High T Cell Receptor Research Articles

Antigen-Specific T Cell Receptor Discovery for Treating Progressive Multifocal Leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a frequently fatal disease of the central nervous system caused by JC virus (JCV). Survival is dependent on early diagnosis and ability to re-establish anti-viral T cell immunity. Adoptive transfer of polyomavirus-specific T cells has shown promise; however, there are no readily available HLA-matched anti-viral T cells to facilitate rapid treatment. Identify epitopes of the JCV major capsid protein VP1 that elicit an immune response in the context of human leukocyte antigen allele A*02:01 (HLA-A2) and isolate cognate T cell receptors (TCRs) from healthy donors. Evaluate individual VP1-specific TCRs for their capacity to be expressed in T cells and clear JCV in vitro . PBMCs from HLA-A2+ healthy donors were stimulated with peptide libraries tiled across the JCV VP1 protein. Multiple rounds of stimulation were performed to identify the antigens that induced the largest expansion and CD8 + T cell response (measured as INF γ , TNF α , CD137, and CD69 expression). High-affinity, antigen-specific CD8 + T cells were isolated based on intensity of tetramer binding for downstream single-cell TCR sequencing. Candidate TCRs were selected based on tetramer binding affinity and activation assays. Promising TCRs were introduced into the T cell genome via viral transduction for in vitro validation including peptide-pulsed K562 cells and astrocyte cells, and JCV-infected astrocytes. Four conserved JCV VP1 epitopes (amino acids 100-108, 251-259, 253-262, and 274-283) presented by HLA-A2 were identified. VP1(100-108) consistently elicited the highest level of IFN- γ production from multiple donors and this peptide is in a highly conserved region of VP1. We next identified fourteen high avidity TCRs specific for VP1(100-108). When virally transduced into primary human T cells, seven of these TCRs demonstrated specific binding to VP1(100-108):HLA-A2 tetramers, and four showed increased IFN- γ response when incubated with peptide. Primary CD8 + T cells expressing two of these TCRs cleared both HLA-A2 positive K562 cells and HLA-A2 positive SVG astrocyte cell line presenting exogenously added VP1 peptide at a range of E:T ratios. In addition, both TCR-transduced T cell populations effectively lysed JCV-infected astrocytes. We identified JCV VP1 epitopes that are immunogenic in the context of HLA-A2 MHC-I, including epitopes that have not been previously described. The VP1(100-108) epitope was used to isolate HLA-A2-restricted TCRs. When cloned into primary human CD8 + T cells, these TCRs recognized VP1 (100-108)-presenting targets, and the transduced T cells conferred cytotoxic activity and eliminated K562 and astrocyte cells displaying the VP1(100-108) peptide and not sham peptide, as well as JCV-infected astrocytes. Taken together, these data suggest that JCV VP1-specific TCRs could be appealing therapeutics for HLA-A2+ individuals with PML in whom intrinsic T cell immunity cannot be rescued.

Single-Cell Antigen Receptor Sequencing in Pigs with Influenza.

Understanding the pulmonary adaptive immune system of pigs is important as respiratory pathogens present a major challenge for swine producers and pigs are increasingly used to model human pulmonary diseases. Single-cell RNA sequencing (scRNAseq) has accelerated the characterization of cellular phenotypes in the pig respiratory tract under both healthy and diseased conditions. However, combining scRNAseq with recovery of paired T cell receptor (TCR) α and β chains as well as B cell receptor (BCR) heavy and light chains to interrogate their repertoires has not to our knowledge been demonstrated for pigs. Here, we developed primers to enrich porcine TCR α and β chains along with BCR κ and λ light chains and IgM, IgA, and IgG heavy chains that are compatible with the 10x Genomics VDJ sequencing protocol. Using these pig-specific assays, we sequenced the T and B cell receptors of cryopreserved lung cells from CD1D-expressing and -deficient pigs after one or two infections with influenza A virus (IAV) to examine whether natural killer T (NKT) cells alter pulmonary TCR and BCR repertoire selection. We also performed paired single-cell RNA and receptor sequencing of FACS-sorted T cells longitudinally sampled from the lungs of IAV-vaccinated and -infected pigs to track clonal expansion in response to IAV exposure. All pigs presented highly diverse repertoires. Pigs re-exposed to influenza antigens from either vaccination or infection exhibited higher numbers of expanded CD4 and CD8 T cell clonotypes with activated phenotypes, suggesting potential IAV reactive T cell populations. Our results demonstrate the utility of high throughput single-cell TCR and BCR sequencing in pigs.

Abstract 16: CD4+ and CD8+ TCRαβ-deficient bioluminescent reporter T cells for screening and characterization of neoantigen-specific TCRs

Abstract Adoptive antigen-specific T cell therapy is currently comprised of chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cells. Clinical results from CAR-T cells have demonstrated promising results in treating leukemias, while TCR-engineered T cells, which have the advantage of recognizing intracellular tumor antigens, is still in early development. Here, we report the development of CD4+ or CD8+ TCRαβ-KO reporter T cell lines for the screening and characterization of transgenic TCRs. A TCRαβ-KO reporter T cell line was first developed by knocking out the endogenous TCR α and β chains in the reporter T cell line using CRISPR/Cas9. Successful knockout was confirmed by phenotypic assays and TCR v chain locus sequencing. We demonstrated that re-introduction of influenza hemagglutinin (HA) peptide-specific TCR (HA1.7) α and β chains into TCRαβ-KO reporter T cell lines results in HA peptide-dependent TCR activation and luciferase reporter expression when HA peptide is presented by a MHCII+ cell line. Similarly, re-introduction of MART-1-specific TCR (DMF5) resulted in MART-1-dependent activation in the presence of MHCI+ cells. The select expression of CD4- or CD8-expressing variants of the TCRαβ-KO reporter T cell line enables the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets. Further development of CD4/CD8 double-positive and double-negative cell lines enables screening of low and high affinity TCRs without MHC bias. Together, this TCRαβ-KO platform represents a powerful tool for the screening and characterization of neoantigen-specific TCRs. Citation Format: Mei Cong, Jamison Grailer, Michael Slater, Pete Stecha, Jim Hartnett. CD4+ and CD8+ TCRαβ-deficient bioluminescent reporter T cells for screening and characterization of neoantigen-specific TCRs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 16.

Intratumoral T-cell receptor repertoire composition predicts overall survival in patients with pancreatic ductal adenocarcinoma

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is refractory to immune checkpoint inhibitor therapy. However, intratumoral T-cell infiltration correlates with improved overall survival (OS). Herein, we characterized the diversity and antigen specificity of the PDAC T-cell receptor (TCR) repertoire to identify novel immune-relevant biomarkers. Demographic, clinical, and TCR-beta sequencing data were collated from 353 patients across three cohorts that underwent surgical resection for PDAC. TCR diversity was calculated using Shannon Wiener index, Inverse Simpson index, and “True entropy.” Patients were clustered by shared repertoire specificity. TCRs predictive of OS were identified and their associated transcriptional states were characterized by single-cell RNAseq. In multivariate Cox regression models controlling for relevant covariates, high intratumoral TCR diversity predicted OS across multiple cohorts. Conversely, in peripheral blood, high abundance of T-cells, but not high diversity, predicted OS. Clustering patients based on TCR specificity revealed a subset of TCRs that predicts OS. Interestingly, these TCR sequences were more likely to encode CD8+ effector memory and CD4+ T-regulatory (Tregs) T-cells, all with the capacity to recognize beta islet-derived autoantigens. As opposed to T-cell abundance, intratumoral TCR diversity was predictive of OS in multiple PDAC cohorts, and a subset of TCRs enriched in high-diversity patients independently correlated with OS. These findings emphasize the importance of evaluating peripheral and intratumoral TCR repertoires as distinct and relevant biomarkers in PDAC.

Reversing immunosuppression in the tumor microenvironment of fibrolamellar carcinoma via PD-1 and IL-10 blockade

Fibrolamellar carcinoma (FLC) is a rare liver tumor driven by the DNAJ-PKAc fusion protein that affects healthy young patients. Little is known about the immune response to FLC, limiting rational design of immunotherapy. Multiplex immunohistochemistry and gene expression profiling were performed to characterize the FLC tumor immune microenvironment and adjacent non-tumor liver (NTL). Flow cytometry and T cell receptor (TCR) sequencing were performed to determine the phenotype of tumor-infiltrating immune cells and the extent of T cell clonal expansion. Fresh human FLC tumor slice cultures (TSCs) were treated with antibodies blocking programmed cell death protein-1 (PD-1) and interleukin-10 (IL-10), with results measured by cleaved caspase-3 immunohistochemistry. Immune cells were concentrated in fibrous stromal bands, rather than in the carcinoma cell compartment. In FLC, T cells demonstrated decreased activation and regulatory T cells in FLC had more frequent expression of PD-1 and CTLA-4 than in NTL. Furthermore, T cells had relatively low levels of clonal expansion despite high TCR conservation across individuals. Combination PD-1 and IL-10 blockade signficantly increased cell death in human FLC TSCs. Immunosuppresion in the FLC tumor microenvironment is characterized by T cell exclusion and exhaustion, which may be reversible with combination immunotherapy.

Abstract A003: Preliminary safety results from a phase I study of autologous transgenic T cells expressing high affinity mesothelin-specific T cell receptor (TCR) (FH-TCR TMSLN) in patients (pts) with metastatic pancreatic ductal adenocarcinoma (mPDA)

Abstract Background: We previously demonstrated murine CD8+ T cells engineered to express a high affinity MSLN-specific TCR exhibit efficacy in the KPC mouse model, particularly with serial infusions, and thus developed a human TCR for expression in autologous T cells for targeting MSLN, which is overexpressed by 80% of PDAs. In this first-in-human phase I trial, we are determining safety, preliminary efficacy, as well as persistence, activation, localization, and functional capacity of autologous FH-TCR-TMSLN (T) cells in refractory, MSLN+ mPDA. Methods: Eligible pts have ECOG PS 0-1, ≥1 prior therapy for mPDA, life expectancy ≥12 weeks, MSLN 2+ expression in ≥30% tumor cells by IHC, HLA-A*2:01, no HLA-B*13:02. Pts undergo leukapheresis, and 3-4 weeks later receive the first T cell infusion. Three T-cell infusions, if sufficient T cells can be manufactured, are being administered every 21 days (d) at 3 dose level cohorts: 1 × 109, 3.3 × 109 and 1 × 1010 T cells, with a 3+3 design. Dose limiting toxicities (DLTs) are assessed during the 21d after each T cell dose. Only pts who receive ≥75% of the assigned T cell dose are DLT evaluable, but all pts are evaluable for safety. Lymphodepleting chemotherapy (LDC) with fludarabine/cyclophosphamide is administered prior to the last T cell infusion (cohorts 1-2) or prior to the first T cell infusion (cohort 3). Primary endpoints are safety and DLTs. Secondary endpoints are response rate, progression-free and overall survival. Exploratory endpoints include TCR-TMSLN cells in tumor biopsies and blood as biomarkers. Results: Between Dec 2021 to July 2023, 7 pts were enrolled, and 6 pts treated (n=3 at dose at level 1, n=3 at dose level 2), 4 women, median age 60 years (range 51-70), median 3 prior systemic therapies. All pts underwent research-related tumor biopsies at baseline, and 2-3 weeks post first T cell infusion, and 2 pts had biopsies after the last T-cell infusion. Treatment exposure was median 3 (range 1-3) T cell infusions. No DLTs occurred among 6 pts treated at dose levels 1 × 109 and 3.3 × 109 T cells. Treatment related adverse events (TRAE) thought at least possibly related to T cells infusions were fever [n=2 grade (gr) 1, n=1 gr 2], and gr 2 chills, cytokine release syndrome (CRS), dizziness, fatigue, hypertension, hypotension, nausea, acute generalized exanthematous pustulosis (AGEP) rash, and sinus tachycardia (n=1 each). TRAE due to LDC were transient leukopenia (n=2 gr 4, n=1 gr 3, n=2 gr 2), neutropenia (n=4 gr 4, n=1 gr 2), lymphopenia (n=3 gr 4), dysgeusia (n=2 gr 2), anemia (n=1 gr 3) and fatigue (n=1 gr 2). One patient had a serious adverse event with gr 2 CRS and gr 2 AGEP, which resolved. Due to anticipated difficulty attaining sufficient T cells doses for three high dose infusions, we amended the protocol to allow treatment with at least one T cell infusion, with the first dose preceded by LDC at the 1 × 1010 T cells dose level. Conclusions: FH-TCR-TMSLN cells are well tolerated with no on-target toxicity, and the study is open to accrual (NCT04809766). Citation Format: Elena G. Chiorean, Aude Chapuis, Andrew Coveler, Cecilia Yeung, Ted Gooley, David B. Zhen, Gentry King, Stacey Cohen, Lindsay Hannan, Rachael Safyan, Anthony Germani, Susan Ra, Jennifer Casserd, Tom Schmitt, Philip Greenberg. Preliminary safety results from a phase I study of autologous transgenic T cells expressing high affinity mesothelin-specific T cell receptor (TCR) (FH-TCR TMSLN) in patients (pts) with metastatic pancreatic ductal adenocarcinoma (mPDA) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A003.

Unrestrained cleavage of Roquin-1 by MALT1 induces spontaneous T cell activation and the development of autoimmunity.

Constitutive activation of the MALT1 paracaspase in conventional T cells of Malt1TBM/TBM (TRAF6 Binding Mutant = TBM) mice causes fatal inflammation and autoimmunity, but the involved targets and underlying molecular mechanisms are unknown. We genetically rendered a single MALT1 substrate, the RNA-binding protein (RBP) Roquin-1, insensitive to MALT1 cleavage. These Rc3h1Mins/Mins mice showed normal immune homeostasis. Combining Rc3h1Mins/Mins alleles with those encoding for constitutively active MALT1 (TBM) prevented spontaneous T cell activation and restored viability of Malt1TBM/TBM mice. Mechanistically, we show how antigen/MHC recognition is translated by MALT1 into Roquin cleavage and derepression of Roquin targets. Increasing T cell receptor (TCR) signals inactivated Roquin more effectively, and only high TCR strength enabled derepression of high-affinity targets to promote Th17 differentiation. Induction of experimental autoimmune encephalomyelitis (EAE) revealed increased cleavage of Roquin-1 in disease-associated Th17 compared to Th1 cells in the CNS. T cells from Rc3h1Mins/Mins mice did not efficiently induce the high-affinity Roquin-1 target IκBNS in response to TCR stimulation, showed reduced Th17 differentiation, and Rc3h1Mins/Mins mice were protected from EAE. These data demonstrate how TCR signaling and MALT1 activation utilize graded cleavage of Roquin to differentially regulate target mRNAs that control T cell activation and differentiation as well as the development of autoimmunity.

Peripheral T-Cell Priming and Micrometastatic Disease Control with Metastasis-Directed Therapy: Multidimensional Immunogenomic Profiling of Oligometastatic Prostate Cancer in the EXTEND Trial

Comprehensive metastasis-directed therapy (MDT) for oligometastatic prostate cancer extended progression-free survival (PFS) and time to new lesion formation in the intermittent hormone therapy (HT) basket of EXTEND. To better understand the mechanism of MDT benefit, we pooled the intermittent and continuous HT baskets of EXTEND and tested the hypothesis that adding MDT to HT would program systemic T-cells to control micrometastatic disease. A total of 174 men were randomized to HT with or without MDT to up to 5 sites of metastases. HT was given for 6 months (intermittent basket, n = 87) or indefinitely (continuous basket, n = 87). Peripheral blood samples were drawn at enrollment, at the end of MDT, at 3 months follow-up (3 mo F/U), and at progression and then analyzed by flow cytometry, T-cell receptor (TCR)-β CDR3 variable region sequencing, multiplex cytokine profiling, and next-generation circulating tumor DNA (ctDNA) sequencing. TCR clonal expansion was determined using a published betabinomial model. Repertoire changes were assessed by Morisita's index, and dominant TCR repertoire motifs were characterized with ImmunoMap. Associations between blood markers and PFS were evaluated with Cox regression adjusted hazard ratios (aHR) accounting for randomization arm and stratifying for intermittent vs continuous HT. Randomization to MDT+HT was associated with T-cell activation, proliferation, and clonal expansion. This response was first observed at end-MDT as upregulated expression of T-cell activation and inhibition markers (i.e., ICOS, Tim-3, and LAG-3) and increases in highly proliferative CD4+ and CD8+ Ki67hi T-cells (all P<0.05). TCR sequencing of 7,678,911 T-cells revealed that MDT+HT was associated with TCR clonal expansion, remodeling of the TCR repertoire, and changes in dominant TCR motifs at end-MDT and 3 mo F/U (all P<0.05). Observed T-cell priming could be driven by signaling networks of canonical T-cell stimulatory cytokines (IL-2, IL-12, and IL-15), which were upregulated at end-MDT and persisted at 3 mo F/U (all P<0.05). This modulation of T-cell phenotype, clonotype, and cytokine concentrations was not observed in the HT-monotherapy arm. At end-MDT, systemic T-cell responses were associated with improved PFS, most notably CD8+ T-cell expression of LAG-3 (aHR 0.22, 95% CI 0.03-0.91) and high TCR clonal expansion (aHR 0.13, 95% CI 0.02-0.52). High ctDNA burden at end-MDT correlated with worse PFS (aHR 1.41, 95% CI 1.04-2.54), as did CD8+ T-cell expression of inhibitory receptor TIGIT at 3 mo F/U (aHR 1.03, 95% CI 1.01-1.06). The addition of MDT to HT induced systemic T-cell activation and expansion, which was not observed in the HT-only arm. This systemic immune response was independently associated with improved PFS. In addition to cytoreduction of macroscopic disease, MDT-induced immune education may be an important complementary mechanism of micrometastatic control in oligometastatic prostate cancer.

Prognosis and therapeutic benefits prediction based on NK cell marker genes through single-cell RNA-seq with integrated bulk RNA-seq analysis for hepatocellular carcinoma.

Tumor-infiltrating immune cells greatly participate in regulating tumorigenesis and metastasis of hepatocellular carcinoma (HCC). Natural killer cell, as an important role of innate immunity, plays an indispensable role in antitumor immunity and regulate tumor development. In this study, we firstly identified 251 NK cell marker genes of HCC based on single-cell RNA sequencing data. Subsequently, an NK cell marker genes-related prognostic signature (NKPS) was developed in the cancer genome atlas (TCGA) cohort for risk stratification and prognosis prediction. The predictive value of the NKPS in prognosis was well validated in different clinical subgroups and three external datasets (ICGC-LIHC cohort, GSE14520 cohort and Guilin cohort). Moreover, multivariate analysis revealed the independent prognostic value of NKPS for OS in HCC. Further functional analysis indicated the NKPS was associated with basic cellular processes, that may contribute to the development and progression of HCC. Thereafter, immune characteristics as well as the therapeutic benefits in NKPS risk score-defined subgroups were analyzed. Patients with low-risk score exhibited immune-active status, manifested as higher immune scores, more infiltration of CD8+ T cells and macrophage M1, and higher T-cell receptor (TCR) richness and diversity. Remarkably, the NKPS was negatively correlated with immunotherapy response-related signatures. In addition, the low-risk group exhibited significantly improved therapeutic benefits, either from immunotherapy or traditional chemotherapy and target therapy. Overall, the NKPS showed an excellent predictive value for prognosis and therapeutic responses for HCC, which might also provide novel insights into better HCC management strategies.

Neoantigen-specific stem cell memory-like CD4+ T cells mediate CD8+ T cell-dependent immunotherapy of MHC class II-negative solid tumors.

CD4+ T cells play key roles in a range of immune responses, either as direct effectors or through accessory cells, including CD8+ T lymphocytes. In cancer, neoantigen (NeoAg)-specific CD8+ T cells capable of direct tumor recognition have been extensively studied, whereas the role of NeoAg-specific CD4+ T cells is less well understood. We have characterized the murine CD4+ T cell response against a validated NeoAg (CLTCH129>Q) expressed by the MHC-II-deficient squamous cell carcinoma tumor model (SCC VII) at the level of single T cell receptor (TCR) clonotypes and in the setting of adoptive immunotherapy. We find that the natural CLTCH129>Q-specific repertoire is diverse and contains TCRs with distinct avidities as measured by tetramer-binding assays and CD4 dependence. Despite these differences, CD4+ T cells expressing high or moderate avidity TCRs undergo comparable in vivo proliferation to cross-presented antigen from growing tumors and drive similar levels of therapeutic immunity that is dependent on CD8+ T cells and CD40L signaling. Adoptive cellular therapy (ACT) with NeoAg-specific CD4+ T cells is most effective when TCR-engineered cells are differentiated ex vivo with IL-7 and IL-15 rather than IL-2 and this was associated with both increased expansion as well as the acquisition and stable maintenance of a T stem cell memory (TSCM)-like phenotype in tumor-draining lymph nodes (tdLNs). ACT with TSCM-like CD4+ T cells results in lower PD-1 expression by CD8+ T cells in the tumor microenvironment and an increased frequency of PD-1+CD8+ T cells in tdLNs. These findings illuminate the role of NeoAg-specific CD4+ T cells in mediating antitumor immunity via providing help to CD8+ T cells and highlight their therapeutic potential in ACT.

Modifications outside CDR1, 2 and 3 of the TCR variable β domain increase TCR expression and antigen-specific function.

T cell receptor (TCR) gene modified T cells are a promising form of adoptive cellular therapy against human malignancies and viral infections. Since the first human clinical trial was carried out in 2006, several strategies have been developed to improve the efficacy and safety of TCR engineered T cells by enhancing the surface expression of the introduced therapeutic TCRs whilst reducing the mis-pairing with endogenous TCR chains. In this study, we explored how modifications of framework residues in the TCR variable domains affect TCR expression and function. We used bioinformatic and protein structural analyses to identify candidate amino acid residues in the framework of the variable β domain predicted to drive high TCR surface expression. Changes of these residues in poorly expressed TCRs resulted in improved surface expression and boosted target cell specific killing by engineered T cells expressing the modified TCRs. Overall, these results indicate that small changes in the framework of the TCR variable domains can result in improved expression and functionality, while at the same time reducing the risk of toxicity associated with TCR mis-pairing.

Analysis of genomic and immune intratumor heterogeneity in linitis plastica via multiregional exome and T-cell receptor sequencing.

The molecular landscape and the intratumor heterogeneity (ITH) architecture of gastric linitis plastica (LP) are poorly understood. We performed whole-exome sequencing (WES) and T-cell receptor (TCR) sequencing on 40 tumor regions from four LP patients. The landscape and ITH at the genomic and immunological levels in LP tumors were compared with multiple cancers that have previously been reported. The lymphocyte infiltration was further assessed by immunohistochemistry (IHC) in LP tumors. In total, we identified 6339 non-silent mutations from multi-samples, with a median tumor mutation burden (TMB) of 3.30 mutations per Mb, comparable to gastric adenocarcinoma from the Cancer Genome Atlas (TCGA) cohort (P = 0.53). An extremely high level of genomic ITH was observed, with only 12.42%, 5.37%, 5.35%, and 30.67% of mutations detectable across 10 regions within the same tumors of each patient, respectively. TCR sequencing revealed that TCR clonality was substantially lower in LP than in multi-cancers. IHC using antibodies against CD4, CD8, and PD-L1 demonstrated scant T-cell infiltration in the four LP tumors. Furthermore, profound TCR ITH was observed in all LP tumors, with no T-cell clones shared across tumor regions in any of the patients, while over 94% of T-cell clones were restricted to individual tumor regions. The Morisita overlap index (MOI) ranged from 0.21 to 0.66 among multi-regions within the same tumors, significantly lower than that of lung cancer (P = 0.002). Our results show that LP harbored extremely high genomic and TCR ITH and suppressed T-cell infiltration, suggesting a potential contribution to the frequent recurrence and poor therapeutic response of this adenocarcinoma.

Peripheral T-cell receptor repertoire dynamics in small cell lung cancer.

Identifying a circulating biomarker predictive of immune checkpoint inhibitor (ICI) benefit in patients with small cell lung cancer (SCLC) remains an unmet need. Characteristics of peripheral and intratumoral T-cell receptor (TCR) repertoires have been shown to predict clinical outcomes in non-small cell lung cancer (NSCLC). Recognizing a knowledge gap, we sought to characterize circulating TCR repertoires and their relationship with clinical outcomes in SCLC. SCLC patients with limited (n=4) and extensive (n=10) stage disease were prospectively enrolled for blood collection and chart review. Targeted next-generation sequencing of TCR beta and alpha chains of peripheral blood samples was performed. Unique TCR clonotypes were defined by identical CDR3, V gene, and J gene nucleotide sequences of the beta chain and subsequently used to calculate TCR diversity indices. Patients with stable versus progressive and limited versus extensive stage disease did not demonstrate significant differences in V gene usage. Kaplan-Meier curve and log-rank analysis did not identify a statistical difference in progression-free survival (PFS) (P=0.900) or overall survival (OS) (P=0.200) between high and low on-treatment TCR diversity groups, although the high diversity group exhibited a trend toward increased OS. We report the second study investigating peripheral TCR repertoire diversity in SCLC. With a limited sample size, no statistically significant associations between peripheral TCR diversity and clinical outcomes were observed, though further study is warranted.

Chemical Complementarity of Breast Cancer Resident, T-Cell Receptor CDR3 Domains and the Cancer Antigen, ARMC3, is Associated With Higher Levels of Survival and Granzyme Expression

Introduction: One of the most pressing goals for cancer immunotherapy at this time is the identification of actionable antigens. Methods: This study relies on the following considerations and approaches to identify potential breast cancer antigens: (i) the significant role of the adaptive immune receptor, complementarity determining region-3 (CDR3) in antigen binding, and the existence cancer testis antigens (CTAs); (ii) chemical attractiveness; and (iii) informing the relevance of the integration of items (i) and (ii) with patient outcome and tumor gene expression data. Results: We have assessed CTAs for associations with survival, based on their chemical complementarity with tumor resident T-cell receptor (TCR), CDR3s. Also, we have established gene expression correlations with the high TCR CDR3-CTA chemical complementarities, for Granzyme B, and other immune biomarkers. Conclusions: Overall, for several independent TCR CDR3 breast cancer datasets, the CTA, ARMC3, stood out as a completely novel, candidate antigen based on multiple algorithms with highly consistent approaches. This conclusion was facilitated by use of the recently constructed Adaptive Match web tool.

TREC И KREC – ПЕРСПЕКТИВНЫЕ МАРКЕРЫ АКТИВНОСТИ ТУБЕРКУЛЕЗНОЙ ИНФЕКЦИИ

Scientists around the Globe are making deliberate attempts to develop the new methods for predicting and diagnosing tuberculosis, but these measures are not enough to end its epidemic. The purpose of the study was to evaluate the excisional rings of the T-cell receptor (TREC) and K-deletion element of B-cells (KREC) in infants and preschool children at high risk of tuberculosis as a promising prognostic marker for tuberculosis infection activity. A cross-sectional study was conducted in Jan. - Sept. 2022. The observation group consisted of 87 children aged 1 to 7 [6 (5‒7)] y/o, of which 13 (14.9%) aged 1 to 3 y/o and 74 aged 4 to 7 y/o; 53 (60.9%) boys/34 (39.1%) girls. The three groups were formed: G1 (n=27) with an established diagnosis of tuberculosis (TB), G2 (n=34) with TB infection diagnosis made based on the results of tuberculin diagnosis and without signs of a local specific process, and [control] G3 (n=26) without signs of TB infection according to the results of specific skin tests and without signs of TB, i.e., conditionally healthy. All children, in addition to methods to confirm or exclude TB and methods of specific immunodiagnostics, have undergone a molecular genetic study: DNA extraction from a dry blood spot and quantitative determination of TREC and KREC by real-time PCR in values of TREC and KREC copies per 105 cells. In order to build the reference values of TREC and KREC quantitative indicators in dry blood spots, the results obtained in G3 were analyzed as well. Results: the reference values of TREC and KREC were established for children of early and preschool ages when extracting DNA from a dry blood spot. When assessing tuberculosis infection in children of early and preschool ages, statistically significant criteria were determined, such as: anti-tuberculosis therapy (F=42.001; p&lt;0.001), positive or negative reaction to the recombinant tuberculosis allergen during an intradermal test (F=39.394; p&lt;0.001), high blood TREC level (F=12.707; p&lt;0.001), tuberculin reaction (F=10.625; p&lt;0.001), and KREC level (F=3.182; p=0.039). Conclusion: high level of TREC can be considered as a marker for the activity or a personalized predictor for the risk of progression of TB infection in children of early and preschool ages with high risk of TB.

Time required for commitment to T cell proliferation depends on TCR affinity and cytokine response.

T cell activation and effector functions are determined by the affinity of the interaction between T cell receptor (TCR) and its antigenic peptide MHC (pMHC) ligand. A better understanding of the quantitative aspects of TCR-pMHC affinity-dependent T cell activation is critical for the development of new immunotherapeutic strategies. However, the role of TCR-pMHC affinity in regulating the kinetics of CD8+ T cell commitment to proliferation and differentiation is unknown. Here, we show that the stronger the TCR-pMHC affinity, the shorter the time of T cell-APC co-culture required to commit CD8+ T cells to proliferation. The time threshold for T cell cytokine production is much lower than that for cell proliferation. There is a strong correlation between affinity-dependent differences in AKT phosphorylation and T cell proliferation. The cytokine IL-15 increases the poor proliferation of T cells stimulated with low affinity pMHC, suggesting that pro-inflammatory cytokines can override the affinity-dependent features of T cell proliferation.

EP16.01-030 Peripheral T-Cell Receptor Repertoire Profiling in Non-small Cell Lung Cancer Using an Amplicon-Based Sequencing Assay

Diversity of the T-cell receptor (TCR) repertoire plays essential roles in the adaptive immune response, including against tumor-derived antigens. Within the tumor microenvironment, high TCR clonality is correlated with response to neoadjuvant chemoimmunotherapy in locally advanced non-small cell lung cancer (NSCLC), and an increase in CD8+ T cells recruitment and expansion results via the restoration of MHC I expression, in EGFR tyrosine kinase inhibitor (TKI) treated tumors. Whether peripheral TCR repertoire diversity indexes are similarly correlated with molecular subtypes of cancer and treatment responses has not yet been examined.

Abstract 2831: High-throughput T cell receptor cloning allows optimization of anti-miHA T cell therapies

Abstract Adoptive T cell therapy with CD8+ T cells engineered to express T cell receptors (TCRs) that recognize hematopoietically-restricted minor histocompatibility antigens (miHAs) is an attractive approach for improving the efficacy of allogeneic hematopoietic stem cell transplantation. We developed a mouse model to determine how to optimally implement anti-miHA T cell therapy, focusing on the key characteristics of anti-miHA TCRs that correlate with in vivo efficacy. We immunized B6 mice to the Kb-restricted miHA H60 and used a high throughput approach to amplify TCR α/β chains from single CD8+ MHC Multimer-H60+ (TetH60+) cells. Based on β chain sequencing of 472 single T cells, a broad repertoire was engaged by H60-vaccination, without a clear β chain preference. We randomly chose 178 unique TCRs, cloned them into retroviral backbones and expressed them in a TCR-CD8+ NFAT-reporter reporter cell lines and screened for H60-reactivity by NFAT-induction after stimulation with H60-pulsed antigen presenting cells (APCs). We chose 22 unique TCRs that had clear H60-specific NFAT-induction for deeper characterization. In similarly-transduced 4G4 reporter cells, these TCRs spanned a range of surface expression levels, affinities for H60 as measured by TetH60 binding, and EC50s in response to H60-pulsed APCs (between 1 and 12 μM peptide concentration). We focused initial functional studies on 2 high avidity TCRs (TCR 61 and TCR 167) with EC50s of 1.25 and 2.5 µM, respectively. These were cloned into an optimized retroviral vector and transduced into B6 CD8 cells concomitant with efficient CRISPR-knock out of the endogenous TCRα/β genes. Engineered T cells bound H60 loaded MHC multimers and killed H60+ targets in vitro. The mean TCR expression of transduced cells was 16-30% of that in unmanipulated T cells, though some had TCR expression similar to or greater than that in unmanipulated T cells. We used TCR 61 and 167 transduced T cell products to treat H60+ blast crisis leukemia cells (BC-CML). TCR 61 and 167 T cell products expanded and were equally effective at reducing BC-CML numbers in spleen and bone marrow (BM). Despite having a greater EC50 and equivalent surface expression in reporter cells, when recovered post-transplant, TCR167 cells had higher TetH60 binding, and produced more IFN-γ. Ongoing studies are testing these and additional TCRs alone and in competition to better elucidate TCR properties that predict for in vivo efficacy. Citation Format: Alexander M. Rowe, Dominic Didiano, Wenzhong Wei, Sawa Ito, Mark Shlomchik, Warren Shlomchik. High-throughput T cell receptor cloning allows optimization of anti-miHA T cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2831.

Heterogeneity of tumor immune microenvironment and real-world analysis of immunotherapy efficacy in lung adenosquamous carcinoma.

e20546 Background: Lung adenosquamous carcinoma (ASC) is an uncommon histological subtype. We aimed to characterize the tumor immune microenvironment (TIME) in lung ASC and estimate patient response to immune checkpoint inhibitors (ICIs), which have never been systematically investigated. Methods: In cohort I, we collected 30 ASCs from a single center for analysis of TIME characteristics, including immuno-phenotyping, tumor mutation burden (TMB), T cell receptor (TCR) repertoires, tumor-infiltrating lymphocytes (TILs), and immune checkpoint expression. In cohort II, the efficacy of ICI-based therapy were estimated using a real-world data of 46 ASCs from 11 centers. Results: In cohort I, 22 (73.3%) patients were EGFR-positive. The TIME were defined by immune-excluded (60%) and immune-desert phenotype (40%). Strikingly, PD-L1 and PD-1 were predominantly expressed in SCCC versus ACC, where enhanced CD4+ FOXP3+ regulatory T cell and attenuated CD57+ natural killer cell infiltration was present, consistent with a landscape of fewer innate immune cells, more immunosuppressive cells. SCCC had higher TMB, higher TCR clonality, and lower TCR diversity than ACC. In cohort II, majority of 46 patients were driver genes negative and unknown mutation status, 18 (39%) and 18 (39%), respectively. Overall objective response rate of 28%, median progression-free survival of 6.0 months (95% CI 4.3-7.7), and median overall survival of 24.7 months (95% CI 7.2-42.2) were observed in the ICI-based treatment. Conclusions: This work ascertains suppressive TIME in lung ASC and genetic and immuno-heterogeneity between ACC and SCCC. Lung ASC patients have a moderate response to ICI-based immunotherapy.

TCR Coexpression Signature Predicts Immunotherapy Resistance in NSCLC.

Background: Lung cancer has the highest morbidity and mortality rate among types of malignant tumors, and as such, research into prolonging the survival time of patients is vital. The emergence of immune checkpoint inhibitors (ICIs) has greatly improved the survival of patients with non-small cell lung cancer (NSCLC), however, the lack of effective biomarkers to predict the prognosis of immunotherapy has made it difficult to maximize the benefits. T cell receptor (TCR) is one of the most important components for recognizing tumor cells, and with this study we aim to clarify the relationship between TCR coexpression and the prognosis of NSCLC patients receiving immunotherapy. Methods: Univariate COX regression, logistics regression, and KM survival analysis were used to evaluate the relationship between TCR coexpression and the prognosis of immunotherapy. Additionally, CIBERSORT, Gene Set Enrichment Analysis (GSEA), and single-sample GSEA (ssGSEA) algorithms were used to evaluate the tumor immune microenvironment (TIME) of NSCLC patients. Results: Univariate Cox regression analysis showed that the TCR coexpression signature can be used as a clinical prognostic indicator for NSCLC patients receiving immunotherapy (p = 0.0205). In addition, those in the NSCLC group with a high TCR coexpression signature had significantly improved progression-free survival (PFS) (p = 0.014). In the ICI treatment cohort (GSE35640). In addition, there was a high infiltration of CD8+T cells, activated memory CD4+T cells, and M1 macrophages in the TIME of those with a high TCR coexpression signature. The results of pathway enrichment analysis showed that patients with a high TCR coexpression signature had significantly activated signal pathways such as lymphocyte proliferation and activation, chemokine binding, and inflammatory cytokine production. Also, we found that patients with a high TCR coexpression signature had an elevated T cell inflammation gene expression profile (GEP). Conclusion: We show that the TCR coexpression signature may be useful as a new biomarker for the prognosis of NSCLC patients undergoing immunotherapy, with high signatures indicating better treatment response. Additionally, we found that patients with a high TCR coexpression signature had tumor immune microenvironments with beneficial anti-tumor characteristics.