T-switch: a specificity-based engineering platform for developing safe and effective t cell therapeutics

Abstract

The technology described herein is directed to methods for producing or selecting self-antigen-specific TCRs and method of using such TCRs.

Description

T-SWITCH: A SPECIFICITY-BASED ENGINEERING PLATFORM FOR DEVELOPING SAFE AND EFFECTIVE T CELL THERAPEUTICSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 591,501 filed October 19, 2023, the contents of which are incorporated herein by reference in their entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Nos. NCI R01CA234600 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 18, 2024 is named 043214-000102WOPT_SL.xml and is 214,330 bytes in size.TECHNICAL FIELD

[0004] The technology described herein relates to adoptive T cell therapy.BACKGROUND

[0005] Adoptive T cell therapy (ACT) has been successfully used to reprogram patients’ own immune cells to target diseased cells, e.g, as in CAR-T technology. Applying this approach to TCRs for self-antigens would be therapeutically beneficial. Self-antigens are antigens found on a patient’s own cells. They may become overexpressed during the development of cancer cells and are therefore possible targets for cancer theapies. However, T cells bearing T cell receptors (TCRs) with high potency and affinity for a self-antigen are eliminated during thymic selection. This means that self- antigen-specific TCRs are not naturally occurring and no such molecules are available to be utilized in ACT.

[0006] The affinity of the low-avidity TCRs that pass through thymic selection can be improved through conventional mutagenesis approaches, but isolating these TCRs is labor intensive, and their subsequent affinity enhancement can generate toxic cross-reactivities. There is therefore a need for new methods that provide rapid and effective production of high affinity self-antigen-specific TCRs for use in ACT.SUMMARY

[0007] The technology described herein is directed to method of producing self-antigen-specific TCRs, e.g., for use in ACT. The methods utilize a self-antigen mimic to raise initial pools of TCRs with high affinity for the mimic. The TCRs are then subjected to mutagenesis and selection. Thisprocess is shown herein to provide rapid, high-throughout, and efficacious self-antigen-specific TCR production which is not plagued by toxic cross-reactivity.

[0008] In one aspect of any of the embodiments, described herein is a method comprising: a) mutagenizing at least one of a CDR3a and a CDR3J3 of a self-antigen mimic-specific TCR to provide a pool of target self-antigen-specific TCR candidates; and b) selecting at least one target self-antigen-specific TCR from the pool of target self- antigen-specific TCR candidates.In one aspect of any of the embodiments, described herein is a method comprising: c) isolating a self-antigen mimic-specific TCR; d) mutagenizing at least one of the CDR3a and CDR3J3 of the self-antigen mimicspecific TCR to provide a pool of target self-antigen-specific TCR candidates; and e) selecting at least one target self-antigen-specific TCR from the pool of target self- antigen-specific TCR candidates.

[0009] In some embodiments of any of the aspects, the isolating comprises injecting an animal or person with the self-antigen mimic. In some embodiments of any of the aspects, the isolating comprises raising naive CD8 T cells from a human donor with the self-antigen mimic. In some embodiments of any of the aspects, the isolating comprises raising naive CD8 T cells, from a human donor HLA-matched with respect to the target self-antigen, with the self-antigen mimic. In some embodiments of any of the aspects, the isolating comprises screening a library of T cells with the selfantigen mimic.

[0010] In some embodiments of any of the aspects, the self-antigen mimic is displayed in a complex with an MHC polypeptide during the isolating step. In some embodiments of any of the aspects, the self-antigen mimic is displayed as part of a fusion protein with an MHC polypeptide during the isolating step. In some embodiments of any of the aspects, the self-antigen mimic is displayed on an antigen presenting cell during the isolating step. In some embodiments of any of the aspects, the antigen presenting cell is a monocyte -derived dendritic cell or an EpiScan cell.

[0011] In some embodiments of any of the aspects, the self-antigen mimic is a polypeptide of no more than 20 amino acids. In some embodiments of any of the aspects, the self-antigen mimic is a polypeptide of more than 20 amino acids. In some embodiments of any of the aspects, the target selfantigen is a polypeptide of no more than 20 amino acids. In some embodiments of any of the aspects, the target self-antigen is a polypeptide of more than 20 amino acids. In some embodiments of any of the aspects, the target self-antigen is a naturally occurring polypeptide.

[0012] In some embodiments of any of the aspects, the at least one of a CDR3a and a CDR3J3 is CDR3a. In some embodiments of any of the aspects, the at least one of a CDR3a and a CDR3J3 is CDR3J3. In some embodiments of any of the aspects, the at least one CDR is CDR3a and CDR3J3.

[0013] In some embodiments of any of the aspects, the mutagenizing comprises mutagenizing a single residue of at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the mutagenizing comprises mutagenizing a single residue of the each of the at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the mutagenizing comprises mutagenizing two residues of at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the mutagenizing comprises mutagenizing three residues of the each of the at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the mutagenizing comprises mutagenizing three residues of at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the mutagenizing comprises mutagenizing at least three residues of at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the mutagenizing comprises mutagenizing at least three residues of the each of the at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the mutagenizing is comprehensive saturation mutagenesis.

[0014] In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of single mutants, double mutants, triple mutants, and CDR3 length changes of at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library comprising all single mutants and all double mutants of at least one of the CDR3a and the CDR3J3.

[0015] In some embodiments of any of the aspects, the selecting comprises selecting target self- antigen-specific TCRs from the pool of target self-antigen-specific candidates. In some embodiments of any of the aspects, the target self-antigen is displayed in a complex with an MHC polypeptide during the selecting step. In some embodiments of any of the aspects, the selecting comprises selecting TCRs that do not bind the self-antigen mimic from the pool of target self-antigen-specific candidates. In some embodiments of any of the aspects, the self-antigen mimic is displayed in a complex with an MHC polypeptide during the selecting step. In some embodiments of any of the aspects, the selecting comprises selecting TCRs that do not bind a plurality of off-target self-antigens from the pool of target self-antigen-specific candidates. In some embodiments of any of the aspects, the selecting comprises selecting TCRs that do not bind a plurality of off-target self-antigens in a human peptidome library from the pool of target self-antigen-specific candidates. In some embodiments of any of the aspects, the selecting comprises selecting TCRs that do not bind a plurality of off-target self-antigens in a human peptidome library presented on T-scan cells from the pool of target self-antigen-specific candidates.

[0016] In one aspect of any of the embodiments, described herein is a TCR polypeptide composition comprising: a) a CDRla comprising the sequence of SEQ ID NO: 1; b) a CDR2a comprising the sequence of SEQ ID NO:2;c) a CDR3a comprising the sequence of one of SEQ ID NOs: 3 and 7-15; d) a CDRip comprising the sequence of SEQ ID NO:4; e) a CDR2J3 comprising the sequence of SEQ ID NO:5; and f) a CDR3P comprising the sequence of SEQ ID NO:6.

[0017] In some embodiments of any of the aspects, the TCR polypeptide composition further comprises one or more of SEQ ID NOs: 16-19.

[0018] In one aspect of any of the embodiments, described herein is a nucleic acid composition comprising one or more nucleic acids encoding the TCR polypeptide composition described herein. In one aspect of any of the embodiments, described herein is a cell comprising the TCR polypeptide composition of any one of the preceding claims and / or the nucleic acid composition described herein. In some embodiments of any of the aspects, the cell is a T cell.

[0019] In one aspect of any of the embodiments, described herein is a method of treating a subject in need of immune stimulation specific for a target self-antigen, the method comprising administering a T cell comprising a target self-antigen-specific TCR selected by the process described herein to the subject.

[0020] In one aspect of any of the embodiments, described herein is a method of treating a subject in need of immune stimulation specific for Tyrosine Hydoxylase (TH), the method comprising administering the T cell described herein to the subject.

[0021] In some embodiments of any of the aspects, the method further comprises a first step of transducing at least one T cell obtained from the subject with the target self-antigen-specific TCR or the TCR polypeptide composition described herein. In some embodiments of any of the aspects, the subject is a subject in need of treatment for cancer. In some embodiments of any of the aspects, the cancer is neuroblastoma. In some embodiments of any of the aspects, the T cell is autologous to the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figs. 1A-1D depict the design of comprehensive saturation mutagenesis libraries and selection strategy to switch the specificity of two model NLV TCRs. Fig. 1 A: Jurkat TCRp) cells lentivirally infected with lentiviruses expressing the NLV2 TCR (left) or NLV3 TCR (right) were stained with tetramers containing variants of the pp65 peptide (NLVPMVATV (SEQ ID NO: 30)). Fig. IB: Summary of tetramer staining properties illustrating differential peptide recognition of NLV2 and NLV3 TCRs. Fig. IB discloses SEQ ID NOS 30, 33, 34, and 32, respectively, in order of appearance. Fig. 1C: A schematic detailing the design of CDR3 libraries. The CDR sequence is AGPMKTSYDKVI (SEQ ID NO: 31) and the amino acids targeted for triple mutants are underlined. Each of the libraries consisted of two different nucleic acids encoding the same peptide variant that serves as internal duplicates generating the library complexity represented under the CDR3 sequence. Fig. 1C discloses SEQ ID NOS 38-41,respectively, in order of appearance. Fig. ID: Worflow of TCR screening strategy. Each quadrant of the tetramer staining reveals the specificity of each TCR.

[0023] Figs. 2A-2I depict NLV2 CDR3a library screening and validation. Fig. 2A: Jurkat cells infected with the library were stained and sorted using an NLV2-specific peptide tetramer (NLVGMVATV (SEQ ID NO: 32)) and an NLV3-specific peptide tetramer (MLVPMVATV (SEQ ID NO: 33)). All four quadrants of the flow plot were sorted separately and three of the quadrants (SP, DP and SW) underwent one round of expansion prior to sequencing. Fig. 2A discloses SEQ ID NOS: 32 and 33, respectively, in order of appearance. Fig. 2B: Amino acid sequence logos showing the frequency of specific residues at each CDR3 position across the different sorting populations. Logo plots are generated using the top 50 enriched CDR3a sequences in each population as measured by the geometric mean of the enrichment of the CDR3 across four total replicates (two screen replicates and two internal barcode replicates each). Fig. 2C: Sequence of TCRs selecte for validation from each of the different sorting populations. Jurkat TCR[37cells were infected separately with lentiviral constructs expressing each of the TCRs. Figs. 2D-2E: Tetramer staining (Fig. 2D) and CD69 activation (Fig. 2E) for each of the validation TCRs. Data are shown as mean ± SDs of three technical replicates. Fig. 2C discloses SEQ ID NOS 31, and 42-50, respectively, in order of appearance. Figs. 2F-2I: The cross-reactivity profiles of the NLV2 WT TCR (Fig. 2F), NLV2 DP TCR (Fig. 2G), and two switch TCRs (Figs. 2H and 21) against the virome wide library. Each dot represents one peptide tile. The y axis represents the geometric meanof the fold enrichment of the peptide aross ten replicates (five screen replicates with two internal barcode replicates each). Peptides highlighted in grey contain the known cognate antigen of the NLV2 TCRs and those highlighted in black contain off-target hits. Fig. 2F discloses SEQ ID NO: 31, Fig. 2G discloses SEQ ID NO: 42, Fig 2H discloses SEQ ID NO: 43, and Fig. 21 discloses SEQ ID NO: 46.

[0024] Figs. 3A-3I depict NLV3 CDR3p library screening and validation. Fig. 3A: The NLV3 CDR36 library was stained and sorted using an NLV3 -specific peptide tetramer (MLVPMVATV (SEQ ID NO: 33)) and an NLV2-specific peptide tetramer (NLCPMVATV (SEQ ID NO: 34)). All four quadrants of the flow plot were sorted separately and three of the quadrants (SP, DP and SW) underwent one round of expansion prior to sequencing. Fig. 3A discloses SEQ ID NOS 33 and 34, respectively, in order of appearance. Fig. 3B: Amino acid sequence logos showing the frequency of specific residues at each CDR36 position across the different sorting populations. Logo plots are generated using the top 50 enriched CDR3b sequences in each population as measured by the geometric mean of the enrichment of the CDR3 across four total replicates (two screen replicates and two internal barcode replicates each). Fig. 3C: Table listing CDR3 sequences chosen for validation experiments. Figs. 3D-3E: Tetramer staining (Fig. 3D) and CD69 activation (Fig. 3E) for each of the validation TCRs expressed in Jurkat TCR[3- / - cells. Data are shown as mean ± SDs of three technical replicates. Fig. 3C discloses SEQ ID NOS 51-56, respectively, in order of appearance. Figs. 3F-3I: The cross-reactivity profiles of NLV3 WT (Fig. 3F), and two switch TCRs (Figs. 3H and 31) againstthe virome-wide library. Each dot represents one peptide tile. The y axis represents the geometric mean of the fold enrichment of the peptide across ten replicates (five screen replicates and two internal barcode replicates each). Peptides highlighted in grey contain the known cognate antigen of the NLV3 TCR. (Fig. 3G) Granzyme B reporter activation in T-Scan HLA-A2+ target cells after coculture with primary T cells expressing either the WT NLV3 TCR (top) or the DP engineered NLV3 TCR variant (bottom). Fig. 3F discloses SEQ ID NO: 51, Fig. 3G discloses SEQ ID NOS 51-52, respectively, in order of appearance, Fig. 3H discloses SEQ ID NO: 54, and Fig. 31 discloses SEQ ID NO: 56.

[0025] Figs. 4A-4D: depict an overview of the T-Switch platform and selection of mutant peptides. Fig. 4A: Schematic of T-Switch workflow. Monocyte-derived dendritic cells (MoDCs) from a healthy donor are pulsed with mutant and self-peptides. Donor naive CD8 T cells will then be cultured with the antigen presenting cells and expanded. HLA tetramer technology is used to identify T cell reactivity against any of the pulsed peptides. Tetramer sorted cells are sorted and sent for 10X TCR sequencing to identify the TCR clonotypes. CDR3 TCR mutagenesis is used to switch the specificity of the mutant-recognizing TCR to a tumor self-reactive TCR and its off-target profile will be assessed via T-Scan. Fig. 4B: Tetramer staining following antigen-specific expansion to unloaded moDCs (left panel), TH WT pulsed moDCs (middle panel) or TH mutant pulsed moDCs (right panel). Fig. 4B discloses SEQ ID NOS 35 and 36, respectively, in order of appearance. Fig. 4C: Tetramer binding validation following 10X TCR sequencing of two expanded clonotypes. Representative flow plot showing one replicate in left panel and bar plot in right panel showing mean ± SDs of three technical replicates. Fig. 4C discloses SEQ ID NOS 35 and 36, respectively, in order of appearance. Fig. 4D: Screening results for TH MUT 10X TCR CDR3a chain to switch its specificity from recognizing the bait (mutant) antigen to recognizing the self (TH WT) antigen. Amino acid sequence logo plots are generated using the top 50 enriched CDR3 sequences in each population as measured by the geometric mean of the enrichment of the CDR3 across four total replicates (two screen replicates and two internal barcode replicates). Fig 4D. discloses SEQ ID NOS 35-36, 57-72, 57, 73- 87, 57, 3, 12, 10, 88-89, 11, 9, 13-14, 90-93, 15, and 7, respectively, in order of appearance.

[0026] Figs. 5A-5L depict the application of T-Switch for engineering TCRs with specificity and functionality to a tumor antigen. Fig. 5A: TH CDR3a sequences chosen for validation experiments. Fig. 5A discloses SEQ ID NOS 58, 67, 73, 84, 76, 74, 3, and 7-15, respectively, in order of appearance. Fig. 5B: Tetramer binding was assessed by infecting Jurkat TCR[37cells infected with lentiviral constructs expressing each of the TCRs in (Fig. 5A). Heatmap shows summary of tetramer staining assay as determined by flow cytometry (n = 3). Fig. 5B discloses SEQ ID NOS 35-36, respectively, in order of appearance. Fig. 5C: CD69 activation of validation TCRs was assessed by individual 24 h co-culture of Jurkat TCR[37cells expressed validation TCRs with 293T-HLA-A2 target cells pulsed with an irrelevant peptide (NEG CTRL), the mutant peptide (ALLSGVRMV (SEQ ID NO:35)), or the WT peptide (ALLSGVRQV (SEQ ID NO: 36)). Fig. 5C discloses SEQ ID NOS 35-36, respectively, in order of appearance. Heatmap shows the percentage of CD69 activation positive Jurkat cells after co-culture as determined by flow cytometry (n=3). Fig. 5D: Schematic representation of functional experiments using two 90aa peptide sequences from the TH protein expressed separately into HLA-A2+ T-scan target cells. Fig 5D discloses SEQ ID NOS 96-97, respectively, in order of appearance. Fig. 5E: CD69 activation of validation TCRs was assessed by individual 24 h co-culture of Jurkat TCRp / _cells expressing validation TCRs with HLA-A2+ T-scan target cells expressing a negative control peptide, the TH WT containing peptide 1 or TH WT containing peptide 2. Heatmap shows the percentage of CD69 activation positive Jurkat cells after co-culture as determined by flow cytometry (n=3). Figs. 5F-5H: Granzyme B activation (Fig. 5F), Caspase 3 / 7 cleavage (Fig. 5G) and percentage of 7-AAD positive (Fig. 5H) of HLA-A2+ T-scan target cells following co-culture with TH-specific TCRs. Fig. 51: Overview of human genome-wide screening strategy via T-Scan. Peptides are tiled across the human proteome in 90aa steps with 22 aa overlaps and lentivirally expressed in HLA- A2+ T-Scan target cells. Figs. 5J-5L: T-Scan screen of TH MUT 10X TCR (Fig. 5J), TH DP TCR (Fig. 5K), and TH SW TCR (Fig. 5L) expressing T cells against the human genome wide library. Each dot represents one peptide tile, with the y axis plotting the geometric mean of the fold change of each peptide across five replicates. Fig. 5J discloses SEQ ID NO: 57, Fig. 5K discloses SEQ ID NO: 76, and Fig. 5L discloses SEQ ID NO: 3.

[0027] Figs. 6A-6G depict cytotoxicity and functionality of engineered TH-specific TCR variants against TH-expressing tumor cell lines. Fig. 6A: Granzyme B activation of 293T T-scan target cells expressing the TH full length ORF (TH ORF) following 24h co-culture with engineered TH WT TCR variant transduced human primary T cells or a negative control TCR at a 1 : 1 T cell to target cell co-culture ratio (left) or a 3: 1 ratio (right). Fig. 6B-6D: Killing of TH expressing cell lines by different TH-specific transduced human primary T cells. Co-culture was performed at a T cell to target cell ratio of 3: 1 and cultured for 24 h. Cell lines that did not express HLA-A2 were transduced with a lentiviral vector expressing HLA-A2. Figs. 6E-6G: cytotoxic granule release (CD107a), IFNg, and TNF staining of different TH-specific TCR variant transduced primary T cells following coculture with TH-expressing tumor cell lines.

[0028] Figs. 7A-7D depict NLV2 TCR screening enriched variants. Fig. 7A: Table showing the top 20 enriched NLV2 TCR CDR3a sequences from each of the three sorted populations clustered using MAFFT to show representative motifs and amino acid usage for each population. Fig 7A. discloses SEQ ID NOS 31, 98-117, 31, 42, 118-136, 31, 43, 45, 137-141, 47, 142-143, 46, 49, 48, 50, 144-148, and 44, respectively, in order of appearance. Fig. 7B: Heatmap showing NLV2 CDR3a positions that demolish binding to the original antigen or that are permissive to binding the original antigen. Each box in the heatmap represents one mutant, where the amino acid along the x axis is mutated to the amino acid indicated on the y axis. Light grey boxes indicate mutations that enrich inthe DN and SW populations and therefore demolish recognition to the original binder and dark grey boxes indicate mutations that enrich in the SP and DP populations and therefore are permissive to the original binder. Fig. 7B discloses SEQ ID NO: 31. Fig. 7C: Heatmap displays the single mutant enrichment of sequencing reads from TCR variants in the NLV2 CDR3a screen SP population. Each box in the heatmap represents the enrichment of one mutant, where the amino acid along the x axis is mutated to the amino acid indicated on the y axis. The enrichment for the SP population depicts the normalized fold change, which was generated by dividing the fold change of each CDR3 by the average fold change of the WT NLV2 CDR3a chain in the library. The enrichment for the DP and SW populations are relative to their occurrence in the starting input or pre-screening library. Fig. 7C discloses SEQ ID NO: 31. Fig. 7D: Tetramer staining of the NLV2 TCR CDR3b library using an NLV2-specific peptide tetramer (NLVGMVATV (SEQ ID NO: 32)) and an NLV3-specific peptide tetramer (MLVPMVATV (SEQ ID NO: 33)). No SP, DP or SW populations were observed for this library (flow plot on the left). Fig. 7D discloses SEQ ID NOS 39, 32, and 33, respectively, in order of appearance.

[0029] Figs. 8A-8E depict NLV2 CDR3a single mutant is sufficient to switch specificity without additional promiscuity. Fig. 8A: Schematic representation of the T-Scan workflow to screen for off- targets. Primary T cells expressing TCRs of interest are co-cultured with T-Scan target cells lentivirally infected with the virome-wide library. 6-8 hours post co-culture, granzyme reporter positive target cells are sorted and sequenced by next generation sequencing (NGS). Fig. 8A discloses SEQ ID NOS 149, 149, and 149, respectively, in order of appearance. Figs. 8B-8C: T-Scan screen of a triple mutant DP NLV2 engineered TCR variant (Fig. 8B), and a double mutant switch TCR variant (Fig. 8C) expressing T cells against the viral peptidome library. Each dot represents one peptide tile. The y axis represents the geometric mean of the fold enrichment of the peptide across ten replicates (five screen replicates with two internal barcode replicates each). Peptides highlighted in grey contain the known cognate antigen of the NLV2 TCRs and those highlighted in black contain off-target hits. Fig. 8D: Heatmap displaying the single mutant enrichment of sequencing reads from TCR variants in the NLV2 CDR3a screen SW population. Each box in the heatmap represents the enrichment of one mutant, where the amino acid along the x axis is mutated to the amino acid indicated on the y axis. Fig. 8E: T-Scan screen of a single mutant SW NLV2 TCR variant expressing T cells against the viral peptidome library. Each dot represents one peptide tile. The y axis represents the geometric mean of the fold enrichment of the peptide across ten replicates (five screen replicates and two internal barcode replicates each). Peptides highlighted in bold contain the known cognate antigen of the NLV2 TCR. Fig. 8B discloses SEQ ID NO: 125, Fig. 8C discloses SEQ ID NO: 45, Fig. 8D discloses SEQ ID NO: 31, and Fig. 8E discloses SEQ ID NO: 150.

[0030] Figs. 9A-9E depict TCR fingerprints of NLV2 engineered TCRs. Fig. 9A: Design of the NLV epitope saturation mutagenesis library. Every position in the original binder (NLVGMVATV(SEQ ID NO: 32)) or the new binder (MLVPMVATV (SEQ ID NO: 33)) is mutated to each of the alternative 19 amino acids. The mutants are expressed as short peptides with an N-terminal tag or in the context of a 56-aa fragment. The two amino acids directly N-terminal and C-terminal to the peptide are also mutagenized in the 56mer version of the fragment. Figs. 9B-9E: Heatmaps represent the TCR recognition fingerprint of mutant peptides of either the original binder (left) or the new binder (right) in the context of 56-aa fragments by NLV2 WT or engineered T cells. Each box in the heatmap represents one mutant, where the amino acid along the x axis is mutated to the amino acid indicated along the axis. The value in the heatmap represents the enrichment of this mutant compared to the cognate binder of the TCR. Heatmaps are plotted for (Fig. 9B) T cells expressing the NLV2 TCR, (Fig. 9C) T cells expressing the DP NLV2 TCR, and (Figs. 9D and 9E) T cells expressing two different SW NLV2 TCRs. Fig. 9A discloses SEQ ID NOS 151-152, respectively, in order of appearance. Fig. 9B discloses SEQ ID NOS 31, and 151-152, respectively, in order of appearance. Fig. 9C discloses SEQ ID NOS 42, and 151-152, respectively, in order of appearance. Fig. 9D discloses SEQ ID NOS 45, and 151-152, respectively, in order of appearance. Fig. 9E discloses SEQ ID NOS 43, and 151-152, respectively, in order of appearance.

[0031] Figs. 10A-10B depict NLV3 TCR screening enriched variants. Fig. 10A: Tetramer staining of the NLV2 TCR CDR3b library using an NLV3-specific peptide tetramer (MLVPMVATV (SEQ ID NO: 33)) and an NLV2-specific peptide tetramer (NLVGMVATV(SEQ ID NO: 32)). No SP, DP or SW populations were observed for this library (flow plot on the left). Fig. 10A discloses SEQ ID NOS 40, 33, and 34, respectively, in order of appearance. Fig. 10B: Table showing the top 20 enriched TCR CDR3J3 sequences from each of the three sorted populations, with exception of the SW population that only had four scoring CDR3J3 sequences. The sequences were clustered using MAFFT to show representative motifs and amino acid usage for each population. Fig. 10B discloses SEQ ID NOS 51, 153-172, 51, 173-189, 52, 190-191, 51, 55, 54, 56, and 53, respectively, in order of appearance.

[0032] Figs. 11A-1 ID depict in silico peptide-MHC modelling for the selection of bait' mutant antigens for T-Switch. Fig. 11A: Structural model of the TH peptide, ALLSGVRQV(SEQ ID NO: 36), binding to HLA-A2. Up arrows are amino acids facing upwards and down arrows are amino acids facing downwards relative to the HLA molecule. Fig. 1 IB: Heatmap of the FoldX derived LLG energy between the original peptide (ALLSGVRQV (SEQ ID NO: 36)) and HLA-A2, as well as all the peptide variants created by substituting each peptide position (x-axis) with all naturally occurring amino acids (n=19). A positive LLG value suggests that the amino acid substitution has decreased the stability of the peptide-MHC interaction, whereas a negative LLG value indicates an improvement in the stability of the interaction. Fig. 11C: Heatmap of NetMHC predicted binding affinity between the original TH peptide and HLA-A2, as well as all the peptide variants created by substituting each peptide position (x-axis) with all naturally occurring amino acids. Fig. 1 ID: Library design and screening schematic for TH MUT 10X TCR. Fig. 11A discloses SEQ ID NO: 36., Fig. 1 IB disclosesSEQ ID NO: 36, Fig 11C discloses SEQ ID NO: 36, and Fig. 1 ID discloses SEQ ID NOS 192-193, respectively, in order of appearance.

[0033] Figs. 12A-12F depict TH CDR3J3 screening and validation. Fig. 12A: Screening results for TH MUT 10X TCR CDR3[3 chain to switch its specificity from recognizing the bait TH mutant antigen to recognizing the WT TH antigen. Amino acid sequence logo plots are generated using the top 50 enriched CDR3J3 sequences in each population as measured by the geometric mean of the enrichment of the CDR3 across four total replicates (two screen replicates and two internal barcode replicates). Fig. 12B: Table showing the top 20 enriched TCR CDR3J3 sequences from each of the three sorted populations clustered using MAFFT to show representative motifs and amino acid usage for each population. Fig. 12B discloses SEQ ID NOS 6, 194-208, 6, 209-223, 6, and 224-238, respectively in order of appearance. Fig. 12C: Table listing CDR3 sequences chosen for validation experiments. Fig. 12C discloses SEQ ID NOS 194, 200, 209, 211, 216, 218, 224, 225, 230, 226, 232, 231, 237, 37, and 94-95, respectively, in order of appearance. Fig. 12D: Tetramer binding was assessed by infecting Jurkat TCR[3 / _cells infected with lentiviral constructs expressing each of the TCRs in Fig. 12C. Heatmap shows summary of tetramer staining assay as determined by flow cytometry (n = 3). Fig.12D discloses SEQ ID NOS 35 and 36, respectively, in order of appearance Fig. 12E: CD69 activation of validation TCRs was assessed by individual 24hr co-culture of Jurkat TCRp7cells expressed validation TCRs with 293T-HLA-A2 target cells pulsed with an irrelevant peptide (NEG CTRL), the mutant peptide (ALLSGVRMV (SEQ ID NO: 35)), or the WT peptide (ALLSGVRQV (SEQ ID NO: 36)). Heatmap shows the percentage of CD69 activation positive Jurkat cells after co-culture as determined by flow cytometry (n=3). Fig. 12F discloses SEQ ID NOS 35 and 36, respectively, in order of appearance Fig. 12F: CD69 activation of validation TCRs was assessed by individual 24hr coculture of Jurkat TCRp7cells expressing validation TCRs with HLA-A2+ T-scan target cells expressing a negative control peptide tile, the TH WT tile 1 or TH WT tile 2. Heatmap shows the percentage of CD69 activation positive Jurkat cells after co-culture as determined by flow cytometry (n=3).

[0034] Figs. 13A-13C depict cross-reactivity profiling of allogeneic-HLA expanded TCRs. The cross-reactivity profiles of three published TCRs identified through expansion on allogeneic-HLA target cells. The off-target profiles of the CD20 A23 TCR (Fig. 13A), TdT T1 TCR (Fig. 13B), and TdT T3 TCR (Fig. 13C) against the human peptidome library. Each dot represents one peptide tile. The y axis represents the geometric mean of the fold enrichment of the peptide across ten replicates (five screen replicates with two internal barcode replicates each). Peptides highlighted in grey contain the known cognate antigen and those highlighted in black contain off-target hits.

[0035] Figs. 14A-14I depict cytotoxicity and functionality of engineered TH-specific TCR variants against TH-positive tumors. Fig. 14A: Granzyme B activation of 293T T-scan target cells expressing the TH full length ORF (TH ORF) following 24 h co-culture with engineered TH WTTCR variant transduced human primary T cells or a negative control TCR at a 1 : 1 T cell to target cell co-culture ratio (left) or a 3: 1 ratio (right). Figs. 14B-14D: Killing of TH expressing cell lines by different TH-specific transduced human primary T cells. Co-culture was performed at a T cell to target cell ratio of 3: 1 and cultured for 24 h. Cell lines that did not express HLA-A2 were transduced with a lentiviral vector expressing HLA-A2. Figs. 14E-14G: Cytotoxic granule release (CD107a), IFNg, and TNF staining of different TH-specific TCR variant transduced primary T cells following co-culture with TH-expressing tumor cell lines. Figs. 14H-14I: The antitumor activity of primary T cells transduced with engineered TH SW TCRs was assessed in a xenogeneic A375 melanoma mouse.DETAILED DESCRIPTION

[0036] The inventors have found that the problems with producing self-antigen-specific TCRs can be avoided by using a self-antigen mimic molecule to create initial pools of TCRs. This avoids the negative selection pressures exerted on self-antigen-specific TCRs during development and greatly improves output. The inventors have shown that these pools of TCRs can then be subjected to targeted mutagenesis in the CDR3 sequences to provide self-antigen-specific TCRs that do not exhibit off-target binding. This methodology provides rapid, effective production of self-antigen-specific TCRs that exhibit high affinity and high specificity and which are therapeutically efficacious when applied to ACT.

[0037] In one aspect of any of the embodiments, described herein is a method comprising mutagenizing at least one of a CDR3a and a CDR3J3 of a self-antigen mimic-specific TCR to provide a pool of target self-antigen-specific TCR candidates; and b) selecting at least one target self-antigen- specific TCR from the pool of target self-antigen-specific TCR candidates. In one aspect of any of the embodiments, described herein is a method comprising: a) isolating a self-antigen mimic-specific TCR; b) mutagenizing at least one of the CDR3a and CDR3J3 of the self-antigen mimic-specific TCR to provide a pool of target self-antigen-specific TCR candidates; and c) selecting at least one target self-antigen-specific TCR from the pool of target self-antigen-specific TCR candidates.

[0038] As described herein, an "antigen" is a molecule that is specifically bound by a B cell receptor (BCR), T cell receptor (TCR), and / or antibody, thereby activating an immune response. An antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof. The term "antigenic determinant" refers to an epitope on the antigen recognized by an antigen-binding molecule (e.g., a a B cell receptor (BCR), T cell receptor (TCR), and / or antibody), and more particularly, by the antigen-binding site of said molecule.

[0039] As used herein, “self-antigen” refers to a molecule produced by a first organism which would act as antigen in a second organism, but which is tolerated by the immune system of the organism in which it occurs. A healthy immune system of the first organism will actively select against immune responses to the self-antigen in a process referred to as central tolerance. Thus,naturally-occuring TCRs with high affinity to a given self-antigen are very rare in the first organism. In some embodiments of any of the aspects, a self-antigen is an antigen encoded in germline cells of the first organism. In some embodiments of any of the aspects, a self-antigen is an antigen not encoded in germline cells of the first organism and encoded in at least one somatic cell, e.g., it is a somatic mutation or an oncogene. An example are mutations of KRAS which promote or give rise to cancer. In some embodiments of any of the aspects, a self-antigen is produced by a first individual organism of a first species, which would act as antigen in a second individual organism of the first species, but which is tolerated by the immune system of the first individual organism in which it occurs. In some embodiments of any of the aspects, a self-antigen is produced by a first organism of a first species, which would act as antigen in a second organism of a second species, but which is tolerated by the immune system of the organisms of the first species in which it occurs.

[0040] Self-antigen can refer to a polypeptide, or any portion thereof that comprises the epitope of the self-antigen. In some embodiments of any of the aspects, a self-antigen is present as a full- length polypeptide as encoded by an organism’s genome. In some embodiments of any of the aspects, a self-antigen is present as polypeptide comprising at least an epitope. In some embodiments of any of the aspects, a self-antigen is present as an epitope. In some embodiments of any of the aspects, a selfantigen is present as an peptide as processed for MHC display. In some embodiments of any of the aspects, a self-antigen is present as an peptide complexed with the MHC. In some embodiments of any of the aspects, a self-antigen is a polypeptide of 6-15 amino acids. In some embodiments of any of the aspects, a self-antigen is a polypeptide of 8-20 amino acids. In some embodiments of any of the aspects, a self-antigen is a polypeptide of 8-11 amino acids. In some embodiments of any of the aspects, a self-antigen is a polypeptide of no more than 20 amino acids. In some embodiments of any of the aspects, a self-antigen is a polypeptide of more than 20 amino acids. In some embodiments of any of the aspects, a self-antigen is a naturally occurring polypeptide.

[0041] As used herein, “target self-antigen” refers to a self-antigen in or on a cell which can be bound TCR and / or processed and displayed in complex with an MHC. Suitable target self-antigens include self-antigens which are known to be highly expressed in diseased tissues, and / or characteristic of diseased tissues. Such self-antigens and their sequences are well known in the art and readily available in public databases such as es, there are multiple databases: IEDB (iedb.org / ), the Cancer Antigenic Peptide Database: caped.icp.ucl.ac.be / peptide / list; and the MHC Motif Atlas: mhcmotifatlas . org / home .

[0042] In some embodiments of any of the aspects, a target self-antigen is present as a full-length polypeptide as encoded by an organism’s genome. In some embodiments of any of the aspects, a target self-antigen is present as polypeptide comprising at least an epitope. In some embodiments of any of the aspects, a target self-antigen is present as an epitope. In some embodiments of any of the aspects, a target self-antigen is present as an peptide as processed for MHC display. In someembodiments of any of the aspects, a target self-antigen is present as a peptide complexed with the MHC. In some embodiments of any of the aspects, a target self-antigen is a polypeptide of 6-15 amino acids. In some embodiments of any of the aspects, a target self-antigen is a polypeptide of 8-20 amino acids. In some embodiments of any of the aspects, a target self-antigen is a polypeptide of 8-11 amino acids. In some embodiments of any of the aspects, a target self-antigen is a polypeptide of no more than 20 amino acids. In some embodiments of any of the aspects, a target self-antigen is a polypeptide of more than 20 amino acids. In some embodiments of any of the aspects, a target selfantigen is a naturally occurring polypeptide.

[0043] As used herein, a “target self-antigen-specific TCR” is a TCR that binds specifically to the target self-antigen. In some embodiments, a target self-antigen-specific TCR binds specifically to the target self-antigen as compared to the self-antigen mimic.

[0044] As used herein, a “target self-antigen-specific TCR candidate” refers to TCR that is to be screened, selected, isolated, and / or analyzed for target self-antigen-specific binding. A “candidate” element can be known to have the relevant activity or structure, but be a candidate in the sense of being a candidate for binding of a particular level of avidity or specificity, a candidate for binding in particular physical conditions, or a candidate for binding as compared to or in direct competition with other candidates. For the methods described herein, candidates may be screened individually, or in groups. Group screening is particularly useful where hit rates for effective candidates are expected to be low such that one would not expect more than one positive result for a given group.

[0045] As used herein, “self-antigen mimic” refers to a polypeptide which has at least 75% sequence identity to a target self-antigen, but which is not itself the target self-antigen. In some embodiments of any of the aspects, the self-antigen mimic is from 8 to 11 amino acids in length and differs from the target self-antigen by only one amino acid. In some embodiments of any of the aspects, the self-antigen mimic differs from the target self-antigen by only one amino acid. In some embodiments of any of the aspects, the self-antigen mimic differs from the target self-antigen by only two amino acids. In some embodiments of any of the aspects, the self-antigen mimic is not a selfantigen. In some embodiments of any of the aspects, the target self-antigen and the self-antigen mimic are the same length. In some embodiments of any of the aspects, the sequence of the target self-antigen and the self-antigen mimic differ only at one amino acid position.

[0046] In some embodiments of any of the aspects, a self-antigen mimic is present as polypeptide comprising at least an epitope. In some embodiments of any of the aspects, a self-antigen mimic is present as an epitope. In some embodiments of any of the aspects, a self-antigen mimic is present as an peptide as processed for MHC display. In some embodiments of any of the aspects, a self-antigen mimic is present as a peptide complexed with the MHC. In some embodiments of any of the aspects, a self-antigen mimic is a polypeptide of 6-15 amino acids. In some embodiments of any of the aspects, a self-antigen mimic is a polypeptide of 8-20 amino acids. In some embodiments ofany of the aspects, a self-antigen mimic is a polypeptide of 8-11 amino acids. In some embodiments of any of the aspects, a self-antigen mimic is a polypeptide of no more than 20 amino acids. In some embodiments of any of the aspects, a self-antigen mimic is a polypeptide of more than 20 amino acids. In some embodiments of any of the aspects, a self-antigen mimic is not a naturally occurring polypeptide. In some embodiments of any of the aspects, a self-antigen mimic is present as a fusion protein with at least one further peptide sequence, e.g., a leader sequence or sequence that promotes processing of the fusion protein for MHC display.

[0047] As used herein, a “self-antigen mimic-specific TCR” is a TCR that binds specifically to the self-antigen mimic. In some embodiments, a self-antigen mimic-specific TCR binds specifically to the self-antigen mimic as compared to the target self-antigen.

[0048] In some embodiments of any of the aspects, the method comprises isolating a target selfantigen mimic-specific TCR. As used herein, “isolating” refers to a) artificially producing an element and / or b) removing the element from a more complex original environment. In some embodiments of any of the aspects, isolating a TCR can comprise isolating the TCR polypeptide from a cellular environment and / or organism. In some embodiments of any of the aspects, isolating a TCR can comprise isolating the T cell expressing the TCR polypeptide from a population of cells and / or an organism. In some embodiments of any of the aspects, isolating a TCR can comprise determing the sequence of the TCR and / or the gene(s) encoding the TCR. In some aspects of any of the embodiments, the method comprises a step of determing the sequence of the target self-antigen mimic-specific TCR and / or the gene(s) encoding the target self-antigen mimic-specific TCR. In some aspects of any of the embodiments, the method comprises a first step of determing the sequence of the target self-antigen mimic-specific TCR and / or the gene(s) encoding the target self-antigen mimicspecific TCR.

[0049] In some embodiments of any of the aspects, isolating a self-antigen mimic-specific TCR comprises raising an immune response against the self-antigen mimic. In some embodiments of any of the aspects, isolating a self-antigen mimic-specific TCR comprises raising a T cell response against the self-antigen mimic. In some embodiments of any of the aspects, isolating a self-antigen mimicspecific TCR comprises raising a CD8+ T cell response against the self-antigen mimic. In some embodiments of any of the aspects, isolating a self-antigen mimic-specific TCR comprises inducing a CD8+ T cell response against the self-antigen mimic. In some embodiments of any of the aspects, isolating a self-antigen mimic-specific TCR comprises injecting an animal with an humanized immune system the self-antigen mimic. In some embodiments of any of the aspects, isolating a selfantigen mimic-specific TCR comprises injecting an animal with humanized TCR-encoding genes with the self-antigen mimic. Methods for raising, stimulating, or selecting TCRs and immune responses are well known in the art.

[0050] In some embodiments of any of the aspects, isolating a self-antigen mimic-specific TCR comprises raising naive CD8+ T cells from a human donor with the self-antigen mimic. In some embodiments of any of the aspects, isolating a self-antigen mimic-specific TCR comprises stimulating naive CD8+ T cells from a human donor with the self-antigen mimic. In some embodiments of any of the aspects, isolating a self-antigen mimic-specific TCR comprises screening naive CD8+ T cells from a human donor with the self-antigen mimic.

[0051] Naive T cells have not yet encountered their specific antigen. In peripheral lymphoid organs, naive T lymphocytes can interact with antigen-presenting cells (APCs), which use MHC molecules to present antigen. Once the T cells recognize specific antigens, they proliferate and differentiate into one of several effector T cell subsets. Effector T cell interact with host cells (rather than the pathogen) to carry out their immune function. T cell use co-receptors, e.g., CD4 or CD8 to bind to the MHC molecules. CD8+ T cells, also known as cytotoxic T cells, mediate direct killing of antigen-presenting target cells. Naive CD8+ T cells are activated upon recognition of antigens presented by MHC class I on dendritic cells in the spleen or lymph nodes. Activated CD8+ T cells expand and become effector CD8+ T cells. CD8+ T cells tend to be evaluated during the study for tumor-infiltrating T cells.

[0052] In some embodiments, the human donor is HLA-matched with respect to the target selfantigen. In some embodiments of any of the aspects, the isolating comprises raising naive CD8 T cells from a human donor with the self-antigen mimic, wherein the human donor is HLA-matched with respect to the target self-antigen. One of skill in the art is aware of which HLA allele displays which self-antigen, and can readily determine the HLA match with respect to the target self-antigen. Lor example, databases of suitable MHC and / or antigen sequences are available on the world wide web at iedb.org; caped.icp.ucl.ac.be / peptide / list; services.healthtech.dtu.dk / services / NetMHC-4.0 / ; mhcmotifatlas.org / home immunespace.org; immgen.org; import / org; peptideatlast.org / repository / ; uniprot.org; ncbi.nlm.nih.gov / protein / ; immunedata.org / index.php; immuneprofiling.org / hipc / ; allergenonline.org / databasebrowe.shtml; and itntrialshare.org. further examples are also provided in Smatti et al. 2019 Viruses 11:762; Beretta-Piccoli et al. 2019 J Autoimmu 94: 1-6; and Cusick et al. 2012 Clinical Reviews in Allergy and Immunology; each of which is incorporated by reference herein in its entirety.

[0053] In some embodiments of any of the aspects, isolating a self-antigen mimic-specific TCR comprises screening a library of T cells with the self-antigen mimic. In some embodiments of any of the aspects, isolating a self-antigen mimic-specific TCR comprises screening a library of TCRs with the self-antigen mimic.

[0054] In some embodiments of any of the aspects, the self-antigen mimic is displayed in a complex with an MHC polypeptide during the step of isolating a self-antigen mimic-specific TCR. In some embodiments of any of the aspects, the self-antigen mimic is displayed in a fusion protein withan MHC polypeptide during the step of isolating a self-antigen mimic-specific TCR. In some embodiments of any of the aspects, the self-antigen mimic is displayed by a TAP-deficient cell during the step of isolating a self-antigen mimic-specific TCR. In some embodiments of any of the aspects, the self-antigen mimic is displayed by a TAP -deficient, ERAP 1 -deficient, and ERAP2-deficicient cell during the step of isolating a self-antigen mimic-specific TCR. Such cells are known in the art and used in EpiScan technology, see, e.g., Bruno et al. Nature Biotechnology 41:980-992 (2023); which is incorporated by reference herein in its entirety. In some embodiments of any of the aspects, the selfantigen mimic is displayed on an antigen-presenting cell (APC) with an MHC polypeptide during the step of isolating a self-antigen mimic-specific TCR.

[0055] An antigen presenting cell or APC is a cell which displays an antigen complexed with major histocompatibility complexes (MHC) on its surface. A T cell will recognize these complexes via a TCR. The role of an APC is to continuously process antigens and present the antigen to a T cell. Non-limiting examples of common, naturally occuring APCs include macrophages, B cells, and dendritic cells. In some embodiments of any of the aspects, the APC is a dendritic cell. In some embodiments of any of the aspects, the APC is a monocyte-derived dendritic cell. In some embodiments of any of the aspects, the APC is an EpiScan cell. In some embodiments of any of the aspects, the APC is a TAP -deficient, ERAP 1 -deficient, and ERAP2-deficicient cell.

[0056] In some embodiments of any of the aspects, the step of isolating a self-antigen mimicspecific TCR comprises the step of isolating a single self-antigen mimic-specific TCR. In some embodiments of any of the aspects, the step of isolating a self-antigen mimic-specific TCR comprises the step of isolating a plurality of self-antigen mimic-specific TCRs.

[0057] In some embodiments of any of the aspects, the method comprises mutagenizing at least one of a CDR3a and a CDR3J3 of a self-antigen mimic-specific TCR to provide a pool of target self- antigen-specific TCR candidates. As used herein “mutatgenizing” of a polypeptide refers to to any change or variation (deletion, substitution, addition, or sequence alteration of one amino acid or more) in the indicated polypeptide relative to the sequence of the polypeptide prior to the mutagenesis. The mutagenesis can be performed directly in a peptide, e.g., by directing synthesis of a mutagenized polypeptide sequence, or by mutagenizing one or more nucleic acids that encode the indicated polypeptide. The mutageneis can be random or directed. Methods of mutagenesis are known in the art and include but are not limited to synthesizing oligonucleotides containing a mutant sequence and ligating the synthesized molecule to other sequences and / or subjecting it to ex vivo expression; oligonucleotide-directed site-specific mutagenesis; site-specific mutagenesis (e.g., Kunkel’s method, cassette mutagenesis, PCR site-directed mutagenesis (e.g., traditional PCR, primer extension, or inverse PCR), whole plasmid mutagenesis, in vivo site-directed mutagenesis, and CRISPR / Cas-guided mutagenesis), transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or byelectroporation), and phage transduction, are known in the art and can be applied by the ordinarily skilled artisan to introduce mutations into specific nucleic acid loci. Techniques for making such alterations are very well established and include, for example, those disclosed by Braman, Jeff, ed. (2002) In Vitro Mutagenesis Protocols, Methods in Molecular Biology, Vol. 182 (2nd ed.); Khudyakov and Fields (2002), Artificial DNA: Methods and Applications, CRC Press; Hsu et al. (2014), Cell 157 (6): 1262-78; Cerchione et al. (2020) PLOS ONE 15 (4): e0231716; and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties.

[0058] In some embodiments of any of the aspects, the method comprises mutagenizing theCDR3a of a self-antigen mimic-specific TCR. In some embodiments of any of the aspects, the method comprises mutagenizing the CDR3J3 of a self-antigen mimic-specific TCR. In some embodiments of any of the aspects, the method comprises mutagenizing the CDR3a and the CDR3J3 of a self-antigen mimic-specific TCR.

[0059] In some embodiments of any of the aspects, the method comprises mutagenizing a single residue of at least one of a CDR3a and a CDR3J3 of a self-antigen mimic-specific TCR. In some embodiments of any of the aspects, the method comprises mutagenizing a single residue of each of a CDR3a and a CDR3J3 of a self-antigen mimic-specific TCR. In some embodiments of any of the aspects, the method comprises mutagenizing two residues of at least one of a CDR3a and a CDR3J3 of a self-antigen mimic-specific TCR. In some embodiments of any of the aspects, the method comprises mutagenizing two residues of each of a CDR3a and a CDR3J3 of a self-antigen mimicspecific TCR. In some embodiments of any of the aspects, the method comprises mutagenizing three residues of at least one of a CDR3a and a CDR3J3 of a self-antigen mimic-specific TCR. In some embodiments of any of the aspects, the method comprises mutagenizing three residues of each of a CDR3a and a CDR3J3 of a self-antigen mimic-specific TCR.

[0060] In some embodiments of any of the aspects, the mutagenizing is comprehensive saturation mutagenesis. In some embodiments of any of the aspects, the mutagenizing is comprehensive saturation mutagenesis of the CDR3a and the CDR3J3 of a self-antigen mimic-specific TCR. In some embodiments of any of the aspects, the mutagenizing is comprehensive saturation mutagenesis of the CDR3a of a self-antigen mimic-specific TCR. In some embodiments of any of the aspects, the mutagenizing is comprehensive saturation mutagenesis of the CDR3J3 of a self-antigen mimic-specific TCR.

[0061] In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of single mutants, double mutants, triple mutants, and CDR3 length changes of at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of single mutants of at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self-antigen- specific TCR candidates comprises a library of single mutants and double mutants of at least one ofthe CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self-antigen- specific TCR candidates comprises a library of all single mutants of at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of at least 90% of all single mutants and double mutants of at least one of the CDR3a and the CDR3p. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of all single mutants and all double mutants of at least one of the CDR3a and the CDR3p. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of single mutants, double mutants, and triple mutants of at least one of the CDR3a and the CDR3p. In some embodiments of any of the aspects, the pool of self- antigen-specific TCR candidates comprises a library of single mutants, double mutants, triple mutants, and CDR3 length changes of at least one of the CDR3a and the CDR3p. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of single mutants, double mutants, and CDR3 length changes of at least one of the CDR3a and the CDR3p.

[0062] In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of single mutants, double mutants, triple mutants, and CDR3 length changes of the CDR3a and the CDR3p. In some embodiments of any of the aspects, the pool of self- antigen-specific TCR candidates comprises a library of single mutants of the CDR3a and the CDR3p. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of all single mutants of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of at least 90% of all single mutants and double mutants of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of single mutants and double mutants of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of all single mutants and all double mutants of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self- antigen-specific TCR candidates comprises a library of single mutants, double mutants, and triple mutants of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self- antigen-specific TCR candidates comprises a library of single mutants, double mutants, triple mutants, and CDR3 length changes of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of single mutants, double mutants, and CDR3 length changes of the CDR3a and the CDR3J3.

[0063] In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of at least 100 mutants of at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of at least 200 mutants of at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of at least500 mutants of at least one of the CDR3a and the CDR3J3. In some embodiments of any of the aspects, the pool of self-antigen-specific TCR candidates comprises a library of at least 1,000 mutants of at least one of the CDR3a and the CDR3J3.

[0064] In some embodiments of any of the aspects, the method comprises selecting at least one target self-antigen-specific TCR from the pool of target self-antigen-specific TCR candidates. As used herein, “selecting” can comprise any means of assaying or screening at least a portion of the pool of target self-antigen-specific TCR candidates for target self-antigen binding activity.

[0065] Methods of detecting and measuring protein-protein interactions are known in the art, e.g., Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA), ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, immunofluorescence assays, sandwich competition assays, titration of a polypeptide agent in a suitable binding assay, and the different variants thereof known in the art; as well as other techniques as mentioned herein.

[0066] In some embodiments of any of the aspects, the selecting comprises selecting target self- antigen-specific TCRs from the pool of target self-antigen-specific candidates. In some embodiments of any of the aspects, the selecting comprises selecting target self-antigen-binding TCRs from the pool of target self-antigen-specific candidates. In some embodiments of any of the aspects, the selecting comprises selecting TCRs that specifically bind the target self-antigen from the pool of target self- antigen-specific candidates. In some embodiments of any of the aspects, the selecting comprises selecting TCRs that specifically bind the target self-antigen, as compared to the self-antigen mimic, from the pool of target self-antigen-specific candidates.

[0067] In some embodiments of any of the aspects, selecting at least one target self-antigen- specific TCR comprises selecting a TCR that binds the target self-antigen with a KD of less than 10 pM. In some embodiments of any of the aspects, selecting at least one target self-antigen-specific TCR comprises selecting a TCR that binds the target self-antigen with a KDof less than 500 nM. In some embodiments of any of the aspects, selecting at least one target self-antigen-specific TCR comprises selecting a TCR that binds the target self-antigen with a KDof less than 100 nM.

[0068] In some embodiments of any of the aspects, the selecting comprises TCRs that do not bind the self-antigen mimic from the pool of target self-antigen-specific candidates. In some embodiments of any of the aspects, the selecting comprises selecting TCRs that do not bind a plurality of off-target self-antigens from the pool of target self-antigen-specific candidates. In some embodiments of any of the aspects, the selecting comprises selecting TCRs that do not bind a plurality of off-target self-antigens in a human peptidome library from the pool of target self-antigen-specific candidates. In some embodiments of any of the aspects, the selecting comprises selecting TCRs that do not bind a plurality of off-target self-antigens in a human peptidome library presented on T-scan cells from the pool of target self-antigen-specific candidates. As used herein, “T-scan cells” refers toa) library of cells expressing a) candidate antigens and / or self-antigens for processing and display on MHC and b) a reporter construct which detects the secretion of cytotoxic granules by bound T cells and generates a detectable signal (e.g., a fluorescent signal). T-sca cells are known in the art and described in further detail in, e.g., Kula et al. Cell 178(4): 1016-1028. el3 (2019); which is incorporated by reference herein in its entirety.

[0069] As used herein, “off-target self-antigen” refers to at least one self-antigen other than the target self-antigen. In some embodiments of any of the aspects, the plurality off-target self-antigens are self-antigens of the same species of organism which is the source of the target self-antigen. In some embodiments of any of the aspects, the plurality off-target self-antigens are self-antigens of the same individual organism which is the source of the target self-antigen. In some embodiments of any of the aspects, the plurality off-target self-antigens are self-antigens obtained from the same organ or tissue which is the source of the target self-antigen. In some embodiments of any of the aspects, the plurality off-target self-antigens are self-antigens obtained from the same diseased organ or diseased tissue which is the source of the target self-antigen. In some embodiments of any of the aspects, the plurality off-target self-antigens are self-antigens obtained from the same diseased organ or diseased tissue which expresses ectopic amounts of the target self-antigen.

[0070] In some embodiments of any of the aspects, the target self-antigen is displayed in a complex with an MHC polypeptide during the selecting step. In some embodiments of any of the aspects, the self-antigen mimic is displayed in a complex with an MHC polypeptide during the selecting step.

[0071] In some embodiments of any of the aspects, the target self-antigen is displayed in a fusion protein with an MHC polypeptide during the selecting step. In some embodiments of any of the aspects, the self-antigen mimic is displayed in a fusion protein with an MHC polypeptide during the selecting step.

[0072] In some embodiments of any of the aspects, the target self-antigen is displayed by a TAP- deficient cell during the selecting step. In some embodiments of any of the aspects, the self-antigen mimic is displayed by a TAP-deficient cell during the selecting step. In some embodiments of any of the aspects, the target self-antigen is displayed by TAP-deficient, ERAP 1 -deficient, and ERAP2- deficicient cell during the selecting step. In some embodiments of any of the aspects, the self-antigen mimic is displayed by a TAP-deficient, ERAP 1 -deficient, and ERAP2-deficicient cell during the selecting step. In some embodiments of any of the aspects, the target self-antigen is displayed by an APC with an MHC polypeptide during the selecting step. In some embodiments of any of the aspects, the self-antigen mimic is displayed by an APC with an MHC polypeptide during the selecting step.

[0073] In some embodiments of any of the aspects, the step of selecting at least one target self- antigen-specific TCR from the pool of target self-antigen-specific TCR candidates comprises isolating a single self-antigen-specific TCR. In some embodiments of any of the aspects, the step of selectingat least one target self-antigen-specific TCR from the pool of target self-antigen-specific TCR candidates comprises isolating a plurality of self-antigen-specific TCRs.

[0074] Described herein are TCR polypeptides which are specific for Tyrosine Hydoxylase (TH). TH is a self-antigen found in cancers, e.g., neuroblastoma. These TCR polypeptides were obtained as a result of a comprehensive saturation mutagenesis approach that resulted in the directed evolution of the TCR specificity towards a desired new target. These sequences had to be engineered and cannot be formed in nature.

[0075] In one aspect of any of the embodiments, described herein is a TCR polypeptide composition comprising: f) a CDRla comprising the sequence of SEQ ID NO: 1; g) a CDR2a comprising the sequence of SEQ ID NO:2; h) a CDR3a comprising the sequence of one of SEQ ID NOs: 3 and 7-15; i) a CDRip comprising the sequence of SEQ ID NON; j) a CDR2J3 comprising the sequence of SEQ ID NO:5; and k) a CDR3J3 comprising the sequence of SEQ ID NO:6.In embodiment of any of the aspects, described herein is a TCR polypeptide composition comprising: a) a CDRla consisting of the sequence of SEQ ID NO: 1; b) a CDR2a consisting of the sequence of SEQ ID NO:2; c) a CDR3a consisting of the sequence of one of SEQ ID NOs: 3 and 7-15; d) a CDRip consisting of the sequence of SEQ ID NON; e) a CDR2P consisting of the sequence of SEQ ID NON; and f) a CDR3P consisting of the sequence of SEQ ID NON.

[0076] In some embodiments of any of the aspects, the the CDR3a comprises the sequence ofSEQ ID NO: 3. In some embodiments of any of the aspects, the the CDR3a comprises the sequence ofSEQ ID NO: 7. In some embodiments of any of the aspects, the the CDR3a comprises the sequence ofSEQ ID NO: 8. In some embodiments of any of the aspects, the the CDR3a comprises the sequence ofSEQ ID NO: 9. In some embodiments of any of the aspects, the the CDR3a comprises the sequence ofSEQ ID NO: 10. In some embodiments of any of the aspects, the the CDR3a comprises the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the the CDR3a comprises the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the the CDR3a comprises the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the the CDR3a comprises the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the the CDR3a comprises the sequence of SEQ ID NO: 15. In some embodiments of any of the aspects, the the CDR3a consists of the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the the CDR3a consists of the sequence of SEQ ID NO: 7. In some embodiments of any of the aspects, the the CDR3a consists of the sequence of SEQ ID NO: 8. In some embodiments of any ofthe aspects, the the CDR3a consists of the sequence of SEQ ID NO: 9. In some embodiments of any of the aspects, the the CDR3a consists of the sequence of SEQ ID NO: 10. In some embodiments of any of the aspects, the the CDR3a consists of the sequence of SEQ ID NO: 11. In some embodiments of any of the aspects, the the CDR3a consists of the sequence of SEQ ID NO: 12. In some embodiments of any of the aspects, the the CDR3a consists of the sequence of SEQ ID NO: 13. In some embodiments of any of the aspects, the the CDR3a consists of the sequence of SEQ ID NO: 14. In some embodiments of any of the aspects, the the CDR3a consists of the sequence of SEQ ID NO: 15.

[0077] In some embodiments of any of the aspects, the TCR polypeptide composition comprises a TCR heterodimer pair, e.g., a TCR a chain polypeptide comprising the CDRla, CDR2a, and CDR3a and a TCR [3 chain polypeptide comprising the CDRip, CDR2J3, and CDR3J3. In some embodiments of any of the aspects, the TCR polypeptide composition comprises a TCR fusion protein comprising a single polypeptide comprising the CDRla, CDR2a, CDR3a, CDR1J3, CDR2J3, and CDR3J3.

[0078] In some embodiments of any of the aspects, the TCR polypeptide composition further comprises a TCR a constant domain and a TCR P constant domain. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises a TCR a variable domain and a TCR P variable domain. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises a TCR a constant domain, a TCR P constant domain, a TCR a variable domain, and a TCR P variable domain.

[0079] In some embodiments of any of the aspects, the TCR polypeptide composition further comprises one or more of SEQ ID NOs: 16-19. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 16. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 17. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 18. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 19. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NOs: 16-19. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NOs: 16 and 17. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NOs: 16 and 18. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NOs: 16 and 19. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NOs: 17 and 18. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NOs: 17 and 19. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NOs: 18 and 19.

[0080] In some embodiments of any of the aspects, the TCR polypeptide composition further comprises one of SEQ ID NOs: 20-29. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 20. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 21. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 22. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 23. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 24. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 25. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 26. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 27. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 28. In some embodiments of any of the aspects, the TCR polypeptide composition further comprises SEQ ID NO: 29.

[0081] In some embodiments of any of the aspects, the TCR polypeptide composition further includes a conservative substitution in a sequence not comprised by a CDR, e.g., a conservative substitution relative to one of SEQ ID NOs: 16-29.

[0082] In some embodiments of any of the aspects, the technology described herein relates to one or more nucleic acids encoding TCR polypeptide composition as described herein. In some embodiments of any of the aspects, the nucleic acid is a cDNA. In some embodiments of any of the aspects, a nucleic acid encoding a TCR polypeptide composition as described herein is comprised by a vector. In some of the aspects of the embodiments described herein, a nucleic acid sequence encoding a TCR polypeptide composition as described herein, or any module thereof, is operably linked to a vector.

[0083] In one aspect of any of the embodiments, described herein is a cell comprising a TCR polypeptide composition as described herein, or a at least one nucleic acid encoding such a TCR polypeptide composition. In one aspect of any of the embodiments, a cell comprising a TCR polypeptide composition as described herein is provided. In some embodiments of any of the aspects, the TCR polypeptide composition as described herein is expressed on the cell surface. In some embodiments of any of the aspects, the cell comprises at least one nucleic acid encoding a TCR polypeptide composition as described herein.

[0084] In some embodiments of any of the aspects, the cell is an immune cell. As used herein, “immune cell” refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B-cells and T-cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the cell is a T-cell; a NK cell; a NKT-cell; lymphocytes, such as B-cells and T- cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, andgranulocytes. In some embodiments of any of the aspects, the cell is a T cell. In some embodiments of any of the aspects, the cell is a CD4+ T cell. In some embodiments of any of the aspects, the cell is a CD8+ T cell.

[0085] In some embodiments of any of the aspects, a cell (e.g., an immune cell) is transduced with a retroviral vector, e.g., a lentiviral vector, encoding a TCR polypeptide. For example, an immune effector cell is transduced with a vector encoding a TCR polypeptie that comprises an antiTyrosine Hydoxylase (TH) TCR that binds a Tyrosine Hydoxylase (TH) polypeptide. Thus, these transduced cells can elicit an immune response against cells comprising or displaying a Tyrosine Hydoxylase (TH) antigen. Trandsuction is well known in the art and described, for example in Ghassemi et al. Nature Biomedical Engineering 6: 118-128 (2022); Pampusch et al. J Vis Exp 18: 10.3791 / 60400 (2020); Kim et al. Scientific Reports 13: 12365 (2023); and Prommsberger et al. Current Protocols in Immunoloy 128:e93 (2020); each of which is incorporated by reference herein in its entirety.

[0086] Retroviruses are a common tool for gene delivery. In some embodiments of any of the aspects, a retrovirus is used to deliver a polynucleotide encoding TCR polypeptide to a cell. As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Once the virus is integrated into the host genome, it is referred to as a “provirus.” The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles.

[0087] Illustrative retroviruses suitable for use in particular embodiments, include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus.

[0088] As used herein, the term “lentivirus” refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV based vector backbones (z.e., HIV cis-acting sequence elements) are preferred. In some embodiments of any of the aspects, a lentivirus is used to deliver a polynucleotide comprising a TCR polypeptide composition to a cell.

[0089] Retroviral vectors and more particularly lentiviral vectors may be used in practicing some embodiments of the present invention. Accordingly, the term “retrovirus” or “retroviral vector”, as used herein is meant to include “lentivirus” and “lentiviral vectors” respectively.

[0090] In one aspect of any of the embodiments, described herein is a composition comprising a TCR polypeptide composition as described herein or a nucleic acid encoding a TCR polypeptide composition as described herein or a cell as described herein. In some embodiments, the composition is a pharmaceutical composition. As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier accepted for use in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which 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 problem or complication, commensurate with a reasonable benefit / risk ratio.

[0091] The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance or maintain the effectiveness of the active ingredient.

[0092] The therapeutic composition as described herein can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

[0093] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active agent used in the invention that will be effective inthe treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[0094] In some embodiments, the composition comprising a TCR polypeptide composition as described herein or a nucleic acid encoding a TCR polypeptide composition as described herein can be a lyophilisate.

[0095] In some embodiments, the technology described herein relates to a syringe or catheter, including an organ-specific catheter (e.g., renal catheter, biliary catheter, cardiac catheter, etc.), comprising a therapeutically effective amount of a composition described herein.

[0096] As used herein, the phrase “therapeutically effective amount”, “effective amount” or “effective dose” refers to an amount that provides a therapeutic or aesthetic benefit in the treatment, prevention, or management of an condition / disorder, e.g., an amount that provides a statistically significant decrease in at least one symptom, sign, or marker of the condition / disorder. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject’s history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents.

[0097] In one aspect, the technology described herein relates to a method comprising administering a TCR polypeptide composition as described herein or a nucleic acid encoding a TCR polypeptide composition, or a cell comprising such a polypeptide composition or nucleic acid to a subject. In some embodiments, the subject is in need of treatment for a cancer. In some embodiments, the method is a method of treating a subject. In some embodiments, the method is a method of treating a cancer in a subject. In some embodiments, the subject is in need of an immune response. In some embodiments, the subject is in need of immune stimulation.

[0098] In one aspect of any of the embodiments, described herein is a method of stimulating an immune response in a subject in need thereof, the method comprising administering a nucleic acid as described herein or an immune cell comprising the nucleic acid to the subject, wherein the subject’s immune cells are caused to express the polypeptide encoded by the nucleic acid. In one aspect of any of the embodiments, described herein is a method of stimulating an immune response specific for a self-antigen in a subject in need thereof, the method comprising administering a nucleic acid encoding a target self-antigen-specific TCR as described herein or an immune cell comprising the nucleic acid to the subject, wherein the subject’s immune cells are caused to express the polypeptide encoded by the nucleic acid. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T-cell. In some embodiments of any of the aspects, the immune cell is autologous to the subject. In some embodiments of any of the aspects, the method comprises a first step of transducing at least one immune cell (e.g., a T cell) obtained from the subject with the target self-antigen-specific TCR or the TCR polypeptide composition as described herein.

[0099] As used herein, an “immune response” refers to a response by a cell of the immune system, such as a B cell, T cell (CD4 or CD8), regulatory T cell, antigen-presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus (e.g., to an antigen and / or adjuvant). In some embodiments of the aspects described herein, the response is specific for a particular antigen (an "antigen-specific response") and refers to a response by a CD4 T cell, CD8 T cell, or B cell via their antigen-specific receptor. In some embodiments of the aspects described herein, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. Such responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response. Stimulation of an immune response refers to an induction or increase of the immune response.

[0100] A "cell-mediated immune response" is elicited by the presentation of antigenic epitopes in association with Class I or Class II molecules of the major histocompatibility complex (MHC), CD1 or other non-classical MHC -like molecules. This activates antigen-specific CD4+ T helper cells or CD8+ cytotoxic lymphocyte cells ("CTLs"). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by classical or non-classical MHCs and expressed on the surfaces of cells. CTLs help induce and promote the intracellular destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide or other antigens in association with classical or non-classical MHC molecules on their surface. A "cell- mediated immune response" also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and / or other white blood cells, including those derived from CD4+ and CD8+ T-cells. The ability of a particular antigen or composition to stimulate a cell- mediated immunological response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, by assaying for T- lymphocytes specific for the antigen in a sensitized subject, or by measurement of cytokine production by T cells in response to re -stimulation with antigen. Such assays are well known in the art. See, e.g., Erickson et al. (1993) J. Immunol. 151:4189-4199; and Doe et al. (1994) Eur. J. Immunol. 24:2369-2376.

[0101] In some embodiments of any of the aspects, the immune response comprises a CD4+ T cell response in the subject. In some embodiments of any of the aspects, an immune response can be cytokine production by CD4+ T cells. In some embodiments of any of the aspects, cytokine production by a CD4+ T cell can comprise production of one or more of IL-2 (proliferation); IL-2, IFN-y, TNF, TNF-J3 (Thl); IL-4, IL-5, IL-9 and IL-13 (Th2); IL-l-p, IL-17A, IL-17E, IL-17F, IL-21, IL-22, IL-23 (Thl7); IL-6, IL21, (Tfh); TGF-J3, IL-10, IL-35 (multiple and Tregs). In someembodiments of any of the aspects, an immune response can be an increase in the level of CD4+ T cells, e.g., antigen-specific CD4+ cells.

[0102] In some embodiments of any of the aspects, the immune response comprises a CD8+ T cell response in the subject. In some embodiments of any of the aspects, an immune response can be cytokine production by CD8+ T cells. In some embodiments of any of the aspects, cytokine production by a CD8+ T cell can comprise production of one or more of IL-2, IFN-y, TNF, and IL-10. In some embodiments of any of the aspects, an immune response can be the release of perforin and / or granzymes by CD8+ T cells. In some embodiments of any of the aspects, an immune response can be an increase in the level of CD8+ T cells.

[0103] In some embodiments of any of the aspects, the immune response stimulates or is an increase of the production of an interferon gamma (IFNy) response from T cells in the subject, e.g., an increase in IFNy levels.

[0104] In some embodiments of any of the aspects, the methods described herein can further comprise administering a second agent and / or treatment to the subject, e.g. as part of a combinatorial therapy.

[0105] In some embodiments of any of the aspects, the immune stimulation is immune stimulation specific for Tyrosine Hydoxylase (TH). In some embodiments of any of the aspects, the immune stimulation is immune stimulation specific for Tyrosine Hydoxylase (TH) and the TCR polypeptide composition comprises a CDRla comprising the sequence of SEQ ID NO: 1; a CDR2a comprising the sequence of SEQ ID NO:2; a CDR3a comprising the sequence of one of SEQ ID NOs: 3 and 7-15; a CDRip comprising the sequence of SEQ ID NO:4; a CDR2J3 comprising the sequence of SEQ ID NO:5; and a CDR3J3 comprising the sequence of SEQ ID NO:6.

[0106] In one aspect of any of the embodiments, described herein is a method of treating cancer in a subject in need thereof, the method comprising administering a cell as described herein, e.g., a cell comprising a TCR polypeptide composition as described herein. In one aspect of any of the embodiments, described herein is a method of treating a cancer in a subject in need thereof, the method comprising administering a nucleic acid as described herein or an immune cell comprising the nucleic acid to the subject, wherein the subject’s immune cells are caused to express the polypeptide encoded by the nucleic acid. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T-cell. In some embodiments of any of the aspects, the method comprises a first step of transducing at least one immune cell (e.g., a T cell) obtained from the subject with the TCR polypeptide composition as described herein.

[0107] In some embodiments of any of the aspects, the cancer expresses Tyrosine Hydoxylase (TH) and the TCR polypeptide composition comprises a CDRla comprising the sequence of SEQ ID NO: 1; a CDR2a comprising the sequence of SEQ ID NO:2; a CDR3a comprising the sequence of one of SEQ ID NOs: 3 and 7-15; a CDRip comprising the sequence of SEQ ID NON; a CDR2comprising the sequence of SEQ ID NO:5; and a CDR3J3 comprising the sequence of SEQ ID NO:6. In some embodiments of any of the aspects, the cancer is a neuroblastoma and the TCR polypeptide composition comprises a CDRla comprising the sequence of SEQ ID NO: 1; a CDR2a comprising the sequence of SEQ ID NO:2; a CDR3a comprising the sequence of one of SEQ ID NOs: 3 and 7-15; a CDRip comprising the sequence of SEQ ID NON; a CDR2P comprising the sequence of SEQ ID NO:5; and a CDR3P comprising the sequence of SEQ ID NO:6.

[0108] Nucleic acids can be targeted to particular cell types by, e.g., use of a cell-type specific promoter and / or a composition that selectively binds to the desired cell type. For example, conjugation of a nucleic acid to an aptamer can permit targeted delivery (McNamara, JO., et al. (2006) Nat. Biotechnol. 24: 1005-1015).

[0109] In an alternative embodiment, the nucleic acid can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of a nucleic acid molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of a nucleic acid by the cell. Cationic lipids, dendrimers, or polymers can either be bound to a nucleic acid, or induced to form a vesicle or micelle (see e.g., Kim SH., et al. (2008) Journal of Controlled Release 129(2): 107-116) that encases a nucleic acid. The formation of vesicles or micelles further prevents degradation of the nucleic acid when administered systemically.

[0110] Methods for making and administering cationic- inhibitory nucleic acid complexes are well within the abilities of one skilled in the art. Some non-limiting examples of drug delivery systems useful for systemic delivery of nucleic acids include DOTAP Oligofectamine, “solid nucleic acid lipid particles”, cardiolipin, polyethyleneimine, Arg-Gly-Asp (RGD) peptides, and polyamidoamines. In some embodiments, a nucleic acid forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of nucleic acids and cyclodextrins can be found in U.S. Patent No. 7, 427, 605, which is herein incorporated by reference in its entirety. Targeted delivery of nucleic acids is described, for example in Ikeda and Taira Pharmaceutical Res 2006 23: 1631-1640; Soutschek et al., Nature 2004 432: 173-8 and Lorenze et al. Bioorg. Med. Chem. Lett. 14, 4975-4977 (2004); each of which is incorporated by reference herein in its entirety. By way of example, the nucleic acid can be targeted to immune cells by encapsulating the inhibitor in a liposome comprising ligands of receptors expressed on immune cells, e.g., TCRs. In some embodiments, the liposome can comprise aptamers specific for immune cells.

[0111] In some embodiments, the methods described herein relate to Adoptive Cell Therapy (ACT). ACT relates to adoptive cell transfer of immune cells (e.g., T-cells) expressing a protein that directs the cell’s activity to a desired target to treat the subject. In some embodiments, the cells administered as part of the therapy can be autologous to the subject. In some embodiments, the cells administered as part of the therapy are not autologous to the subject. In some embodiments, the cellsare engineered and / or genetically modified to express a TCR polypeptide composition as described herein. Further discussion of ACT can be found, e.g., in Tsimberidou et al. Journal of Hematology & Oncology 14: 102 (2021); Yarza et al. Oncologist 28(6):e406-415 (2023); Maus et al. Blood 2014 123:2624-35; Reardon et al. Neuro-Oncology 2014 16: 1441-1458; Hoyos et al. Haematologica 2012 97: 1622; Byrd et al. J Clin Oncol 2014 32:3039-47; Maher et al. Cancer Res 2009 69:4559-4562; and Tamada et al. Clin Cancer Res 2012 18:6436-6445; each of which is incorporated by reference herein in its entirety.

[0112] As used herein, the term “cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.

[0113] In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer. As used herein, the term “malignant” refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (z.e., division beyond normal limits), invasion (z.e., intrusion on and destruction of adjacent tissues), and metastasis (z.e., spread to other locations in the body via lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor. As used herein, the term “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.

[0114] A “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre -malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0115] As used herein the term "neoplasm" refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues. Thus, a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm.

[0116] A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.

[0117] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, nonsmall cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g, lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade / follicular non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome

[0118] A “cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation / cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.

[0119] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having cancer with a TCR polypeptide composition, or nucleic acid encoding the TCR polypeptide composition, or a cell comprising the TCR polypeptide composition or nucleic acid. Subjects having cnacer can be identified by a physician using current methods of diagnosing cancer. Symptoms and / or complications of cancer which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, fatigue, weight changes, lumps, etc. Tests that may aid in a diagnosis of cancer include, but are not limited to, CT canc, MIR, ultrasound, xrays, blood chemistry tests, complete blood counts, and biopsies. A family history of cancer, or exposure to risk factors for cancer can also aid in determining if a subject is likely to have cancer or in making a diagnosis of cancer.

[0120] The compositions and methods described herein can be administered to a subject having or diagnosed as having cancer. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein to a subject in order to alleviate a symptom of a disease, e.g., cancer. As used herein, "alleviating a symptom" is ameliorating any condition or symptom associated with the disease. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.

[0121] The term “effective amount" as used herein refers to the amount of the active ingredient (e.g., cells as described herein) needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of the active ingredient that is sufficient to provide a particular effect, e.g, anti-tumor effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0122] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route ofadministration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the active ingredient, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor growth or growth rate, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0123] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the minimal effective dose and / or maximal tolerated dose. The dosage can vary depending upon the dosage form employed and the route of administration utilized. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a dosage range between the minimal effective dose and the maximal tolerated dose. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor growth and / or size among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0124] In some embodiments, the pharmaceutical composition as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.

[0125] Suitable vehicles that can be used to provide parenteral dosage forms as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt can also beincorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.

[0126] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the composition can be administered in a sustained release formulation.

[0127] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0128] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0129] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and6,365,185 Bl ; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.

[0130] Im some embodiments of any of the aspects, the ACT therapy described herein is administered as a monotherapy, e.g., another treatment for the cancer or immune response is not administered to the subject.

[0131] In some emboidments of any of the aspects, the methods described herein can further comprise administering a second agent and / or treatment to the subject, e.g. as part of a combinatorial therapy. Non-limiting examples of a second agent and / or treatment can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1- TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor- free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0132] In addition, the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.

[0133] In certain embodiments, an effective dose of a composition comprising a TCR polypeptide composition (or nucleic acid encoding same, or cell comprising same) as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition comprising a cell comprising a TCR polypeptide, such as, e.g. O.OOOlxlO9cells to 10xl09cells, O.OOlxlO9cells to 5xl09cells, O.OOlxlO9cells to IxlO9cells, O.OOlxlO9cells to O.lxlO9cells, 0. OlxlO9cells to 10xl09cells, or O.OlxlO9cells to 5xl09cells.

[0134] In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. tumor size, or tumor growth, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.

[0135] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the active ingredient. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and / or treatments daily over a period of weeks or months. Examples of dosing and / or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition described herien can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

[0136] The dosage ranges for the administration of a composition described herein, according to the methods described herein depend upon, for example, the form of the active ingredient, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for tumor size or the extent to which, for example, an immune response are desired to be induced. The dosage should not be so large as to cause adverse side effects, such as autoimmunity. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0137] The efficacy of a composition described herien in, e.g. the treatment of a condition described herein, or to induce a response as described herein (e.g. an immune response) can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or theincidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. T cell counts, T cell activity, tumor size, rate of tumor growth, etc. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, forthat disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. T cell counts, T cell activity, tumor size, rate of tumor growth, etc). It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of cancer and / or immune responses. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. T cell counts, T cell activity, tumor size, rate of tumor growth, etc.

[0138] In vitro and animal model assays are provided herein which allow the assessment of a given dose of the compositions described herein.

[0139] In one respect, the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not ("comprising). In some embodiments of any of the aspects, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments of any of the aspects, the compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.

[0140] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0141] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0142] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0143] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[0144] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments,the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0145] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cancer. A subject can be male or female.

[0146] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.

[0147] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0148] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect, e.g. antigen-binding activity, of the full length polypeptide, e.g., of a full-length wildtype TCR or a TCR as described herein, (e.g., one of SEQ ID NOs: 20-29). Conservative substitution variants that maintain the activity of a wildtype TCR or reference TCR will include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wildtype is guided by, for example, sequence alignment with TCR homologs or paralogs from other species. Amino acids that are identical between TCR homologs are less likely to tolerate change, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with nonconservative differences are less likely to be critical to function and more likely to tolerateconservative substitution in an artificial variant. Variants, fragments, and / or fusion proteins can be tested for activity, for example, by administering the variant to an appropriate animal model of cancer as described herein. Further discussion of the structure of TCRs can be found, e.g. in Szeto et al. Int J Mol Scie 22:68 (2020); and Davis et al. Nature 334:395-402 (1988); which are incorporated by reference herein in its entirety.

[0149] In some embodiments, a polypeptide, e.g., a TCR polypeptide, can be a variant of a sequence described herein, e.g. a variant of a TCR polypeptide comprising the amino acid sequence of one of SEQ ID NOs: 20-29. In some embodiments, the variant is a conservative substitution variant. Variants can be obtained by mutations of native nucleotide sequences, for example. A “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Polypeptide -encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains the relevant biological activity relative to the reference protein, e.g., can bind a target antigen at least 50% as well as wildtype or reference TCR. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3% or fewer, or 1% or fewer) of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. It is contemplated that some changes can potentially improve the relevant activity, such that a variant, whether conservative or not, has more than 100% of the activity of wildtype or reference TCR, e.g. 110%, 125%, 150%, 175%, 200%, 500%, 1000% or more.

[0150] One method of identifying amino acid residues which can be substituted is to align, for example, human TCR to a TCR homolog from one or more non-human species. Alignment can provide guidance regarding not only residues likely to be necessary for function but also, conversely, those residues likely to tolerate change. Where, for example, an alignment shows two identical or similar amino acids at corresponding positions, it is more likely that that site is important functionally. Where, conversely, alignment shows residues in corresponding positions to differ significantly in size, charge, hydrophobicity, etc., it is more likely that that site can tolerate variation in a functional polypeptide. The variant amino acid or DNA sequence can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence, e.g. a TCR as described herein or a nucleic acid encoding one of those amino acid sequences. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web. The variant amino acid or DNA sequence can be at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an “original” sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp or BLASTn (available on the world wide web at blast.ncbi.nlm.nih.gov), with default parameters set.

[0151] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0152] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. antigen-binding activity and specificity of a native or reference polypeptide is retained.

[0153] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity of a native or reference polypeptide is retained. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0154] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1)non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu. Typically conservative substitutions for one another also include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0155] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to the assays described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0156] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide- encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

[0157] In some embodiments, a polypeptide, e.g., a TCR polypeptide can comprise one or more amino acid substitutions or modifications. In some embodiments, the substitutions and / or modifications can prevent or reduce proteolytic degradation and / or prolong half-life of the polypeptide in a subject. In some embodiments, a polypeptide can be modified by conjugating or fusing it to other polypeptide or polypeptide domains such as, by way of non-limiting example,transferrin (WO06096515A2), albumin (Yeh et al., 1992), growth hormone (US2003104578AA); cellulose (Levy and Shoseyov, 2002); and / or Fc fragments (Ashkenazi and Chamow, 1997). The references in the foregoing paragraph are incorporated by reference herein in their entireties.

[0158] In some embodiments, a polypeptide, e.g., a TCR polypeptide, as described herein can comprise at least one peptide bond replacement. A TCR polypeptide as described herein can comprise one type of peptide bond replacement or multiple types of peptide bond replacements, e.g. 2 types, 3 types, 4 types, 5 types, or more types of peptide bond replacements. Non-limiting examples of peptide bond replacements include urea, thiourea, carbamate, sulfonyl urea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid, para-(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)- phenylacetic acid, thioamide, tetrazole, boronic ester, olefinic group, and derivatives thereof.

[0159] In some embodiments, a polypeptide, e.g., a TCR polypeptide, as described herein can comprise naturally occurring amino acids commonly found in polypeptides and / or proteins produced by living organisms, e.g. Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M), Gly(G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q), Asp (D), Glu (E), Lys (K), Arg (R), and His(H). In some embodiments, a TCR polypeptide as described herein can comprise alternative amino acids. Non-limiting examples of alternative amino acids include, D-amino acids; beta-amino acids; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gammacarboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1, 2,3,4, - tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3-mercapto-D-valine), ornithine, citruline, alpha-methyl-alanine, para-benzoylphenylalanine, para-amino phenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylgly cine), diaminobutyric acid, 7-hydroxy- tetrahydroisoquinoline carboxylic acid, naphthylalanine, biphenylalanine, cyclohexylalanine, aminoisobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylglycine, 1- amino-1 -cyclopentanecarboxylic acid, 1-amino-l -cyclohexanecarboxylic acid, amino-benzoic acid, amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine, nipecotic acid, alpha-amino butyric acid, thienyl-alanine, t-butylglycine, trifluoro valine; hexafluoroleucine; fluorinated analogs; azide-modified amino acids; alkyne -modified amino acids; cyano-modified amino acids; and derivatives thereof.

[0160] In some embodiments, a polypeptide, e.g. a TCR polypeptide, can be modified, e.g. by addition of a moiety to one or more of the amino acids that together comprise the peptide. In some embodiments, a polypeptide as described herein can comprise one or more moiety molecules, e.g. 1 or more moiety molecules per polypeptide, 2 or more moiety molecules per polypeptide, 5 or more moiety molecules per polypeptide, 10 or more moiety molecules per polypeptide or more moiety molecules per polypeptide. In some embodiments, a polypeptide as described herein can comprise one more types of modifications and / or moieties, e.g. 1 type of modification, 2 types of modifications,3 types of modifications or more types of modifications. Non-limiting examples of modifications and / or moieties include PEGylation; glycosylation; HESylation; ELPylation; lipidation; acetylation; amidation; end-capping modifications; cyano groups; phosphorylation; albumin, and cyclization. In some embodiments, an end-capping modification can comprise acetylation at the N-terminus, N- terminal acylation, and N-terminal formylation. In some embodiments, an end-capping modification can comprise amidation at the C-terminus, introduction of C-terminal alcohol, aldehyde, ester, and thioester moieties. The half-life of a polypeptide can be increased by the addition of moieties, e.g. PEG, albumin, or other fusion partners (e.g. Fc fragment of an immunoglobin).

[0161] Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0162] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established. Alterations of the original amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by Khudyakov et al. “Artificial DNA: Methods and Applications” CRC Press, 2002; Braman “In Vitro Mutagenesis Protocols” Springer, 2004; and Rapley “The Nucleic Acid Protocols Handbook” Springer 2000; which are herein incorporated by reference in their entireties. In some embodiments, a polypeptide as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.

[0163] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single -stranded or double-stranded. A single -strandednucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.

[0164] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and / or to the translation of mRNA into a polypeptide.

[0165] In some embodiments, the expression of a biomarker(s), target(s), or gene / polypeptide described herein is / are tissue-specific. In some embodiments, the expression of a biomarker(s), target(s), or gene / polypeptide described herein is / are global. In some embodiments, the expression of a biomarker(s), target(s), or gene / polypeptide described herein is systemic.

[0166] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g.5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0167] “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0168] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0169] In some embodiments of any of the aspects, the TCR polypeptide described herein is exogenous. In some embodiments of any of the aspects, the TCR polypeptide described herein isectopic. In some embodiments of any of the aspects, the TCR polypeptide described herein is not endogenous.

[0170] The term "exogenous" refers to a substance present in a cell other than its native source. The term "exogenous" when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell. As used herein, “ectopic” refers to a substance that is found in an unusual location and / or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and / or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.

[0171] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g. a TCR polypeptide) is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably linked to a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non- viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0172] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).

[0173] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optomized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but will be transcribed and / ortranslated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native / wild-type sequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.

[0174] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[0175] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and / or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0176] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0177] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. cancer. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected inthe absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0178] In some embodiments of any of the aspects, treatment is effective if tumor size is reduced. In some embodiments of any of the aspects, treatment is effective if tumor growth rate is reduced. In some embodiments of any of the aspects, treatment is effective if T cell activity is increased. In some embodiments of any of the aspects, treatment is effective if target antigen-specific T cell activity is increased.

[0179] In some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein “prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom. Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to tumor metastasis. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.

[0180] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which 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 problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.

[0181] As used herein, the term “nanoparticle” refers to particles that are on the order of about 1 to 1,000 nanometers in diameter or width. The term “nanoparticle” includes nanospheres; nanorods; nanoshells; and nanoprisms; these nanoparticles may be part of a nanonetwork. The term “nanoparticles” also encompasses liposomes and lipid particles having the size of a nanoparticle. Exemplary nanoparticles include lipid nanoparticles or ferritin nanoparticles. Lipid nanoparticles cancomprise multiple componenents, including, e.g., ionizable lipids (such as MC3, DLin-MC3-DMA, ALC-0315, or SM-102), pegylated lipids (such as PEG2000-C-DMG, PEG2000-DMG, ALC-0159), phospholipids (such as DSPC), and cholesterol.

[0182] Exemplary liposomes can comprise, e.g., DSPC, DPPC, DSPG, Cholesterol, hydrogenated soy phosphatidylcholine, soy phosphatidyl choline, methoxypolyethylene glycol (mPEG-DSPE) phosphatidyl choline (PC), phosphatidyl glycerol (PG), distearoylphosphatidylcholine, and combinations thereof.

[0183] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and / or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and / or the subject being treated.

[0184] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell or subject. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine. In some embodiments, the contacting provides direct physical contact of the agent and the at least one cell or subject, e.g., the agent and the at least one cell are touching, e.g., at least one surface of the agent is touching or forming a junction with at least one aspect of the at least one cell.

[0185] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0186] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0187] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0188] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0189] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0190] As used herein, the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.

[0191] T cell receptors (TCR) are found in nature as a heterodimeric receptor complex found on the surface of a T cell that recognizes and binds an antigen bound to / displayed upon the MHC or an APC. Binding of MHC -displayed antigen by the TCR initiates signal transduction by the TCR necessary for activation of the T cell. The majority of the T cells bear a and [3 chains in their T cell receptor (TCR). The structure of TCRs is known in the art, and a TCR comprises 6 CDRs - 3 CDRs on the a chain (CDRla, CDR2a, and CDR3a) and 3 CDRs on the [3 chain (CDR1[3, CDR2[3, and CDR3J3). The a and [3 chains, in the context of chains or CDRs, may also be referred to interchangeably herein as “a” or “b” chains, respectively.

[0192] Each chain is composed of a variable region of said chain (and a constant region of said chain. The variable regions may be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs) and interspersed with conserved sequences. This structure is well known to those skilled in the art.

[0193] A TCR, as that term is used herein, refers to one or more portions of a full-length heterodimeric receptor complex, said one or more portions binding specifically to the indicated antigen. A TCR can be present as a complex of the individual a and [3 chains polypeptides, or as a single polypeptide comprising the sequences of both the a and [3 chains, e.g., joined by a linker sequence. In som embodiments of any of the aspects, a TCR comprises at least a CDRla, CDR2a, CDR3a, CDR1J3, CDR2J3, and CDR3J3.

[0194] As used herein, the term “CDR” refers to the complementarity determining regions within TCR variable sequences. The exact boundaries of these CDRs have been defined differently according to different systems. CDRs may be defined according to the Kabat system (see Kabat, E. A.et al., 1991, “Sequences of Proteins of Immunological Interest”, 5th edit., NIH Publication no. 91- 3242, U.S. Department of Health and Human Services). Other systems may be used to define CDRs, which as the system devised by Chothia et al (see Chothia, C. & Lesk, A. M., 1987, “Canonical structures for the hypervariable regions of immunoglobulins”, J. Mol. Biol., 196, 901-917) and the IMGT system (see Lefranc, M. P., 1997, “Unique database numbering system for immunogenetic analysis”, Immunol. Today, 18, 50). See also Johnson and Wu “Kabat Database and its applications: future directions” Nucleic Acids Reearch 29:205-206 (2001). A TCR typically contains 3 heavychain CDRs and 3 light chain CDRs. The term CDR or CDRs is used here to indicate one or several of these regions. A person skilled in the art is able to readily compare the different systems of nomenclature and determine whether a particular sequence may be defined as a CDR. The methods and compositions used herein may utilize CDRs defined according to any of these systems. Each of the foregoing reference is incorporated by reference herein in its entirety.

[0195] The term “antigen-binding portion” of an TCR refers to one or more portions of TCR as described herein, said one or more portions still having the binding affinities as defined above herein.

[0196] In some embodiments, the TCR is a fully human TCR. In some embodiments, the TCR is a humanized TCR. In some embodiments, the TCR is a fully humanized TCR. In some embodiments, the TCR is a chimeric TCR. In some embodiments, the TCR is a recombinant TCR.

[0197] The term “human TCR” refers to TCRs whose variable and constant regions correspond to or are derived from sequences of the human germ line. However, the human TCRs can contain amino acid residues not encoded by human germ line sequences (for example mutations which have been introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular in CDR3. Recombinant TCRs as described herein have variable regions and may also contain constant regions derived from sequences of the human germ line. According to particular embodiments, however, such recombinant human TCRs are subjected to in-vitro mutagenesis (or to a somatic in-vivo mutagenesis, if an animal is used which is transgenic due to human Ig sequences) so that the amino acid sequences of the variable regions, particularly the CDRs are sequences which although related to or derived from sequences of the human germ line, do not naturally exist in vivo within the human TCR germ line repertoire. According to some embodiments, recombinant antibodies of this kind are the result of selective mutagenesis or back mutation or of both. Preferably, mutagenesis leads to an affinity to the target which is greater, and / or an affinity to non-target structures which is smaller than that of the parent TCR.

[0198] Additionally, a polypeptide having functional activity means the polypeptide exhibits activity similar, but not necessarily identical to, an activity of a reference TCR, as described herein, including mature forms, as measured in a particular assay, such as, for example, a biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the reference TCR, but rather substantially similar to the dose -dependence in a given activity as compared to the reference TCR, as described herein (i.e., the candidate polypeptide will exhibit greater activity, or not more than about 25 -fold less, about 10-fold less, or about 3 -fold less activity relative to the TCR described herein).

[0199] In some embodiments, the TCRs described herein are not naturally-occurring biomolecules. For example, a TCR that specifically binds a self-antigen would not occur in nature absent human intervention and manipulation, e.g., manufacturing steps carried out by a human. Chimeric TCRs are also not naturally-occurring biomolecules, e.g. , in that they comprise sequencesobtained from multiple species and assembled into a recombinant molecule. In certain particular embodiments, the human TCRs described herein are not naturally-occurring biomolecules, e.g., fully human TCRs directed against a human self-antigen would be subject to negative selection in nature and are not naturally found in the human body.

[0200] In some embodiments, the TCR is an isolated polypeptide. In some embodiments, the TCR is a purified polypeptide. In some embodiments, the TCR is an engineered polypeptide.

[0201] As used herein, an “epitope” can be formed on a polypeptide both from contiguous amino acids, or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. An “epitope” includes the unit of structure conventionally bound by a TCR. Epitopes define the minimum binding site for an TCR, and thus represent the target of specificity of an TCR. The terms “antigenic determinant” and “epitope” can also be used interchangeably herein. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and / or specific charge characteristics.

[0202] “Avidity” is the measure of the strength of binding between a TCR and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the TCR, and the number of pertinent binding sites present on the TCR. Typically, a TCR will bind to its cognate or specific antigen with a dissociation constant (KD of 10 to 1012moles / liter or less, such as 107to 1012moles / liter or less, or 102to 1012moles / liter (z.e., with an association constant (KA) of 105to 1012liter / moles or more, such as 107to 1012liter / moles or 108to 1012liter / moles). Any KD value greater than 104mol / liter (or any KA value lower than 104M ') is generally considered to indicate non-specific binding. The KDfor biological interactions which are considered meaningful (e.g, specific) are typically in the range of 10 '" M (0.1 nM) to I O2M (10000 nM). The stronger an interaction, the lower is its KD. For example, a binding site on TCR described herein will bind to the desired antigen with an affinity less than 500 nM, such as less than 200 nM, or less than 10 nM, such as less than 500 pM. Specific binding of TCR to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as other techniques as mentioned herein.

[0203] Accordingly, as used herein, “selectively binds” or “specifically binds” refers to the ability of a peptide (e.g., TCR) described herein to bind to a target, such as an antigen present on thecell-surface, with a KD10~5M (10,000 nM) or less, e.g., 10 " M, 107M, 10sM, 10 " M, 10 '" M, 10 " M, 1012M, or less. Specific binding can be influenced by, for example, the affinity and avidity of the polypeptide agent and the concentration of polypeptide agent. The person of ordinary skill in the art can determine appropriate conditions under which the polypeptide agents described herein selectively bind the targets using any suitable methods, such as titration of a polypeptide agent in a suitable cell binding assay. A polypeptide specifically bound to a target is not displaced by a nonsimilar competitor. In certain embodiments, a TCR is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0204] The MHC (Major Histocompatibility Complex), which is also referred to as the human leukocyte antigen (HLA), is comprised of a set of genes that code for cell surface proteins essential for the acquired, e.g., adaptive immune system to recognize foreign molecules in vertebrates, which in turn determines histocompatibility. The MHC gene family is divided into three subgroups: MHC class I, MHC class II, and MHC class III. Class I MHC molecules have the [32 microglobulin subunit which can only be recognised by CD8 co-receptors. Class II MHC molecules have [31 and [32 subunits and can be recognized by CD4 co-receptors. In this way MHC molecules chaperone which type of lymphocytes bind to the given antigen with high affinity, since different lymphocytes express different T-Cell Receptor (TCR) co-receptors. Components of the MHC are known in the art and can be readily identified by a skilled person. The MHC is further described in, e.g., Janeway CA Jr, Travers P, Walport M, et al, Immunobiology: The Immune System in Health and Disease, 5th edn (New York: Garland Science, 2001); Vigneron N, Stroobant V, Chapiro J, Ooms A, Degiovanni G, Morel S, et al. (April 2004). "An antigenic peptide produced by peptide splicing in the proteasome". Science. 304 (5670): 587-90; and K. Murphy, “Antigen recognition by T cells,” in Janeway's Immunobiology, 8th, Ed., Garland Science, 2012, pp. 138-153; which are incorporated herein by reference in their entireties. A complete MHC class I complex comprises one MHC class I heavy chain, one peptide ligand sequence, and a beta 2 microglobulin. A complete MHC class II complex comprises an MHC class II alpha chain, MHC class II beta chain, and one peptide ligand sequence.

[0205] The term “complex” refers to an association between at least two moieties (e.g. chemical or biochemical) that have an affinity for one another. “Protein complex” or “polypeptide complex” refers to a complex comprising at least one polypeptide.

[0206] As used herein, the term “specific binding” refers to an interaction between two molecules, compounds, cells and / or particles wherein the first entity binds to the second entity with greater specificity and affinity than it binds to a third entity. A first entity specifically bound to a second entity is not displaced by a non-similar competitor. In certain embodiments, a first entity is said to specifically bind a second entity when it preferentially recognizes the second entity in a complex mixture of proteins and / or macromolecules. In some embodiments, specific binding can refer to an affinity of the first entity for the second entity which is at least 10 times, at least 50 times, atleast 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third entity. In some embodiments, specific binding refers to the ability of a first entity to bind to a second entity with a KD10~5M (10000 nM) or less, e.g., 10~6M, 10~7M, 10~8M, 10~9M, 10~10M, 10~nM, 10~12M, or less. The person of ordinary skill in the art can determine appropriate conditions under which a first entity (e.g., an TCR described herein) selectively binds a second entity (e.g., an antigen) using any suitable methods, such as titration of an entity in a suitable binding assay. Specific binding can comprise ionic bonding, hydrogen bonds, ionic bonds, van der Waals interactions, and / or London dispersion forces. In some embodiments, specific binding does not refer to covalent bonding. In some embodiments, specific binding does not refer to a peptide bond. In some embodiments, specific binding does not refer to a phosphodiester bond.

[0207] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0208] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0209] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Bruce Alberts et al., Molecular Biology of the Cell, published by W.W. Norton & Company, 2022 (ISBN 0393884821, 978-0393884821); John M. Lackie eat al.(eds.), The Dictionary of Cell and Molecular Biology, 5thEdition, published by Academic Press, 2013 (ISBN 0123849314, 978-0123849311); Nalini Chandar et al., Lippincott Illustrated Reviews: Cell and Molecular Biology, 3rdEdition, published by LWW, 2023 (ISBN 1975180895, 978-1975180898);Teresa Atwood et al., Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006; Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); Johnathan Law et al., (eds.), A Dictionary of Chemistry, 8thEdition, published by Oxford University Press, 2020 (ISBN 9780198841227, 9780191876783); Robert C. King et al. (eds.), A Dictionary of Genetics, 8thEdition, published by Oxford University Press, 2013 (ISBN 9780199766444, 9780199376865); Richard Cammack et al. (eds.), Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006 (ISBN 9780198529170, 9780191727641); John Lackie et al. (eds.), A Dictionary of Biomedicine, 2ndEdition, published by Oxford University Press, 2019 (ISBN 9780191829116); Lodish et al., Molecular Cell Biology, 8thEdition, published by W.H. Freeman, 2016 (ISBN 1464183392, 978- 1464183393); Abul K. Abbas et al., Cellular and Molecular Immunology, 10thEdition, published by Elsevier, 2021 (ISBN 0323757480, 978-0323757485); Kenneth M. Murphy et al., Janeway's Immunobiology, 10thEdition, published by W. W. Norton & Company, 2022 (ISBN 0393884899, 978-0393884890); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 0444569464); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Frederick M. Ausubel (ed.), Current Protocols in Molecular Biology (CPMB), John Wiley and Sons, 1987-2010 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0210] In all embodiments where a sample is obtained or has been obtained or provided, the sample can be sample taken, obtained, or provided via minimally invasive methods and / or involves only a minor intervention. In some embodiments of any of the aspects, a sample is taken, obtained, or provided by one or more of a blood draw or prick, an epidermal or mucus membrane swab, buccal sampling, saliva sample, a epidermal skin sampling technique, and / or collection of a secreted or expelled bodily fluid (e.g., mucus, urine, sweat, etc), fecal sampling, semen / seminal fluid sampling, or clippings (e.g., of hair or nails). In some emodiments of any of the aspects, the sample comprises, consists of, or consists essentially of blood (or any fraction or component thereof), serum, urine, mucus, epithelial cells, saliva, buccal cells, a secreted or expelled bodily fluid, and / or hair or nail clippings.

[0211] Other terms are defined herein within the description of the various aspects of the invention.

[0212] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0213] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0214] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0215] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A method comprising: a) mutagenizing at least one of a CDR3a and a CDR3J3 of a self-antigen mimic-specific TCRto provide a pool of target self-antigen-specific TCR candidates; andb) selecting at least one target self-antigen-specific TCR from the pool of target self- antigen-specific TCR candidates. A method comprising: a) isolating a self-antigen mimic-specific TCR; b) mutagenizing at least one of the CDR3a and CDR3P of the self-antigen mimicspecific TCR to provide a pool of target self-antigen-specific TCR candidates; and c) selecting at least one target self-antigen-specific TCR from the pool of target self- antigen-specific TCR candidates. The method of paragraph 2, wherein the isolating comprises injecting an animal or person with the self-antigen mimic. The method of paragraph 2, wherein the isolating comprises raising naive CD8 T cells from a human donor with the self-antigen mimic. The method of paragraph 2, wherein the isolating comprises raising naive CD8 T cells, from a human donor HLA-matched with respect to the target self-antigen, with the self-antigen mimic. The method of any one of paragraphs 2-5, wherein the isolating comprises screening a library of T cells with the self-antigen mimic. The method of any one of the preceding paragraphs, wherein the self-antigen mimic is displayed in a complex with an MHC polypeptide during the isolating step. The method of any one of the preceding paragraphs, wherein the self-antigen mimic is displayed as part of a fusion protein with an MHC polypeptide during the isolating step. The method of any one of the preceding paragraphs, wherein the self-antigen mimic is displayed on an antigen presenting cell during the isolating step. The method of paragraph 9, wherein the antigen presenting cell is a monocyte -derived dendritic cell or an EpiScan cell. The method of any one of the preceding paragraphs, wherein the self-antigen mimic is a polypeptide of no more than 20 amino acids. The method of any one of the preceding paragraphs, wherein the self-antigen mimic is a polypeptide of more than 20 amino acids. The method of any one of the preceding paragraphs, wherein the target self-antigen is a polypeptide of no more than 20 amino acids. The method of any one of the preceding paragraphs, wherein the target self-antigen is a polypeptide of more than 20 amino acids. The method of any one of the preceding paragraphs, wherein the target self-antigen is a naturally occurring polypeptide.The method of any one of the preceding paragraphs, wherein the at least one of a CDR3a and a CDR3P is CDR3a. The method of any one of the preceding paragraphs, wherein the at least one of a CDR3a and a CDR3P is CDR3p. The method of any one of the preceding paragraphs, wherein the at least one CDR is CDR3a and CDR3p. The method of any one of the preceding paragraphs, wherein the mutagenizing comprises mutagenizing a single residue of at least one of the CDR3a and the CDR3p. The method of any one of the preceding paragraphs, wherein the mutagenizing comprises mutagenizing a single residue of the each of the at least one of the CDR3a and the CDR3p. The method of any one of the preceding paragraphs, wherein the mutagenizing comprises mutagenizing two residues of at least one of the CDR3a and the CDR3p. The method of any one of the preceding paragraphs, wherein the mutagenizing comprises mutagenizing three residues of the each of the at least one of the CDR3a and the CDR3p. The method of any one of the preceding paragraphs, wherein the mutagenizing comprises mutagenizing three residues of at least one of the CDR3a and the CDR3p. The method of any one of the preceding paragraphs, wherein the mutagenizing comprises mutagenizing at least three residues of at least one of the CDR3a and the CDR3p. The method of any one of the preceding paragraphs, wherein the mutagenizing comprises mutagenizing at least three residues of the each of the at least one of the CDR3a and the CDR3p. The method of any one of the preceding paragraphs, wherein the mutagenizing is comprehensive saturation mutagenesis. The method of any one of the preceding paragraphs, wherein the pool of self-antigen-specific TCR candidates comprises a library of single mutants, double mutants, triple mutants, and CDR3 length changes of at least one of the CDR3a and the CDR3p. The method of any one of the preceding paragraphs, wherein the pool of self-antigen-specific TCR candidates comprises a library comprising all single mutants and all double mutants of at least one of the CDR3a and the CDR3p. The method of any one of the preceding paragraphs, wherein the selecting comprises selecting target self-antigen-specific TCRs from the pool of target self-antigen-specific candidates. The method of any one of the preceding paragraphs, wherein the target self-antigen is displayed in a complex with an MHC polypeptide during the selecting step.The method of any one of the preceding paragraphs, wherein the selecting comprises selecting TCRs that do not bind the self-antigen mimic from the pool of target self-antigen- specific candidates. The method of any one of the preceding paragraphs, wherein the self-antigen mimic is displayed in a complex with an MHC polypeptide during the selecting step. The method of any one of the preceding paragraphs, wherein the selecting comprises selecting TCRs that do not bind a plurality of off-target self-antigens from the pool of target self-antigen-specific candidates. The method of any one of the preceding paragraphs, wherein the selecting comprises selecting TCRs that do not bind a plurality of off-target self-antigens in a human peptidome library from the pool of target self-antigen-specific candidates. The method of any one of the preceding paragraphs, wherein the selecting comprises selecting TCRs that do not bind a plurality of off-target self-antigens in a human peptidome library presented on T-scan cells from the pool of target self-antigen-specific candidates. A TCR polypeptide composition comprising: a) a CDRla comprising the sequence of SEQ ID NO: 1; b) a CDR2a comprising the sequence of SEQ ID NO:2; c) a CDR3a comprising the sequence of one of SEQ ID NOs: 3 and 7-15; d) a CDRip comprising the sequence of SEQ ID NO:4; e) a CDR2P comprising the sequence of SEQ ID NO:5; and f) a CDR3P comprising the sequence of SEQ ID NO:6. The TCR polypeptide composition of any one of the preceding paragraphs, further comprising one or more of SEQ ID NOs: 16-19. A nucleic acid composition comprising one or more nucleic acids encoding the TCR polypeptide composition of any one of the preceding paragraphs. A cell comprising the TCR polypeptide composition of any one of the preceding paragraphs and / or the nucleic acid composition of paragraph 38. The cell of paragraph 39, wherein the cell is a T cell. A method of treating a subject in need of immune stimulation specific for a target selfantigen, the method comprising administering a T cell comprising a target self-antigen- specific TCR selected by the process of any one of paragraphs 1-35 to the subject. A method of treating a subject in need of immune stimulation specific for Tyrosine Hydoxylase (TH), the method comprising administering the T cell of paragraph 40 to the subject.43. The method of any one of the preceding paragraphs, further comprising a first step of transducing at least one T cell obtained from the subject with the target self-antigen-specific TCR or the TCR polypeptide composition of any one of paragraphs 36-37.44. The method of any one of the preceding paragraphs, wherein the subject is a subject in need of treatment for cancer.45. The method of paragraph 44, wherein the cancer is neuroblastoma.46. The method of any one of the preceding paragraphs, wherein the T cell is autologous to the subject.

[0216] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLESExample 1

[0217] The invention described is called T-Switch, an in vitro T cell receptor (TCR) engineering platform that creates, modifies, and comprehensively profiles TCRs for use in adoptive T cell therapy (ACT). The technology is intended to create TCR therapeutics that target tumor-associated selfproteins that are typically overexpressed self-antigens. T-Switch bypasses T cell tolerance by raising T cells from the natural repertoire that recognize a related ‘foreign’ peptide that differs by one amino acid from a self-antigen. The fine specificity of the TCR is then modulated through directed evolution of the peptide binding region (CDR3 chain of the TCR) to switch its specificity towards the selfantigen of interest. T-Switch enables the engineering of TCR specificity while avoiding the pitfalls of affinity enhancement by selecting against a closely related epitope and thereby making stepwise changes to fine-tune TCR potency and specificity.

[0218] The invention provides a valuable technology for the creation of collections of highly sensitive synthetic TCRs for T cell-based immunotherapies. The technology has been validated through multiple proof-of-concept experiments to switch the specificity of two viral TCRs and was applied to a clinically relevant tumor-associated antigen to show TCR therapeutic safety and efficacy. T-Switch emphasizes engineering specificity and sensitivity without the need to isolate a known T cell clone and eliminates toxic cross-reactivities by selecting against closely related epitopes. This technology represents a significant advancement in ACT and provides safe and potent T cell therapeutics for the treatment of solid tumors. Specifically, we have used T-Switch to generate tyrosine hydroxylase (TH) -specific TCRs. These TCRs recognize the TH antigen ALLSGVRQV (SEQ ID NO: 36) bound to HLA-A2. TH is a tumor-associated target antigen highly expressed in neuroblastomas but not in normal tissues and has relevance to neuroblastoma pathology based on published literature.

[0219] This invention consists of a comprehensive saturation mutagenesis TCR library design directed to the CDR3a or CDR3J3 chains of the TCR to switch its specificity to a closely related epitope. It also consists of therapeutic TCRs targeting a self-antigen (TH) for the treatment of neuroblastomas.

[0220] The invention has been made and tested through multiple proof-of-concept experiments and it has also been applied to a therapeutic target. First, we demonstrate the value of our approach by the creation of libraries of viral-specific TCRs and the subsequent in vitro selection of TCRs that switched specificity to a closely related epitope. The engineered TCRs showed robust T-cell activation after ligand recognition. Importantly, the engineered TCRs displayed no additional promiscuity or off-target specificities as compared to the parental TCRs.

[0221] Finally, we apply Tswitch to a clinically relevant cancer self-target highly expressed in neuroblastoma. This resulted in engineered TCR variants that displayed target specificity and no additional off-target cross-reactivities as measured against the whole peptidome; thus, representing therapeutic candidates.

[0222] We have developed a methodology to change the specificity of TCRs to a self antigen. In principle this technology could be used to generate a TCR to any epitope of interest whether it be a self antigen or a cancer-relevant antigen. As examples we created TCRs specific for the TH antigen (ALLSGVRQV (SEQ ID NO: 36)) through amino acid mutations in the CDR3a or CDR3[3 chains of a TCR that recognizes a closely related peptide that differs by one amino acid (ALLSGVRMV (SEQ ID NO: 35)). These TCRs can be commercialized for use in ACT for neuroblastomas. We have also generated TCRs that recognize IgE and NUTM1 self antigens that could be used for allergy or cancer respectively.Example 2: T-Switch: A specificity-based engineering platform for developing safe and effective T cell therapeutics

[0223] Many promising targets for adoptive T cell therapy (ACT) are overexpressed self-antigens. However, T cells bearing T cell receptors (TCRs) with high potency and affinity for a self-antigen are eliminated during thymic selection. The affinity of the remaining low-avidity TCRs can be improved through conventional mutagenesis approaches, but isolating these TCRs is labor intensive, and their subsequent affinity enhancement can generate toxic cross-reactivities. To bypass T cell tolerance and obtain potent and safe T cell therapeutics, we developed T-Switch, an in vitro TCR engineering platform for the creation, modification, and comprehensive profiling of TCRs that can target tumor- associated self-proteins for ACT. In this approach, we bypass T cell tolerance by first raising T cells from the natural repertoire that recognize a related ' foreign1peptide that differs by one amino acid from a self-antigen. Then, we modulate the fine specificity of the TCR by directed evolution of the peptide binding region to switch its specificity toward the self-antigen of interest. T-Switch emphasizes thecapacity to engineer TCR specificity while avoiding the pitfalls of affinity enhancement by selecting against a closely related epitope and thereby making stepwise changes to fine tune TCR potency and specificity. We applied T-Switch to successfully engineer synthetic TCRs to a clinically relevant tumor-associated antigen (TH) and validated their translational potential through multiple assays of safety and efficacy. Crucially, the candidate T-Switch variants displayed no off-target specificities as measured against the whole peptidome. Therefore, T-Switch represents a valuable technology for the creation of collections of highly sensitive synthetic TCRs for T cell-based immunotherapies.

[0224] Introduction

[0225] T cells play several major roles in cellular immunity, including the elimination of cancer cells with tumor associated antigens. T cell receptors (TCRs) expressed on T cells mediate this action by recognition of peptides bound to MHC molecules. Advances in immunotherapy utilizing TCRs has led to the development of adoptive cell therapy (ACT). ACT involves the isolation and genetic modification of patient-derived T cells to enhance their anti-tumor activity, followed by their infusion back into the cancer patient. In this approach, T cells are transduced with a tumor-antigen specific TCR or a chimeric antigen receptor (CAR), leading to the selective destruction of cancer cells. One major advantage of using TCRs over CARs is their ability to recognize internal antigens at very low antigen densities on tumors, providing higher sensitivity and expanding the breadth of antigens that can be targeted.

[0226] The two main classes of internal antigens are neoantigens and self-antigens. The downside to targeting neoantigens is that their occurrence is rare, with each patient expressing a unique set of neoantigens, which requires antigen identification and designing a therapy tailored towards each individual patient. Alternatively, certain self-antigens are common and shared among cancer patients, allowing for the development of more standardized and broadly applicable therapies. Clinical trials of ACT using TCRs targeting self-antigens have demonstrated tremendous success in inducing durable and complete responses in patients with refractory or relapsed solid tumors. However, finding TCRs targeting tumor self-antigens is complicated by the process of central tolerance, which is the negative selection of developing T cells that recognize and react with high affinity to self-antigens in the thymus. Consequently, naturally occurring TCRs with high-affinity to their target self-antigen are extremely rare, and the remainder of T cells are of low-affinity for recognition and destruction of tumor cells.

[0227] To improve the properties of low-affinity TCRs to self-antigens, directed evolution and protein engineering methods (ex. phage and yeast display) have been employed to enhance TCR affinity to these antigens. These methods target the six complementarity determining regions (CDRs) of the TCR: three (CDR1, CDR2 and CDR3) in each a and b chain, which are involved in binding peptide-MHC ligands. The CDR1 and CDR2 loops are germline encoded and are typically positioned over the MHC helices. The CDR3 loops are somatically encoded by nucleotide rearrangements at the junction of gene segments, and these hypervariable regions are typically positioned over the peptide.Without structural data for a particular TCR, it is difficult to delineate the precise residues that are in direct contact with the peptide due to the complexity of interloop interactions. Therefore, protein engineering directed to the CDR1 and CDR2 loops has a substantial chance of enhancing interactions between the TCR and the MHC helices leading to TCR cross-reactivity and toxicity. Indeed, one notable example is an HLA-A1 restricted TCR that targeted MAGE-A3, which was affinity-enhanced by altering four residues in its CDR2 region via phage display. This TCR developed an off-target reactivity to a self-antigen from the heart muscle protein titin, which resulted in treatment-induced cardiotoxicity and patient deaths. Moreover, it is now well-appreciated that affinity enhancement alone does not correlate with improved TCR potency. Even though TCRs with robust affinities in the range of 1-5 mM tend to display high functionality, TCRs with high supraphysiological affinities (<1 mM) can lead to impaired function (T cell dysfunction and poor serial TCR triggering) and reactivity to the presenting MHC molecule. Thus, the development of alternative engineering platforms that focus on engineering TCR specificity and sensitivity while simultaneously avoiding off-target promiscuity are needed. Moreover, given the complexities of identifying natural TCRs to engineer, a more generalizable strategy is desired for the creation of T cell therapeutics.

[0228] Here, we present T-Switch, an in vitro selection strategy aimed at facilitating small-step specificity changes, for the creation, modification and high-throughput profiling of TCRs. First, to circumvent T cell tolerance, we isolate antigen-specific T cells to mutant peptides that differ from a selfantigen by one amino acid; thus, stimulating a neoantigen-like response. Second, we switch the specificity of the TCR to the self-peptide and away from the mutant peptide by mutagenesis directed to the CDR3 regions, which play a large part in determining peptide specificity; thereby generating engineered cancer reactive TCRs. Finally, we use T-Scan, a genetic pooled screening platform for epitope discovery, to comprehensively assess the off-target specificities of the engineered TCRs. We first successfully demonstrate that we can use a directed evolution approach to switch the specificity of TCRs to closely related peptides without altering the off-target profile of the TCRs. Then, we applied T- Switch to a clinically relevant cancer self-target highly expressed in neuroblastoma. This resulted in engineered TCR variants that displayed on-tumor specificity and no additional off-target crossreactivities as measured against the whole peptidome; thus, representing therapeutic candidates. Therefore, T-Switch represents an off-the-shelf platform for the potential discovery of highly potent TCRs against the large array of candidate internal antigens.

[0229] Results

[0230] A directed evolution strategy to switch the specificity of two model NLV TCRs. The general goal of this study was to devise a method to generate TCRs to self-antigens, which are limited by central tolerance, while avoiding a loss of specificity. To achieve this, we reasoned that we could select a TCR to a neoantigen version of the self-peptide that was different in only a single residue, then mutagenize the TCR to reverse the specificity to the wild-type peptide. The swap in specificityshould guarantee that the gained specificity was not simply through enhanced MHC interaction. Although changing specificity of TCRs to completely different peptides has been achieved, there has been no reports of using directed evolution to systematically change the specificity of TCRs between closely related peptides. To do this, we employed two well characterized viral TCRs: the NLV2 and NLV3 TCRs. These TCRs specifically recognize the immunodominant HLA-A2 restricted cytomegalovirus (CMV) epitope (NLVPMVATV (SEQ ID NO: 30)) (Fig. 1A, top panel). They were chosen because of prior knowledge that they differentially recognize distinct one amino acid variants of the immunodominant NLV epitope. We selected some of these variants to validate via tetramer staining. Jurkat TCRb7cells lentivirally infected with NLV2 showed tetramer binding to an NLVV497C variant at position three of the epitope and an NLVP498G variant at position four of the epitope. Conversely, cells expressing NLV3 showed specificity to an NLVN495M variant at position one (Figs. 1A and IB). Having two TCRs that had overlapping but distinct specificities allowed us to ask the question as to whether we can switch the specificity from one epitope to another using directed evolution to discriminate between closely related epitopes without altering their off-target profile.

[0231] To alter peptide specificity without generating TCRs that are broadly cross-reactive, we focused our library design on the CDR3 loop of the TCR. Based on structural data, the CDR3 is the major determinant of peptide specificity where the CDR3 of the TCRa chain (CDR3a) is primarily in contact with the N-terminal portion of the peptide and the CDR3 of the TCRb chain (CDR3b) is primarily in contact with the C-terminal portion. Since the variant peptides differ in their N-terminal half, we expected the CDR3a chain libraries to drive the generation of novel specificities. To systematically and productively manipulate TCR specificity, we generated comprehensive saturation mutagenesis libraries that consist of all single mutants, all double mutants, a subset of triple mutants and CDR3 length changes targeted to the CDR3a orb chains of the NLV2 and NLV3 TCRs (Fig. 1C). We synthesized oligonucleotides encoding these libraries, which range from -16K - 400K mutants depending on the size of the CDR3. CDR3 libraries were separately sub-cloned into aNLV2 orNLV3 TCR-expressing lentiviral backbone vector. The Jurkat TCRb7cell line was separately infected with each of the libraries at low multiplicity of infection (MOI) to generate a library of T cells with variant CDR3 loops. Antigen-specific TCR variants recognizing either the original epitope, the novel epitope or both epitopes were isolated by tetramer staining and fluorescence-activated cell sorting (FACS) and characterized by next generation sequencing (Fig. ID).

[0232] We first screened the NLV2 TCR library. The NLV2 a chain containing the CDR3a library (diversity: 346K oligos) was first paired with the wild-type (WT) NLV2 b chain in the transduced Jurkat cells. The library was then screened with peptide-MIC loaded tetramers to identify variants that switched binding specificity from recognizing the NLV2-specific permissive mutant (NLVP498G) to binding the NLV3-specific mutant (NLVN495M). More than 95% of the library lost specificity to bothpeptides, leaving -3% that retained binding to the NLV2-specific mutant, -1% that bound both peptides and -1% that completely switched specificity to the NLV3-specific mutant (Fig. 2A, left panel). Each population was sorted and expanded twice prior to next generation sequencing (Fig. 2A, right panel). The single positive (SP) population refers to the variant TCRs that retain binding to the original TCR specificity. The double positive (DP) population refers to the variants that bind to both the original specificity and the novel specificity, and the switched population (SW) signifies the TCR variants that lost specificity to the original NLV2 permissive binder and gained novel specificity to the NLV3- specific mutant.

[0233] Analysis of the allowable substitutions revealed certain patterns. CDR3 substitutions in the TCRs maintaining or improving binding to the original peptide (SP) showed permissivity for mutation of the lysines at position 5 and 10 to more bulky residues, especially P in position 5 and conservative substitutions at position 11 (Fig. 2B and Fig. 7A). Analysis of the DP and SW populations revealed a significant reliance on position 7 or 8 for recognition of the new binder (Fig. 2B and Fig. 7A). Moreover, our library design allows us to dissect detrimental changes that demolish TCR binding to its original antigen. For example, single mutations at positions 4 and 5 tend to be permissive for binding the original antigen, but all other positions tend to demolish recognition to the original binder (Fig. 7B). We can also better understand the SP enrichments that enhance recognition to the original binder by normalizing the enrichments to that of the WT CDR3a enrichment (Fig. 7C). For example, acidic residues at positions 4 or 5 enhance recognition to the original binder (Fig. 7C). This type of resolution could be useful in future studies to better understand the determinants of TCR specificity.

[0234] The top eight enriched SW TCRs were selected for validation by binding and functional assays and included the WT TCR as a control and one of the DP enriched TCRs (Fig. 2C). All SW NLV2 TCR-infected Jurkat cells acquired novel peptide (NLVN495M) recognition by tetramer binding (Fig. 2D) and upregulated the early activation marker CD69 (Fig. 2E) following co-culture with target cells pulsed with the NLV3-specific mutant. All TCRs retained activation to the NLVwTimmunodominant epitope that was not selected against during screening. Five of eight SW TCRs showed no cross-reaction to the NLV2-specific mutant (original binder), demonstrating a complete switch in specificity. The second library of the NLV2 CDR3b chain paired with the WT NLV2 a chain did not show detectable DP or SW populations (Fig. 7D), suggesting that the WT b chain CDR3 is not in contact with the N-terminal part of the peptide as expected from structural data. These experiments show that comprehensive mutagenesis libraries targeted to the CDR3 region of the TCR allow discrimination between closely related peptides and yield receptors with novel specificities and high potency.

[0235] Comprehensive off-target profiling displays favorable specificity of NLV TCR switch variants. Each time a TCR is engineered, it poses a potential threat to its specificity profile. To gain a sense of whether we drastically altered the recognition profile of the TCRs while changing theirspecificity, we comprehensively assessed their off-target reactivities via T-Scan. The advantage of the T-Scan method is its ability to interrogate large numbers of highly complex candidate antigens in a high- throughput screen. Another major advantage is the granzyme reporter readout, which offers sensitivity over traditionally used activation markers in identifying the targets of TCRs. We selected the NLV2 WT TCR, a variant NLV2 TCR that can recognize both peptides (DP TCR) and two SW TCRs, expressed them in primary T cells, and screened them with the virome-wide library, which consists of 93,904 56 amino acid fragments that collectively tile across the proteomes of all viruses known to infect humans (Fig. 8A). The library was expressed in cells that expresses only a single MHC, HLA-A2. If we drastically enhanced the affinity, we would expect enhanced TCR recognition of multiple peptides on HLA-A2. As expected, the two most enriched peptides in the screen with the WT TCR were the only two tiles that contained the known NLVwTepitope (Fig. 2F). In contrast, the DP TCR recognized 16 additional off-targets, becoming very promiscuous and recognizing multiple peptides on HLA-A2 (Fig. 2G). Conversely, the switched TCRs, one with two mutations in the CDR3a (SW1), and another with three mutations (SW4) both retained recognition of only the NLVwTpeptide tiles, with no additional cross-reactivity (Fig. 2H and 21). This phenomenon was further confirmed by performing additional virome-wide screens with additional NLV2 variants that had different sequences, DP? (Fig. 8B) SW3 (Fig. 8C). Similarly, the second DP TCRPHDacquired promiscuity, recognizing a different set of off- target tiles than those recognized with the first DP TCR556screened (Fig. 8B). Interestingly, several single amino acid changes at position Y8 of CDR3a were sufficient to switch specificity of the NLV2 TCR (Fig. 8D). To test whether single mutants can also be specific, we selected one of the single mutant CDR3s to examine via T-Scan. A single amino acid change at position 8 to an alanine - a mutation that is expected to add flexibility to the CDR3 region - resulted in no additional promiscuity or crossreactivity (Fig. 8E). This suggests that the complete switch in specificity from a closely related epitope is key to avoid drastic changes to the off-target profile of the variant TCRs.

[0236] To further characterize the footprint of the engineered TCRs, we performed epitope saturation mutagenesis T-Scan screens. To accomplish this, we generated a comprehensive single mutant library of both the NLV2-specific permissive mutant and the NLV3 -specific-mutant, with each mutant epitope present in the context of both a 56-aa fragment and a 9-aa fragment (Fig. 9A). As a control for the 56-aa version, we also mutagenized the two amino acids immediately upstream and downstream of the epitopes since they are not predicted to affect TCR recognition (Fig. 9A). This mutant library was introduced into HLA-A2 T-scan target cells and co-cultured with the primary T cells expressing the four different TCRs. Specificity profiling of the NLV2 WT TCR on the NLV2- specific epitope showed that most NLVP498G peptide mutations, with the exception of a few relatively conservative substitutions at positions 2,3, 4 and 6, were detrimental for WT NLV2 TCR activation and killing of target cells (Fig, 9B, left). As expected, the footprint of the WT NLV2 TCR on the non-cognate NLV3-specific peptide only gave one mutant that activated at position 1, whichreverted the sequence back to the primary NLV immunodominant epitope (Fig. 9B, right). This was in stark contrast to the footprints of the DP TCRFFs, where most mutations at all positions for both epitopes were activating, especially at position 1 where the mutation lies (Fig. 9C). This extensive substitution permissivity explains the ability of the DP TCR to recognize so many different epitopes in the virome-wide screen. For the SW TCR footprints, we see a very specific footprint on the novel specificity NLV3-specific peptide and no footprint on the original binder (Figs. 9D and 9E). Most mutations identified in the SW TCR footprints appear to be chemically similar to the unmutated residue, thereby preserving the recognition profile of the SW TCRs.

[0237] To examine the generality of this method, we similarly engineered a second NLV TCR: NLV3. The NLV3 TCR is unique because of its short CDR3a chain (only 3 amino acids) and its long CDR3b chain (13 amino acids long) (Fig. 1C). This is interesting because the non-binding peptide we wish to evolve it to recognize (NLVv497c) has its mutation in the N-terminal part of the peptide; and therefore, we would expect to rely on the a chain to switch specificity. However, when we screened the NLV3 CDR3a chain library to switch its specificity from its original binder (NLVN495M) to the closely related NLV2-specific peptide (NLVv497c), no population enrichments were observed (Fig. 10A). In contrast, after three rounds of sorting, the NLV3 CDR3b library identified four variant TCRs with redirected specificity to the NLVv497c peptide (Figs. 3A, 3B and 10B). This suggests that the CDR3b chain of the NLV3 TCR is in contact with the N-terminal part of the peptide, in contrast to what is generally expected structurally from most TCR-epitope interactions. The four rare switch variants identified were triple mutants that contained a LY motif at positions 5 and 6, suggesting that we needed complex mutations for this switch in specificity. For the DP population, the major enrichment was a proline to alanine change at position 9 of the CDR3b chain coupled with a change at position 3 or 5 (Figs. 3B and 10B). Validation of the SW TCR variants (Fig. 3C) revealed binding (Fig. 3D) and activation (Fig. 3E) to the NLVv497c peptide.

[0238] Next, we assessed the off-target landscape of these TCRs via virome-wide T-Scan. We selected four TCRs to screen: the WT NLV3 TCR, the single mutant proline to alanine DP variant and two SW TCRs. Each of the TCRs were expressed separately in primary T cells and co-cultured with target T-Scan cells expressing the virome-wide library. Interestingly, co-culture of the DP TCR with the target cells revealed a high background of activation and so we co-cultured the WT or DP TCRs with HLA-A2 target cells without expression of the virome-wide library and tested for granzyme B reporter activation. We found that there is a high basal activation (-3%) of the reporter (Fig. 3G), suggesting that the DP TCR has either acquired promiscuity to an antigen expressed in 293T cells or the engineering has enhanced the binding between the TCR and the HLA helices themselves. Thus, we were unable to screen this TCR because of its extreme background activation. Crucially, the SW TCR screens revealed no additional off-targets or promiscuity (Fig. 3H and 31). This confirms thatthe DP population tends to generate promiscuous TCRs and that the switch in specificity between closely related epitopes is crucial to create safe and effective TCRs.

[0239] Application of T-Switch for engineering TCRs with specificity and functionality to a tumor antigen. Next, we wanted to translate our approach into the generation of cancer reactive TCRs. A major advantage of our approach is that it presents a strategy for the high-throughput generation of TCRs against potential cancer targets without the need to extensively screen for T cell clones from patient samples. Instead, we can use well established assays for in vitro antigen specific activation and expansion of naive, healthy human CD8 T cells (Fig. 4A). After selection of a self-antigen target, we generate mutant peptides that differ by one amino acid from the self-antigen. The goal of these neoantigen-like peptides is to bypass T cell tolerance and act as foreign peptides that can stimulate and expand naive CD8 T cells, similar to a neoantigen-like response. Monocyte-derived dendritic cells (MoDCs) from a healthy donor are then pulsed with both selected mutant and self-peptides and cocultured with autologous naive CD8 T cells and expanded for 10 d. Next, antigen-specific T cells are identified by HLA tetramers loaded with peptides of interest. We expect to see rapid expansion to the mutant peptides as that anticipated with neoantigens and no or weak expansion to self-antigens. Expanded TCR clonotypes will then be identified by 10X TCR sequencing. After identification of a TCR clonotype recognizing a mutant peptide of interest, we use that as the starting material for the generation and screening of TCR mutagenesis libraries, as described above, to switch the specificity of the TCR towards the self-antigen. We can then assess the cross-reactivity profile of the engineered self- TCRs by T-scan against the human genome-wide library (Fig. 4A). We refer to this bait and switch platform as T-Switch, where we bait the T cells with the mutant epitope and then switch its specificity for the self-antigen.

[0240] We applied T-Switch to a published self-antigen (ALLSGVRQV (SEQ ID NO: 36)) from the protein tyrosine hydroxylase (TH), a tumor-associated target antigen highly expressed in neuroblastomas but not in normal tissues and has relevance to neuroblastoma pathology based on published literature. Importantly, TH exhibits a developmentally restricted expression pattern, where it is expressed during fetal development and is down-regulated or silenced in normal tissues before birth. Additionally, the chosen peptide was selected based on its relative abundance on MHC compared to other peptides and its optimal pMHC binding affinity on HLA-A2 (the most frequently expressed allele in the US). To identify mutant peptides of the TH antigen, we first generated an in silico structure-based model of the TH peptide bound to HLA-A2 (Fig. 11A). This served to visualize the best fit' of the TH peptide in the MHC pocket and allowed us to determine the orientation of the peptide amino acids relative to the HLA molecule. Arrows pointing upwards represent the amino acid side chains that face toward the TCR and arrows pointing downwards are in the direction of the MHC base (Fig. 11A). An unfavorable change in energy when substituting with any amino acid at the positions pointing downward, suggests that positions 2,3,6 and 9 may be important to maintaining the peptide in aconformation that promotes TCR engagement, and are less likely to be direct interaction points for the TCR (Fig. 11A and 11B). In contrast, the positions pointing upwards (1,4,5 and 8) and have no or minimal change in energy when substituting with different amino acids are most likely TCR interaction points (Fig. 11A and 11B). Another consideration in electing positions to mutate is to ensure there is no drastic change in pMHC affinity as a result. Therefore, we generated a NetMHC predicted pMHC affinity matrix for each substitution across the 9-aa peptide (Fig. 11C). Based on this, we selected amino acid substitutions that (i) are in amino acids pointing upwards, (ii) do not significantly affect the overall stability or affinity of the predicted pMHC complex, and (ii) are chemically dissimilar to the original amino acid to allow for bypass of T cell tolerance (boxes with heavy borders in 11C). From these mutants, we selected the glutamine to methionine mutation at position 8 (ALLSGVRMV(SEQ ID NO: 35)) as our 'bait' antigen. Co-culture of naive CD8 T cells against moDCs pulsed with either the WT or mutant TH antigen showed antigen specific T cell expansion only to the mutant peptide as measured by tetramer staining (Fig. 4B). Next, we validated our in vitro expansion by reconstituting Jurkat TCRb7cells with the top two expanded clonotypes and stained the cells with tetramers loaded with either the WT peptide or the mutant peptide. The second topmost expanded clonotype bound the mutant-loaded tetramer but not the WT-loaded tetramer, as expected from the naive CD8 expansion (Fig. 4C). As a result, this clonotype serves as a candidate for T-Switch in order to redirect its specificity towards the TH self-antigen (ALLSGVRQV(SEQ ID NO: 36)).

[0241] We applied the same comprehensive library design, as before, to both the CDR3a and CDR3b chains of Clonotype 2 (Fig. HD). The Jurkat TCRb7cell line was separately infected with each of the libraries to generate a library of T cells with variant CDR3 loops (diversity of CDR3a library: 300K and diversity of CDR3b library: 430K). Antigen-specific TCRs for the SP, DP and SW populations were isolated by tetramer staining and characterized by next generation sequencing. Analysis of the top enriched TCRs from each population of the CDR3a library revealed a requirement for a serine to glycine mutation at position three as well as a second mutation at position 6, to switch its specificity towards the TH WT peptide by replacing a glycine (Fig. 4D). With the exception of one double mutant, all CDR3a variants that switched specificity were triple mutants (Fig.4D). Given that the bait mutation is in the C-terminal portion of the peptide, at position 8, it was surprising to see a switch in specificity from the CDR3a chain library as we would expect the CDR3b chain to be the primary contact to that region. This capitalizes on the complexity of antigen specificity and the need to use comprehensive directed evolution libraries to identify those rare variants. Nevertheless, we also identified variants from the CDR3b chain that switched specificity to the TH WT peptide (Figs. 12A and 12B). In order to validate our results experimentally, we reconstituted Jurkat TCRb7cells with the top enriched variants from each population (Fig. 5A). Consistent with sequence enrichment data, T- Switch variants from the SW population of the CDR3a chain library displayed tetramer binding and CD69 activation exclusively to the TH self-antigen and not to the mutant antigen (Figs. 5B and 5C).Additionally, the DP population bound and activated to both antigens and the SP population interacted exclusively with the mutant antigen (Figs. 5B and 5C). Similarly, validation of the SW population CDR3b chain variants (Fig. 12C) revealed exclusive tetramer binding to the WT TH peptide and loss of binding to the mutant peptide (Fig. 12D). However, when we measured CD69 activation post coculture of Jurkat TCRb7cells with target cells pulsed with the mutant or WT antigen, we found that only 3 / 10 TCRs had functional activation (Fig. 12E). This displays one of the limitations of engineering TCRs and further highlights the discordance between binding and activation that has been observed in previous reports.

[0242] To ensure that the variant TCRs can recognize the peptide following antigen processing and presentation, we reconstituted HLA-A2+ T-Scan target cells with two 90-mer peptides that encode part of the TH protein and contain the target antigen (Fig. 5D). We co-cultured the target cells with Jurkat TCRb7cells expressing our variant TCRs to measure CD69 upregulation or with primary T cells expressing a subset of the variant TCRs to measure target cell killing following recognition of the endogenously processed and presented antigen (Fig. 5D). In response to target cells expressing the TH peptides, the engineered TCR variant expressing Jurkat cells showed potent CD69 activation, with the exception of the SP population TCRs (SP1 and SP2) that activate to the mutant TH peptide and not the WT, as expected (Fig. 5E). Moreover, engineered switched variant expressing primary T cells show potent target cell killing when co-cultured with target cells expressing the 90mer TH peptides and not when co-cultured with target cells expressing an irrelevant tile as measured by granzyme B activation (Fig. 5F), caspase 3 / 7 cleavage (Fig. 5G), and 7-AAD uptake (Fig. 5H). This serves as the first example of applying the T-Switch platform to redirect a TCR recognizing a bait antigen toward recognition of an endogenously processed and presented self-antigen. As a result, the generation of additional de novo TCRs targeting cancer-specific self-antigens, whose production are normally prevented by tolerance mechanisms in vivo, is feasible with this approach.

[0243] T-Switch variants display high specificity and mediate potent target cell killing. To examine the specificity of the engineered TCR variants and whether they exhibited cross-reactivity, we performed whole genome-wide T-Scan screens. The human peptidome library consists of -500K total peptide tiles that are 90-aa fragments with 22-aa overlaps that tile across the entire human proteome (Fig. 51). The library was infected in HLA-A2 positive T-Scan target cells and co-cultured with primary T cells expressing either the original unmutated 10X clonotype that recognizes the mutant TH peptide, the TH DP3 TCR variant with 3 mutations in the CDR3a chain, or the SW 1 TH TCR variant with 2 mutations in the CDR3a chain (Fig. 51). The original TCR failed to enrich any overlapping peptide tiles, which was expected due to its recognition of the mutant TH peptide, which is not present in the library (Fig. 5J). In contrast, the DP TCR enriched many off-target overlapping tiles from numerous self-proteins (Fig. 5K). Crucially, the SW TCR had strong and reproducible enrichment of 3 peptide tiles that contained our target TH WT epitope and no additional promiscuity (Fig. 5L). This isin line with our previous data, confirming that a complete switch in specificity, following T-Switch engineering, does not introduce off-target specificities corresponding to known sequences in the human proteome.

[0244] Self-antigen-reactive TCRs generated by allo-reactivity show less specificity than T Switch. We wished to examine how the off-target profiles of these methods compare with some of the alternative methodologies used to isolate self-TCRs, such as the identification of allo-HLA restricted TCRs by expansion of T cells with self-antigens presented in the context of foreign HLA. Since the naive T cells used have a different MHC, the TCRs recognizing the MHC -epitope combination will not have been eliminated by central tolerance. We picked three TCRs from the literature to test via T-Scan and identified five or more off-targets for each of the allo-HLA restricted TCR screens (Figs. 13A- 13C). Therefore, we predict that allo-HLA restricted TCRs may be inherently more cross-reactive, possibly in part by stronger recognition of the HLA molecule itself, although a larger scale analysis with additional examples will be needed to fully confirm that possible interpretation.

[0245] T-Switch generated anti-TH TCRs kill tumor cells expressing TH. We next explored whether the TH WT TCR variants could efficiently kill HLA-A2-TH7tumor cells. We transduced human primary T cells with the 10X TCR clonotype or three SW TCR variants and first performed a co-culture with T-Scan target cells expressing the full-length TH ORF (Fig. 6A). Efficient granzyme B reporter activation was observed in target cells in response to co-culture with the SW TCR variants but not the original 10X clonotype (Fig. 6A, left) and that response was amplified when a 3: 1 ratio of T cell to target cell was used (Fig. 6A, right). Next, we selected a highly expressing TH glioblastoma cell line (KS-1) and saw efficient cytotoxicity following coculture with primary T cells expressing SW TCR variants (Fig. 6B). Similar results were seen in response to SKNBE and KPNYN cells, which express lower levels of the TH antigen (Figs. 6C and 6D). Furthermore, additional T cell metrics of functionality were also regulated in response to coculture with TH-expressing target cells including the degranulation marker CD 107a, and cytokines IFNg and TNFa (Figs. 6E-6G). Combined, this data shows that our T-Switch variants can efficiently kill tumor cells expressing endogenously processed and presented antigen, representing therapeutic candidates.

[0246] Discussion

[0247] Here, we describe the development and application of T-Switch, an engineering platform that utilizes a two-step strategy for TCR evolution to systematically and productively direct its specificity toward desired new targets, especially self-antigens. The key element of our strategy is to evolve a TCR against a neo-epitope peptide with a single amino acid change that would allow central tolerance escape. Once TCRs to the neo-epitope have been identified, we systematically mutagenize the CDR3 region of the TCR to generate a diverse library of related TCRs and then screen for binding specificity swaps from the neoantigen to the desired target. Swapping the specificity, i.e. losing theability to recognize the initial epitope and instead recognize a new epitope. Switching specificity rather than gaining a new specificity appears to be the key to avoiding deleterious cross-reactivity.

[0248] In our initial proof of principle experiments we chose two viral NLV TCRs as our initial proof-of-concept experiments based on our previous knowledge that they have differential binding to closely related epitopes that differ by one amino acid from the immunodominant CMV epitope. Moreover, the NLV2 and NLV3 TCRs exhibit no sequence identity in their variable regions, providing us with valuable insights into the behavior of diverse TCRs on our platform. Our library design, targeted exclusively to the hypervariable CDR3 region, is unique and comprehensive and provides a diversity approaching that of the human naive TCR repertoire. We were able to switch specificities for both TCRs to recognize the epitopes normally only seen by the other TCR. With the exception of the NLV2 CDR3a library where a single mutant at position 8 was sufficient to switch specificity, we found that complex double or triple mutants are required for specificity switches. Interestingly, diverse motif group enrichments in the different populations underscoring the challenges of computationally predicting the determinants of TCR specificity and shows that this library design lends itself well to a general approach for TCR development without requiring specialized structural insights. The most important lesson learned by these preliminary experiments is that switching the specificity as opposed to gaining a new specificity was a feature critical to the success of this strategy. T Scan analysis of the NLV2a DP and the THa DP TCRs revealed recognition of multiple diverse peptides on HLA-A2 and in the case of the NLV3b DP TCR and THb DP TCRs, we saw what could be interpreted as either improved interaction between the TCR and HLA molecule or promiscuity to a self-antigen on the target cells, two caveats typically seen by affinity-enhanced TCRs. Importantly, by engineering the switch in specificity away from the closely related epitope, we eliminate the predisposition toward off- target cross-reactivity compared with affinity-matured TCRs. These small-step specificity changes are therefore essential in obtaining safe and effective TCRs.

[0249] The use of T-scan is another important component of the T-switch platform. Off-target toxicity is a major concern regarding therapeutic TCRs, especially after the lethal cross-reactivity resulting from the affinity enhanced MAGE-A3 TCR. Therefore, it is now key to incorporate a comprehensive cross-reactivity profiling method to assess the specificity of engineered TCRs. T-scan offers significant advantages over existing profiling methods: 1) It preserves essential aspects of the TCR-pMHC interaction, such as the natural TCR recognition and signaling and the physiological antigen processing and presentation of antigens. 2) The use of primary T cells and the granzyme B reporter in target cells offer a sensitive readout of T cell activation and killing as compared with existing methodologies that use CD69 or the NF AT reporter. 3) It has the ability to interrogate highly complex sets of candidate antigens in a high-throughput screen.

[0250] Finally, we were able to show the therapeutic potential of T-Switch by applying it to generate TCR variants for the neuroblastoma self-antigen, TH. This was achieved by first bypassing Tcell tolerance and raising naive CD8 T cells to recognize a closely related neoantigen form of the TH peptide. After identifying a clonotype that binds to the mutant TH peptide, we used our mutagenesis approach to find variants that switched specificity to the wild-type antigen. Surprisingly, both the CDR3a and CDR3b chains switched specificity, which shows divergence from structural data expectations. This again emphasizes the challenge in using computational prediction algorithms to anticipate TCR specificity. Next, we performed several preclinical development assays showing that engineered TCR variants expressed in primary T cells: 1) can target and kill endogenously processed and presented TH self-antigen, 2) lacked cross-reactivity with a T-scan screen against the whole proteome, and 3) demonstrated potent tumor cell killing.

[0251] Overall, the T Switch platform has demonstrated the ability to change the recognition of a TCR to a distinct related antigen while maintains its specificity, a major goal of direct T cell evolution. It appears to out-perform allo-generated TCRs to self-antigens with respect to the frequency of off target recognition. It also outperforms straight affinity enhancement strategies to the extent they have been validated so far. Finally, the strategy to circumvent self-tolerance mechanisms promises to have many application in the future. In summary, we anticipate the T Switch platform developed here to facilitate the de novo generation of collections of potent and effective cancer reactive TCRs for ACT.Example 3: T-Switch generated anti-TH TCRs kill tumor cells expressing TH

[0252] We explored whether the TH WT TCR variants could efficiently kill HLA-A2-TH+ tumor cells. We transduced human primary T cells with the TH MUT TCR or three SW TCR variants and first performed a co-culture with T-Scan target cells expressing the full-length TH open reading frame (ORF) (Fig. 14A). Efficient granzyme B reporter activation was observed in target cells in response to co-culture with the SW TCR variants but not the TH MUT TCR (Fig. 14A, left) and that response was amplified when a different effector-to-target ratio (3: 1) was used (Fig. 14A, right).

[0253] Next, we tested killing against three cell lines that express TH endogenously (Fig. 14G). We first selected a highly expressing TH glioblastoma cell line (KS-1) that also expresses HLA-A2 and saw efficient cytotoxicity following co-culture with primary T cells expressing SW TCR variants (Figure 6B). Similar results were seen in response to neuroblastoma cells lines, such as SKNBE and KPNYN cells, which express lower levels of the TH antigen (Figs. 14C and 14D). It is important to note that apart from the potential for cross-reactivity, the alloreactivity of TCRs towards different HLA alleles also poses a safety concern. Therefore, it is promising that our T-Switch TCRs do not activate towards different HLA molecules expressed on the HLA-A2 negative neuroblastoma cell lines (Figs. 14C and 14D). Furthermore, additional T cell metrics of functionality were also upregulated in response to co-culture with TH-expressing target cells including the degranulation marker CD107a, and cytokines IFNg and TNFa (Figs. 14E-14G).Combined, this data shows that our T-Switch variants can efficiently kill tumor cells expressing endogenously processed and presented antigen, representing therapeutic candidates.

[0254] We next determined the anti-tumor activity in a A375 melanoma xenogeneic tumor model with or without expression of the TH ORF using immunocompromised NSG mice. A single i.v. infusion of T-Switch engineered THa SW1-SW3 TCRs showed a substantial and significant improvement in antitumor activity relative to primary T cells expressing a non-cognate TCR when injected into mice bearing A375 tumor cells expressing the TH ORF but not in mice bearing A375 tumor cells alone (Figs. 14H and 141). Overall, our findings demonstrate how T-Switch can be used to identify synthetic TCR variants with promising therapeutic properties.Example 4: T-Sw tch TH Screen sequences

[0255] TCR THa SWl (SEQ ID NO: 20)MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVELQTAPIDGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRKRSGSGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAYGGGRADGLTFGKGTHLIIQPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS

[0256] TCR_THa_SW2 (SEQ ID NO: 21)MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVELQTAPIDGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRKRSGSGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAYGGGFADGPTFGKGTHLIIQPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS

[0257] TCR_THa_SW3 (SEQ ID NO: 22)MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVELQTAPIDGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRKRSGSGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCASGGGYADGLTFGKGTHLIIQPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS

[0258] TCR_THa_SW4 (SEQ ID NO: 23)MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVELQTAPIDGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRKRSGSGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAYGGGSADGLDFGKGTHLIIQPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS

[0259] TCR_THa_SW5 (SEQ ID NO: 24)MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVELQTAPIDGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRKRSGSGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAYGGGKDDGLTFGKGTHLIIQPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS

[0260] TCR_THa_SW6 (SEQ ID NO: 25)MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVELQTAPIDGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRKRSGSGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAYGGGHADGLGFGKGTHLIIQPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS

[0261] TCR_THa_SW7 (SEQ ID NO: 26)MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVELQTAPIDGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRKRSGSGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAYGGGAADGITFGKGTHLIIQPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS

[0262] TCR_THa_SW8 (SEQ ID NO: 27)MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVELQTAPIDGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRKRSGSGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAYGGGYADGLDFGKGTHLIIQPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS

[0263] TCR_THa_SW9 (SEQ ID NO: 28)MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVELQTAPIDGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRKRSGSGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAYGGGFADGLDFGKGTHLIIQPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS

[0264] TCR THa SWIO (SEQ ID NO: 29)MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVELQTAPIDGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRKRSGSGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAYGGGYKDGLTFGKGTHLIIQPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS

[0265] Sequences

Claims

What is claimed herein is:

1. A method comprising: a) mutagenizing at least one of a CDR3a and a CDR3P of a self-antigen mimic-specific TCRto provide a pool of target self-antigen-specific TCR candidates; and b) selecting at least one target self-antigen-specific TCR from the pool of target self- antigen-specific TCR candidates.

2. A method comprising: a) isolating a self-antigen mimic-specific TCR; b) mutagenizing at least one of the CDR3a and CDR3P of the self-antigen mimicspecific TCRto provide a pool of target self-antigen-specific TCR candidates; and c) selecting at least one target self-antigen-specific TCR from the pool of target self- antigen-specific TCR candidates.

3. The method of claim 2, wherein the isolating comprises injecting an animal or person with the self-antigen mimic.

4. The method of claim 2, wherein the isolating comprises raising naive CD8 T cells from a human donor with the self-antigen mimic.

5. The method of claim 2, wherein the isolating comprises raising naive CD8 T cells, from a human donor HLA-matched with respect to the target self-antigen, with the self-antigen mimic.

6. The method of any one of claims 2-5, wherein the isolating comprises screening a library of T cells with the self-antigen mimic.

7. The method of any one of the preceding claims, wherein the self-antigen mimic is displayed in a complex with an MHC polypeptide during the isolating step.

8. The method of any one of the preceding claims, wherein the self-antigen mimic is displayed as part of a fusion protein with an MHC polypeptide during the isolating step.

9. The method of any one of the preceding claims, wherein the self-antigen mimic is displayed on an antigen presenting cell during the isolating step.

10. The method of claim 9, wherein the antigen presenting cell is a monocyte-derived dendritic cell or an EpiScan cell.

11. The method of any one of the preceding claims, wherein the self-antigen mimic is a polypeptide of no more than 20 amino acids.

12. The method of any one of the preceding claims, wherein the self-antigen mimic is a polypeptide of more than 20 amino acids.

13. The method of any one of the preceding claims, wherein the target self-antigen is a polypeptide of no more than 20 amino acids.

14. The method of any one of the preceding claims, wherein the target self-antigen is a polypeptide of more than 20 amino acids.

15. The method of any one of the preceding claims, wherein the target self-antigen is a naturally occurring polypeptide.

16. The method of any one of the preceding claims, wherein the at least one of a CDR3a and a CDR3P is CDR3a.

17. The method of any one of the preceding claims, wherein the at least one of a CDR3a and a CDR3P is CDR3p.

18. The method of any one of the preceding claims, wherein the at least one CDR is CDR3a and CDR3p.

19. The method of any one of the preceding claims, wherein the mutagenizing comprises mutagenizing a single residue of at least one of the CDR3a and the CDR3p.

20. The method of any one of the preceding claims, wherein the mutagenizing comprises mutagenizing a single residue of the each of the at least one of the CDR3a and the CDR3p.

21. The method of any one of the preceding claims, wherein the mutagenizing comprises mutagenizing two residues of at least one of the CDR3a and the CDR3p.

22. The method of any one of the preceding claims, wherein the mutagenizing comprises mutagenizing three residues of the each of the at least one of the CDR3a and the CDR3p.

23. The method of any one of the preceding claims, wherein the mutagenizing comprises mutagenizing three residues of at least one of the CDR3a and the CDR3p.

24. The method of any one of the preceding claims, wherein the mutagenizing comprises mutagenizing at least three residues of at least one of the CDR3a and the CDR3p.

25. The method of any one of the preceding claims, wherein the mutagenizing comprises mutagenizing at least three residues of the each of the at least one of the CDR3a and the CDR3p.

26. The method of any one of the preceding claims, wherein the mutagenizing is comprehensive saturation mutagenesis.

27. The method of any one of the preceding claims, wherein the pool of self-antigen-specific TCR candidates comprises a library of single mutants, double mutants, triple mutants, and CDR3 length changes of at least one of the CDR3a and the CDR3p.

28. The method of any one of the preceding claims, wherein the pool of self-antigen-specific TCR candidates comprises a library comprising all single mutants and all double mutants of at least one of the CDR3a and the CDR3p.

29. The method of any one of the preceding claims, wherein the selecting comprises selecting target self-antigen-specific TCRs from the pool of target self-antigen-specific candidates.

30. The method of any one of the preceding claims, wherein the target self-antigen is displayed in a complex with an MHC polypeptide during the selecting step.

31. The method of any one of the preceding claims, wherein the selecting comprises selecting TCRs that do not bind the self-antigen mimic from the pool of target self-antigen-specific candidates.

32. The method of any one of the preceding claims, wherein the self-antigen mimic is displayed in a complex with an MHC polypeptide during the selecting step.

33. The method of any one of the preceding claims, wherein the selecting comprises selecting TCRs that do not bind a plurality of off-target self-antigens from the pool of target self- antigen-specific candidates.

34. The method of any one of the preceding claims, wherein the selecting comprises selecting TCRs that do not bind a plurality of off-target self-antigens in a human peptidome library from the pool of target self-antigen-specific candidates.

35. The method of any one of the preceding claims, wherein the selecting comprises selecting TCRs that do not bind a plurality of off-target self-antigens in a human peptidome library presented on T-scan cells from the pool of target self-antigen-specific candidates.

36. A TCR polypeptide composition comprising: a) a CDRla comprising the sequence of SEQ ID NO: 1; b) a CDR2a comprising the sequence of SEQ ID NO:2; c) a CDR3a comprising the sequence of one of SEQ ID NOs: 3 and 7-15; d) a CDRip comprising the sequence of SEQ ID NO:4; e) a CDR2P comprising the sequence of SEQ ID NO:5; and f) a CDR3P comprising the sequence of SEQ ID NO:6.

37. The TCR polypeptide composition of any one of the preceding claims, further comprising one or more of SEQ ID NOs: 16-19.

38. A nucleic acid composition comprising one or more nucleic acids encoding the TCR polypeptide composition of any one of the preceding claims.

39. A cell comprising the TCR polypeptide composition of any one of the preceding claims and / or the nucleic acid composition of claim 38.

40. The cell of claim 39, wherein the cell is a T cell.

41. A method of treating a subject in need of immune stimulation specific for a target selfantigen, the method comprising administering a T cell comprising a target self-antigen- specific TCR selected by the process of any one of claims 1-35 to the subject.

42. A method of treating a subject in need of immune stimulation specific for Tyrosine Hydoxylase (TH), the method comprising administering the T cell of claim 40 to the subject.

43. The method of any one of the preceding claims, further comprising a first step of transducing at least one T cell obtained from the subject with the target self-antigen-specific TCR or the TCR polypeptide composition of any one of claims 36-37.

44. The method of any one of the preceding claims, wherein the subject is a subject in need of treatment for cancer.

45. The method of claim 44, wherein the cancer is neuroblastoma.

46. The method of any one of the preceding claims, wherein the T cell is autologous to the subject.

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