T-cell Receptor Alpha Chain Research Articles

Assessment of the piroxicam-incited model of synchronized colitis in T-cell receptor alpha chain-deficient mice.

A multitude of mouse models are utilized to emulate and study intestinal inflammation. T-cell receptor alpha chain (TCRα)-deficient mice are used as a model of spontaneous colitis that has similarities with human ulcerative colitis. However, colitis is triggered late in the life of the mouse (age: 4-5 months), and inflammation does not develop at the same time in different mice. A previously conducted study reported that the administration of the drug piroxicam triggered predictable and early colitis in TCRα-deficient mice at the age of 6-8 weeks. However, a detailed characterization of ensuing inflammation was not provided. We conducted an in-depth examination of piroxicam-triggered colitis in TCRα-deficient mice, with emphasis on spatial histopathologic changes and analysis of expression of inflammatory markers. Furthermore, we tested amelioration of colitis with dexamethasone. We confirmed that piroxicam induced a time-prescribed colitis and did so in the proximal colon as well as the cecum of TCRα-deficient mice. Piroxicam administration was observed to induce epithelial hyperplasia, goblet cell loss, and leukocyte infiltration with occasional ulceration. A Swiss roll technique was used to examine the colon and cecum in its entirety. Importantly, we observed that inflammation was multifocal segmental, with areas of tissue damage in between healthy tissue. In addition, we observed variability in the severity of inflammation among replicate animals and treatments, and that the administration of dexamethasone only partially ameliorated inflammation in the proximal colon. Piroxicam consistently induced multifocal segmental colitis in the proximal colon and cecum, although the degree of inflammation was reduced in the latter. Importantly, spatial variability in inflammation in the large intestine and the inter-replicate variation in the severity of inflammation must be taken into consideration when utilizing this murine model of synchronized colitis.

Using combined single-cell gene expression, TCR sequencing and cell surface protein barcoding to characterize and track CD4+ T cell clones from murine tissues.

Single-cell gene expression analysis using sequencing (scRNA-seq) has gained increased attention in the past decades for studying cellular transcriptional programs and their heterogeneity in an unbiased manner, and novel protocols allow the simultaneous measurement of gene expression, T-cell receptor clonality and cell surface protein expression. In this article, we describe the methods to isolate scRNA/TCR-seq-compatible CD4+ T cells from murine tissues, such as skin, spleen, and lymph nodes. We describe the processing of cells and quality control parameters during library preparation, protocols for multiplexing of samples, and strategies for sequencing. Moreover, we describe a step-by-step bioinformatic analysis pipeline from sequencing data generated using these protocols. This includes quality control, preprocessing of sequencing data and demultiplexing of individual samples. We perform quantification of gene expression and extraction of T-cell receptor alpha and beta chain sequences, followed by quality control and doublet detection, and methods for harmonization and integration of datasets. Next, we describe the identification of highly variable genes and dimensionality reduction, clustering and pseudotemporal ordering of data, and we demonstrate how to visualize the results with interactive and reproducible dashboards. We will combine different analytic R-based frameworks such as Bioconductor and Seurat, illustrating how these can be interoperable to optimally analyze scRNA/TCR-seq data of CD4+ T cells from murine tissues.

Barcoding intracellular reverse transcription enables high-throughput phenotype-coupled T cell receptor analyses

Assays linking cellular phenotypes with Tcell or B cell antigen receptor sequences are crucial for characterizing adaptive immune responses. Existing methodologies are limited by low sample throughput and high cost. Here, we present INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS), an approach that combines molecular indexing of receptor repertoires within intact cells and fluorescence-activated cell sorting (FACS). We demonstrate that INCERTS enables efficient processing of millions of cells from pooled human peripheral blood mononuclear cell (PBMC) samples while retaining robust association between Tcell receptor (TCR) sequences and cellular phenotypes. We used INCERTS to discover antigen-specific TCRs from patients with cancer immunized with a novel mutant KRAS peptide vaccine. After exvivo stimulation, 28 uniquely barcoded samples were pooled prior to FACS into peptide-reactive and non-reactive CD4+ and CD8+ populations. Combining complementary patient-matched single-cell RNA sequencing (scRNA-seq) data enabled retrieval of full-length, paired TCR alpha and beta chain sequences for future validation of therapeutic utility.

Non-viral TRAC-knocked-in CD19KICAR-T and gp350KICAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency.

The ubiquitous Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8+ T cell exhaustion and dysregulates CD4+ T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen. We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain (TRAC) locus. Applying upscaled methods with the ExPERT ATx® MaxCyte system, KI efficacy was ~20% of the total ~2 × 108 TCR-knocked-out (KO) generated cells. KOTCRKICAR-T cells were co-cultured in vitro with the gp350+CD19+ BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro. CD8+ KICAR-T cells showed higher persistency than CD4+ KICAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 × 105 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19KICAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4+ CD19KICAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (Tregs) and TIM-3+CD4+ T cells. Administration of gp350KICAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8+PD-1+LAG-3+ gp350KICAR-T cells were predominant in the bone marrow. The two types of KOTCRKICAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.

Tumor resident, TRA anti-viral CDR3 chemical sequence motifs are associated with a better breast cancer outcome.

While for certain cancers, such as cervical cancer, the link to viral infections is very strong and very clear, other cancers represent a history of links to viral infections that are either co-morbidities or drive the cancer in ways that are not yet fully understood, for example the "hit and run" possibility. To further understand the connection of viral infections and the progress of breast cancer, we identified the chemical features of known anti-viral, T-cell receptor alpha chain (TRA) complementarity determining region-3 (CDR3) amino acid sequences among the CDR3s of breast cancer patient TRA recombinations and assessed the association of those features with patient outcomes. The application of this novel paradigm indicated consistent associations of tumor-derived, anti-CMV CDR3 chemical sequence motifs with better breast cancer patient outcomes but did not indicate an opportunity to establish risk stratifications for other cancer types. Interestingly, breast cancer samples with no detectable TRA recombinations represented a better outcome than samples with the non-anti-CMV CDR3s, further adding to a rapidly developing series of results allowing a distinction between positive and possibly harmful cancer immune responses.

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.

A CD3 Fusion Receptor (CD3-FR) Uniquely Enables Compatibility of Allogeneic CAR-T and -NK Cells with T Cell Engagers to Enhance Antitumor Function and Limit Antigen Escape

Chimeric antigen receptor (CAR) T cells have shown remarkable clinical success in the treatment of many malignancies. However, certain challenges remain, including broad patient access and durability of response. We have shown that a scalable manufacturing platform, where genetically edited induced pluripotent stem cell (iPSC) master cell lines are created to serve as a renewable starting material for the derivation of uniformly engineered CAR T or NK cells, can facilitate broad patient access in an off-the-shelf manner. To improve durability of response, often plagued by antigen heterogeneity found in cancer, we are pursuing several combination strategies, merging cell therapy with therapeutic agents such as checkpoint blockade therapy and monoclonal antibodies to increase anti-tumor activity and multi-antigen targeting of cancer. T cell engagers have also shown remarkable advancements in the treatment of various cancer types, but they are also afflicted by their own challenges and would equally benefit from combination strategies. Unfortunately, T cell engagers are not compatible with allogeneic adoptive cell therapy, as the cells used in the allogenic setting do not express the CD3 molecule used for interaction with the engagers, as the T cell receptor (TCR) that supports CD3 surface expression, is either not expressed in NK cells or has been ablated in T cells to prevent graft-versus-host disease. To investigate the anti-tumor synergy between off-the-shelf cell therapy and T cell engagers, we developed a novel CD3ε fusion receptor (CD3-FR) to uniquely support the expression of a functional CD3 on TCR-less allogeneic T and NK cells and to enable compatibility between allogeneic cell therapy and T cell engagers. Initially, CD3-FR constructs with various co-stimulating endodomains were confirmed to elicit T cell engager-dependent NFAT activity in TCR alpha chain (TRAC) knockout (T-KO) Jurkat cells. Next, iPSCs already containing a CAR inserted into the TRAC locus were engineered to contain CD3-FR. CAR+ CD3-FR+ iPSCs were differentiated into CAR-iT cells, uniformly expressing both modalities (>95% CAR+, >90% CD3ε+, TCR not detected). To demonstrate that CD3-FR+ CAR-iT cells can elicit T cell engager-dependent antitumor activity, CAR antigen (1° Ag) negative but T cell engager (2° Ag) positive target cells were used in various cytotoxicity assays. As expected, control CAR-iT cells failed to kill the 1° Ag negative tumors despite addition of T cell engager targeting 2° Ag. On the other hand, CD3-FR+ CAR-iT cells showed potent activity toward target cells but only in the presence of the T cell engager (>80% cytolysis). Furthermore, when comparing the expression of activation markers in CD3-FR+ CAR-iT cells in co-cultures with or without the T cell engager, we observed an increased proportion of CD69+ (61% vs. 28%) and CD25+ (37% vs. 2%) cells in the CD3-FR+ CAR-iT cell co-cultures containing the T cell engager. Cytokine production was also tested, with increased TNF (5-fold) and IFNγ (7-fold) measured in supernatants from CD3-FR+ co-cultures upon the addition of T cell engager, further demonstrating T cell engager-dependent functionality and specificity of the CD3-FR construct. To test the ability of CD3-FR CAR-iT cells to mitigate 1° Ag escape, we used a target cell population heterogenous for the CAR-specific target (1° Ag, 50% positive) but homogenous for EpCAM (2° Ag, 100% positive). After three days, the standard CAR-iT cells failed to control tumor cell growth due to antigen escape. In contrast, CD3-FR+ CAR-iT cells exhibited robust control (80% cytolysis) of the 1° Ag heterogenous targets in the presence of αEpCAM, indicating mitigation of 1° Ag escape. Lastly, CD3-FR was edited into iPSC-derived NK (iNK) cells to demonstrate compatibility of CD3-FR+ NK cells with T cell engagers. An xCelligence-based killing assay was performed in the presence of a T cell engager, where CD3-FR+ iNK cells showed superior target cell killing (~80% lysis) after 96 hours compared to parental iNK cells (~30% lysis), demonstrating the potential for synergy between T cell engagers and NK cells (Figure 1). Taken together, these studies present a novel opportunity to combine T cell engager technology with off-the-shelf CAR-iT or iNK cell products, uniquely merging two powerful therapeutics whose combination was previously underappreciated in improving cancer therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prediction of immunodominant CD4+ SARS-CoV-2 epitopes with TCR repertoire sequencing data

Abstract The T cell receptor (TCR) is a hypervariable molecule defining the specificity of T cells for peptide-MHC complex. Defining exact epitopes is crucial to profile T cell responses in COVID-19 and yet very few MHC-II restricted SARS-CoV-2 epitopes are currently known. Here, we propose a reverse epitope discovery technique, which, instead of using large pools of peptides to identify reactive T cells, utilizes TCR repertoire sequencing data as the means to predict immunodominant epitopes. The core idea of the approach is to combine information from large, publicly available TCR repertoire datasets: TCRbeta repertoires from cohorts of COVID-19 patients and controls, and paired TCR repertoires of single T cells activated by SARS-CoV-2 peptides. Our pipeline allows us to predict the HLA-restriction of public SARS-CoV-2 specific TCR clonotypes, which in turn allows us to predict binding to a specific peptide. We applied this approach to single cell TCR repertoires of CD4+ T cells from COVID-19 patients, and predicted six MHC-II restricted immunodominant epitopes and alpha-beta TCR motifs recognising them and tested our predictions experimentally. We further applied this technique to bulk TCRalpha repertoires from T follicular helper cells from draining lymph nodes of healthy donors after BNT162b2 mRNA vaccination. We found that the DPB1*04 restricted S167–180 epitope is responsible for the largest public CD4+ response, and recognition of this epitope is driven by a semi-invariant TCR alpha chain. This finding led to generation of the DPB1*04 S167–180 tetramer, allowing to track SARS-CoV-2 specific CD4 cells in peripheral blood and lymph node samples with flow cytometry. This work was partially supported by R01AI136514 grant. This work was partially supported by R01AI136514 grant.

A novel unconventional T cell population enriched in Crohn’s disease

ObjectiveOne of the current hypotheses to explain the proinflammatory immune response in IBD is a dysregulated T cell reaction to yet unknown intestinal antigens. As such, it may be possible...

TT52CAR19: Phase 1 Trial of CRISPR/Cas9 Edited Allogeneic CAR19 T Cells for Paediatric Relapsed/Refractory B-ALL

Background‘Off-the-shelf’ CAR T cell therapies are being investigated as alternatives to autologous CAR therapy, and can be generated using genome editing from allogeneic donors. Strategies to address HLA barriers include disruption of T cell receptor expression to prevent GVHD and removal of cell surface HLA expression to obviate host mediated rejection, as well as removal of CD52 to create a survival advantage in the presence of Alemtuzumab. In this study, lentiviral mediated CAR19 expression is linked to CRISPR editing through the incorporation of CRISPR guide RNA sequences within the vector 3'Long terminal repeat (LTR). The trial is in progress using pre-manufactured batches of TT52CAR19 T cells.Investigational medicinal product (IMP)Three allogeneic non-HLA matched donor derived CAR19 T cell banks were manufactured from steady state apheresis harvests from registry volunteer donors, using a semi-automated process under compliant conditions. Cells were activated with anti-CD3/CD28 (Transact) reagent and transduced with a lentiviral vector, TT52CAR19, and dual guide sgRNA cassettes targeting the T cell receptor alpha chain (TRAC) and CD52 loci. Next electroporation of Cas9 mRNA elicited transient genome editing, and automated magnetic bead depletion removed remaining TCRαβ+ T cells (mean 0.7%) and enriched CAR19+ T cells (mean 92.8%). Cells were cryopreserved in aliquots suitable for dose-banding and subjected to release testing, including flow cytometry, quantification of copy number (mean 3.83) and exclusion of replication competent lentivirus. Potency of each bank was confirmed in human:murine chimera experiments.Trial sponsorship & approvalsThe study is open at a single site and is sponsored by Great Ormond Street Hospital NHS trust and is supported by the Medical Research Council and National Institute for Health Research. Clinical trial authorisation was awarded by the MHRA after expert review, and ethical approval including gene therapy advisory committee (GTAC) review, and health research authority (HRA) approval. The study is open label, single arm and non-randomised.Study aims and ObjectivesThe study aims to establish the safety and feasibility of TT52CAR19 for the induction of molecular remission in children with relapsed /refractory CD19-positive B-cell acute lymphoblastic leukaemia (B-ALL) within 28 days, ahead of planned allogeneic haematopoietic stem cell transplantation (allo-SCT). Assessments include time to remission, duration of remission, disease-free survival and overall survival. Expansion, persistence and elimination of TT52CAR19 cells and tracking of immune recovery after allo-SCT is monitored. Recording of complications and toxicities, including possible genotoxic side effects from CRISPR/Cas9 modification provides safety profile information.Eligibility, infusion and outcomesThe study plans to treat 10 children aged between 6 months and < 18 years with CD19+ B-ALL quantified at >10 -4 in marrow (by flow or PCR) who are ineligible for autologous therapy. To date 2/4 children screened were found eligible and proceeded to lymphodepletion comprising Fludarabine, Cyclophosphamide and Alemtuzumab followed by a single infusion of 0.8-2.0x10 6 CAR19 T cells and a maximum of 5x10 4/kg TCRαβ T cells. In both cases, there was no GVHD or CRS >grade 1 and recovery of neutrophil counts by d28. Molecular remission was achieved in one child, where TT52CAR19 cells persistence was tracked by chimerism and copy number until further conditioning and allo-SCT. This child remains in remission >6 months later.ConclusionsFeasibility of pre-manufacturing off-the-shelf CRISPR/Cas9 edited CAR19 T cells is demonstrated and the trial has provided first in human safety data and preliminary indications of potent anti-leukaemic activity in one of two subjects dosed. DisclosuresQasim: Autolus: Current equity holder in publicly-traded company; Novartis: Honoraria; Servier: Research Funding; Tessa: Membership on an entity's Board of Directors or advisory committees.

Previously Unknown Stage of TCR Beta VDJ Recombination As a Novel Rich Source of Markers for Clonality Assessment and MRD Monitoring in T-ALL

Background. Genomic rearrangements of T and B cell receptor genes (BCR and TCR) have become the gold standard marker for clonality assessment and minimal residual disease monitoring (MRD) in acute lymphoblastic leukemia (ALL). B-ALL can bear both TCR and BCR rearrangements, whereas T-ALL contains mainly TCR rearrangements. The limitation on the number of potential markers decreases T-ALL cases suitable for MRD monitoring and its reliability. We discovered a missed stage of TRB locus VDJ-recombination during which rearrangement between D1 and D2 segments occurs. Here we examined this new type of rearrangement in pediatric T-ALL as a potential clonal marker for MRD diagnostics for the first time.Methods. Identification of TRB D1-D2 rearrangements was performed using targeted high-throughput sequencing on the Illumina MiSeq instrument [1]. Genomic libraries for sequencing were obtained using PCR with intronic primers flanking the TRBD1 and TRBD2 genes. Genomic DNA for the libraries was extracted from T-ALL patients' bone marrow and peripheral blood of healthy volunteers. DNA from normal CD4+ and CD8+ T-cells was used as a positive control. DNA from normal B-cells and the RMS cell line was used as a negative control. Extraction of TRB D1-D2 rearrangements from sequencing data was performed using modified MiXCR software. Post-analysis was performed using VDJtools software.Results. This study analyzed a cohort of 144 children aged 2-16 years with T-ALL and 12 healthy individuals aged 25-40 years. We identified TRB D1-D2 rearrangements in all analyzed samples except negative controls (B-cells and RMS cell line). The dominant TRB D1-D2 clone in all normal samples occupied 25-30% and had zero length of the D1-D2 junction. The clonal size of the second clone in the top was 3-8%. Based on this observation, we set a 30% cut-off for zero-length D1-D2 junction rearrangements and a 10% cut-off for others to identify abnormally proliferated T-ALL specific clones.We identified 221 different leukemia-related D1-D2 rearrangements in 124 T-ALL samples in total. We intersected them with those in normal samples to test the uniqueness of leukemia-related rearrangements as potential markers. 89% of analyzed rearrangements were specific for leukemia samples, so they can be used for tracking leukemic cells. 11% of rearrangements were present in at least one normal sample and can be used for clonality analysis but are useless for MRD monitoring due to its relatively high generation probability. Zero-length D-D junction was detected as a major clonal marker (25-100%) in 42 T-ALL samples. Nevertheless, 23 out of these 42 samples contained additional unique rearrangements useful for MRD. 29 out of the other 82 samples included one major rearrangement (clonal rate 80-90%), six samples had two major rearrangements with equal clonal rates (30-50%), and the rest of the samples contained 3-5 rearrangements representing oligoclonality.The length of the D-D junction among detected rearrangements in both leukemic and normal samples has a bimodal distribution which is probably linked to two different levels of TdT or exonuclease activity during D1-D2 recombination. Additionally, we observed a strong correlation (Mann-Whitney-U-test, p-value < 0.001) between the presence of TRB D1-D2 rearrangements in T-ALL samples and the CD117 negative immunophenotype of leukemic cells.Conclusions. Obtained results show that TRB D1-D2 rearrangements are a common feature of normal and malignant T-cells. Unique leukemia-related TRB D1-D2 rearrangements can be detected in 73% of pediatric T-ALL cases. It can be routinely detected by PCR with subsequent NGS and can be easily integrated into an existing multiplex system for T-ALL clonality and MRD analysis.References. 1.Komkov et al. High-throughput sequencing of T-cell receptor alpha chain clonal rearrangements at the DNA level in lymphoid malignancies. British Journal of Haematology. 2020 Mar. 188(5): 723-731.Acknowledgment. This work was supported by Russian Science Foundation grant № 20-75-10091 to AK. DisclosuresNo relevant conflicts of interest to declare.

Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering for Cell Therapy Development

Gene-editing technology has revolutionized molecular therapeutics, enabling DNA-engineering-based approaches to treat disease. Despite this, development of medicines using gene editing has been hampered by technological, immunological, and legal limitations. We described previously the discovery of novel type II and type V gene-editing systems from metagenomic data, the characterization of these systems in vitro, and a demonstration of their activity in immortalized cell lines. Here, we substantially advance this work with three separate, novel gene-editing systems, demonstrating their utility for cell therapy development. We express and purify the nuclease components of both type II and type V effectors and show that all three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain constant region occurred in up to 95% of cells, both copies of the TCR beta-chain constant region in up to ~90% of cells, and beta-2 microglobulin in up to 95% of cells. Simultaneous double knock-out of TRAC and TRBC was obtained at an equal frequency. Gene editing with our systems had no effect on T cell viability. Further, we use our novel gene-editing systems to exploit homology-dependent DNA repair to integrate a CAR construct into the TCR alpha-chain locus (in up to ~60% of T cells), and demonstrate high-level CAR expression and antigen-dependent CAR-T cytotoxicity. Such robust editing activity at the TCR loci will permit efficient engineering and manufacture of allogeneic chimeric antigen receptor- (CAR) and TCR-based cell therapies.We next applied our novel gene-editing tools to NK cells and B cells. We achieved almost 100% gene disruption at the CD38 locus in NK cells and integrated a chimeric antigen receptor into the NK cell genome at a frequency of ~40%. Such CAR-NK cells displayed robust CAR-directed cellular cytotoxicity. B cell editing occurred in approximately 80% of target cells with successful transgene integration.Cas9 is notorious for tolerating mismatches and bulges in its target, resulting in high levels of unwanted DNA double-strand break formation. In contrast, interrogation of our gene editing systems using GUIDE-seq reveals no or very few off-target sites even at doses greatly in excess of those required for high-frequency gene editing. Finally, as our systems are taken from microbes found in environmental samples rather than from human pathogens, we expect pre-existing immunity to our nucleases will be quite rare. In all, we show that three new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development. DisclosuresNo relevant conflicts of interest to declare.

Abstract 226: Tumor-infiltrating lymphocyte diversity and clear cell renal cell carcinoma

Abstract The availability of systemic treatments of metastatic clear cell renal cell carcinoma (ccRCC) has evolved steadily over the past decade, particularly with the availability of drugs that work in the microenvironment through immune checkpoint blockade (ICB). However, various studies have indicated that response to ICB is not likely driven only by mutational burden, neoantigen burden, or PD-L1 status. We here employed genomics approaches to recover V(D)J recombination sequences from primary tumor bulk RNA sequencing from two ccRCC cohorts; Moffitt Total Cancer Care Cohort (n=105) and the Clinical Proteomic Tumor Analysis Consortium Cohort (n=110). We applied an ecological generalized diversity index (GDI) to quantify tumor infiltration by CDR3 sequences from patient tumors using three point-estimate descriptions: richness, evenness, and inflection point of the GDI. GDI has multiple properties that render it more flexible than the commonly used Shannon index, e.g. the inflection point q, which can be understood as a dataset specific diversity estimate that specifically balances richness and evenness of the distribution that defines diversity. Individuals with larger and more aggressive tumors had increased richness diversity of tumoral CDR3 sequences, specifically in sequences recovered from T-cell receptor alpha (TRA) and B-cell immunoglobulin lambda light chain. Furthermore, the TRA inflection point q diversity stratified patients based on survival, with individuals that had a larger inflection point q diversity having improved overall survival (hazard ratio of 0.526, p-value 0.036 from multivariate Cox regression). We also found significant discrimination of diversity between racial and gender differences in tumor-infiltrating lymphocyte diversity. Non-white patients showed a 2.1-fold increase in evenness in B-cell receptors compared to white patients; T-cell receptors gamma and delta inflection point q diversity was increased at least 1.7-fold in female patients compared to male patients. These demographic differences highlight the need to further investigate race- and gender-related differences in immune infiltration in ccRCC. With ICB being first line therapy of metastatic ccRCC, it is critical to define and evaluate novel analytical tools to characterize the level of immune cell infiltration and diversity. We have demonstrated here that the ecological generalized diversity index shows promise as a novel quantitative, prognostic biomarker for ccRCC, with the potential to extend to other malignancies. Citation Format: Meghan C. Ferrall-Fairbanks, Nicholas Chakiryan, Boris I. Chobrutskiy, Youngchul Kim, Jamie K. Teer, Anders Berglund, James Mulé, Michelle Fournier, Erin M. Siegel, Esther N. Katende, George Blanck, Brandon J. Manley, Philipp M. Altrock. Tumor-infiltrating lymphocyte diversity and clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 226.

Gene Editing of TRAC Locus Utilizing Megatal Nucleases Increases Expression of Transgenic TCRs Delivered Via Lentiviral Vector-Mediated Gene Transfer

T-cell receptor (TCR) transgenic T cell therapies have demonstrated efficacy in late stage and refractory oncologic malignancies, including melanoma and synovial cell sarcoma. However, these responses are generally either short in duration or occur in a minority of patients. One of the limitations of transgenic TCR therapies may be related to the mispairing of the introduced transgenic TCR alpha and beta chains with the endogenous TCR chains, resulting in non-productive TCR transgenic T cells. Using genome editing, endogenous TCR genes can be eliminated, potentially reducing mispairing with a subsequent increase in the activity of the transgenic TCR. It is essential that therapeutic genome editing be achieved with sufficient specificity to minimize off-target editing events. MegaTALs, derived by fusing a DNA binding array to an engineered meganuclease, can be iteratively refined in response to off-target detection, achieving a genome-wide increase in on-target versus off-target discrimination. To this end, we developed a megaTAL targeting the T-cell receptor alpha chain (TRAC) locus and identified a prominent off-target site using unbiased double strand break detection methods coupled with targeted amplicon sequencing. Optimization of this megaTAL using a combination of protein engineering approaches resulted in a reagent achieving >90% editing of the TRAC locus in primary human T cells and the off-target site was eliminated. The TRAC specific megaTAL resulted in a 92% reduction in CD3 surface expression a surrogate marker of TCR expression (5.6±0.4% vs 97.6±0.2%, p<0.0001). To further evaluate this nuclease we transduced primary human T cells with a lentiviral vector encoding an anti-NY-ESO-1 transgenic TCR. Expression of the transgenic TCR was followed by staining for the TCR Vbeta13.1 protein and we observed 74% transduction efficiency accounting for Vbeta13.1 usage in the endogenous TCR repertoire (79.4±2.6% vs 5.1±0.7%, p<0.0001). Of the transduced Vbeta13.1 positive cells, 64.3% were positive for the transgenic TCR as determined by tetramer staining, suggesting 35.7% of transduced cells exhibit TCR mispairing with components of the endogenous TCR. In parallel, T cells were treated with a combination of megaTAL and TCR vector transduction. Transgenic TCR expression in TRAC megaTAL edited T cells restores CD3 surface expression in all Vbeta13.1-positive transduced cells (74.0±2.4% vs 5.6±0.4%, p < 0.0001). In addition, 96% of the TRAC edited Vbeta13.1 positive cells were now positive for the transgenic TCR as shown by the tetramer staining (95.5±1.0% vs 64.3±4.8%, p<0.01). Treatment of transgenic TCR expressing human T cells with the TRAC specific megaTAL resulted in a 31% reduction in mispairing of the transgenic TCR (4.4±1.0% vs 35.7±4.8%, p<0.01). Moreover, TRAC megaTAL treatment produced a 1.7-fold increase in the expression of the transgenic TCR in tetramer positive cells (gMFI 6292±662 vs 3800±200, p <0.05). TRAC edited TCR-transgenic T cells exhibit a similar phenotypic profile as un-edited TCR transgenic T cells and TRAC megaTAL editing does not result in a significant impact on T cell expansion, CD4 to CD8 T cell ratios, T cell memory subsets, or lentiviral integration during manufacturing. These results demonstrate that the megaTAL architecture facilitates the iterative refinement of nuclease specificity and activity, generating exquisitely selective, single-component reagents, and highlight the potential of megaTAL driven TRAC gene editing to further improve expression of transgenic TCRs. DisclosuresMagee:bluebirdbio: Employment, Equity Ownership. Diaconu:bluebirdbio: Employment, Equity Ownership. Pogson:bluebirdbio: Employment, Equity Ownership. Gupta:bluebirdbio: Employment, Equity Ownership. Jensen-Smith:bluebirdbio: Employment, Equity Ownership. Collette:bluebirdbio: Employment, Equity Ownership. Chavkin:bluebirdbio: Employment, Equity Ownership. Pasackow:bluebirdbio: Employment, Equity Ownership. Edwards:bluebirdbio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Astrakhan:bluebird bio: Employment, Equity Ownership. Certo:bluebird bio: Employment, Equity Ownership. Jarjour:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership, Patents & Royalties.

Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo

Although chimeric antigen receptor T (CAR-T) cell therapy achieves high remission rates, challenges (e.g., toxicity management and relapse prevention) remain. The major risks are cytokine release syndrome and related neurological toxicity. The influence of the CAR surface density on the efficacy/safety of CAR-T cell therapy and the factors determining CAR density were not elucidated comprehensively. Here, we discovered that the use of the MND promoter increased the transduction rate and reduced the CAR surface density. Additionally, MND-driven CAR-T cells had prolonged antileukemia activity in a mouse model. In an initial dual-armed anti-CD19 CAR-T cell pilot study (ClinicalTrials.gov: NCT03840317), eight and six subjects were infused with MND and EF1α promoter-driven autologous CAR-T cells (3 × 105 CAR-T cells/kg), respectively. MND subjects developed mild fever and lower interferon gamma (IFN-γ) concentrations than in the EF1A19 group. All but one subject in each cohort reached minimal residual disease (MRD)-negative complete remission after the first month of evaluation. These results represent the first comprehensive study on the promoter-driven modulation of CAR-T cell functionality. These findings encourage further evaluation of the potential of the MND promoter to drive CAR-T cells as a broadly applicable cellular product for anticancer immunotherapy.

Current challenges for unseen-epitope TCR interaction prediction and a new perspective derived from image classification.

The prediction of epitope recognition by T-cell receptors (TCRs) has seen many advancements in recent years, with several methods now available that can predict recognition for a specific set of epitopes. However, the generic case of evaluating all possible TCR-epitope pairs remains challenging, mainly due to the high diversity of the interacting sequences and the limited amount of currently available training data. In this work, we provide an overview of the current state of this unsolved problem. First, we examine appropriate validation strategies to accurately assess the generalization performance of generic TCR-epitope recognition models when applied to both seen and unseen epitopes. In addition, we present a novel feature representation approach, which we call ImRex (interaction map recognition). This approach is based on the pairwise combination of physicochemical properties of the individual amino acids in the CDR3 and epitope sequences, which provides a convolutional neural network with the combined representation of both sequences. Lastly, we highlight various challenges that are specific to TCR-epitope data and that can adversely affect model performance. These include the issue of selecting negative data, the imbalanced epitope distribution of curated TCR-epitope datasets and the potential exchangeability of TCR alpha and beta chains. Our results indicate that while extrapolation to unseen epitopes remains a difficult challenge, ImRex makes this feasible for a subset of epitopes that are not too dissimilar from the training data. We show that appropriate feature engineering methods and rigorous benchmark standards are required to create and validate TCR-epitope predictive models.

RNA and TCR Sequencing Shed Light on Mechanisms of Treg Suppression in a Murine Model of Acute GvHD

Introduction Regulatory T cell (Treg) based therapies are a promising approach for graft-versus-host disease (GvHD) prevention and treatment. However, mechanisms of Treg suppression of alloreactive conventional T cells (Tcon) in GvHD are incompletely understood. In this study, we performed paired RNA and T cell receptor (TCR) sequencing analysis on Tcon and Treg before and after transplantation to further elucidate Treg suppressive function during in vivo suppression of acute GvHD in an MHC major-mismatch mouse model. Methods CD45.2 Thy1.2 BALB/c mice were lethally irradiated (8.8 Gy) and transplanted with 5x106 T cell- depleted bone marrow cells (TCD-BM) from CD45.1 Thy1.2 C57Bl/6 mice alone or together with CD45.2 Thy1.2 C57Bl/6 FoxP3/GFP+ Treg (1x106) on day 0. On day 2, CD45.1 Thy1.1 C57Bl/6 Tcon (1x106; CD4:CD8 ratio = 2:1) were injected to induce GvHD. Irradiated (11 Gy) syngeneic C57Bl/6 recipients receiving C57Bl/6 TCD-BM and CD45.1 Thy1.1 Tcon alone were used as controls. On day 8, donor Thy1.1+ CD45.1+ CD4 and CD8 Tcon and Thy1.2+CD45.2+ FoxP3/GFP+ Treg were isolated by FACS from spleens and lymph nodes. T cell subsets before injection and recovered at day 8 were analyzed by paired RNA and TCR sequencing. Results The transcriptomic analysis revealed a dominant effect of the allogeneic transplant procedure on the clustering of the different T cell populations. Principal component analysis (PCA) of the top 1000 most differentially expressed genes revealed that 68% of the variance was explained by PC1, which clearly segregated CD4and CD8Tcon recovered at day 8 from allogeneic recipients from cells before injection or recovered from syngeneic recipients (Fig. 1). PC1 was mainly driven by naïve T cell genes (Ccr7, Sell, Il6ra, Il6st, Foxo1) that were progressively downregulated along PC1. Analysis of the TCR repertoire based on sequencing of the TCR alpha chain revealed a progressive clonal restriction along PC1 in CD4 and CD8 T cells (Fig. 1). Accordingly, clonal overlap between cells collected at day 8 and cells analyzed before injection were reduced in allogeneic recipients (Morisita Index [MI], CD4: 0.06±0.01; CD8: 0.02±0.01) compared to syngeneic controls (MI, CD4: 0.23±0.26; CD8: 0.57±0.05). Treg administration did not affect CD4 or CD8 T cell segregation along PC1, suggesting that they minimally interfered with cell activation and differentiation during GvHD. Accordingly, Treg did not inhibit clonal restriction (Fig.1) nor the reduction in clonal overlap (MI, CD4: 0.04±0.01; CD8: 0.01±0.01), indicating that Treg did not inhibit the initial activation of alloreactive T cells clones. Treg impact on CD4 but not CD8 Tcon transcriptome was revealed by PC2 (Fig.1). Treg induced the downregulation of TH1-signature genes (Tbx21, Il12rb1, Il12rb2, Stat4) and proinflammatory genes (Il18rap) while promoting up-regulation of anti-inflammatory genes (Il18bp), TH2 signature genes (Ccr4, Il4) and Il2 in CD4Tcon. Moreover, gene set enrichment analysis (GSEA) revealed that Treg treatment significantly impacted gene sets involved in metabolic processes in CD4and CD8Tcon, leading to a global up-regulation of genes encoding for enzymes involved in oxidative phosphorylation (OXPHOS) and downregulation of genes encoding for enzymes contributing to glycolysis (Slc2a1, Hk1, Pfkl, Pfkp, Pkm). Treg recovered at day 8 preserved a distinct transcriptomic signature observed before injection and further enhanced by the up-regulation of genes involved in Treg activation and suppressive function (Gata3, Tnfrsf18, Tnfrsf4, Icos, Ccr1, Ccr4, Il9r). GSEA in Treg revealed significant up-regulation of genes in the OXPHOS signature. TCR repertoire analysis showed clonal restriction of Treg during GvHD. Direct comparison of clone frequencies in Treg and CD4Tcon showed smaller clonal overlap on day 8 (MI=0.005±0.006) compared to day 0 (MI= 0.11±0.004) suggesting that Treg and CD4Tcon responses during GvHD are engaging different cell clonotypes triggered by different epitopes or antigens. Conclusion Our results indicate that Treg treatment did not interfere with Tcon activation and differentiation of alloreactive Tcon clones in a model of acute GvHD. Treg predominantly affected CD4Tcon and to a lesser extent CD8Tcon transcriptome, modulating the transcription of genes encoding pro- and anti-inflammatory molecules as well as enzymes involved in metabolic processes. Disclosures Turkoz: Adicet Bio: Current Employment, Current equity holder in private company. Negrin:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; BioEclipse Therapeutics: Current equity holder in private company; Amgen: Consultancy; KUUR Therapeutics: Consultancy; Biosource: Current equity holder in private company; UpToDate: Honoraria.

Transgenic lymphocytes targeting minor histocompatibility antigens for post-transplant immunotherapy

Background & Aim The relapse of acute leukemia occurs in one third of the patients after HLA-matched allogeneic stem cell transplantation (HSCT), resulting in high mortality. Disparities in minor histocompatibility antigens (MiHA, MHC-presented peptides derived from polymorphic self-proteins) can result in the development of allogeneic immune response. Several MiHA (HA-1, HA-2, and ACC-1Y) are encoded by the genes predominantly expressed in hematopoietic tissues. They represent promising targets for post-HSCT relapse therapy. The immune response towards them selectively targets patient hematopoietic cells sparing hematopoietic cells of donor origin and patients healthy tissues. Methods, Results & Conclusion Naïve CD8+ T cells from MiHA-negative donors were expanded in vitro using autologous dendritic cells, pulsed with MiHA peptides. Antigen-specific cells were sorted, and the sequences of T-cell receptor (TCR) alpha and beta chains were determined by NGS. 8, 10, and 1 TCRs specific for HA-1, HA-2, and ACC-1Y antigens, respectively, were cloned in the lentiviral vectors containing coreceptor CD8. T cell lines were generated for further analysis. To prevent off-target effects caused by the mispairing of endogenous and recombinant TCR chains, the endogenous TCR knockout was made using gRNA/Cas9 ribonucleoprotein complexes. Transgenic alpha and beta-chain constant domains were modified to be resistant to Cas9 cleavage and to form more stable TCR. Antigen-specificity was measured by binding to MHC-tetramers loaded with the MiHA peptide and by TCR-mediated activation of the Jurkat 76 triple parameter reporter cell line upon antigen stimulation. Most of the generated cell lines were specific to the desired antigen, and none were activated in response to irrelevant peptides presented in the same HLA. After analysis of cross-reactivity to allogeneic HLA on the panel of Epstein-Barr virus-transformed lymphoblastoid cell lines, the best receptors would be selected for further therapeutic application. Adoptive transfer of MiHA-specific genetically modified T cells holds great promise for the treatment of leukemia relapse after allo-HSCT. The work was supported by the Russian Foundation for Basic Research grant 19-29-04156. The relapse of acute leukemia occurs in one third of the patients after HLA-matched allogeneic stem cell transplantation (HSCT), resulting in high mortality. Disparities in minor histocompatibility antigens (MiHA, MHC-presented peptides derived from polymorphic self-proteins) can result in the development of allogeneic immune response. Several MiHA (HA-1, HA-2, and ACC-1Y) are encoded by the genes predominantly expressed in hematopoietic tissues. They represent promising targets for post-HSCT relapse therapy. The immune response towards them selectively targets patient hematopoietic cells sparing hematopoietic cells of donor origin and patients healthy tissues. Naïve CD8+ T cells from MiHA-negative donors were expanded in vitro using autologous dendritic cells, pulsed with MiHA peptides. Antigen-specific cells were sorted, and the sequences of T-cell receptor (TCR) alpha and beta chains were determined by NGS. 8, 10, and 1 TCRs specific for HA-1, HA-2, and ACC-1Y antigens, respectively, were cloned in the lentiviral vectors containing coreceptor CD8. T cell lines were generated for further analysis.

Abstract A92: Assessing the T-cell exhaustion status of TCR discovery outputs

Abstract Identification of antigen-specific T cell receptors (TCRs) is an essential component of the ImmTAC (Immune mobilizing monoclonal TCRs Against Cancer) platform at Immunocore. ImmTAC molecules are formed of a soluble TCR fused to an anti-CD3 effector function that engages, redirects and activates T cells to kill cancerous cells. TCRs recognize peptide antigens presented on the surface of the cancer cell by human leukocyte antigen (HLA). Thus, a fundamental step in our pipeline is to isolate T-cell receptors specific for validated cancer antigens for downstream development. T cells are isolated from whole blood of healthy donors to identify antigen-specific T cell clones using an in vitro model that consists of several stimulation steps. Activated CD8+ T cells are sorted into single cells for subsequent analysis and characterization. Despite the relative success of this approach, often analysis of T-cell clones is limited by low or absent mRNA expression for either or both TCR alpha (TRAV) and beta (TRBV) chains. We predict that TCR downregulation may result from overstimulation of T cells, leading to exhaustion. Here, we aimed to establish an in vitro model of T-cell exhaustion that would enable us to better understand the molecular mechanisms behind this process and thus facilitate method development. We induced extreme conditions of stimulation using CD3/CD28 beads and recombinant human IL-2 (rhIL-2) for 2-3 weeks. Whole-transcriptome analysis of the resulting activated T cells was compared to rested cells using a 5′ single-cell RNA-seq method from 10X Genomics (California, USA). Our preliminary analysis indicates that the activated T cells express several checkpoint inhibitors at higher levels. Our findings will be further investigated by performing full-length RNA sequencing of previously isolated single T-cell clones with low TRAV and/or TRBV detection rates. By identifying a molecular signature that represents the phenotype of exhausted T cells, we can potentially assess the state of T cells in TCR discovery outputs. Most importantly, determination of the expression levels of inhibitory receptors that are responsible for the decrease or loss of TCR expression is expected to enhance our understanding of the potential impact of T-cell exhaustion. Citation Format: Paraskevi Mallini, Filipa Bravo-Lopes, Karolina Lech, Sunir Malla, Maria Busz, Nathaniel Davies, Jacob Hurst, Luke Williams, Sterenn Davis, Michelle Teng. Assessing the T-cell exhaustion status of TCR discovery outputs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A92.

Abstract B62: Evaluation of systemic RNA-based cancer vaccine induced T-cell responses via mouse T-cell receptor (TCR) profiling

Abstract Identification of biomarkers is pivotal towards the proper development of cancer immunotherapies, in particular for designing the most effective regimens and identifying patient subgroups most likely to benefit from a given treatment. T-cell receptor (TCR) repertoire emerged as a promising biomarker in this respect, as it allows measurement of the dynamics of the T cell response both in time and in different compartments. In this work, we developed and evaluated an NGS-based mouse TCR profiling method (TCR alpha and TCR beta) for discovery and validation of biomarkers related to the antitumoral T-cell response in different compartments upon systemic administration of an RNA-based cancer vaccine (RNA-lipoplex). The in-house established mouse TCR profiling protocol was able to detect blood-spiked known-TCR sequences down to a frequency of 0,001% spiked cells for both TCR alpha and TCR beta chains and the relative frequencies of detected TCRs in the tumor correlated well with the degree of T-cell infiltration into the same tumors assessed by flow cytometry. Comparative analysis of changes in the TCR repertoire between blood and tumor upon repetitive vaccination with RNA-lipoplexes encoding immunodominant epitope of chicken ovalbumin (OVA257-264) revealed that richness as well as diversity of endogenous TCRs were generally lower in B16-OVA tumors compared to blood and decreased with further vaccination. Interestingly, top clones overlapped increasingly in blood and tumor until after the second vaccination, but drifted apart after the third vaccination, with the TCR repertoire in the tumor narrowing down. This overlap was less evident when mice were inoculated with B16-F10 tumors. Top clones within the same compartment were found to be highly variable between individual animals, complicating comparative analyses. Monitoring intra-individual TCR repertoire changes in the blood of a given mouse over time, however, indicated a tendency for decreasing richness and diversity following a single RNA vaccination, which may be pointing at repertoire adaptation and antigen focus. Supporting this notion, tumors isolated at late time-points exhibited a lower diversity than those investigated at earlier time-points and at baseline. In contrast to RNA vaccination, richness and diversity were reduced in response to treatment with an agonistic anti-CD40 antibody in blood and spleen but were similar among tumors. Consequently, richness and diversity in the periphery may not necessarily be predictive of corresponding repertoire changes in the tumor microenvironment. Citation Format: Lena Mareen Kranz, Barbara Schrörs, Thomas Bukur, Christian Albrecht, Lorieta Leppin, Martin Suchan, Marta Faryna, Eileen Powalsky, Rainer Hipfel, Özlem Türeci, Ugur Sahin, Mustafa Diken. Evaluation of systemic RNA-based cancer vaccine induced T-cell responses via mouse T-cell receptor (TCR) profiling [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B62.