specific binding of decoy tetramers is observed (as represented as an increase in Log2 UMI reads) as decoy concentration increases, the specific binding of top 3 barcodes remain readily identifiable above the noise.

[0014] Fig. 4 shows identification of graft-vs-leukemia antigens and TCRs from patient samples using the disclosed method. Panel A shows a heatmap showing tetramer- associated barcode UMI on individual cells. A large number of graft-vs-leukemia antigen-specific cells (GvU) are highlighted. p:HUA #1 represents a minor histocompatibility antigen. Panel B shows that TCR-T cells expressing TCRs identified in Panel A were stained with APC / PE conjugated tetramers, confirming the antigen specificity identified in Panel A. Each plot represents a unique TCR; p:HLA #l-#4 represent 4 distinct peptide:HLA complexes that were found to bind these patient TCRs. In Panel C, p:HLA #1 specific TCR-T cells were cultured with HLA-A2+ cancer cell lines that express (TF1, U937) or do not express (THP1) the minor histocompatibility antigen. CD 137 expression on T cells shows that TCR-T cells recognize the antigen on the surface of target cells. In Panel D, TCR-T cells were cultured with cancer cell lines. TCR-T cells kill pMHC#l expressing cell lines. Representative data, 6 distinct TCRs tested with similar results.

[0015] Fig. 5 shows part 1 of a workflow overview of the disclosed high throughput, pooled method to simultaneously identify immunogenic pMHC and cognate paired TCRa and TCRP sequences. Panel A. Monocyte derived DCs are prepared from PBMCs, and are electroporated with synthetic mRNA encoding antigens of interest. These are cultured with autologous T cells. Panel B. A tiled peptide library containing all possible 8-11 amino acids containing / composing the antigen(s) of interest. The red star signifies a somatic mutation (e.g., DNMT3A R882H) Panel C. The peptides in B are empirically tested for MHC binding using the UV exchange method. Binding of a peptide to MHC is read out using an ELISA which measures the association of MHC to B2M (which only occurs in the presence of a binder peptide). Panel D. Synthesis of tetramer pool. All binder peptides identified in C are used, and DNA-barcoded peptide:MHC tetramers are produced. Each peptide:MHC combination has a unique DNA signature in the barcoding scheme. Panel E and Panel F. Plots showing NetMHC4.0 predicted peptide affinity (x-axis) vs. empirically determined recovery of monomers in the UV exchange assay (y-axis). Panel E shows peptide binding to HLA-A*02:01 and Panel F shows peptide binding to HLA-B*07:02. Dotted lines show binding cutoffs, with predicted binders set at %rank<2, and empirical binders set at a z-score of 3 from the mean. Solid line represents7US_ACTIVE\131538682W-1monomer recover in ELISA when monomer is not exposed to UV light. Peptides in the upper left quadrant represent peptides that were experimentally found to bind HLA using the UV exchange assay but were not predicted to do so. Circles around data points represent nonpredicted peptides that have additionally been detected as HLA binders in the literature using immunopcptidomics.

[0016] Fig. 6 shows part 1 of a workflow overview of the disclosed high throughput, pooled method to simultaneously identify immunogenic pMHC and cognate paired TCRa and TCRP sequences. Panel A) DNA-barcoded tetramer libraries are used to stain T cell cultures. Cultures are subsequently stained with a fluorescent anti-Streptavidin antibody, which makes all tetramer positive cells (regardless of p:MHC specificity) fluorescent. Tetramer positive cells are sorted, and single cell sequencing is performed. The TCR sequences of each cell, and the bound tetramer-associated DNA barcodes are recovered. Panel B) Representative data visualization. Each column is a cell, each row a DNA barcode. In this experiment, the antigen specificity is encoded by a unique combination of 2 or 3 DNA barcodes. Cells with the same antigen specificity have the same barcode binding pattern.

[0017] Fig. 7 shows an example of identification of neoantigen specific TCRs using the methods disclosed herein. T cells specific for common cancer driver neoantigens were generated. DNA barcoded tetramers (dual barcoded) were created. In the plot, each row represents a TCR. Columns are DNA barcodes, and column labels denote the antigen specificity of the identified TCRs. TCRs were only assigned an antigen specificity if the tetramer binding was concordant across the T cell clone; that is, only if every cell with the same TCR bound the same tetramer. In some cases (e.g. DNMT3A R882H:HLA-A*0L01), multiple antigen binding TCRs were identified.

[0018] Fig. 8 demonstrates that TCRs identified by the disclosed methods recognize endogenously processed / presented antigens. Panel A) TCR-T cells expressing DNMT3A R882X-HLA-A:01 :01 -specific TCRs were tested for potency and specificity in a peptide pulse experiment. Panel B) The TCRs in Panel A demonstrate recognition of endogenously processed and presented DNMT3A R882C / H-HLA-A*01:01 on cancer cells. Panel C) An IDH2 R140Q-HLA-B *07:02 specific TCR has modest (22nM) potency in a peptide dose response assay. Panel D) IDH2 R140Q-HLA-B*07:02 is expressed by TF1 cells and activates the TCR in Panel C.8US_ACTIVE\131538682W-1

[0019] Fig. 9 demonstrates that the disclosed method identified FLT3 D835X-HLA-A*02:01- specific TCRs. Panel A) TCR-T cells expressing FLT3 D835X-HLA-A*02:01 specific TCRs were prepared and stained with FLT3 D835Y, D835V, and D835H tetramers. One TCR, 1-2- la, bound to all 3 tetramers. 1-2-4 favored D835V, and 1-1-12 favored D835Y. (B) TCR-T cells were evaluated for potency in a peptide dose titration assay. TCRs were highly potent. 1-1-12 had an ECso of ~200pM, which was more potent than the NLV2 anti-viral TCR control response to CMV pp65 peptide (gray lines).

[0020] FIG. 10 shows an example of a tetramer CITEseq workflow in an embodiment of the method as disclosed herein and exemplary data produced therefrom. FIG. 10, Panel A, shows a schematic of a tetramer CITEseq workflow. DNA-barcoded peptide:MHC tetramers are produced and used to stain T cell cultures. Many different tetramer specificities can be pooled, and the tetramer ID can be determined via the DNA barcode sequences. One of the tetramers in the pool was SF3B1 K700E:HLA-A*02:01. Tetramer binding cells are sorted and single cell sequenced. FIG. 10, Panel B, shows heatmaps showing tetramer barcode sequencing for several cultures. Each column is a DNA barcode, and each row is a cell. SF3B 1 K700E-HLA- A*02:01 binding T cells are shown. Tetramers were dual-barcoded such that each antigen specificity is encoded by a combination of 2 unique barcodes. Other non-SF3Bl neoantigens were tested in same experiment (and can be seen by unique binding patterns of barcodes).

[0021] FIG. 11 demonstrates the specificity of seven SF3B 1 K700E-HLA-A*02:01 TCRs. FIG. 11, Panels A to Panel G, show expression of GDI 37 (an activation marker) on CD8+ TCR-T cells 18-24h after co-culture with SF3B 1 K700E mutant or WT peptide pulsed T2 target cells. All TCRs show higher sensitivity to mutant peptide (filled circles) compared to WT peptide (open circles). Of note, TCR5 (FIG. 11, Panel E) was particularly sensitive to the mutant peptide, with a >4 order of magnitude selectivity. FIG. 11 , Panel H shows specific lysis of peptide loaded T2 target cells by SF3B1 K700E TCR5 transduced cells and CMV NLV3 anti-viral TCR transduced cells after 4-hour co-culture. SF3B1 K700E specific cells demonstrate high specificity and potency.BRIEF DESCRIPTION OF THE SEQUENCES

[0022] SEQ ID NO: 1 - Representative antigenic peptide derived from SF3B1 comprising the K700E mutation.9US_ACTIVE\131538682W-1

[0023] SEQ ID NO:2 - An amino acid sequence of the CDR3 of the TCRP chain of TCR1 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.

[0024] SEQ ID NO:3 - An amino acid sequence of the CDR3 of the TCRa chain of TCR1 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.

[0025] SEQ ID NO:4 - An amino acid sequence of the CDR3 of the TCRP chain of TCR2 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.

[0026] SEQ ID NO:5 - An amino acid sequence of the CDR3 of the TCRa chain of TCR2 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.

[0027] SEQ ID NO:6 - An amino acid sequence of the CDR3 of the TCRP chain of TCR3 that specifically binds epitopes SF3B 1 comprising the K700E mutation presented on HLA- A*02:01.

[0028] SEQ ID NO:7 - An amino acid sequence of the CDR3 of the TCRa chain of TCR3 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.

[0029] SEQ ID NO:8 - An amino acid sequence of the CDR3 of the TCRP chain of TCR4 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.

[0030] SEQ ID NO:9 - An amino acid sequence of the CDR3 of the TCRa chain of TCR4 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.

[0031] SEQ ID NOTO - An amino acid sequence of the CDR3 of the TCRP chain of TCR5 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.

[0032] SEQ ID NO: 11 - An amino acid sequence of the CDR3 of the TCRa chain of TCR5 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.10US_ACTIVE\131538682W-1

[0033] SEQ ID NO: 12 - An amino acid sequence of the CDR3 of the TCRP chain of TCR6 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.

[0034] SEQ ID NO: 13 - An amino acid sequence of the CDR3 of the TCRa chain of TCR6 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.

[0035] SEQ ID NO: 14 - An amino acid sequence of the CDR3 of the TCRP chain of TCR7 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.

[0036] SEQ ID NO: 15 - An amino acid sequence of the CDR3 of the TCRa chain of TCR7 that specifically binds epitopes SF3B1 comprising the K700E mutation presented on HLA- A*02:01.

[0037] SEQ ID NO: 16 - a representative antigenic peptide from influenza matrix protein 1.DETAILED DESCRIPTION OF THE INVENTION

[0038] A T cell-mediated immune response is initiated when T cells interact with immune cells or antigen-presenting cells via T cell receptors (TCRs). In certain processes, this interaction is mediated by major histocompatibility complex (MHC) molecules, including human leukocyte antigens (HLA), that are specialized to present peptide antigens at the cell surface as peptide:MHC (pMHC) antigens. Understanding which pMHC antigens can be targeted by T cells, and which TCRs mediate interaction with specific pMHC antigens is an important objective for cancer immunology, infectious disease research, and autoimmunity research. However, there is an enormous number of possible TCR sequences and pMHC combinations, making the process of identifying antigen-specific T cells and the TCRs mediating their specificity extremely complex.A. Fluorescent pMHC Tetramers

[0039] Fluorescent pMHC tetramers are reagents that can be used to identify antigen-specific T cells. pMHC tetramers comprise four pMHC monomers, which are the physiological ligands for a TCR. In fluorescence-based assays, pMHC tetramers provide a useful increase in signal-to-noise ratio to compared to fluorescently labeled pMHC monomers. When a population of cells is incubated with a specific pMHC tetramer (e.g. Influenza matrix protein 1-derived G1LGFVFTL(SEQ ID NQ: 16):HLA-A*02:01), T cells whose TCR can recognize11US_ACTIVE\131538682W-1the specific pMHC bind it, and in doing so, are tagged with a fluorophore. These cells can then be analyzed or sorted using flow cytometry for downstream experimentation.

[0040] The conventional tetramer assay is a workhorse for identifying antigen-specific T cells, however, it is bound by the limitations of flow cytometry. There are a limited number of fluorophores that can be used to label tetramers, and the consequence of this is that only a few (usually 1 or 2, but possibly up to 20 with advanced strategies) fluorescent pMHC tetramers can be used to stain a single sample. This limits the utility of flow cytometry-based pMHC tetramer assays for antigen discovery purposes, especially given that only about 1% of predicted pMHC antigens are experimentally confirmed in most cases.

[0041] Attempts have been made to develop further assays for identifying antigen-specific T cells, however previously known methods are not capable of determining TCR sequences together with pMHC sequences, are only capable of evaluating a small number of antigens simultaneously, result in unacceptably high rates of false positive identification of pMHC, or provide unacceptably low sensitivity. Therefore, there is a continued need for improved high throughput methods of effectively identifying antigen-specific T cells and their TCR sequences.B. Methods for Determining TCR Specificity and Sequence

[0042] To overcome this deficiency, the present inventors have developed a pMHC tetramer assay in which pMHC tetramers are DNA-barcoded rather fluorescence encoded. Fig. 1, Panel A, shows an embodiment of the methods for determining TCR specificity and sequence provided herein. The disclosed methods allow thousands of pMHC tetramers to be screened using a single sample. In certain embodiments of the methods disclosed herein, a panel of thousands of biotinylated pMHC monomers is made, and biotinylated pMHC monomers are conjugated to DNA-barcoded streptavidin to form DNA-barcoded tetramers. These are pooled, and used to stain a population of cells. Tetramer binding cells are sorted, and single cell sequencing is performed. For each cell sequenced, both the TCRot and TCR sequences and the antigen specificity (via DNA barcodes) is determined. In certain embodiments, RNA gene expression may also optionally be recovered.

[0043] The methods for determining TCR specificity and sequence provided by the instant disclosure further offer uniquely high sensitivity and throughput. In certain embodiments of the methods described herein, tetramer panels are designed such that each pMHC antigen is labeled by at least two unique DNA barcodes, requiring binding between a specific pMHC12US_ACTIVE\131538682W-1antigen and a specific TCR to be assessed twice (once for each barcode label) and thereby confirmed. This increases the specificity of the assay and greatly lowers the likelihood of false positives as multiple independent binding events are required for detection.

[0044] The methods provided herein further enhance specificity and sensitivity because all cells with the same TCR sequence are required to bind the same pMHC antigen, which can both be determined from the assay. This results in very high confidence for any clones that have two or more cells. The methods provided herein therefore allow for enumeration of multiple clonotypes that bind the same pMHC, unlike methods that lack TCR sequence output.

[0045] In other embodiments of the methods provided by the instant disclosure, cross reactive TCRs (those that truly bind more than one pMHC) are identified.

[0046] The description and examples provided herein demonstrate that antigen specific cells detected via flow cytometry are robustly detected using the methods for determining TCR specificity and sequence provided herein. Furthermore, method development experiments demonstrate that antigen specific cells can be identified when 3000 or more tetramers are used in a single panel.

[0047] The instant disclosure therefore provides methods for determining TCR specificity and sequence which are significantly more specific, sensitive, and higher throughput than conventional methods. The methods described herein further provide sequence information for both the pMHC and bound TCR.13US_ACTIVE\131538682W-1

[0048] In an exemplary embodiment, a method for determining TCR specificity and sequence provided herein includes one or more of the following steps.• Step 1: DNA-barcoded streptavidin is generated, using conjugation methods or kits. In certain examples, this could be done using protein-oligo conjugation kit (e.g., Product S-9011-1, Vector Labs, Newark, CA). Pools of up to 100, up to 200, up to 300, up to 400, up to 500, up to 750, up to 1000, or more streptavidin-oligo conjugates with unique oligos may be generated.• Step 2: Arrayed pools of biotinylated pMHC monomers are generated. This can be done, for example, by using UV-mediated peptide exchange. In certain examples, peptides for use in creating pools of monomers can be designed using prediction algorithms or by using all possible peptides within a smaller protein sequence.• Step 3: A DNA-barcoded tetramer library is created by pooling streptavidin-oligo conjugates, such that there is a unique combination of at least two DNA-barcoded streptavidin-oligos in each pool, and adding biotinylated pMHC monomers to each pool. In some cases a fluorophore conjugated, non-DNA oligo conjugated streptavidin may be additionally added to the streptavidin pool prior to pMHC monomer addition. Streptavidin has 4 biotin binding sites. Biotinylated monomer binds the SA-ohgo to form tetramers. Soluble biotin is subsequently added to the tetramers to block any remaining unoccupied biotin binding sites on the streptavidin-oligos.• Step 4: A tetramer pool is created by combining the arrayed tetramers, which is then concentrated. This allows for staining of thousands of tetramers simultaneously on a single sample.• Step 5: Cells are stained with the tetramer pool. If no fluorophore conjugated SA was used in the tetramer pool, cells are stained with a fluorescent anti-streptavidin antibody, and all tetramer positive cells (regardless of tetramer ID) are sorted. This reduces higher order multimer production and background staining which results from most fluorescent SA-conjugates being multivalent (more than 1 SA per fluor). If a fluorescent SA- conjugate was included in the production of the tetramer pool, this step can be skipped.• Step 6: Cells may be sorted for tetramer positive cells (using aforementioned tetramer- associated fluorophores). In some embodiments the tetramer associated fluorophore is conjugated to a anti-SA antibody. In certain embodiments, the tetramer associated fluorophore is conjugated to streptavidin.• Step 7: Single cells are partitioned, for example by capturing single cells in gel emulsion beads using a 10X genomic controller.• Step 8: A library is constructed and sequenced. For each cell, paired TCRa and TCRP sequences, a pcptidc / MHC barcode, and gene expression data can be determined.• Step 9: Computational Analysis is conducted to ensure that tetramer barcode binding is significant compared to a background distribution and / or that all cells with the same TCR have the same tetramer barcode binding pattern.C. Methods for Identification of Cancer Cell-Specific pMHC

[0049] Cancer cell-specific pMHC antigens are attractive drug targets, as they provide an opportunity to target cancer cells while sparing healthy cells. These antigens may arise from14US_ACTIVE\131538682W-1somatic mutations in oncogenes (e.g., KRAS G12 mutations), or they may be the consequence of aberrant / overexpression of non-mutated proteins (e.g., cancer testis antigens, ERVs). pMHC antigens are formed when proteins are degraded into small peptides and loaded onto MHC molecules within the cell, and are subsequently expressed on the cell surface. These antigens can be targeted by T cells, T cell receptor (TCR) bascd / TCR-likc antibody therapeutics, and / or vaccines.

[0050] Identification of cancer cell-specific pMHC is challenging because most peptides are not processed and presented by MHC. Prediction algorithms are useful but they are known to be inaccurate. In addition, in the human population, HLA are highly polymorphic; each individual expresses a specific set of HLA-A, -B, and -C alleles, and it is the distinct combination of a peptide and specific HLA molecule constitutes the pHLA antigen. A functional consequence of this is that most pMHC discovery work is performed only on the most common HLA allele, HLA-A*02:01 (which most people still do not express). Peptide:MHC discovery on more MHC types provides an opportunity to address the larger population.

[0051] Existing strategies employed by the field to identify cancer- specific pMHC are labor intensive and low sensitivity. For example, immunopeptidomics, where peptides are eluted off of MHC and identified using mass spectrometry, only detects highly abundant pMHC. However, most pMHC are very rare (<20 copies per cell). A TCR can identify and initiate an immune response against a single pMHC molecule, meaning that immunologically relevant pMHC are routinely missed by immunopeptidomics. Methods to rapidly, efficiently, and robustly identify “immunologically visible" cancer cell-specific pMHC are needed.

[0052] The present disclosure therefore provides improved methods for identification of cancer cell-specific pMHC. The instant disclosure provides high throughput, pooled methods to simultaneously identify immunogenic pMHC and cognate paired TCRa and TCRP sequences. For example, in humans, thousands of peptides can be screened against unique HLA-I alleles simultaneously. Demonstration of pMHC immunogenicity occurs inline and is the feature of the system.

[0053] In certain embodiments, methods for identification of cancer cell-specific pMHC provided in the instant disclosure include following steps.Step 1: Generating a pool of antigen- specific T cellsStep 2: Generating a DNA-barcoded peptide / MHC library15US_ACTIVE\131538682W-1• Step 3: Using the DNA barcoded library created in step 2 to identify antigen specific TCRs generated in step 1 using methods provided herein.D. DNA-barcoded Streptavidin

[0054] In certain embodiments of the invention, DNA-barcoded streptavidin contains streptavidin which is covalently linked to DNA oligonucleotides. There are many ways to generate streptavidin-oligo conjugates, including commercial sourcing, using commercially available kits for barcode labeling, or other methods to covalently linked DNA oligonucleotides to proteins . In certain examples, this could be done using protein-oligo conjugation kit (e.g., Product S-9011-1, Vector Labs, Newark, CA). When this kit is used, 5’ amino-oligonucleotides are reacted with succinimidyl 4-formylbenzoate (s-4FB) to yield 5’ 4FB-tagged oligos. Amino groups on streptavidin (e.g., lysine) are reacted with succinimidyl 6-hydrazinonicotinate acetone hydrazine (S-HyNic) to yield HyNic modified streptavidin. 5’ 4FB-tagged oligos and HyNic-modified streptavidin are then mixed in the presence of an aniline catalyst, such that a covalent bond forms between the 4FB moiety and a HyNic moiety yielding DNA-barcoded streptavidin. In certain embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80 at least 85, at least 90, at least 95, at least 100, at least 200, at least 300, at least 400, at least 500, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2100, at least 2200, at least 2300, at least 2400, at least 2500, at least 2600, at least 2700, at least 2800, at least 2900, at least 3000, at least 3100, at least 3200, at least 3300, at least 3400, at least 3500, at least 3600, at least 3700, at least 3800, at least 3900, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500, at least 10000, or more, streptavidin-oligo conjugates with unique DNA barcode oligos may be generated.E. Biotinylated pMHC Monomers

[0055] In certain embodiments of the invention, arrayed pools of biotinylated pMHC monomers are generated. Biotinylated MHC or HLA monomers may be generated using UV exchange methodology as described by Rodenko (Nat Protoc. 2006; 1 (3) : 1120-32) or by other methods known in the art.16US_ACTIVE\131538682W-1

[0056] Peptides for use in generating biotinylated pMHC or HLA monomers may be any peptides of interest and may be of any origin. For example, peptides may designed using prediction algorithms or may be designed to include all possible peptides found within a protein sequence. Peptide sequences may be of any length, for example comprising at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids, at least 18 amino acids, at least 19 amino acids, at least 20 amino acids, at least 21 amino acids, at least 22 amino acids, at least 23 amino acids, at least 24 amino acids, at least 25 amino acids, at least 26 amino acids, at least 27 amino acids, at least 28 amino acids, at least 29 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, at least 50 amino acids, or more, in length.F. DNA-Barcoded Tetramer Libraries

[0057] In certain embodiments, the methods provided herein include generation of an arrayed DNA-barcoded tetramer library. Barcoded streptavidin-oligo conjugates are matrixed such that each pool is unique combination of two or more DNA-barcoded streptavidin-oligo conjugates. In some embodiments, a first streptavidin-oligo conjugate having a first DNA barcode is pooled with a second streptavidin-oligo conjugate having a second DNA barcode. In further embodiments, a first streptavidin-oligo conjugate having a first DNA barcode and a second streptavidin-oligo conjugate having a second DNA barcode may be pooled with a third streptavidin-oligo conjugate having a third DNA barcode, a fourth streptavidin-oligo conjugate having a fourth DNA barcode, a fifth streptavidin-oligo conjugate having a fifth DNA barcode, a sixth streptavidin-oligo conjugate having a sixth DNA barcode, a seventh streptavidin-oligo conjugate having a seventh DNA barcode, an eighth streptavidin-oligo conjugate having an eighth DNA barcode, a ninth streptavidin-oligo conjugate having a ninth DNA barcode, or a tenth streptavidin-oligo conjugate having a tenth DNA barcode. A pool of DNA-barcoded streptavidin-oligo conjugates may further comprise an number of additional streptavidin-oligo conjugates having further DNA barcodes.

[0058] In certain embodiments, biotinylated pMHC monomers as described herein are added to a pool of DNA-barcoded streptavidin-oligo conjugates to form a DNA-barcoded tetramer library. Biotinylated pMHC monomers bind to streptavidin-oligo conjugates at the four biotin binding sites, forming DNA barcoded pMHC tetramers.17US_ACTIVE\131538682W-1

[0059] In certain embodiments, DNA barcoded tetramers are made in an arrayed format (that is, a single type of monomer is mixed with SA-oligo). The S A-oligo may be a pool of multiple SA-oligo types, but the monomer is a homogenous / single type). After the monomer is reacted to make tetramers, then the different tetramers in the array may be pooled to make a pooled tetramer library.

[0060] In one example of the disclosed method, a first streptavidin-oligo conjugate having a first DNA barcode is pooled with a second streptavidin-oligo conjugate having a second DNA barcode. Biotinylated pMHC monomers are then added to the pool of streptavidin-oligo conjugates to form DNA-barcoded pMHC tetramers having the first and / or the second DNA barcode. This can be used to confirm binding of the specific pMHC to a specific TCR when both DNA-barcodes are observed to be associated with the specific TCR after cell staining and sequencing.G. Tetramer Pools

[0061] In some embodiments of the disclosed methods, a tetramer pool is created by adding free biotin to the arrayed tetramer library as described herein, followed by pooling the matrix of DNA- barcoded tetramers and concentrating them. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80 at least 85, at least 90, at least 95, at least 100, at least 200, at least 300, at least 400, at least 500, at least 700, at least 800, at least 900, at least 1000, at least 1 100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2100, at least 2200, at least 2300, at least 2400, at least 2500, at least 2600, at least 2700, at least 2800, at least 2900, at least 3000, at least 3100, at least 3200, at least 3300, at least 3400, at least 3500, at least 3600, at least 3700, at least 3800, at least 3900, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500, at least 10000, or more, DNA-barcoded pMHC tetramers arc pooled.H. Cell Staining

[0062] In some embodiments of the disclosed methods, cells are stained with the DNA-barcoded tetramer pool, which is non-fluorescent. In further embodiments, cells may be stained with a DNA-barcoded tetramer pool which is fluorescent due to the covalent linkage of a streptavidin18US_ACTIVE\131538682W-1molecule to a fluorophore. Without wishing to be bound by theory, the DNA-barcoded pMHC tetramers are able to bind to TCRs on the cells during staining.

[0063] In further embodiments, cells are stained with a fluorescent anti-streptavidin antibody, and all tetramer-positive cells (identified by the fluorescent antibody) are sorted. Fluorescent antibodies may be labeled with any fluorescent label or fluorophore known in the art, such as for example phycoerythrin (PE), allophycocyanin (APC), PE-Cy5, PE-Cy7, APC-Cy7, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705, Brilliant® Violet (BV) 421, BV 605, BV 510, BV 711, BV786, PerCP, PerCP / Cy5.5, AlexaFluor® 488, AlexaFluor® 647, FITC, BV570, BV650, DyLignt® 488, Dylight® 649, PE / Dazzle® 594.I. Cell Sorting

[0064] In certain embodiments of the disclosed methods, cells are sorted using any method known in the art, for example a flow sorter, to partition all pMHC tetramer-positive cells. Single cells are then partitioned using any known methods, for example by capturing single cells in gel emulsion beads using a 10X Chromium Controller (10X Genomics, Pleasanton, CA).J. Library Construction and Sequencing

[0065] For each cell, paired TCRa and TCR0 sequences, as well as pMHC ID are obtained. In further embodiments, gene expression data is also obtained from single cells. During single cell partitioning by the 10X Chromium Controller, cells are deposited in emulsion droplets that additionally contain a 10X gel bead. Within the emulsion droplet, mRNA are reverse transcribed in the presence of both a poly-dT primer (which additionally contain a non-poly- dT PCR handle) and a 5’ binding template switch oligo. This results in the conversion of cellular mRNA to cDNA linked to a cellular barcode, a unique molecular identifier (UMI), and 5’ and 3’ PCR handles. Concurrently, tetramer associated barcodes are also linked cellular barcodes, a unique molecular identifier, a 5’ PCR handle (which was not originally templated by the tetramer associated barcode), and a 3’ PCR handle (which was templated by the tetramer barcode.

[0066] To construct sequencing libraries, cDNA is first amplified using PCR. After amplification, PCR products are size selected using SPRIselect beads, at a 0.6X SPRIselect reagent to sample ratio. The SPRI beads are harvested and “long” nucleic acids, which containing the TCR and gene expression libraries are eluted. The supernatant, which contains “short” tetramer barcode nucleic acids, is also harvested.19US_ACTIVE\131538682W-1

[0067] For TCR library construction, TCR sequences are PCR amplified from “long” size selected amplified cDNA. Two rounds of PCR are performed - TCR amplification 1 utilizes the “long” size selected cDNA as a template, and a forward primer complementary to the 5’ end of the cDNA and additionally contains sequencing adapters, and “outer” reverse primers complementary to constant TRAC and TRBC1 / TRBC2 sequences. TCR amplification 2 utilizes the product from TCR amplification 1 as a template, the same forward primer as in PCR 1, and “inner” primers complementary to the a more 5’ sequence contained within the constant TRAC and TRBC1 / TRBC2 genes. After two rounds of TCR amplifications, enzymatic fragmentation and size selection are used to generate variable length fragments that collectively span the entire V(D)J segments of the amplified TCR transcripts prior to library construction. P5, P7, i5 and i7 sample indexes, and an Illumina R2 sequence (read 2 primer sequence) are collectively and sequentially added via enzymatic reactions including End Repair, A-tailing, Adaptor Ligation, and eventually typically 6-8 cycles of Sample Index PCR depending on the ng of cDNA carry forward V(D)J-amplified product input calculated post V(D)J amplification. The final TCR libraries contain the P5 and P7 primers used in Illumina amplification for sequencing.

[0068] For tetramer barcode library construction, a single PCR is performed. In this PCR, the an aliquot of the “short” size selected fraction of the amplified cDNA is used as a template. The forward primer binds the 5’ end of the template, and additionally contains the sequencing adaptor P5 sequence and i5 sample index. The reverse primer binds the 3' end of the template, and additionally contains the sequencing adaptor P7 sequence and i7 sample index.

[0069] For gene expression library construction, an aliquot of the “long” fraction of the amplified cDNA is used. This cDNA is enzymatically fragmented, end repaired, and A-tailed. The resultant DNA pool is double sided SPRI size selected. In the first selection, the nucleic acids are mixed with SPRIselect reagent at a 0.6X SPRIselect to sample ratio, and the supernatant is recovered. In the second selection, the supernatant from the first selection is mixed with SPRIselect reagent at a 0.8X SPRIselect to sample ratio, and the SPRI bead bound nucleic acids are recovered and eluted. Sequencing adaptors containing illumina read 2 sequence are ligated to the 3’ end of the nucleic acids, and the product is SPRI selected using a 0.8X ratio of SPRIselect ratio to sample, recovering the bead bound fraction of nucleic acids. Subsequently index PCR is carried out. In this reaction, the eluted nucleic acid pool is the template, the forward primer is complementary to the 5’ end of the nucleic acid pool and additionally contains P5 primer and i5 indexes, and the reverse primer is complementary to the20US_ACTIVE\131538682W-13’ end of the nucleic acid pool and contains P7 primers and i7 indexes. Libraries are cleaned up using double sided SPRI bead size selection (first selection at 0.6X reagent: sample ratio, recover supernatant fraction, and second selection at 0.8X reagent:sample ratio, recover bead bound fraction) .All libraries are standard Illumina paired end libraries, containing P5 and P7 sequences. Libraries arc sequenced using an Illumina Novoscq 6000, on SI flow cells with vl.5 150 cycle sequencing kits. All libraries utilize Truseq read 1 sequencing primers to recover 10X cell barcodes and UM1. TCR and gene expression libraries utilize Truseq read 2 primers to recover TCR / gene sequences. Tetramer barcode libraries utilize Nextera read 2 primers to recover tetramer barcode sequences. Typically 5,000 reads per cell is targeted for TCR and tetramer barcode libraries, while 20,000 reads per cell is targeted for gene expression libraries.K. Computational Analysis

[0070] To avoid false positive results due to non-specific binding between pMHC and TCRs, computational analysis is conducted to ensure that tetramer binding is significant compared to a background distribution. Further, all cells comprising the same TCR sequences are expected to have the same tetramer barcode binding pattern.L. Peptide Library Creation for Methods for Identification of Cancer Cell-Specific pMHC

[0071] In methods for identification of cancer cell-specific pMHC provided in the instant disclosure may include generating a pool of antigen- specific T cells. CD 14-positive monocytes and naive CD8 T cells are isolated from healthy donor PBMCs. Monocytes are then differentiated into dendritic cells (moDCs) using cytokine cocktail containing GM-CSF, IL-4, IFNg and LPS. Mature moDCs are then electroporated with mRNA, which contains the antigen(s) of interest. In some embodiments, mRNA may contain concatenated minigenes, each minigene encoding a 25 amino acid long peptide with a mutation associated residue at position 13. In other embodiments, mRNA encoding other candidate antigens, such as tumor- associated antigens, cancer testis antigens, aberrant expressed antigens, or self-antigens may be used. Autologous naive CD8 T cells are stimulated by transfected moDCs with the goal of expanding antigen-specific T cells. T cell-expansion is firstly driven by IL-21 followed by the combination of IL-7 and IL- 15 for a total of 10 to 14 days21US_ACTIVE\131538682W-1M. Pharmaceutical and Therapeutic Compositions

[0072] In certain aspects, the present disclosure provides pharmaceutical and therapeutic compositions comprising the peptides, TCRs, and / or cells of the present disclosure. In some embodiments, the peptides, TCRs, and / or cells of the present disclosure may be combined with a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier,” “pharmaceutically acceptable adjuvant,” or “adjuvant” refers to reagents, cells, compounds, materials, compositions, and / or dosage forms that are not only compatible with the polynucleotide molecules, polypeptide molecules, cells, and / or or other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit / risk ratio. Also included may be an agent that modifies the effect of other agents and is useful in preparing a therapeutic compound or composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable. Such an agent may be added to a therapeutic composition to modify the immune response of a subject by boosting the response such as to give a higher amount of polynucleotide molecules, polypeptide molecules, and or cells and longer-lasting protection from degradation. Such an agent may include any excipient, diluent, carrier, or adjuvant that is acceptable for pharmaceutical use. Such an agent may be non-naturally occurring, or may be naturally occurring, but not naturally found in combination with other agents in the immunogenic composition.

[0073] As used herein, a “therapeutic compound” or “therapeutic composition” refers to a composition comprising a peptides TCR, and / or cell of the present disclosure. In one embodiment, the composition is capable of eliciting an immune response. Such a compound or composition is meant to encompass a composition suitable for administration to a subject, such as a mammal, particularly a human subject. In general, a therapeutic composition is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the immunogenic composition is pharmaceutical grade). Therapeutic compositions may be designed for administration to subjects in need thereof via a number of different routes of administration including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalational, and the like. The appropriate dosage of a composition, as described herein, may be determined based on the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, clinical22US_ACTIVE\131538682W-1history, response to the treatment, and the discretion of the attending physician. In some embodiments, therapeutic compositions provided by the present disclosure may include various “unit doses.” A unit dose is defined as containing a predetermined quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0074] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0075] As used herein, “subject” or “patient” refers to animals, including humans, who are treated with the therapeutic compounds or compositions or in accordance with the methods described herein. For diagnostic or research applications, a wide variety of mammals may be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine, such as inbred pigs and the like.

[0076] A composition, as described herein, may include, in particular embodiments, a combination of therapeutic agents. In some embodiments, a composition as described here may be administered as a single composition or as more than one composition. Different compositions as provided herein, in certain embodiments, may be administered by the same route of administration or by different routes of administration.

[0077] In certain embodiments, the compositions, and methods for treating an individual described herein may be combined with any other composition or method of treatment known in the art. The compositions and methods may be administered in any suitable manner known in the art. For example, a first and a second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time). In some aspects, a first and a second cancer treatment may be administered in separate compositions. In certain embodiments, a first and a second cancer treatment may be administered in the same composition.

[0078] Non-limiting examples of additional treatment modalities that may be included in combination with the compositions and methods provided herein include immunotherapy,23US_ACTIVE\131538682W-1chemotherapy, radiation therapy, molecular targeted therapy, hormone therapy, transplantation, and surgery.N. Detection and Therapeutic Agents

[0079] In some aspects, the present disclosure provides methods and compositions for detection and therapeutic labeling of the peptides, TCRs, and / or cells provided by the present disclosure. Methods for labeling and detection of polypeptide molecules are well-known in the art, and any such method known in the art may be used to label or detect the polypeptides described herein. As a non-limiting example, polypeptides may be labeled with a detectable moiety, such as a radioactive atom, a chromophore, a fluorophore, or the like, and then detected using methods known in the art. Such labeled polypeptides may be used, in some embodiments, for in vivo or in vitro diagnostic techniques.

[0080] As used herein, the term “label” refers to a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected. In certain embodiments, a polynucleotide molecule, protein, or cell may be labeled to generate a labeled composition. In particular embodiments, labeled compositions also include sequences which are conjugated a polynucleotide molecule that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable. Labels may be suitable for small scale detection or for high- throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Labels may be simply detected or may be quantified. In certain embodiments, labels that may be quantified provide numerically reportable value. In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.O. Kits

[0081] Certain aspects of the present disclosure further provide kits containing compositions of the disclosure or compositions to implement methods of the invention. In some aspects, kits can be used to evaluate one or more biomarkers or MHC / HLA types. In certain aspects, a kit contains, contains at least or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,24US_ACTIVE\131538682W-115, 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, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therein.

[0082] Kits may comprise, in certain embodiments, components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.

[0083] Individual components, in particular embodiments, may also be provided in a kit in concentrated amounts. In one embodiment, a component is provided individually in the same concentration as it would be in a solution with other components. In certain embodiments, concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more.

[0084] In certain aspects, negative and / or positive control nucleic acids, probes, and inhibitors are included in some kit aspects. In some embodiments, a kit may include a sample that is a negative or positive control for methylation of one or more biomarkers.P. Definitions

[0085] As used herein, a “cell” may refer to a bacterial, mammalian, yeast, or insect cell, specifically to a mammalian cell such as human cell. In certain embodiments, a call may be a cancer cell or a precancerous cell. In another embodiment, the cancer or the precancerous condition may be selected from the group consisting of: myelodysplastic syndrome, acute myeloid leukemia, chronic lymphocytic leukemia, breast cancer, lung cancer, colorectal cancer, pancreatic cancer, appendiceal cancer, small bowel adenocarcinoma, hepatobiliary cancer, a gynecological malignancy, hematopoietic cancer a bladder cancer, prostate cancer, or skin cancer, or any combination thereof. In particular embodiments, the cancer or the precancerous condition is selected from the group consisting of myelodysplastic syndrome, acute myeloid leukemia, chronic lymphocytic leukemia, breast cancer, or any combination thereof.

[0086] As used herein, a “T cell” or “T-lymphocyte” refers to an immune cell involved in the adaptive immune response.

[0087] As used herein, a “T cell receptor” or “TCR” refers to a receptor found on the surface of T cells that is capable of recognizing antigens bound to major histocompatibility complex (MHC) molecules including human leukocyte antigen (HLA). A TCR may have highly variable a and chains (TCRa and TCR0) linked by a disulfide bond.25US_ACTIVE\131538682W-1

[0088] As used herein, a “barcode” or “DNA barcode” refers to a unique nucleic acid sequence useful identifying a molecule to which it is linked, such as an oligo nucleotide or streptavidin molecule.

[0089] As used herein, “major histocompatibility complex” or “MHC” may refer genes encoding MHC class I and class II glycoproteins, or MHC1 and MHC2 molecules, that can present peptides to T-cell receptors. In humans, “human leukocyte antigen” or “HLA” refer to proteins encoded by MHC genes.

[0090] As used herein, “peptide antigen” or “peptide” refers to an amino acid sequence that is recognized by a TCR. A peptide antigen may be derived from a larger molecule, and may be host-derived (e.g., tumor antigen, autoimmune antigen) or exogenously derived (e.g., bacterial, viral). The antigenic peptide may be of any length that is capable of being presented by class I or class II MHC molecule. In particular embodiments, the antigenic peptide may be 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, or 40 amino acid residues in length. An antigenic peptide of the present disclosure may be isolated from the organism in which it naturally occurs, produced by recombinant expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods known in the art.

[0091] Complexes of peptide and MHC class I are recognized by CD8-positive T cells bearing the appropriate TCR, whereas complexes of peptide and MHC class II molecules are recognized by CD4-positive-helper-T cells bearing the appropriate TCR.

[0092] As used herein, “peptide / MHC” or “pMHC” refers to a peptide antigen complexed with an MHC. A “peptide / HLA” or “pHLA” refers to a peptide antigen complexed with an HLA.

[0093] The term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. When used in conjunction with the word “comprising” or other open language in the claims, the words “a” and “an” denote “one or more,” unless specifically noted otherwise. The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” are also open- ended. For example, any method that “comprises,” “has,” or “includes” one or more steps is26US_ACTIVE\131538682W-1not limited to possessing only those one or more steps and also covers other unlisted steps. Similarly, any system or method that “comprises,” “has,” or “includes” one or more components is not limited to possessing only those components and covers other unlisted components. As used herein, the term “consists essentially of’, when used in reference to a nucleotide or amino acid sequence of the present disclosure, means that the nucleotide sequence or amino acid sequence, may contain additional nucleotides or amino acids so long as the additional nucleotides or amino acids do not materially alter the function of the recited sequences. The term “materially alter,” as applied to a nucleotide sequence or amino acid sequence of the present disclosure, refers to a decrease in the antigen binding activity of the encoded polypeptide or polypeptide sequence of at least 25%. For example, additional nucleotides or amino acids added to a nucleotide or an amino acid sequence of the present disclosure may be deemed to “materially alter” the encoded polypeptide or polypeptide sequence, if such additions decrease the antigen-specific binding activity by at least 25%.

[0094] Other objects, features, and advantages of the present disclosure are apparent from detailed description provided herein. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. Any embodiment of the present disclosure may be used in combination with any other embodiment described herein.

[0095] All references herein are incorporated herein by reference in their entirety.EXAMPLES

[0096] The following examples are included to illustrate embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and27US_ACTIVE\131538682W-1modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.Example 1: A High Throughput Method of Identifying Antigens and Antigen-Specific TCRs

[0097] Methods for identifying antigens and antigen-specific TCRs with greater specificity and sensitivity were designed and tested. In an exemplary method, referred to as Tetramer CITEseq, a panel of thousands of biotinylated peptide:MHC monomers was made. Biotinylated peptide- MHC monomers were conjugated to DNA-barcoded streptavidin to form DNA-barcoded tetramers. DNA-barcoded tetramers were pooled and used to stain a population of cells. Cells binding tetramers were sorted, and single cell sequencing was performed using the 10X Genomics 5’ Immune profiling workflow.

[0098] An exemplary workflow is shown in Fig 1, Panel A. Custom DNA barcoded tetramers are made and used to stain T cells. Tetramer positive cells are sorted and single-cell sequenced, yielding the TCR sequences and identity of the bound pMHC. Fig. 1 , Panel B, shows a cell sample stained individually with pMHC tetramers and analyzed via flow cytometry (left) or in a pooled fashion using the disclosed method (right). All tetramer positive populations detected by flow cytometry were detected by the method disclosed herein. Using this method, both the TCRa and TCRP sequences and the antigen specificity (via DNA barcodes) was determined. RNA gene expression can also optionally be determined.Example 2: Comparison with Fluorescent Tetramer Staining

[0099] Experiments were performed to demonstrate that the method disclosed herein can recover validated antigen-binding TCRs from a single cell. Fig. 2, Panel A, shows Experiment 1 in which antigen-specific TCRs were identified in patient samples using the disclosed method. TCR sequences were introduced into retroviral vectors and primary human CD8 T cells were transduced to create TCR-T cells. Cells were then stained with APC / PE fluorescent tetramers to validate the ability of the TCR to bind tetramer.

[0100] TCR-T validation on a set of TCRs is shown in Fig. 2, Panel A. The number in the bottom right of the plot signifies the clone size (the number of single cells with the identical TCR identified).28US_ACTIVE\131538682W-1

[0101] Fig. 2, Panel B, shows a plot of the number of times a cell was observed in the disclosed method for both validated and non-validated TCRs. Most TCRs observed two or more times corresponded to validated TCRs. About 50% of cells observed a single time corresponded to validated TCRs. One large clone (13 cells) did not validate. This may have been due to the presence of a second TCR alpha or beta chain that the assay did not detect, or due to poor expression of TCR in TCR-T construct.

[0102] Fig. 2, Panel C, shows Experiment 2: TCR-T validation as in Fig. 2, Panel A, using another set of TCRs identified using the disclosed method. Fig. 2, Panel D, shows a plot of the number of times the TCRs in Fig. 2, Panel C, were observed. All TCRs shown in Fig. 2, Panel C, were validated binders.

[0103] These experiments show that the ability of the method disclosed herein to recover TCR sequences greatly enhances specificity / sensitivity, as all cells with the same TCR are required to bind the same peptide:MHC in the disclosed workflow. This results in very high confidence for any clones whose cells are observed 2 or more times (Fig. 2). The method disclosed herein further allows for enumeration of multiple clonotypes that bind the same peptide:MHC, unlike methods that do not provide the TCR sequence as output (Fig. 2, Panel A, Fig. 2, Panel C). These experiments further demonstrate that antigen-specific cells detected via flow cytometry are robustly detected using the method disclosed herein (Fig IB).

[0104] Additional experiments carried out with the disclosed method further demonstrate that antigen-specific cells can be identified when as many as 3000 tetramers are used in single panel (Fig. 3). To assess the tetramer capacity of the disclosed method, a population of cells containing known antigen-specific TCRs against 10 pMHC was stained with those 10 pMHC using a triple DNA barcoding scheme, in the absence (Fig. 3, Panel A) or presence (Fig. 3, Panel B) of up to 3000 tests worth of irrelevant tetramers. A test of tetramer is defined as 0.1 ug of monomer, in tetramerized format. The DNA barcode UMI heatmap plots show that antigen specific cells (signified by cells with 3 distinct ’’dark gray” bands) are readily identifiable in the presence of the decoy tetramers. A modest increase in “non-specific” signal is seen in the presence of decoy tetramers. (Fig. 3, Panel C) and (Fig. 3, Panel D) show binding patterns of two antigen- specific TCR clonotypes in (A) and (B). Each line represents a specific DNA barcode. Binding (Log2 UMI) of the barcodes associated with the tetramer known to bind the clone’s TCR (black triangles) remained relatively constant with added decoy tetramer. Open circles show Log2 UMI of the barcodes associated with other tetramers contained within the 10 p:HLA pool. Black closed circles show the UMI counts of decoy29US_ACTIVE\131538682W-1tetramer associated barcodes. Binding of decoy tetramers increases with increasing decoy concentration, but the top 3 barcodes remain readily identifiable.Example 3: Methods for Antigen Discovery

[0105] An example of the application of the disclosed method to antigen discovery is shown in Fig. 4. PBMCs from an AML patient ~90 days post transplant were incubated with a panel of -500 unique labeled DNA barcoded peptide:MHC tetramers, with the goal of identifying candidate anti-leukemia TCRs. The tetramer panel was designed to include common HLA- A*02:01 restricted minor histocompatibility antigens, neoantigens, leukemia associated antigens, and cancer testis antigens. Tetramer positive cells were sorted, and single cells sequenced. In Fig. 4, Panel A, clusters of cells with unique DNA barcode binding patterns are shown. To validate TCR antigen specificities, TCRs were introduced into viral vectors and expressed in TCR-T cells, which were then stained with dual color fluorescent tetramers to confirm binding (Fig. 4, Panel B). 10 exemplary TCR-Ts are shown, in which binding of the tetramer CITEseq identified p:HLA was validated by fluorescent tetramer staining. Two of the TCR-T cell lines specific for p:HLA#l (a novel minor histocompatibility antigen) were activated by (Fig. 4, Panel C) and killed (Fig. 4, Panel D) cancer cell lines expressing the minor antigen allele (TF1, U937) while ignoring / sparing THP1 cells, which do not have the minor antigen allele. Four other TCRs specific for p:HLA#l were identified and displayed similar activity to those shown in Fig 4C and 4D (data not shown).Example 4: Method of Identifying Immunogenic Antigens and Antigen-Specific TCRs Simultaneously

[0106] Cancer cell-specific peptide:MHC (pMHC) antigens are attractive drug targets, as they provide an opportunity to target cancer cells while sparing healthy cells. These antigens can be targeted by T cells, T cell receptor (TCR) based / TCR-like antibody therapeutics, and / or vaccines.

[0107] Existing strategies employed by the field to identify pMHC are labor intensive and low sensitivity. Methods to rapidly, efficiently, and robustly identify “immunologically visible” cancer cell-specific pMHC are needed.

[0108] In order to address this, the present disclosure provides a high throughput, pooled method to simultaneously identify immunogenic pMHC and cognate paired TCRa and TCRP sequences. For example, in humans, thousands of peptides can be screened against up to 630US_ACTIVE\131538682W-1unique HLA-I alleles simultaneously. Demonstration of pMHC immunogenicity occurs inline and is the feature of the system.

[0109] The disclosed method includes three parts as follows:• Generating a pool of antigen specific T cells.• Generating a DNA-barcoded peptide / MHC library.• Using the DNA barcoded library created in step 2 to identify antigen specific TCRs generated in step 1 according to the currently disclosed methods.

[0110] In the first part of this method (Fig. 5 A), pMHC-specific cells are generated in a pooled culture. Monocyte-derived dendritic cells (DCs) are transfected with a synthetic mRNA encoding the candidate protein antigens of interest. This mRNA can encode a single protein, or a series of peptides of interest (i.e., a tandem minigene). The mRNA is translated by the DC and the resultant protein is exposed to the antigen processing machinery. If the resultant protein is capable of being processed and loaded onto an MHC molecule within the cell, the resultant pMHC is expressed on the surface of the cell.

[0111] The DCs are then co-cultured with a large number of autologous T cells, each with a unique TCR. If a T cell has a TCRs that recognizes the DC-expressed pMHC, it receives an agonistic signal and undergoes clonal expansion. This occurs for all possible peptide:MHC and TCRs simultaneously. After 10-14 days of culture, the pool of cells contains a mixture of clonally expanded T cells which are reactive to naturally processed and presented (but as yet undefined) pMHC.

[0112] In the second part of this method, a DNA-barcoded p:MHC library is created. A peptide library is constructed comprising every possible 8-11 amino acid peptide contained within each candidate antigen (Fig. 5, Panel B). As an example - for a single amino acid mutant (e.g., DNMT3A R882H) where the mutation was centered in the middle of a 25aa encoding mRNA, this would constitute 38 unique peptides ranging from 8-11 amino acids. These peptides are then empirically assessed for their ability to bind the MHC of interest using the well described UV-mediated peptide exchange method (Fig. 5, Panel C). Binding peptides are selected, and biotinylated pMHC monomers are generated containing these peptides in arrayed format. These monomers are then conjugated to DNA-barcoded streptavidin, such that tetramers are formed (Fig. 5, Panel D). The DNA-barcodes are designed to be compatible with the 10X Genomics 5’ immune profiling workflow (10X Genomics, Pleasanton, CA).31US_ACTIVE\131538682W-1Each unique pMHC tetramer is assigned a unique DNA-barcode (or combination of barcodes) which can be used to track the pMHC identity.

[0113] FIG. 5, Panel E (HLA-A*02:01) and FIG. 5, Panel F (HLA-B*07:02) plot NetMHC4.0-predicted peptide affinity against empirically measured monomer recovery in the UV-mediated peptide exchange ELISA, demonstrating that a substantial fraction of peptides exhibit robust empirical binding despite falling outside standard prediction cutoffs. Notably, numerous peptides in the “empirical binder, non-predicted” quadrant were also observed in the literature by immunopeptidomics, underscoring that they are genuinely presented despite being missed by algorithmic prediction. Accordingly, the methods of the present disclosure provide superior sensitivity and real-world relevance compared to reliance on HLA-binding algorithms, such as NetMHC4.0.

[0114] In the third part of this method the DNA-barcoded tetramer library created in the second step is pooled and used to identify the clonally expanded cells made in the first step using the method of staining cells with a barcoded tetramer library followed by sequencing as described in Example 1. The workflow for this step is shown in Fig. 6, Panel A. The tetramer library is incubated with the cells. If a cell has a TCR that can bind a given pMHC, it does, and is labeled with the corresponding DNA barcode. Tetramer binding cells are then sorted and sequenced using the 10X Genomics 5’ immune profiling workflow. After sequencing, for each cell, the following information is recovered: 1) the identity of the pMHC (as revealed by the tetramer associated DNA barcodes), and 2) the paired alpha / beta TCR sequences of the cells).Example 5: Identification of Immunogenic Antigens and Antigen-Specific TCRs

[0115] An example of the data output from the disclosed method of method of identifying immunogenic antigens and antigen-specific TCRs simultaneously is shown in Fig. 7. Monocyte-derived dendritic cells (moDCs) were transfected with mRNA that contained common cancer drive mutations. The cells were stained with dual DNA-barcoded tetramers, and sequenced. Highlighted are identified TCR clonotypes specific for RUNX-RUNX1T1 (HLA-B *07:02), IDH2 R140Q (HLA-B*07:02), NRAS Q61R (HLA-A*01 :01), NRAS Q61K (HLA-A*01:01), PTPN11 G60V (HLA-A*01:01), DNMT3A R882H (HLA-A*01 :01), DNMT3A R882C (HLA-A*01:01), PTPN11 G61Y (HLA-A*01:01), FLT3 D835V (HLA- A*02:01), FLT3 D835Y (HLA-A*02:01), and FLT3 D835H (HLA-A*02:01). Of note, several cross reactive TCRs were identified, in particular ones that bind FLT3 D835V / Y / H,32US_ACTIVE\131538682W-1and DNMT3 A R882H / C. Additionally, for some antigens (e.g. DNMT3 A R882H) TCRs were identified that responded to different peptide:HLA derived from the same mutation. In the case of DNMT3A R882H, TCRs reactive to a 9mer, lOmer, or 1 Imer peptide were identified.

[0116] Cells with the same TCR (that is, cells that are part of the same clonotype) are expected to bind the same tetramer, if the binding is TCR dependent. The automated analysis pipeline developed for this method scores a clonotype for binding to each possible tetramer, and assigns antigen specificity to the best scoring tetramer. If all members of a clonotype do not bind the same tetramer, the clonotype is scored as “non-concordant” and is excluded. This eliminates non-specific binding tetramers / TCRs from analysis, and is an important step because a well-known limitation of pMHC tetramers is a degree of TCR-independent, nonspecific binding. The remaining clonotypes are observed to bind specifically to a single tetramer specificity, or in rare cases multiple similar specificities (Fig. 7, Fig. 9).Example 6: Validation of TCR Specificities

[0117] To validate the TCR specificities from Example 5, TCRs identified using the method were inserted into retroviral constructs, and primary CD8 T cells were transduced to create TCR-T cells. These TCR T cells were then tested for potency, and for recognition of endogenously processed and presented pMHC on target cells. First, TCRs that were specific for two analogous 11 -amino acid peptides derived from DNMT3A R882H and DNMT3A R882C bound to HLA-A*01:01 were evaluated. TCR-T cells were cocultured with peptide pulsed IILA-A*01:01+B-LCL cells, and T cell activation was determined by CD137 upregulation 24 hours post co-culture (Fig. 8, Panel A).

[0118] One TCR, 2-3- la, was determined to be equipotent against both the DNMT3A R882C and DNMT3A R882H peptides, with an EC50 of 3.1nM, which was more than 1000 times higher than the sensitivity observed to the WT “R882R” peptide. The other TCR, 2-4-10, preferred the DNMT3A R882C peptide over the DNMT3A R882H peptide. When cultured with (non-peptide pulsed) cancer cells, both TCRs recognized an IILA-A*01:01-expressing OCI-AML3 cell line, which carries the DNMT3A R882C mutation. Additionally, TCR 2-3- la recognized HLA-A*01 :01 expressing SET-2 cells, which carry the DNMT3A R882H mutation (Fig. 8, Panel B). HLA-A*01:01 expressing K562 cells, which have a lack DNMT3A R882 mutations, were not recognized by the 2-3-la TCR. This demonstrates that the disclosed platform can specifically identify naturally processed and presented pMHC. Another example is a TCR that is specific for IDH2 R140Q-HLA*B07:02 (Fig. 8, Panel C),33US_ACTIVE\131538682W-1and was able to recognize TF1 cancer cell lines that carry 1 allele of the IDH2 R140Q mutation, while ignoring isogenic TF1 cancer cell that lack the mutation (Fig. 8, Panel D). Highly potent TCRs (sub InM ECso) were discovered that responded to FLT3 D835V, D835Y, and D:835H as well (Fig. 9). These findings demonstrate that immunologically relevant cancer ncoantigcns can be discovered using the disclosed platform.Example 7: Identification of SF3B1 K700E:HLA-A*02:01-specific T cell receptors from healthy donor T cell repertoires.

[0119] This example demonstrates an embodiments of the disclosed method in which SF3B1 K700E-HLA-A*02:01-specific TCRs were identified. In the first approach, a pool of peptide / HLA tetramers was prepared for candidate neoantigens of interest (of which SF3B1 K700E:HEA-A*02:01 was one), and these were used to isolate tetramer binding T- cells from healthy donor PBMCs. These T cells were expanded by culturing them with irradiated feeder PBMC, anti-CD3 antibody (OKT3), and IL-2, and it was confirmed that the cultures contain tetramer binding cells using fluorescent tetramers.

[0120] In the second approach, PBMCs were collected from HLA-A*02:01+ healthy donors and cultured in the presence of SF3B 1 K700E peptide (GLVDEQQEV (SEQ ID NO:1)) and recombinant human IL-2 for about 5 days, and then IL-2 alone for about 7-10 more days.

[0121] For both approaches, after cell culture the paired alpha beta TCR sequences of SF3B 1 K700E-HLA-A*02:01 binding cells were recovered using an embodiment of the disclosed tetramer CITEseq method (FIG. 10, Panel A). In this method, cells are incubated with a pool of DNA-barcoded tetramers (one of which was SF3B1 K700E-HLA-A*02:01 tetramer). The tetramer positive cells were then sorted and single cell sequencing using the 10X Genomics 5’ immune profiling workflow / Illumina sequencing was performed. This workflow produces the paired alpha beta TCR sequences of the cells, as well as the ID of the tetramers that the cells bind (via the associated tetramer DNA barcodes). SF3B1 K700E-HLA-A*02:01 specific TCRs were identified as those clones which has expanded and bound the SF3B1 tetramer barcodes but not the other tetramer barcodes (FIG. 10, Panel B). Seven unique TCRs were identified and the TCR sequences are shown in Table 1 .34US_ACTIVE\131538682W-1Table 1: TCR sequences of SF3B1 K700E HLA-A*02:01 specific TCRs.

[0122] To confirm the specificity of the SF3B1 K700E-HLA-A*02:01 TCRs, the TCR sequences were used to generate retroviral constructs encoding the TCRs (linked to TCR murine constant regions, to facilitate detection of transgenic TCR). Retroviral supernatants with these TCRs were prepared and healthy donor T cells were transduced (individually) with each TCR to make TCR-T cells. TCR reactivity and potency were tested using the T2 -peptide pulse assay. In this assay, HLA-A*02:01+target cells (T2 cells) are incubated with titrated amounts of peptides overnight. This facilitates loading of the peptide onto the HLA-A*02:01 on the surface of the cells, in a peptide dose dependent manner. These cells are then washed and cultured with the TCR-T cells. After 18-24 hours of coculture, T cell activation can be determined by expression of interferon gamma, and / or expression of CD 137+ on the surface of the T cells.

[0123] The results showed that all 7 TCRs were functional and that they all were specific for SF3B1 K700E over the WT sequence (FIG. 11, Panels A to G). Most TCRs were of modest potency (ECsos >10nM). One TCR in particular however, had a ~300pM ECso (FIG. 4, Panel E). This is on par with what is observed with anti-viral TCRs and would be considered a potent TCR. This same TCR had a 4+order of magnitude preference for the SF3B1 K700E peptide over the SF3B 1 WT peptide.* * *35US_ACTIVE\131538682W-1All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments or aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.36US_ACTIVE\131538682W-1

Claims

CLAIMS1. A method for producing a nucleic acid-barcoded peptide / MHC tetramer, the method comprising contacting a set of biotinylated peptide MHC monomers with a pool of streptavidin conjugated nucleic acids to produce a nucleic acid-barcoded peptide MHC tetramer, wherein the pool comprises a unique combination of at least two distinct streptavidin conjugated nucleic acids, each comprising a distinct barcode sequence.

2. The method of claim 1, further comprising producing a nucleic acid-barcoded peptide MHC tetramer library by performing said method using a plurality of sets of biotinylated peptide MHC monomers, wherein each set of biotinylated peptide MHC monomers is contacted with a unique pool of streptavidin conjugated nucleic acids.

3. The method of claim 1, further comprising producing the set of biotinylated peptide MHC monomers by UV peptide exchange prior to contacting the set with the pool of streptavidin conjugated nucleic acids.

4. The method of claim 1 , further comprising contacting the peptide MHC tetramer with free biotin.

5. The method of claim 1, wherein the peptide MHC tetramer is non-fluorescent.

6. The method of claim 1, wherein the streptavidin conjugated nucleic acids further comprise a capture sequence.

7. A nucleic acid-barcoded peptide MHC tetramer produced by the method of claim 1.

8. A nucleic acid-barcoded peptide MHC tetramer library produced by the method of claim 2.

9. The nucleic acid-barcoded peptide MHC tetramer library of claim 8, wherein the peptide MHC tetramers of the library are non-fluorescent.

10. A method of determining a specificity of at least one T cell receptor, the method comprising:(a) contacting a plurality of T cells with the nucleic acid-barcoded peptide MHC tetramer library of claim 8;(b) contacting the plurality of T cells with an anti-streptavidin antibody;37US_ACTIVE\131538682W-1(c) separating at least one peptide MHC tetramer bound T cell from unbound T cells; and(d) determining the barcode sequence of at least one peptide MHC tetramer and a sequence of a polynucleotide encoding a TCRa variable region or a polynucleotide encoding a TCRP variable region of the at least one peptide MHC tetramer bound T cell.

11. The method of claim 10, wherein said step (d) comprises determining the barcode sequence of a plurality of peptide MHC tetramers.

12. The method of claim 10, wherein said step (d) comprises determining the sequence of the polynucleotide encoding the TCRa variable region and the polynucleotide encoding the TCRP variable region of the at least one peptide MHC tetramer bound T cell.

13. The method of claim 10, wherein the anti-strepta vidin antibody comprises a fluorescent label.

14. The method of claim 13, wherein said separating comprises fluorescent activated cell sorting (FACS).

15. The method of claim 10, wherein the streptavidin conjugated nucleic acids further comprise a capture sequence.

16. The method of claim 15, wherein the capture sequence is complementary to a nucleic acid sequence of a polynucleotide molecule coupled to a substrate, and the method further comprises contacting the at least one peptide MHC tetramer bound T cell with the substrate prior to said step (d).

17. The method of claim 10, wherein said step (c) comprises separating a plurality of peptide MHC tetramer bound T cells from unbound T cells.

18. The method of claim 17, wherein said step (d) comprises determining the barcode sequence of a plurality of peptide MHC tetramers and the sequence of the polynucleotide encoding the TCRa variable region or the polynucleotide encoding a TCRP variable region of the plurality of peptide MHC tetramer bound T cells.

19. The method of claim 18, wherein said step (d) comprises determining the sequence of the polynucleotide encoding the TCRa variable region and the polynucleotide encoding the TCRP variable region of the plurality of peptide MHC tetramer bound T cells.38US_ACTIVE\131538682W-120. A nucleic acid-barcoded peptide MHC tetramer library comprising a plurality of nucleic acid-barcoded peptide MHC tetramers each comprising four biotin molecules, at least one streptavidin conjugated nucleic acid comprising a barcode sequence, and four peptide MHC monomers, wherein: each peptide MHC monomer of each MHC peptide tetramer comprises an identical peptide, the library comprises a plurality of MHC tetramers comprising identical peptides, and at least two MHC tetramers of the plurality of MHC tetramers comprising identical peptides comprise distinct barcode sequences.

21. The nucleic acid-barcoded peptide MHC tetramer library of claim 20, wherein the library comprises a plurality of MHC tetramers comprising a plurality of distinct peptides.

22. A method of determining a specificity of at least one T cell receptor, the method comprising:(a) contacting a plurality of T cells with the nucleic acid-barcoded peptide MHC tetramer library of claim 20;(b) contacting the plurality of T cells with an anti-streptavidin antibody;(c) separating at least one peptide MHC tetramer bound T cell from unbound T cells; and(d) determining the barcode sequence of at least one peptide MHC tetramer and a sequence of a polynucleotide encoding a TCRa variable region or a polynucleotide encoding a TCR variable region of the at least one peptide MHC tetramer bound T cell.

23. The method of claim 22, wherein said step (d) comprises determining the barcode sequence of a plurality of peptide MHC tetramers.

24. The method of claim 22, wherein said step (d) comprises determining the sequence of the polynucleotide encoding the TCRa variable region and the polynucleotide encoding the TCRP variable region of the at least one peptide MHC tetramer bound T cell.

25. The method of claim 22, wherein the anti-streptavidin antibody comprises a fluorescent label.39US_ACTIVE\131538682W-126. The method of claim 25, wherein said separating comprises fluorescent activated cell sorting (FACS).

27. The method of claim 22, wherein the streptavidin conjugated nucleic acid further comprises a capture sequence.

28. The method of claim 27, wherein the capture sequence is complementary to a nucleic acid sequence of a polynucleotide molecule coupled to a substrate, and the method further comprises contacting the at least one peptide MHC tetramer bound T cell with the substrate prior to said step (d).

29. The method of claim 22, wherein said step (c) comprises separating a plurality of peptide MHC tetramer bound T cells from unbound T cells.

30. The method of claim 29, wherein said step (d) comprises determining the barcode sequence of a plurality of peptide MHC tetramers and the sequence of the polynucleotide encoding the TCRa variable region or the polynucleotide encoding a TCRP variable region of the plurality of peptide MHC tetramer bound T cells.

31. The method of claim 30, wherein said step (d) comprises determining the sequence of the polynucleotide encoding the TCRa variable region and the polynucleotide encoding the TCR variable region of the plurality of peptide MHC tetramer bound T cells.

32. A kit comprising:(a) a set of biotinylated peptide MHC monomers; and(b) a pool of streptavidin conjugated nucleic acids, wherein the pool comprises a unique combination of at least two distinct streptavidin conjugated nucleic acids, each comprising a distinct barcode sequence.

33. The kit of claim 32, further comprising free biotin, fluorophore conjugated streptavidin, or an anti-streptavidin antibody.

34. The kit of claim 32, wherein the streptavidin conjugated nucleic acids further comprise a capture sequence.

35. The kit of claim 34, further comprising a substrate coupled to a polynucleotide molecule comprising a nucleic acid sequence that is complementary to said capture sequence.

36. A kit comprising the nucleic acid-barcoded peptide MHC tetramer library of claim 20.

37. The kit of claim 36, further comprising an anti-streptavidin antibody.40US_ACTIVE\131538682W-138. The kit of claim 36, wherein the streptavidin conjugated nucleic acid further comprises a capture sequence.

39. The kit of claim 38, further comprising a substrate coupled to a polynucleotide molecule comprising a nucleic acid sequence that is complementary to said capture sequence.

40. The method of claim 1, further comprising contacting the pool of streptavidin conjugated nucleic acids with fluorophore-conjugated streptavidin.

41. The method of claim 40, wherein the fluorophore-conjugated streptavidin is not conjugated to a nucleic acid molecule.

42. A nucleic acid-barcoded peptide MHC tetramer library produced by the method of claim 40.

43. A method of determining a specificity of at least one T cell receptor, the method comprising:(a) contacting a plurality of T cells with the nucleic acid-barcoded peptide MHC tetramer library of claim 42;(b) separating at least one peptide MHC tetramer bound T cell from unbound T cells; and(c) determining the barcode sequence of at least one peptide MHC tetramer and a sequence of a polynucleotide encoding a TCRa variable region or a polynucleotide encoding a TCR variable region of the at least one peptide MHC tetramer bound T cell.

44. The method of claim 43, wherein said step (c) comprises determining the barcode sequence of a plurality of peptide MHC tetramers.

45. The method of claim 43, wherein said step (c) comprises determining the sequence of the polynucleotide encoding the TCRa variable region and the polynucleotide encoding the TCRP variable region of the at least one peptide MHC tetramer bound T cell.

46. The method of claim 43, wherein said separating comprises fluorescent activated cell sorting (FACS).

47. The method of claim 43, wherein the streptavidin conjugated nucleic acids further comprise a capture sequence.41US_ACTIVE\131538682W-148. The method of claim 47, wherein the capture sequence is complementary to a nucleic acid sequence of a polynucleotide molecule coupled to a substrate, and the method further comprises contacting the at least one peptide MHC tetramer bound T cell with the substrate prior to said step (c).

49. The method of claim 43, wherein said step (b) comprises separating a plurality of peptide MHC tetramer bound T cells from unbound T cells.

50. The method of claim 49, wherein said step (c) comprises determining the barcode sequence of a plurality of peptide MHC tetramers and the sequence of the polynucleotide encoding the TCRa variable region or the polynucleotide encoding a TCR variable region of the plurality of peptide MHC tetramer bound T cells.

51. The method of claim 50, wherein said step (c) comprises determining the sequence of the polynucleotide encoding the TCRa variable region and the polynucleotide encoding the TCR0 variable region of the plurality of peptide MHC tetramer bound T cells.

52. The nucleic acid-barcoded peptide library MHC tetramer library of claim 20, wherein the plurality of nucleic acid-barcoded peptide MHC tetramers each further comprise at least one streptavidin conjugated fluorophore.

53. A method of determining a specificity of at least one T cell receptor, the method comprising:(a) contacting a plurality of T cells with the nucleic acid-barcoded peptide MHC tetramer library of claim 52;(b) separating at least one peptide MHC tetramer bound T cell from unbound T cells; and(c) determining the barcode sequence of at least one peptide MHC tetramer and a sequence of a polynucleotide encoding a TCRa variable region or a polynucleotide encoding a TCR variable region of the at least one peptide MHC tetramer bound T cell.

54. The method of claim 53, wherein said step (c) comprises determining the barcode sequence of a plurality of peptide MHC tetramers.

55. The method of claim 53, wherein said step (c) comprises determining the sequence of the polynucleotide encoding the TCRa variable region and the polynucleotide encoding the TCRP variable region of the at least one peptide MHC tetramer bound T cell.42US_ACTIVE\131538682W-156. The method of claim 53, wherein said separating comprises fluorescent activated cell sorting (FACS).

57. The method of claim 53, wherein the streptavidin conjugated nucleic acid further comprises a capture sequence.

58. The method of claim 57, wherein the capture sequence is complementary to a nucleic acid sequence of a polynucleotide molecule coupled to a substrate, and the method further comprises contacting the at least one peptide MHC tetramer bound T cell with the substrate prior to said step (c).

59. The method of claim 53, wherein said step (b) comprises separating a plurality of peptide MHC tetramer bound T cells from unbound T cells.

60. The method of claim 59, wherein said step (c) comprises determining the barcode sequence of a plurality of peptide MHC tetramers and the sequence of the polynucleotide encoding the TCRa variable region or the polynucleotide encoding a TCRP variable region of the plurality of peptide MHC tetramer bound T cells.

61. The method of claim 60, wherein said step (c) comprises determining the sequence of the polynucleotide encoding the TCRa variable region and the polynucleotide encoding the TCRP variable region of the plurality of peptide MHC tetramer bound T cells.43US_ACTIVE\131538682W-1

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