T-cell receptor sequences for targeted cancer immunotherapy and methods for identification of novel therapeutic tcrs

Abstract

Disclosed are methods and compositions for identifying and utilizing antigen-specific T cell receptors (TCRs) for therapeutic applications. The invention includes high-throughput screening techniques using peptide–MHC single-chain trimer (SCT) tetramers and single-cell sequencing to isolate paired alpha and beta chain TCRs specific for target antigens, including human papillomavirus (HPV) E6 and E7. Identified TCRs are validated for functional activity and may be introduced into CD4+ or CD8+ T cells for use in adoptive cell therapy. Compositions include nucleic acids, vectors, engineered T cells, and pharmaceutical formulations comprising the disclosed TCRs. The invention also provides methods for treating HPV-associated diseases and other conditions by administering modified T cells. Variants of the disclosed TCRs that retain antigen specificity are also contemplated. This platform supports the discovery and deployment of TCRs for infectious diseases, cancer, and immune monitoring.

Description

T-CELL RECEPTOR SEQUENCES FOR TARGETED CANCER IMMUNOTHERAPYAND METHODS FOR IDENTIFICATION OF NOVEL THERAPEUTIC TCRSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 640,828, filed April 30, 2024, and U.S. Provisional Patent Application No. 63 / 756,710, filed February 10, 2025. Each of these applications is hereby incorporated by reference in its entirety for all purposes.FIELD

[0002] This disclosure relates the sequences of T cell receptors and methods to identify potentially therapeutic T cells.ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under R01 CA264090-01 awarded by the National Cancer Institute of the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] Cancer remains a leading cause of death worldwide, with many tumor types demonstrating resistance to conventional therapies such as chemotherapy, radiation, and surgery. The immune system plays a central role in the surveillance and elimination of malignant cells (Luckheeram et al., 2012), and immunotherapies that harness or enhance immune responses have emerged as promising treatment strategies.

[0005] Among these, T cells have demonstrated particular potential in mediating tumorspecific immune responses (Tay et al., 2021; and Oh et al., 2021). T cells can recognize and eliminate cancer cells by detecting tumor-associated antigens presented by majorhistocompatibility complex (MHC) molecules. Advances in genetic engineering have enabled the development of adoptive cell therapies, including the modification of T cells to express antigen-specific receptors, offering the possibility of durable and targeted anti-tumor activity.

[0006] Despite progress in this field, there remain significant challenges in identifying and deploying tumor-reactive T cell receptors (TCRs), particularly in ways that ensure broad population coverage and functional potency. WO 2021168122 (Heath et al., 2021) discloses single chain trimer MHC Class II nucleic acids and proteins. WO 2022236102 (Chour et al., 2022) discloses single chain trimer MHC Class I nucleic acids and proteins. WO 2023205728 (Chour et al., 2023) discloses soluble single chain dimers from single chain trimers.

[0007] There is a continuing need for effective strategies to discover, validate, and utilize TCRs that recognize tumor-associated antigens across diverse human leukocyte antigen (HLA) backgrounds.SUMMARY

[0008] In some embodiments, a method of facilitating adoptive cell treatment of a subject is provided, the method comprising: identifying one or more T cell receptors predicted to be effective to treat the subject if used to genetically modify T cells administered to the subject, wherein the identification includes selecting one or more T cell receptors directed against human papilloma virus (HPV) E6 or E7 epitopes, wherein the one or more T cell receptors includes: at least one T cell receptor identified as TCR 1-101; at least one T cell receptor identified as TCR 102-400; at least one T cell receptor specific for an antigen identified as TCR 1-101; and / or at least one T cell receptor specific for an antigen identified as TCR 102-400; and outputting an identification of the one or more T cell receptors.

[0009] In some embodiments, a method of facilitating adoptive cell treatment of a subject is provided, the method comprising: isolating T cells from a blood sample of the subject; genetically modifying the T cells to express one or more T cell receptors, wherein the one or more T cell receptors includes: at least one T cell receptor identified as TCR 1-101; at least one T cell receptor identified as TCR 102-400; at least one T cell receptor specific for an antigen identified as TCR 1-101; and / or at least one T cell receptor specific for an antigen identified as TCR 102-400; and providing the modified T cells for administration to the subject.

[0010] With respect to part or all of one or more methods disclosed herein:• the one or more T cell receptors may comprise at least one TCR associated with a cell type with a cytotoxic phenotype and at least one TCR from a cell type with a memory T cell phenotype;• the one or more T cell receptors may be selected by predicting that the one or more peptides are configured to replicate tumor-infiltrating T cells of the subject.• the one or more T cell receptors may comprise an isoform that retains antigen specificity for HPV E6 or E7; and / or• the genetic modification may be conducted by viral transfections, plasmids, CRISPR- CAS9 or any suitable molecular biology or genetic engineering technique.

[0011] In some embodiments a T cell receptor comprising an amino acid sequence selected from TCR 1-101 is provided, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor.

[0012] In some embodiments, a T cell receptor comprising an amino acid sequence selected from TCR 102-400 is provided, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor.

[0013] In some embodiments, an antigen identified in FIG. 6 recognized by a T-cell receptor identified as TCR 1-101 is provided.

[0014] In some embodiments, an antigen identified in FIG. 7 recognized by a T-cell receptor identified as TCR 102-400 is provided.

[0015] In some embodiments, a nucleic acid molecule encoding part or all of (i) a T cell receptor identified as TCR 1-400, or (ii) a T cell receptor specific for an antigen identified in, and selected from, FIG. 6 or FIG. 7 is provided. A vector comprising the nucleic acid molecule may be provided. A library comprising the plurality of nucleic acid molecules may be provided. In some embodiments, a T cell, such as a CD8+ T cell or a CD4+ T cell, transformed with the nucleic acid molecule is provided.

[0016] In some embodiments, a composition comprising and / or encoding a T cell receptor comprising the amino acid sequence of TCR 401, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor is provided. The composition may include the variant, and wherein the variant may be optimized for increased expression, stability, or reduced immunogenicity in human cells. The composition may include the variant, and wherein the variant may retain specificity for the same HPV E6 epitope as the receptor comprising TCR 401.

[0017] In some embodiments, a composition comprising and / or encoding a T cell receptor comprising the amino acid sequence of TCR 402, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor is provided. The composition may include the variant, and wherein the variant may be optimized for increased expression, stability, or reduced immunogenicity in human cells. The composition may include the variant, and wherein the variant may retain specificity for the same HPV E6 epitope as the receptor comprising TCR 402.

[0018] In some embodiments, a composition comprising and / or encoding a T cell receptor comprising the amino acid sequence of TCR 403, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor is provided. The composition may include the variant, and wherein the variant may be optimized for increased expression, stability, or reduced immunogenicity in human cells. The composition may include the variant, and wherein the variant may retain specificity for the same HPV E6 epitope as the receptor comprising TCR 403.

[0019] In some embodiments, a composition comprising and / or encoding a T cell receptor comprising the amino acid sequence of TCR 404, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor is provided. The composition may include the variant, and wherein the variant may be optimized for increased expression, stability, or reduced immunogenicity in human cells. The composition includes the variant, and wherein the variant may retain specificity for the same HPV E6 epitope as the receptor comprising TCR 404.

[0020] In some embodiments, a composition comprising and / or encoding a T cell receptor comprising the amino acid sequence of TCR 405, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor is provided. The composition may include the variant, and wherein the variant may be optimized for increased expression, stability, or reduced immunogenicity in human cells. The composition may include the variant, and wherein the variant may retain specificity for the same HPV E6 epitope as the receptor comprising TCR 405.

[0021] In some embodiments, a method is provided for identifying an antigen-specific T cell receptor (TCR) is provided, the method comprising: (a) obtaining a biological sample comprising CD4+ or CD8+ T cells from a subject; (b) contacting the T cells with one or more peptide-MHC single-chain trimer (SCT) tetramers, each comprising a peptide of interest and an HLA class I or class II molecule; (c) isolating tetramer-positive T cells; (d) performing singlecell RNA sequencing and TCR sequencing to obtain paired alpha and beta chain sequences and corresponding transcriptomic profiles from individual tetramer-positive T cells; and (e) identifying one or more TCRs specific for an antigen based on tetramer binding, clonal enrichment, or functional profile. The SCT tetramers may be barcoded with unique oligonucleotides to allow multiplexed antigen identification. The antigen may be derived from a human papillomavirus (HPV) E6 or E7 protein. The method may further include cloning the TCR alpha and beta chains into a nucleic acid construct and introducing the construct into a T cell or reporter cell line. The method may comprise validating antigen specificity using a peptide stimulation, cytokine production assay, tetramer staining, or cytotoxicity assay. The method may include the transcriptomic profile indicating a phenotype selected from the group consisting of: naive, effector memory, regulatory, cytotoxic, Th l, Th 17, or proliferative. The method wherein the subject may have a disease selected from the group consisting of: cervical cancer, oropharyngeal cancer, SARS-CoV-2 infection, or melanoma. The method wherein the identified TCR may be selected based on clonal expansion or persistence across longitudinal timepoints.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] The present disclosure is described in conjunction with the appended figures:

[0024] FIG. 1 is a table listing CD8+ TCR clonotypes with corresponding CDR3 sequences,HLA restriction, and target antigens.

[0025] FIG. 2 is a table summarizing cancer types and tumor sites associated with HPV antigens relevant to the disclosed TCRs.

[0026] FIG. 3 is a table showing the population frequency of selected HLA haplotypes across multiple ethnic groups, highlighting limited global coverage of HLA-A*02:01.

[0027] FIG. 4 displays UMAP projections of CD4+ T cells colored by gene expression and by clonotype, illustrating transcriptional heterogeneity and donor-specific clustering.

[0028] FIG. 5 is a table listing the full alpha and beta chain sequences of five validated CD4+ TCRs (H1-H5) used in therapeutic development.

[0029] FIGs. 6A-6D are tables listing CD4+ TCR clonotypes with full alpha and beta chain sequences, peptide reactivity, phenotypic and functional annotations, and HLA restriction across multiple patients and timepoints.

[0030] FIG. 6E shows UMAP plots visualizing the spatial and temporal distribution of clonally expanded HPV-specific CD4+ T cells, annotated by functional phenotype and donor identity.

[0031] FIGs. 7A-7G are tables listing CD8+ TCR clonotypes with corresponding CDR3 sequences, V(D)J gene usage, cell type, clone ID, and antigen specificity annotations derived from high-throughput sequencing and functional profiling.

[0032] FIGs. 7H-7W are tables presenting dominant phenotypes, clonal sizes, full TCR sequences, and HLA allele restrictions for CD8+ TCR clonotypes identified in FIGs. 7A-7G.

[0033] FIGs. 7X-7AA are tables annotating the same CD8+ TCR clonotypes from FIGs. 7A-7W with their corresponding pathogen targets, recognized antigens, and HLA-peptide complexes across multiple viral infections including HPV, EBV, CMV, and influenza.

[0034] FIGs. 8A-8C are tables providing the full-length nucleotide and amino acid sequences of five validated CD4+ TCRs, along with their CDR3 regions, HLA restriction, and mapped antigenic peptides and epitopes.

[0035] FIG. 9 illustrates the design and functional validation of the SCT-based platform for TCR screening, including construct design, tetramer-based enrichment, single-cell sorting, and antigen-specific activation assays.

[0036] FIG. 10 illustrates the assembly, expression, and functional characterization of SCT constructs used for antigen presentation, including plasmid design, protein production, and tetramer validation through flow cytometry and biochemical analysis.

[0037] FIG. 11 shows flow cytometry plots from multiple donors demonstrating doublepositive tetramer staining of CD4+ T cells specific for SCT-presented antigens.

[0038] FIG. 12 presents the application of the SCT-based screening platform to SARS-CoV- 2, including longitudinal CD4+ T cell profiling from patient samples, epitope-specific TCR identification, clonal diversity analysis, and functional validation via tetramer staining and peptide activation assays.

[0039] FIG. 13 presents quantitative analyses of antigen-specific CD4+ T cell clonotypes, including epitope mapping, clonal expansion and persistence, functional phenotyping, and crosspatient TCR sharing.

[0040] FIG. 14 presents longitudinal transcriptomic and phenotypic analysis of antigenspecific CD4+ T cells across recovery stages, including UMAP clustering, gene expression heatmaps, antigen cluster immunodominance, and phenotype distributions over time.

[0041] FIG. 15 shows the knock-in efficiency of TCR constructs in primary CD4+ versus CD8+ T cells, demonstrating higher transduction rates in the CD4+ subset.

[0042] FIG. 16 summarizes the discovery, engineering, and functional validation of HPV- specific CD4+ TCRs, including antigen binding, cytokine secretion, and cytotoxic activity in engineered primary T cells.SEQUENCES

[0043] Any nucleic acid and amino acid sequences listed herein are shown using standard letter abbreviations for nucleotide bases and amino acids, as defined in 37 C.F.R. § 1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

[0044] SEQ ID NOs. for amino acid sequences and nucleic acid sequences are listed in 57ISB19-PCT_SL.xml.DETAILED DESCRIPTION

[0045] The present invention provides compositions and methods for identifying, engineering, and applying T cell receptors (TCRs) that target tumor-associated antigens, including antigens derived from human papillomavirus (HPV). In particular, the invention discloses a panel of both CD4+ and CD8+ TCRs that have been isolated and characterized for their specificity against HPV epitopes. These TCRs recognize viral peptides presented by major histocompatibility complex (MHC) molecules, including both class I and class II alleles, enabling cytotoxic and helper T cell responses in adoptive immunotherapy applications.

[0046] A total of five CD4+ TCRs were fully validated in this work, each showing antigenspecific binding, surface expression, and functional activity following TCR gene transfer into primary T cells. These validated TCRs — referred to herein as Hl through H5 or TCR 401-405 — target HPV-derived peptides presented by HLA-DRBl*01 :01 and exhibit cytokine secretion and cytotoxic potential in vitro. Additionally, a set of CD8+ TCR clonotypes are disclosed, with full alpha and beta chain sequences, phenotypic annotations, and defined HLA class I restrictions. Together, these TCRs form a foundation for the development of T cell therapies with broad clinical relevance, including for HPV-positive cancers such as cervical, head and neck, and anal carcinomas.

[0047] In some embodiments, the invention provides a high-throughput platform for the discovery and characterization of antigen-specific T cell receptors (TCRs). The platform utilizes synthetic class II single-chain trimer (SCT) constructs encoding viral, tumor-associated, or other immunologically relevant antigens to enable MHC-restricted tetramer staining and single-cell sorting of reactive T cells from primary donor samples. Notably, the use of SCT designs to classII MHC molecules for high-throughput screening and TCR discovery represents a novel advancement. By integrating tetramer-based enrichment with transcriptome-coupled TCR sequencing, the methodology preserves the native transcriptional phenotype of captured T cells and allows for rapid identification of antigen-specific TCR clonotypes across diverse donors and longitudinal timepoints. In certain embodiments, the platform may be used not only to isolate TCRs with therapeutic potential, but also to characterize adaptive immune responses in the context of vaccination, infectious disease, or cancer immunotherapy.

[0048] FIG. 1 shows a table listing CD8+ T cell receptor (TCR) clonotypes identified using the disclosed high-throughput screening method. Each entry includes the associated cell type, clone identifier, CDR3 amino acid sequences for both the alpha and beta chains, and the V, D, and J gene segments used in TCR assembly. These clonotypes were isolated based on antigenspecific reactivity to HPV-derived peptides and restricted by MHC class I alleles. The data demonstrate a diverse range of CD8+ TCR sequences with unique V(D)J recombination patterns and CDR3 motifs, supporting the capacity of the platform to capture broad clonal diversity from donor-derived peripheral blood mononuclear cells (PBMCs). These sequences form the basis for downstream engineering, phenotypic validation, and therapeutic application.

[0049] FIG. 2 shows a table summarizing the anatomical distribution of HPV-associated malignancies, listing cancer types and tumor sites known to be driven by HPV infection. The table categorizes cancers such as cervical, head and neck, anal, vulvar, vaginal, and penile carcinomas, linking each to HPV etiology. This figure provides clinical context for the disclosed TCRs by illustrating the range of malignancies for which HPV-specific T cell responses may be therapeutically relevant. The broad tissue tropism of HPV underlines the importance of developing TCR-based immunotherapies that are applicable across multiple cancer types and patient populations.

[0050] FIG. 3 shows a table presenting the population frequencies of selected HLA class I haplotypes across five major ethnic groups. The data highlight the uneven distribution of common alleles such as HLA-A*02:0I, which is highly prevalent in individuals of European ancestry but less frequent in African American, Asian Pacific Islander, Hispanic, and Native American populations. This disparity underscores a key limitation of immunotherapies that rely exclusively on A02:01 -restricted TCRs. By contrast, the present invention includes TCRsrestricted by additional alleles with broader population coverage, thereby improving the accessibility and applicability of TCR-based therapies across genetically diverse patient populations.

[0051] FIG. 4 shows UMAP projections of CD4+ T cells colored by the expression of representative genes and by TCR clonotype. The left panel displays the distribution of cells according to the expression levels of GZMB, CCR7, GZMK, and SELL, revealing functional heterogeneity among the antigen-specific population. Cells expressing cytotoxic markers such as GZMB and GZMK occupy distinct clusters compared to those enriched for lymphoid homing markers such as CCR7 and SELL. The right panel highlights specific TCR clonotypes mapped onto the same transcriptional space, illustrating the spatial localization and phenotypic diversity of individual TCR-expressing cells. This integrated analysis enables the pairing of TCR identity with functional state, facilitating the selection of therapeutic candidates with defined phenotypes.

[0052] FIG. 5 shows a table listing the full-length T cell receptor (TCR) alpha and beta chain sequences of five validated CD4+ TCRs, designated Hl through H5 or TCR 401 through 405. These sequences were derived from single-cell sequencing of tetramer-positive CD4+ T cells obtained from human donors screened using the SCT-based discovery platform. Each TCR includes defined V and J gene segments, along with the unique CDR3 regions that confer antigen specificity. The sequences disclosed in this figure represent complete, functional receptors capable of recognizing HPV-derived peptides presented by HLA class II molecules, specifically HLA-DRB 1*01 :01. These five receptors are disclosed herein as compositions of matter, and may be used for adoptive T cell therapy, antigen-specific immune monitoring, or the generation of engineered cell products.

[0053] The functional activity of TCRs Hl through H5 was confirmed through a multi-step validation process. Each TCR was cloned into lentiviral vectors or introduced by CRISPR knock-in into primary CD4+ T cells from healthy donors. Transduced T cells were assessed for surface expression of the TCR, antigen-specific tetramer binding, and cytokine production following stimulation with HPV-derived peptides. Functional assays included quantification of IL-2, IFNy, TNFa, and GZMB secretion, as well as cytotoxicity assays using peptide-pulsed target cells. All five TCRs showed reproducible antigen recognition and activation profiles, confirming their capacity to mediate class Il-restricted immune responses. These receptorstherefore represent clinically relevant, fully characterized TCRs with defined specificity, sequence, and function, and can be used individually or as a panel in therapeutic or diagnostic applications. It will be appreciated that variants of these TCRs containing minor amino acid substitutions that do not materially alter antigen specificity or HLA restriction may also be suitable for use, and are encompassed within the scope of the present disclosure.

[0054] FIG. 6A-6E disclose, in various embodiments, a set of antigen-specific CD4+ T cell receptors (TCRs) identified and characterized in the context of human papillomavirus (HPV) infection. These figures collectively illustrate the identity, antigen specificity, phenotypic features, and transcriptional context of the disclosed TCRs. In particular, FIG. 6A through 6D provide tabular data for 101 distinct CD4+ TCR clonotypes, while FIG. 6E contextualizes their phenotypic localization within the CD4+ T cell landscape using single-cell transcriptomic projections.

[0055] FIG. 6A displays metadata for the disclosed CD4+ TCRs. The column labeled T Cell Reference provides a unique identifier for each clonotype (e.g., SEQ l, SEQ 2). The T cell type column specifies CD4 as the T cell lineage. Clonally persistent or expanded indicates whether the TCR clonotype was found to be persistent across timepoints or expanded in frequency, suggesting immunological relevance. The TCR sequence field lists the full amino acid sequence of the paired TCR a and P chains. # Occurrences refers to the number of times that specific TCR was recovered across the sampled data. Phenotype reflects the differentiation status of the cell (e g., naive, effector memory, cytotoxic), and is determined from single-cell RNA sequencing and protein expression data. Leiden refers to the Leiden cluster number derived from unsupervised clustering of the transcriptomic data. Clonotype ID and Patient (optional) indicate the subject from which the TCR was derived, and may include an anonymized patient identifier. Timepoint refers to the sampling interval (e.g., screen, wkl5, 7dpm) relative to diagnosis or treatment.

[0056] FIG. 6B provides information on antigen specificity. Peptides 0 through 4 correspond to viral epitopes shown to stimulate T cells bearing the respective TCR. Antigen 0 through Antigen 4 identify the peptide’s source (e.g., E6_129-142), referencing the HPV protein (e.g., E6 or E7) and the position of the epitope. Core peptide (hpv) refers to the core epitope believed to be presented in the HLA groove, often informed by motif analysis. The HLA allele column (e.g.,DRB1*O1 :O1) denotes the presenting class II major histocompatibility complex (MHC) restriction, based on tetramer validation or inference. Note (optional) may include further annotation regarding peptide validation or sequence overlaps.

[0057] FIG. 6C and FIG. 6D continue this tabular presentation, collectively covering TCRs SEQ 26 through SEQ 101. These figures follow the same column format as described above and continue to disclose peptide reactivity, HLA restriction, and antigen breadth. Across the entire dataset, TCRs show specificity for multiple epitopes within HPV E6 and E7 proteins, and are restricted predominantly to HLA-DRB 1*01 :01. Some TCRs are shown to cross-recognize additional viral peptides (e.g., influenza, EBV), highlighting potential applications beyond HPV.

[0058] FIG. 6E shows UMAP (Uniform Manifold Approximation and Projection) plots that visualize the transcriptomic positioning of individual T cells bearing select clonotypes across donors and timepoints. The first six panels highlight longitudinal tracking of clonally expanded TCRs from specific patients across timepoints (e.g., screen, wkl5, 7 / 8m), while the final panel overlays Leiden cluster annotations, identifying transcriptionally defined states such as naive, cytotoxic, and effector memory (TEM). These phenotypic states were inferred from gene expression and marker profiling, offering insight into functional orientation of TCR-bearing cells.

[0059] It will be appreciated that the sequences disclosed in FIG. 6A-6D represent only exemplary embodiments of TCRs that are specific for HPV-derived antigens. It will be appreciated that other TCRs bearing high sequence similarity, including conservative amino acid substitutions, insertions, deletions, or recombined variable (V), diversity (D), or joining (J) gene segments, that retain binding specificity for the same antigens and MHC restriction, fall within the scope of the present disclosure. Functionally similar TCRs may be generated through natural immune processes or by rational engineering and are intended to be encompassed by the claimed embodiments.

[0060] FIG. 7, comprising panels 7A through 7AA, discloses, in some embodiments, a comprehensive dataset describing a large panel of CD8+ T cell receptors (TCRs) identified as specific to human papillomavirus (HPV) antigens. In various embodiments, these TCRs are derived from donor samples analyzed using the methods described herein, including high- throughput sequencing, HLA typing, and functional validation. Each panel in FIG. 7 presentsdifferent aspects of these TCRs, encompassing their sequence composition, phenotypic characteristics, antigen specificity, and contextual HLA restriction.

[0061] FIG. 7A through 7G provide a listing of the full-length TCR a and P chain sequences for each CD8+ TCR identified. The table includes the clone ID, the CDR3a and CDR3P amino acid sequences, and the associated TCR gene segments used, including TRAV (T cell receptor alpha variable), TRAJ (T cell receptor alpha joining), TRBV (T cell receptor beta variable), and TRBJ (T cell receptor beta joining) gene usage. These data enable reconstruction of the full receptor sequence for therapeutic, diagnostic, or screening use. In some embodiments, the sequences disclosed in FIG. 7A-G may be used to synthetically reconstruct TCRs for clinical or research application. It will be appreciated that TCRs with similar or homologous sequences capable of recognizing the same antigen-HLA complex would fall within the scope of this disclosure.

[0062] FIG. 7H through 7W characterize functional and phenotypic properties of the same TCRs. For each TCR, this table includes the clone size (i.e., the number of cells from which the same TCR was recovered), the dominant T cell phenotype (e.g., cytotoxic, memory), and the restricting HLA allele. The dominant phenotype was determined using single-cell transcriptomic or flow cytometry profiling, and includes functional designations such as cytotoxic, memory, or naive phenotypes. The HLA field indicates one or more class I HLA alleles (e.g., A0201, Al 101) inferred to restrict antigen recognition for that particular TCR, as determined through HLA typing and tetramer or peptide-MHC multimer analysis. The presence of clonally expanded cells with a cytotoxic phenotype across multiple donors supports the immunological relevance and therapeutic potential of the disclosed TCRs.

[0063] FIG. 7X through 7AA provide functional antigen specificity for each CD8+ TCR shown in the previous panels. This includes the source organism from which the antigen is derived (e.g., HPV, Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), or Influenza), the specific antigenic protein (e.g., HPV E6 or E7 oncoproteins), and the precise HLA-restricted peptide epitope recognized. These panels disclose the HLA:peptide:TCR triplet required for antigen recognition. For example, certain TCRs are shown to recognize the HP VI 6 E6 129-142 peptide in the context of HLA-A*02:01. The tables further disclose multi-antigen specificity in some cases, reflecting TCR cross-reactivity or dual specificity across pathogens. It will beunderstood that TCRs with minor amino acid variations that retain recognition of the same antigen-HLA complexes are considered within the scope of the invention, particularly where such variants arise from natural allelic variation, somatic mutation, or engineered optimization.

[0064] Taken together, FIG. 7 presents a comprehensive atlas of CD8+ TCRs with relevance to HPV-specific immunity. The TCRs disclosed herein, in some embodiments, may be used for T cell engineering, vaccine design, immune monitoring, or as therapeutic agents in adoptive cell therapy. The level of sequence and contextual detail disclosed enables both replication and extension of these findings to new clinical applications.

[0065] FIG. 8A-C discloses exemplary embodiments of T cell receptors (TCRs) that have been functionally validated for recognition of human papillomavirus (HPV) antigens. These figures present both the nucleotide and amino acid sequences for the TCR alpha and beta chains, their respective complementarity-determining region 3 (CDR3) sequences, and the antigenic peptides recognized by each TCR. In particular, FIG. 8A provides the full-length alpha chain amino acid sequences and the corresponding DNA sequences (TCRa dna) for five validated TCRs (TCRs: 401-405). FIG. 8B shows the corresponding beta chain DNA sequences (TCRp dna), along with the CDR3a and CDR3P regions that contribute to antigen specificity. FIG. 8C details the cognate peptide epitopes, HLA restriction (in this case, DRBl*01:01), and alternate peptides tested for recognition by these TCRs.

[0066] These TCRs recognize antigenic peptides derived from the HPV E6 protein, including but not limited to sequences such as KQRFHNIRGRWTG, GTITLEQQYNKPLCDLL, and HLDKKQRFHNIRGRWTGR. Each of the peptides is presented in the context ofHLA- DRB 1*01 :01, and recognition has been demonstrated through tetramer staining and peptide stimulation assays. As such, these sequences may be used in therapeutic, diagnostic, or research applications where specific targeting of HPV-positive cells is desired, including but not limited to HPV-associated cancers such as cervical, anal, vulvar, penile, and oropharyngeal cancers.

[0067] It will be appreciated that the invention is not limited to the exact TCR sequences shown in FIG. 8A-C. In some embodiments, functionally equivalent variants are also contemplated, including TCRs comprising conservative amino acid substitutions within the CDR3 regions or synonymous nucleotide substitutions that do not alter the encoded protein.TCRs that differ slightly but retain specificity for the same peptide-HLA complex, and exhibit similar binding and / or functional activity, fall within the scope of this disclosure.

[0068] The TCRs disclosed in FIG. 8A-C may be used in engineered T cell therapies, including TCR-transduced T cells, wherein a patient's T cells are modified to express one or more of the disclosed sequences. Additionally, these sequences may be incorporated into soluble TCR constructs, bispecific molecules, or other antigen-recognition platforms. In some embodiments, they may serve as reference sequences for screening, optimizing, or validating other HPV-targeted receptors using the high-throughput methods also disclosed herein. These TCRs thus form a part of a broader strategy for identifying, characterizing, and applying TCRs directed against viral antigens in the context of cancer or other disease.HIGH-THROUGHPUT PLATFORMFOR ANTIGEN- SPECIFIC TCR DISCOVERY

[0069] To further support the broad screening aspects of the present disclosure, the following section describes in detail the general methodology for constructing and applying SCT-based libraries to identify antigen-specific T cells. Representative figures are briefly referenced herein for illustrative purposes; however, detailed descriptions of these figures and associated experimental data are provided in the subsequent Examples section. This methodology is applicable across a range of Class I and Class II MHC targets and is not limited to the specific examples set forth below.METHODSSCT Construction

[0070] General SCT construction and use are disclosed in WO 2021168122 (Heath et al., 2021), WO 2022236102 (Chour et al., 2022), and WO 2023205728 (Chour et al., 2023), which references are incorporated herein in their entirety.

[0071] In some embodiments, single-chain trimers (SCTs) may be engineered by genetically linking antigenic peptides directly to the extracellular domains of MHC molecules to form a stabilized complex capable of T cell receptor (TCR) engagement. For Class II SCTs, the design may involve fusing the N-terminus of a selected peptide to the a-chain via a flexible polypeptidelinker (Linker 1 or LI), and the C-terminus of the peptide to the P-chain via a second flexible polypeptide linker (Linker 2 or L2). A partial invariant chain (pli) segment may also be incorporated adjacent to the P-chain to promote correct folding and stabilization of the class II heterodimer. Class I SCTs may be constructed using a similar approach, where the peptide is fused between p2-microglobulin and the MHC class I a-chain through analogous linkers.

[0072] In certain embodiments, the SCT designs may be optimized for enhanced expression, stability, and biotinylation efficiency. Linker sequences may be adjusted to allow appropriate peptide flexibility while maintaining MHC binding groove conformation. Stabilizing mutations may be optionally introduced within the a- or P-chains to further promote proper folding during recombinant expression. The SCT constructs may be designed to include affinity purification tags, such as a C-terminal His-tag, to facilitate downstream processing.

[0073] In one illustrative example, Class II SCT constructs were generated encoding overlapping peptide sequences spanning the receptor-binding domain (RBD) of the SARS-CoV- 2 spike protein. The peptide sequences were genetically linked to HLA-DRB 1*04:01 a- and P- chain domains through flexible glycine-serine rich linkers. A pli segment was incorporated adjacent to the P-chain to stabilize the heterodimeric structure. Plasmid constructs encoding the SCTs were cloned into mammalian expression vectors and transiently transfected into Expi293 cells to achieve high-yield recombinant expression. Purification was performed using immobilized metal affinity chromatography (IMAC), exploiting the incorporated His-tag. As shown in FIG. 9, the resulting SCTs exhibited correct folding and were amenable to downstream multimerization via biotinylation.

[0074] The SCTs produced by these methods demonstrated enhanced stability compared to traditional peptide-MHC complexes, retaining the ability to specifically bind cognate TCRs under staining and sorting conditions. The stabilized, covalently-linked configuration of the SCTs reduced dissociation rates during multimer staining procedures, improving the sensitivity and reproducibility of antigen-specific T cell detection across multiple donors and experimental platforms.Library Assembly

[0075] In some embodiments, libraries of SCT constructs may be assembled to enable broad, multiplexed screening of antigen-specific T cell responses. SCT libraries may comprise panels ofconstructs encoding distinct peptide antigens derived from pathogenic organisms, tumor- associated proteins, autoantigens, or other relevant targets. Peptides may be selected to tile across full-length proteins, specific domains, or epitopic hotspots, typically using overlapping windows of approximately 13-25 amino acids in length to ensure full coverage of potential MHC class II binding registers. Each SCT construct within a library may incorporate a unique peptide while maintaining a standardized MHC framework to allow consistent expression and analysis.

[0076] In certain embodiments, library design may be informed by genomic, proteomic, or immunopeptidomic data sources to prioritize antigen candidates. Libraries may be customized for specific HLA alleles, patient cohorts, or disease settings. Computational tools may be employed to optimize peptide tiling strategies, reduce redundancy, and ensure inclusion of immunologically relevant sequences. Libraries may also be subdivided into smaller pools based on functional annotation, protein localization, or predicted immunogenicity to facilitate targeted screening approaches.

[0077] In one illustrative example, a 54-element Class II SCT library was constructed encoding overlapping peptide sequences spanning the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Each peptide was genetically fused to HLA-DRB 1*04:01 a- and 0- chain domains using flexible glycine-serine linkers, with a partial invariant chain segment incorporated to enhance stability. Plasmid constructs for the full library were generated by high- throughput cloning, using standardized backbones compatible with mammalian expression systems. SCT proteins were produced by transient transfection of Expi293 cells and subsequently purified using immobilized metal affinity chromatography (IMAC). Biotinylation was performed post-purification to enable multimer assembly for downstream T cell staining. The construction and production of the SCT library are further illustrated in FIG. 9.

[0078] The resulting library enabled parallel interrogation of dozens of antigenic epitopes in a single staining and sequencing workflow. This approach facilitated broad, unbiased identification of SARS-CoV-2-specific CD4+ T cells without reliance on computational binding predictions or pre-screened epitopes, demonstrating the scalability and flexibility of the SCT- based library generation strategy.Labeling and Staining:

[0079] In some embodiments, purified SCT proteins may be multimerized by conjugation to streptavidin-fluorophore complexes to facilitate high-avidity binding to antigen-specific T cells. SCTs may be biotinylated during or following purification, allowing site-specific attachment to streptavidin molecules labeled with fluorescent dyes such as phycoerythrin (PE), allophycocyanin (APC), or Brilliant Violet (BV) variants. Multimerization may enhance the apparent binding affinity of the SCT constructs for cognate TCRs, improving detection sensitivity in heterogeneous T cell populations.

[0080] In certain embodiments, multimerized SCTs may be pooled to create staining cocktails comprising multiple specificities. Cells to be stained may include peripheral blood mononuclear cells (PBMCs), tumor-infiltrating lymphocytes (TILs), or other lymphoid-derived populations. Prior to staining, cell populations may be enriched for CD4+ or CD8+ subsets using magnetic bead selection or fluorescence-based gating strategies to increase assay sensitivity. Cells may be incubated with the SCT multimers under optimized conditions to preserve TCR conformational integrity and minimize non-specific interactions. Negative control SCTs presenting irrelevant peptides may be included in the staining panel to assess background binding.

[0081] In one illustrative example, biotinylated Class II SCTs encoding SARS-CoV-2 spike- derived peptides were multimerized with streptavidin-PE conjugates and pooled for T cell staining. PBMCs enriched for CD4+ T cells were incubated with the SCT multimer pools at 4°C to minimize internalization and off-target binding. Following incubation, cells were washed and subjected to flow cytometric analysis to identify multimer-positive T cells based on PE fluorescence intensity. Negative controls included SCTs loaded with non-coronavirus peptides and samples stained with streptavidin-PE alone. Representative staining and sorting procedures are illustrated in FIG. 10.

[0082] The optimized labeling and staining workflow enabled robust, reproducible detection of low-frequency antigen-specific T cells across multiple donors. High multimer stability and minimized non-specific staining improved signal-to-noise ratios, facilitating downstream sorting and sequencing applications.Single-Cell Sorting and Sequencing:

[0083] In some embodiments, following staining with SCT multimers, antigen-specific T cells may be isolated through fluorescence-activated cell sorting (FACS) based on fluorescent multimer binding profiles. Cells demonstrating positive binding to SCT multimers may be individually captured into wells, tubes, or microfluidic droplets for subsequent single-cell analysis. Surface marker staining panels may optionally be incorporated during FACS to allow simultaneous phenotypic characterization of antigen-specific T cell subsets, including memory, activation, or exhaustion markers.

[0084] In certain embodiments, sorted cells may be processed for single-cell RNA sequencing to profile transcriptomic states at high resolution. Concurrent recovery of paired TCRa and TCRp chain sequences may also be performed to establish clonotypic identities and link specific TCR sequences to corresponding transcriptomic profiles. Technologies such as droplet-based capture platforms, microwell arrays, or plate-based single-cell systems may be utilized depending on experimental design preferences and throughput requirements.

[0085] In one illustrative example, multimer-positive CD4+ T cells from SARS-CoV-2- infected individuals were sorted using high-parameter FACS after staining with pooled Class II SCT multimers. Cells were stained simultaneously with a panel of antibodies against key phenotypic markers (e.g., CD45RA, CCR7, PD-1) to allow subset discrimination during analysis. Sorted cells were processed using a droplet-based single-cell RNA sequencing platform, yielding paired transcriptome and TCRa / p sequence information. To enable multiplexing across individuals and time points, cells were labeled with sample-specific DNA hashtags prior to pooling. Whole genome sequencing (WGS) of donor material was utilized to resolve genetic identity and facilitate demultiplexing. The workflow for multimer-based sorting and single-cell analysis is depicted in FIGS. 10 and 11.

[0086] This integrated approach enabled comprehensive characterization of antigen-specific T cells at the clonal, transcriptional, and phenotypic levels, providing detailed insights into immune dynamics across longitudinal samples and diverse patient cohorts.Data Analysis:

[0087] In some embodiments, data obtained from single-cell sequencing of antigen-specific T cells may be analyzed to characterize the clonal architecture, phenotypic diversity, and functional states of the immune response. Captured TCRa and TCRp sequences may beassembled and clustered to define clonotypes based on shared CDR3 regions. Clonal expansion, persistence over time, and repertoire diversity metrics may be calculated to assess the magnitude and quality of the antigen-specific T cell response. Single-cell transcriptomic data may further be utilized to assign T cells to functional subsets based on gene expression signatures.

[0088] In certain embodiments, T cells may be categorized into subsets such as naive, central memory, effector memory, Thl, Thl7, regulatory (Treg), exhausted, or cytotoxic lineages by mapping transcriptional profiles against reference atlases or through unsupervised clustering. Integration of clonotype and phenotype information may allow tracking of clonal trajectories over time, identification of dominant antigen-specific TCRs, and correlation of T cell function with clinical outcomes. Epitope-specific immunogenicity may be inferred by evaluating the breadth and persistence of TCR clonotypes associated with individual SCT antigens.

[0089] In one illustrative example, single-cell transcriptomic profiles and TCRa / p sequences were analyzed from multimer-positive CD4+ T cells sorted after SARS-CoV-2 infection. TCR clonotypes expanded during acute infection were tracked longitudinally across memory time points. Single-cell RNA-seq data enabled assignment of cells into Thl, Treg, effector memory, and exhausted subsets, revealing phenotypic shifts over time. Immunogenicity assessments showed that certain spike-derived epitopes elicited broader and more durable TCR responses compared to others. Exemplary data analysis approaches and representative findings are shown in FIG. 11.

[0090] This comprehensive analytical framework provided high-resolution insights into the antigen-specific T cell landscape, revealing dynamic changes in clonal composition, functional phenotypes, and immune memory over time, and validating the power of SCT-based screening platforms for longitudinal immunological studies.Validation Techniques

[0091] In some embodiments, validation of the SCT-based screening methodology may be performed to confirm the specificity, stability, and functional integrity of the generated SCT reagents. Validation steps may include binding specificity assays, comparative multimer binding studies, structural quality assessments, and functional activation assays. These procedures ensure that the SCT constructs maintain critical biological properties necessary for effective antigenspecific T cell identification.

[0092] In certain embodiments, SCT tetramers may be constructed using well-characterized antigenic peptides paired with corresponding T cell receptors (TCRs) to verify selective binding. Binding assays may involve incubating the SCT tetramers with Jurkat cells engineered to express the cognate TCR and assessing fluorescent multimer binding via flow cytometry. Non-cognate TCR-expressing cells and irrelevant SCT tetramers may be included as negative controls to evaluate non-specific binding background.

[0093] In one illustrative example, SCT tetramers incorporating known viral peptides were generated and tested for binding against cognate TCR-expressing Jurkat cells. The resulting tetramers specifically bound their matching TCRs without detectable non-specific interactions. Additionally, SCT tetramers encoding influenza-derived peptides were compared to conventional peptide-MHC (pMHC) multimers. Binding efficiency, staining brightness, and stability were found to be comparable or superior in the SCT reagents relative to standard multimers.

[0094] Structural quality and post-translational modifications of SCT constructs may also be assessed. In certain embodiments, glycosylation status, including the presence of N-linked glycosylation motifs, may be evaluated using biochemical assays or mass spectrometry. Maintenance of native-like glycosylation patterns may enhance physiological relevance for TCR binding fidelity. In one example, SCT constructs were confirmed to preserve critical glycosylation profiles, supporting their structural authenticity.

[0095] Functional validation of captured TCRs may further be performed to ensure biological relevance. In some embodiments, captured TCR sequences may be cloned into NFAT- GFP reporter cell lines, enabling assessment of TCR activation upon antigen exposure. Coculture experiments may be conducted with antigen-presenting cells pulsed with cognate peptides to determine the functionality of the TCRs identified through SCT capture. In one illustrative example, TCRs isolated using SCT multimers exhibited specific NF AT -mediated GFP activation upon recognition of the appropriate antigen stimulus, confirming the functional competence of the selected T cell clones.

[0096] Validation data supporting the specificity, functionality, and stability of SCT constructs are illustrated in FIGS. 12-16.

[0097] In some embodiments, the SCT-based screening platform described herein offers multiple advantages over conventional techniques for identifying antigen-specific T cellreceptors (TCRs). By covalently linking antigenic peptides to major histocompatibility complex (MHC) domains, SCTs provide enhanced stability, reduced dissociation rates, and improved yield compared to traditional peptide-MHC (pMHC) complexes. This stabilized configuration permits multimerization and staining protocols that are more robust and reproducible, particularly when screening rare antigen-specific T cell populations.

[0098] In certain embodiments, the use of SCT libraries enables broad, unbiased antigen discovery without reliance on computational epitope prediction or prior knowledge of immunodominant regions. Libraries can be rapidly constructed and scaled to include hundreds of candidate antigens, allowing for simultaneous interrogation of diverse peptide repertoires in a single experimental workflow. The SCT platform supports the use of both Class I and Class II MHC molecules, enabling comprehensive profiling of CD8+ and CD4+ T cell responses across infectious disease, oncology, autoimmunity, and other fields.

[0099] In one illustrative application, the platform enabled the identification and phenotypic characterization of over 2,000 SARS-CoV-2-specific CD4+ T cells across a longitudinal cohort, defining clonal trajectories, persistence patterns, and epitope-specific TCR repertoires up to two years post-infection. In another embodiment, SCT libraries targeting human papillomavirus type 16 (HPV-16) oncogenic proteins were used to isolate tumor-reactive CD4+ TCRs with therapeutic potential. These examples demonstrate the general applicability, seal ability, and versatility of the SCT-based screening methodology.

[0100] Unless otherwise indicated, all techniques, procedures, and methodologies described herein, including those in the examples, are carried out using conventional and well-established protocols in the relevant fields of molecular biology, biochemistry, pharmacology, or related disciplines. Such methods are either explicitly described or are readily understood and accessible to those of ordinary skill in the art, and are deemed to be within the scope of the present disclosure. The mention of such techniques is not intended to imply that they are prior art or known to be part of the public domain unless specifically stated.

[0101] The following examples describe specific applications and implementations of the SCT-based screening methodology disclosed herein. These examples are intended to further illustrate the advantages and versatility of the platform for identifying antigen-specific T cells.However, the scope of the present disclosure is not limited by the examples, but rather is defined by the appended claims.EXAMPLES

[0102] The following examples illustrate specific applications and embodiments of the high- throughput SCT-based platform described above. Unless otherwise indicated, the methods employed were carried out substantially as described in the Methodology section. These examples are provided for illustrative purposes only and are not intended to limit the scope of the disclosure, which is defined by the appended claims. It will be appreciated that variations in the experimental design, including modifications to the antigen screening platform, TCR capture techniques, and validation assays, may be implemented without departing from the spirit or scope of the invention. Functionally equivalent approaches for isolating, characterizing, or validating antigen-specific T cell receptors (TCRs) are considered as falling within the scope of the present disclosure.

[0103] In one embodiment, a high-throughput platform was developed to identify antigenspecific CD4+ and CD8+ TCRs using single-chain trimer (SCT) libraries encoding peptide- MHC class II or class I complexes. Synthetic SCT constructs were designed to present HPV- derived peptides in the context of specific HLA alleles, such as HLA-DRB101 :01 for class II and HLA-A02:01 for class I. These SCTs were expressed, biotinylated, and assembled into tetramers for antigen-specific cell capture. Peripheral blood mononuclear cells (PBMCs) from multiple human donors were stained with tetramer libraries, and antigen-specific CD4+ and CD8+ T cells were sorted for downstream analysis.

[0104] Tetramer-positive cells were subjected to single-cell RNA sequencing (scRNA-seq) to obtain paired TCRa and TCRP chain sequences along with transcriptomic profiles. This enabled the simultaneous identification of antigen-specific clonotypes and characterization of their transcriptional phenotype, including naive, effector, memory, and cytotoxic subtypes. Clonotypes that were persistent across timepoints or enriched following antigen exposure were prioritized for further analysis.

[0105] A subset of CD4+ TCRs identified through this approach were selected for experimental validation. These receptors were cloned into lentiviral vectors or introduced into primary human T cells via CRISPR-mediated knock-in. Engineered cells were tested for tetramerbinding, cytokine secretion following peptide stimulation, and cytotoxic activity against antigenexpressing target cells. Five TCRs (designated H1-H5 and also referred to as TCR 401-405) were confirmed to exhibit robust, antigen-specific responses and are disclosed herein with full- length sequences and functional annotations.

[0106] FIG. 9 shows an exemplary embodiment of a high-throughput screening platform used to identify antigen-specific T cell receptors (TCRs). The figure demonstrates the design and utility of a single-chain trimer (SCT) approach for isolating functional TCRs through tetramer binding and peptide activation analysis.

[0107] Panel a depicts the structure of a representative class II SCT construct used in the screening process. The SCT encodes an MHC class II molecule composed of a covalently linked a and P chain connected via flexible linkers (LI and L2), with a target antigen inserted between the chains. The construct further includes an AviTag and HisTag to facilitate biotinylation and purification.

[0108] Panel b illustrates the experimental workflow used to validate candidate TCRs. TCRa and TCR chains were cloned and introduced into Jurkat reporter cells via lentiviral transduction. Cells expressing the engineered TCRs were stained with SCT tetramers presenting cognate antigens, enabling assessment of antigen-specific binding.

[0109] Panel c shows the specificity of SCT tetramers for various HLA alleles. Tetramer binding was evaluated in TCR-expressing Jurkat cells using different HLA class II alleles, including DRB 101 :01, DRB111 :01, DRB113:01, and DRB 104:01. Binding efficiency varied across alleles, with DRB 1*01 :01 yielding the highest percentage of tetramer-positive cells, highlighting the utility of this allele for broad population coverage in certain embodiments.

[0110] Panel d presents the generation and validation of SCT tetramer libraries. Multiple SCT constructs were synthesized, expressed, and biotinylated to form tetramers. The figure illustrates SDS-PAGE validation of expression and tetramer integrity, along with a schematic showing the general tetramer screening workflow applied to primary CD4+ T cells derived from peripheral blood mononuclear cells (PBMCs). Cells were enriched and stained with pooled SCT tetramers, sorted, and profiled by single-cell RNA and TCR sequencing.

[0111] Panel e shows data from the screening of CEFT-specific CD4+ TCRs. In the upper graph, candidate TCRs were tested for SCT tetramer binding, and percent tetramer binding is reported for each receptor, indicating the identification of multiple CEFT-reactive clones. The lower graph reports peptide activation using an NFAT-GFP Jurkat assay, where engineered Jurkat cells expressing individual TCRs were co-cultured with K562 antigen-presenting cells loaded with the cognate CEFT peptide. Activation was quantified via GFP expression. As shown, most TCRs were activated only in the presence of cognate peptide and not by a negative control, confirming their antigen specificity.

[0112] Panel f shows a schematic representation of the NFAT-GFP activation assay. Jurkat cells engineered with a reporter construct for nuclear factor of activated T-cells (NFAT) driving GFP expression were used to test activation upon recognition of peptide-presenting target cells.

[0113] In some embodiments, the screening strategy illustrated in FIG. 9 may be used to identify TCRs against any antigen of interest, including infectious disease antigens, tumor- associated antigens, or neoantigens. It will be appreciated that variations in SCT linker design, epitope insertion site, or tetramer generation may be employed without departing from the scope of the invention. Likewise, alternative cell lines or primary cells may be substituted in the TCR validation steps.

[0114] This figure illustrates a robust and scalable method for identifying and functionally validating antigen-specific TCRs, which may be used in therapeutic, diagnostic, or research applications.

[0115] FIG. 10 shows an exemplary workflow and experimental validation for the synthesis, expression, and use of class II single-chain trimer (SCT) tetramers, as disclosed herein. This figure demonstrates a scalable molecular pipeline for the generation of class II SCT libraries for use in identifying antigen-specific T cell receptors (TCRs).

[0116] Panel a illustrates the molecular construction of class II SCTs. DNA fragments encoding antigenic peptides were designed via primer extension and cloned into plasmid constructs containing sequences for MHC class II a- and P-chain domains, separated by linker sequences (LI and L2). These DNA constructs were assembled by Gibson assembly and amplified in bacterial cultures. The resulting plasmids were transfected into mammalian cells for protein expression. SCT proteins were then harvested, biotinylated, and purified to formmultimeric complexes for antigen-specific T cell screening. This approach enables high- throughput production of diverse SCT libraries presenting multiple antigens across different human leukocyte antigen (HLA) alleles.

[0117] Panel b presents a functional assay for SCT specificity using flow cytometry-based tetramer staining. Jurkat T cells expressing engineered TCRs (TCR001-TCR004) were incubated with two different SCT constructs (SCT2 and SCT4). The percentage of tetramerpositive cells was quantified, revealing distinct differences in binding efficiency depending on the SCT-TCR pairing. The bar graph summarizes these findings, showing robust binding for TCR001 in the presence of SCT2 and lower binding for SCT4, confirming antigen-specific recognition by the TCR-SCT interaction.

[0118] Panel c compares the staining efficacy of SCT tetramers with that of conventional peptide-major histocompatibility complex (pMHC) class II tetramers. Influenza-specific CD4+ T cells were stained with both SCT and pMHC tetramers. The resulting flow cytometry plots show that SCT tetramers are highly effective in labeling antigen-specific T cells, with over 98% of cells double positive for influenza SCT and control tetramer, indicating high sensitivity and specificity of the SCT format.

[0119] Panel d presents biochemical characterization of SCT proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SCT constructs loaded with various peptides were subjected to gel electrophoresis under reduced and reduced + PNGase treatment conditions. PNGase F digestion removes N-linked glycans and confirms the glycosylation status of SCTs. The resulting mobility shifts verify proper folding and post-translational processing of SCTs. The peptide sequences tested in this panel are provided in the lower inset, and include known epitopes from influenza and SARS-CoV-2 receptor binding domain (RBD) antigens.

[0120] As disclosed herein, SCTs may be used to screen, identify, and validate TCRs specific for antigens from infectious diseases, cancer, or autoimmune conditions. In some embodiments, SCTs may be synthesized with customized peptide sequences of interest and tailored to specific HLA alleles for use in personalized immunotherapy. It will be appreciated that modifications to the linker sequences, biotinylation sites, or SCT expression vectors may be made without departing from the scope of the present invention.

[0121] FIG. 10 illustrates that class II SCTs are robust, flexible, and amenable to high- throughput synthesis and screening platforms, providing an efficient tool for the discovery of therapeutically relevant TCRs.

[0122] FIG. 11 shows representative flow cytometry plots demonstrating the identification of rare, antigen-specific CD4+ T cells from multiple human donors using class II single-chain trimer (SCT) tetramers as disclosed in the present invention. These plots illustrate one application of the SCT tetramers in high-sensitivity detection of epitope-specific T cells ex vivo, without prior expansion or stimulation, thereby validating their use for immune monitoring and therapeutic development.

[0123] Each panel corresponds to staining performed on peripheral blood mononuclear cells (PBMCs) from three different human donors using SCT tetramers loaded with specific peptide- MHC complexes (SCT-A through SCT-E). The x- and y-axes represent fluorescence intensity for two different fluorophore-labeled tetramers, and cells falling in the upper-right quadrant of each plot (boxed region) are considered double-positive for binding to the tetramer complex. These cells are thus identified as specific for the antigen-HLA complex encoded by the SCT.

[0124] The fraction of tetramer-positive CD4+ T cells is reported numerically in each panel, with values ranging from 0.00896% to 0.772%. Although rare in frequency, these antigenspecific populations were consistently detected across different donors and SCT constructs, highlighting the sensitivity and reproducibility of the disclosed SCT staining method. For example, SCT-E staining in Donor 2 resulted in 0.772% of cells being double-positive, while SCT-A in Donor 1 yielded 0.013%, reflecting natural variation in epitope-specific T cell prevalence among individuals.

[0125] Importantly, these results demonstrate that SCT tetramers generated according to the methods described in this invention are capable of identifying clinically relevant antigen-specific TCRs directly from primary human samples. In some embodiments, the invention may be used for immunoprofiling in the context of infection, cancer, vaccination, or autoimmune disease. It will be appreciated that similar detection methods using alternative fluorophores, staining conditions, or gating strategies are also within the scope of this invention.

[0126] Furthermore, it will be appreciated that SCTs bearing modified peptide sequences, truncated linkers, or different biotinylation strategies may be employed for similar purposes, andthat minor variations in construct design that do not materially affect tetramer performance should be covered by the scope of the present disclosure. The invention thus provides a versatile and high-throughput platform for antigen-specific T cell discovery, immune monitoring, and therapeutic development.

[0127] FIG. 12 shows an illustrative example of a high-throughput antigen-specific T cell discovery and characterization approach in accordance with certain embodiments of the invention. This approach demonstrates the scalability of the disclosed technology to profile immune responses across multiple individuals and over extended timepoints using single-cell sequencing and SCT tetramer-based enrichment.

[0128] Panel a outlines a schematic of the study workflow. CD4+ T cells were enriched from peripheral blood mononuclear cells (PBMCs) collected from 22 HLA-DRB 1*01 :01 -positive patients enrolled in the longitudinal UNCOVR study. Samples were collected at multiple timepoints spanning the acute phase through long-term convalescence (6, 12, 18, 24, and 36 months post-infection), totaling 50 samples. Following CD4+ enrichment, cells were stained with a pool of SCT-based tetramers displaying SARS-CoV-2-derived peptides, each conjugated to a unique DNA barcode. Flow cytometric sorting of tetramer-positive cells was followed by single-cell RNA sequencing to obtain paired transcriptomic and TCR-antigen specificity information.

[0129] Panel b discloses representative peptide sequences derived from the SARS-CoV-2 spike receptor-binding domain (RBD), mapped across the RBD amino acid sequence (positions 317-543). For each peptide, biophysical properties including inverse binding affinity (1 / Affinity in nM), epitope prediction scores (l / %Rank_EL), SCT expression efficiency, and number of antigen-specific cells captured are shown. These values enabled prioritization of immunogenic epitopes for further validation.

[0130] Panel c displays a horizontal bar plot of clonal diversity and patient diversity for SARS-CoV-2-specific CD4+ T cell responses. Each bar represents a unique T cell clonotype (identified by its unique TCR sequence), and the vertical stacking of bars indicates clonotype frequency. Colors correspond to the viral protein of origin (RBD, non-RBD Spike, Nucleocapsid [N], or Membrane [M]), demonstrating broad epitope coverage and heterogeneity in individual immune responses.

[0131] Panels d and e provide validation of SARS-CoV-2-specific TCRs identified through this workflow. Panel d shows the percentage of tetramer-positive cells following transduction of individual TCRs into Jurkat reporter cells, confirming specific binding to SARS-CoV-2 SCTs. Panel e shows functional activation (measured by NFAT-GFP reporter activity) in response to peptide-loaded antigen-presenting cells. TCRs activated in response to their cognate peptide, but not to negative control peptides, demonstrating functional recognition.

[0132] Together, these panels demonstrate a scalable, reproducible platform for mapping CD4+ T cell immune responses at the single-cell level, including isolation of paired TCR sequences and identification of antigen-specific clonotypes. In some embodiments, the methods disclosed herein may be applied to immune monitoring, vaccine evaluation, or therapeutic TCR engineering. It will be appreciated that alternative antigens, SCT designs, or sequencing platforms may also be employed within the scope of the invention. Furthermore, minor modifications to SCT tetramer staining, TCR cloning, or cell activation assays that do not materially alter antigen-specific T cell detection or validation are considered within the spirit and scope of this disclosure.

[0133] FIG. 13 illustrates further characterization of SARS-CoV-2-specific CD4+ T cell responses across diverse epitopes and donors, in accordance with certain embodiments of the invention. The figure discloses a comprehensive analysis of antigen-specific TCR clonotypes, epitope mapping, persistence, clonal expansion, and phenotypic distribution.

[0134] Panel a shows a comprehensive peptide library of SARS-CoV-2-derived sequences used for stimulation and tetramer generation. Each peptide is mapped to its corresponding domain (e.g., RBD for receptor binding domain, S for spike protein outside RBD, N for nucleocapsid, and M for membrane protein), along with the amino acid position of the antigenic region. These peptides were synthesized and cloned into SCT constructs for presentation to T cells in high-throughput assays. The lower section of panel a includes gel electrophoresis validation of peptide incorporation across multiple SCT clones. These validation blots confirm successful construction and expression of SCTs spanning the breadth of SARS-CoV-2 antigenic diversity.

[0135] Panel b discloses the number of cells associated with each antigen-specific TCR clonotype. Blue bars represent non-expanded clonotypes (i.e., clonotypes observed at lowfrequency), whereas orange bars denote expanded clonotypes that underwent proliferation in vivo. The inset chart quantifies the proportion of expanded clonotypes across disease phases (acute [AC], convalescent [CV], and long-term memory [3-36M]), indicating that clonal expansion is prominent during acute and early convalescent stages and persists in select lineages over time.

[0136] Panel c includes four subpanels that map epitope-level features of CD4+ T cell responses. The upper left subpanel shows the number of unique TCR clonotypes targeting each antigen, indicating the breadth of response. The upper right subpanel shows the number of donors contributing antigen-specific responses to each epitope. The lower left subpanel quantifies the persistence of T cell clonotypes across timepoints. The lower right subpanel shows the percentage of clonotypes that underwent clonal expansion in vivo. Taken together, these charts highlight both immunodominant epitopes and those associated with long-term memory responses.

[0137] Panel d provides a correlation matrix summarizing the relationship among various immunological features across antigens. Metrics include the number of unique clonotypes per antigen, the number of patients who responded, the percentage of clonotypes that persisted over time, and the fraction of clonotypes that expanded. Notably, clonotype diversity positively correlates with patient response rate, whereas clonal expansion and persistence appear as partially independent features.

[0138] Panel e explores the phenotypic properties of clonally expanded and persistent T cell populations. Bar plots show the frequency of memory subsets (Naive / Tem, Effector Memory [TEM], Thl, Cytotoxic, Th 17, Treg, Proliferative) among clonotypes exhibiting clonal expansion or persistence. The rightmost subpanel displays the extent to which TCR clonotypes are shared across patients, highlighting examples of public T cell responses to SARS-CoV-2 that may represent conserved protective immunity.

[0139] As disclosed, FIG. 13 provides insights into the immunological properties of CD4+ T cell responses at the clonal and epitope levels. In some embodiments, antigens and TCR clonotypes identified through this methodology may be used to guide vaccine design, identify biomarkers of durable immunity, or engineer TCR-based immunotherapies. It will be appreciated that small modifications to antigen sequences, TCRs, or assay platforms that do not materiallyaffect antigen specificity or immune functionality remain within the scope of the present invention.

[0140] FIG. 14 discloses transcriptomic and phenotypic profiling of antigen-specific CD4+ T cells over time and across multiple SARS-CoV-2 epitopes, according to certain embodiments of the invention. This figure illustrates how the disclosed single-cell immune profiling methods may be used to characterize the functional state and longitudinal behavior of T cell clonotypes following viral infection or vaccination.

[0141] Panel a illustrates a schematic workflow in which CD4+ T cells from SARS-CoV-2- infected donors were enriched and stained with SCT (single-chain trimer) tetramers bearing SARS-CoV-2-derived peptides. Cells were also labeled with timepoint-specific DNA barcodes to allow longitudinal tracking. Following fluorescence-activated cell sorting (FACS), single cells were subjected to RNA sequencing, enabling simultaneous recovery of transcriptome data and antigen-specific TCR sequences from each individual cell.

[0142] Panel b shows a Uniform Manifold Approximation and Projection (UMAP) plot in which CD4+ T cells are clustered according to transcriptional phenotype. Each dot represents a single cell, color-coded by functional subtype including Naive / central memory (Tcm), T helper 1 (Thl), Exhausted, Cytotoxic, Effector memory (Tern), T helper 17 (Th 17), Regulatory T cells (Treg), and Proliferative phenotypes. This panel provides an overview of the functional diversity present within antigen-specific CD4+ T cell populations.

[0143] Panel c presents a heatmap of gene expression profiles corresponding to the major phenotypic clusters. Columns represent different T cell clusters, while rows denote selected genes. Expression levels range from low (dark purple) to high (bright yellow), indicating clusterspecific transcriptional programs. These data validate the assignment of functionally distinct T cell subsets and facilitate high-resolution phenotypic classification.

[0144] Panel d highlights three distinct antigen clusters (i.e., groups of peptides with shared TCR reactivity profiles), showing how cells specific for each cluster localize to different functional regions of the UMAP. Each row corresponds to a representative antigen cluster (e.g., cluster 1 : ADSF...QTKGA; cluster 2: FFNR...ALINT; cluster 3: KIAD...GVKG). Columns represent sampling timepoints: acute (AC), convalescent (CV), and long-term recovery (3-36 months). Cells specific to cluster 1 are enriched in Naive / Tcm regions, cluster 2 in the Exhaustedregion, and cluster 3 in the Tern region. These data reveal that epitope specificity can correlate with distinct differentiation states over time.

[0145] Panel e summarizes immunogenicity scores for each SARS-CoV-2 antigen tested. Bars are color-coded to show the number of unique clonotypes (blue), the number of patients who responded to a given antigen (orange), the proportion of persistent responses (gray), and the proportion undergoing clonal expansion (red). Antigens are classified into High, Medium, and Low immunogenicity tiers, illustrating that some epitopes elicit broader, more durable T cell responses than others.

[0146] Panel f characterizes the phenotype distribution of antigen-specific T cells responding to high, medium, and low immunogenicity epitopes across timepoints. Stacked bar plots indicate the relative proportion of Naive / Tcm, Exhausted, Tern, Thl, Cytotoxic, Thl7, Treg, and Proliferative cells during acute (AC), convalescent (CV), and long-term (3-36M) stages. Notably, responses to high-immunogenicity antigens are enriched for Tern and Cytotoxic phenotypes during early timepoints and show persistence over time.

[0147] As disclosed herein, FIG. 14 illustrates an embodiment in which single-cell immune profiling is combined with antigen-specific tetramer staining and longitudinal sampling to uncover the transcriptional programs and differentiation trajectories of TCR clonotypes. In certain embodiments, the invention may include TCRs, SCTs, or antigens that preferentially engage specific phenotypes or are associated with long-term persistence. It will be appreciated that minor modifications to epitope sequences, TCR structures, or clustering methods that retain specificity and function fall within the scope of this invention.

[0148] FIG. 15 illustrates knock-in efficiency of T cell receptors (TCRs) into primary human CD4+ and CD8+ T cells. In this example, multiple TCRs, including the five validated sequences designated Hl through H5, as well as additional TCRs (e.g., TCR1, TCR11, and TCR17), were introduced into CD4+ and CD8+ primary human T cells to assess relative knock-in efficiency. Knock-in efficiency is presented as a percentage of cells expressing the introduced TCR (TCR+%) following gene editing and expansion. This figure enables comparison of the efficiency of TCR integration across T cell subsets.

[0149] The data show that knock-in efficiency is consistently higher in CD4+ T cells (shown in dark blue) compared to CD8+ T cells (shown in orange) across all tested TCRs. In severalcases, CD4+ T cell knock-in efficiencies exceeded 60%, whereas CD8+ T cell efficiencies remained below 20% for the same TCR constructs. These findings underscore the differential responsiveness of T cell subsets to gene modification, and suggest that CD4+ T cells may be more amenable to knock-in approaches for therapeutic TCR delivery using the disclosed methods.

[0150] It will be appreciated that while specific TCRs are shown in this figure, any TCR, including variants or derivatives thereof, may be introduced into CD4+ or CD8+ T cells using the described techniques. The knock-in approach disclosed herein is broadly applicable to engineered T cell therapies, and may be used with receptors targeting any disease-associated antigen, including viral, cancer, or autoimmune antigens. Minor alterations to the TCR nucleotide or amino acid sequence, such as conservative substitutions that retain antigen specificity, are also contemplated within the scope of the invention.

[0151] FIG. 16 illustrates an example of functional validation and application of HPV- specific T cell receptors (TCRs) in the setting of therapeutic vaccination and adoptive T cell transfer, further demonstrating their immunogenic potential and therapeutic relevance. These data provide non-limiting embodiments of the disclosed invention and support the utility of the disclosed TCRs in immunotherapeutic contexts, including HPV-associated diseases and cancers. It will be appreciated that minor variations in TCR sequences or experimental conditions that do not materially alter TCR antigen specificity or function fall within the scope of this disclosure.

[0152] Panel a shows a vaccination timeline in which subjects received priming and booster DNA vaccines targeting HPV antigens at scheduled intervals, followed by functional assessment of HPV-specific T cells. Panel b presents tetramer staining and peptide activation data. The left graph demonstrates the percentage of tetramer-positive CD4+ T cells across a panel of TCRs, confirming their ability to bind HPV 16 peptide-MHC complexes. The right bar graph demonstrates activation of Jurkat NFAT-GFP reporter cells transduced with various TCRs in response to cognate HPV16 peptide stimulation, showing that multiple validated TCRs (including Hl, H2, H3, H4, H5) are capable of specific activation. Statistical comparisons highlight differences in functional strength among these TCRs.

[0153] Panel c illustrates the generation of HPV TCR+ primary CD4+ T cells from healthy donors via CRISPR-mediated knockout of endogenous TCRs followed by lentiviral knock-in ofselected TCR constructs. The resulting cells were tested for tetramer binding, as shown in the adjacent bar graph. Robust surface expression and antigen recognition were observed for a number of HPV-specific TCRs, confirming their stability and functionality in primary T cells.

[0154] Panel d shows cytokine secretion data following co-culture of TCR-expressing primary CD4+ T cells with DRB 1 *01 :01-positive K562 target cells pulsed with HPV peptide. Panels display production of IL-2, IFNy, TNFa, and Granzyme B (GZMB) by ELISA. These data demonstrate that the TCR-transduced CD4+ T cells can mediate polyfunctional effector responses upon antigen-specific stimulation, supporting their use in adoptive immunotherapy for HPV-associated malignancies or other relevant indications.

[0155] Panel e shows real-time killing activity over time using impedance-based cytotoxicity assays. TCR-transduced CD4+ T cells were co-cultured with target cells pulsed with HPV peptides. Multiple replicates of TCR-transduced effector cells (notably HI-FI, H1-F2, H2-F1, etc.) showed consistent and significant lytic activity, with distinct kinetics depending on the TCR. These results support the feasibility of applying the disclosed TCRs in contexts requiring cytotoxic CD4+ responses, such as therapeutic vaccination or adoptive T cell therapy.

[0156] Together, FIG. 16 provides experimental evidence that the disclosed TCRs are functional, specific, and effective in both artificial and primary immune contexts. The demonstrated cytokine secretion, cytotoxic potential, and stability of expression underscore their relevance for therapeutic use in HPV-related indications and potentially in other diseases where similar immunological targeting strategies are employed.

[0157] The foregoing disclosure describes non-limiting embodiments of systems and methods for the identification, characterization, and application of antigen-specific T cell receptors (TCRs), with a focus on high-throughput workflows for TCR discovery, validation, and potential therapeutic deployment. These embodiments include both nucleic acid and amino acid sequences of functional TCRs specific to human papillomavirus (HPV) antigens, as well as illustrative examples of their use in cellular assays and engineered immune cell platforms.Validated TCRs disclosed herein demonstrate robust and specific recognition of defined peptide- HLA complexes and functional effector activity, including cytokine production and cytotoxic responses.

[0158] While many of the disclosed TCRs are specific to HPV, it will be appreciated that the methods described are broadly applicable to the identification of TCRs for other infectious agents, malignancies, or autoimmune targets. For example, in one embodiment, the platform was applied to the study of SARS-CoV-2, and resulting TCRs specific to viral antigens, including the spike protein receptor-binding domain (RBD), were characterized and functionally validated. These results illustrate the flexibility of the disclosed approach for identifying clinically relevant TCRs across a wide range of disease settings.

[0159] Accordingly, the invention provides a framework for isolating antigen-specific TCRs suitable for use in diagnostic, therapeutic, or research applications. Identified TCRs may be used to engineer T cells for adoptive cell therapies, to generate TCR-mimic reagents for antigen detection, or to study immune responses in vaccinated or infected individuals. The disclosed sequences, along with methods for TCR validation and therapeutic engineering, may be adapted to target additional tumor-associated antigens, viral epitopes, or neoantigens, thus enabling personalized or population-based immunotherapeutic strategies. It will further be appreciated that minor modifications to the TCR sequences, including conservative amino acid substitutions or codon optimizations, that do not materially affect antigen specificity or function, fall within the scope of this disclosure.

[0160] In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention.

[0161] Some embodiments of the present disclosure include a system including one or more data processors. In some embodiments, the system includes a non-transitory computer readable storage medium containing instructions which, when executed on the one or more data processors, cause the one or more data processors to perform part or all of one or more methods and / or part or all of one or more processes disclosed herein. Some embodiments of the present disclosure include a computer-program product tangibly embodied in a non-transitory machine- readable storage medium, including instructions configured to cause one or more data processors to perform part or all of one or more methods and / or part or all of one or more processes disclosed herein.

[0162] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention as claimed has been specifically disclosed by embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0163] The present description provides preferred exemplary embodiments only, and is not intended to limit the scope, applicability or configuration of the disclosure. Rather, the present description of the preferred exemplary embodiments will provide those skilled in the art with an enabling description for implementing various embodiments. It is understood that various changes may be made in the function and arrangement of elements without departing from the spirit and scope as set forth in the appended claims.

[0164] Specific details are given in the present description to provide a thorough understanding of the embodiments. However, it will be understood that the embodiments may be practiced without these specific details. For example, circuits, systems, networks, processes, and other components may be shown as components in block diagram form in order not to obscure the embodiments in unnecessary detail. In other instances, well-known circuits, processes, algorithms, structures, and techniques may be shown without unnecessary detail in order to avoid obscuring the embodiments.REFERENCES (each of which is hereby incorporated by reference in its entirety for all purposes)1. Luckheeram, R. V., Zhou, R., Verma, A. D. & Xia, B. CD4+T Cells: Differentiation and Functions. Clin. Dev. Immunol. 2012, 925135 (2012).2. Tay, R. E., Richardson, E. K. & Toh, H. C. Revisiting the role of CD4+ T cells in cancer immunotherapy — new insights into old paradigms. Cancer Gene Ther. 28, 5-17 (2021).3. Oh, D. Y. & Fong, L. Cytotoxic CD4+ T cells in cancer: Expanding the immune effector toolbox. Immunity 54, 2701-2711 (2021).4. WO 2021168122 (Heath et al., 2021) “Single Chain Trimer MHC Class II Nucleic Acids and Proteins and Methods of Use.”5. WO 2022236102 (Chour et al., 2022) “Single Chain Trimer MHC Class I Nucleic Acids and Proteins and Methods of Use.”6. WO 2023205728 (Chour et al., 2023) “Soluble Single-Chain Dimers From SingleChain Trimers.”

Claims

CLAIMSWhat is claimed:

1. A method of facilitating adoptive cell treatment of a subject, the method comprising: identifying one or more T cell receptors predicted to be effective to treat the subject if used to genetically modify T cells administered to the subject, wherein the identification includes selecting one or more T cell receptors directed against human papilloma virus (HPV) E6 or E7 epitopes, wherein the one or more T cell receptors includes: at least one T cell receptor identified as TCR 1-101; at least one T cell receptor identified as TCR 102-400; at least one T cell receptor specific for an antigen identified as TCR 1-101; and / or at least one T cell receptor specific for an antigen identified as TCR 102-400; and outputting an identification of the one or more T cell receptors.

2. A method of facilitating adoptive cell treatment of a subject, the method comprising: isolating T cells from a blood sample of the subject; genetically modifying the T cells to express one or more T cell receptors, wherein the one or more T cell receptors includes: at least one T cell receptor identified as TCR 1-101; at least one T cell receptor identified as TCR 102-400; at least one T cell receptor specific for an antigen identified as TCR 1-101; and / or at least one T cell receptor specific for an antigen identified as TCR 102-400; and providing the modified T cells for administration to the subject.

3. The method of Claim 1 or Claim 2, wherein the one or more T cell receptors comprise at least one TCR associated with a cell type with a cytotoxic phenotype and at least one TCR from a cell type with a memory T cell phenotype.

4. The method of Claim 1 or Claim 2, wherein the one or more T cell receptors are selected by predicting that the one or more peptides are configured to replicate tumor-infiltrating T cells of the subject.

5. The method of Claim 1 or Claim 2, wherein the one or more T cell receptors comprise an isoform that retains antigen specificity for HPV E6 or E7.

6. The method of Claim 1 or Claim 2, wherein the genetic modification is conducted by viral transfections, plasmids, CRISPR-CAS9 or any suitable molecular biology or genetic engineering technique.

7. A T cell receptor comprising an amino acid sequence selected from TCR 1-101, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor.

8. A T cell receptor comprising an amino acid sequence selected from TCR 102- 400, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor.

9. An antigen identified in FIG. 6 recognized by a T-cell receptor identified as TCR 1-101.

10. An antigen identified in FIG. 7 recognized by a T-cell receptor identified as TCR 102-400.

11. A nucleic acid molecule encoding part or all of (i) a T cell receptor identified as TCR 1-400, or (ii) a T cell receptor specific for an antigen identified in, and selected from, FIG. 6 or FIG. 7.

12. A vector comprising a nucleic acid molecule of claim 11.

13. A library comprising a plurality of nucleic acid molecules of claim 11.

14. A T cell, such as a CD8+ T cell or a CD4+ T cell, transformed with a nucleic acid molecule of claim 11.

15. A composition comprising and / or encoding a T cell receptor comprising the amino acid sequence of TCR 401, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor.

16. The composition of claim 15, wherein the composition includes the variant, and wherein the variant is optimized for increased expression, stability, or reduced immunogenicity in human cells.

17. The composition of claim 15, wherein the composition includes the variant, and wherein the variant retains specificity for the same HPV E6 epitope as the receptor comprising TCR 401.

18. A composition comprising and / or encoding a T cell receptor comprising the amino acid sequence of TCR 402, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor.

19. The composition of claim 18, wherein the composition includes the variant, and wherein the variant is optimized for increased expression, stability, or reduced immunogenicity in human cells.

20. The composition of claim 18, wherein the composition includes the variant, and wherein the variant retains specificity for the same HPV E6 epitope as the receptor comprising TCR 402.

21. A composition comprising and / or encoding a T cell receptor comprising the amino acid sequence of TCR 403, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor.

22. The composition of claim 21, wherein the composition includes the variant, and wherein the variant is optimized for increased expression, stability, or reduced immunogenicity in human cells.

23. The composition of claim 21, wherein the composition includes the variant, and wherein the variant retains specificity for the same HPV E6 epitope as the receptor comprising TCR 403.

24. A composition comprising and / or encoding a T cell receptor comprising the amino acid sequence of TCR 404, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor.

25. The composition of claim 24, wherein the composition includes the variant, and wherein the variant is optimized for increased expression, stability, or reduced immunogenicity in human cells.

26. The composition of claim 24, wherein the composition includes the variant, and wherein the variant retains specificity for the same HPV E6 epitope as the receptor comprising TCR 404.

27. A composition comprising and / or encoding a T cell receptor comprising the amino acid sequence of TCR 405, or a variant thereof comprising one or more amino acid substitutions, insertions, or deletions that do not substantially alter the antigen specificity of the receptor.

28. The composition of claim 27, wherein the composition includes the variant, and wherein the variant is optimized for increased expression, stability, or reduced immunogenicity in human cells.

29. The composition of claim 27, wherein the composition includes the variant, and wherein the variant retains specificity for the same HPV E6 epitope as the receptor comprising TCR 405.

30. A method of identifying an antigen-specific T cell receptor (TCR), the method comprising:(a) obtaining a biological sample comprising CD4+ or CD8+ T cells from a subject;(b) contacting the T cells with one or more peptide-MHC single-chain trimer (SCT) tetramers, each comprising a peptide of interest and an HLA class I or class II molecule;(c) isolating tetramer-positive T cells;(d) performing single-cell RNA sequencing and TCR sequencing to obtain paired alpha and beta chain sequences and corresponding transcriptomic profiles from individual tetramer-positive T cells; and(e) identifying one or more TCRs specific for an antigen based on tetramer binding, clonal enrichment, or functional profile.

31. The method of claim 30, wherein the SCT tetramers are barcoded with unique oligonucleotides to allow multiplexed antigen identification.

32. The method of claim 30, wherein the antigen is derived from a human papillomavirus (HPV) E6 or E7 protein.

33. The method of claim 30, further comprising cloning the TCR alpha and beta chains into a nucleic acid construct and introducing the construct into a T cell or reporter cell line.

34. The method of claim 30, further comprising validating antigen specificity using a peptide stimulation, cytokine production assay, tetramer staining, or cytotoxicity assay.

35. The method of claim 30, wherein the transcriptomic profile indicates a phenotype selected from the group consisting of: naive, effector memory, regulatory, cytotoxic, Thl, Thl7, or proliferative.

36. The method of claim 30, wherein the subject has a disease selected from the group consisting of: cervical cancer, oropharyngeal cancer, SARS-CoV-2 infection, or melanoma.

37. The method of claim 30, wherein the identified TCR is selected based on clonal expansion or persistence across longitudinal timepoints.