TCR Alpha Chain Research Articles

Ultrasensitive allele inference from immune repertoire sequencing data with MiXCR.

Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), is of critical importance for immune responses to pathogens and vaccines. Adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread in immunology research making it the most readily available source of information about allelic diversity in immunoglobulin (IG) and T cell receptor (TR) loci. Here we present a novel algorithm for extra-sensitive and specific variable (V) and joining (J) gene allele inference, allowing reconstruction of individual high-quality gene segment libraries. The approach can be applied for inferring allelic variants from peripheral blood lymphocyte BCR and TCR repertoire sequencing data, including hypermutated isotype-switched BCR sequences, thus allowing high-throughput novel allele discovery from a wide variety of existing datasets. The developed algorithm is a part of the MiXCR software. We demonstrate the accuracy of this approach using AIRR-seq paired with long-read genomic sequencing data, comparing it to a widely used algorithm, TIgGER. We applied the algorithm to a large set of IG heavy chain (IGH) AIRR-seq data from 450 donors of ancestrally diverse population groups, and to the largest reported full-length TCR alpha and beta chain (TRA; TRB) AIRR-seq dataset, representing 134 individuals. This allowed us to assess the genetic diversity within the IGH, TRA and TRB loci in different populations and to establish a database of alleles of V and J genes inferred from AIRR-seq data and their population frequencies with free public access through an online database.

Abstract 2062: Identification of functional MAIT-specific TCRs using MR1 dCODE Dextramer® reagents and evaluating antigen presentation

Abstract Objectives: To support TCR discovery and enable evaluation of TCR target expression on the surface of cells, we have established a streamlined workflow allowing TCR Discovery, validation and evaluation of MHC-antigen expression on target cells. As proof of concept, MR1 dCODE Dextramer® reagents were used to identify antigen-specific MAIT cells using single-cell sequencing, TCR sequences were extracted from the resulting data and then cloned and expressed as TCR monomers. The specificity of the TCRs was subsequently validated as TCR Dextramer® reagents and used to evaluate target MHC-peptide presentation on cell surface. Methods: Mixed PBMC samples from three healthy donors were stained with antibodies targeting relevant phenotypic markers as well as MR1 dCODE Dextramer® reagents carrying the MAIT cell-specific ligand 5-OP-RU. Following staining, sorting, and partitioning on a BD Rhapsody™ Single-Cell Analysis System, the cells underwent sequencing for a characterization of gene and surface marker expression as well as paired alpha-beta TCR sequences. Single alpha and beta chains were individually expressed, refolded together and purified. Refolded TCR monomer functionality was validated using an artificial cell assay. The functional TCRs can be multimerized on a Dextramer® backbone and used to evaluate expression of target on cell surface. Results: The majority of the identified CD161+ MAIT cells (~70%) expressed typical TCRs consisting of TRAV1-2 and TRAJ33 as well as TRBV6/20, TRBD1/2, and TRBJ2 gene segments. Three alpha-beta TCR sequences were selected: Two TCRs having common TRAV+TRAJ, TRBV+TRBJ combinations and one having a rare TRAV+TRAJ one. TCR alpha and beta chains were successfully expressed for all selected TCRs but only 2 TCRs showed a correctly refolded TCR as demonstrated by proper recognition of MR1/5-OP-RU. Based on these two functional TCRs, TCR Dextramer® reagents were generated and used to evaluate expression of target antigen on cell surface. Conclusion: We demonstrate a workflow allowing: (i) Identification of MAIT cells and their corresponding TCR sequences. (ii) Generation of soluble TCR molecules based on the identified sequences and validation of their specificity. (iii) Generation of TCR Dextramer® reagents allowing identification of target expression on surface of cells. This workflow is also useful for identification of other antigen-specific T cells and creates a new tool for evaluation of antigen-expression by MHC on surface of cells. By combining the high sensitivity of dCODE Dextramer® technology with TCR sequencing and creation of TCR Dextramer® reagents, both low-affinity interactions and rare cell types can be detected and studied in unique detail. Citation Format: Kevin Lenogue, Bjarke Hansen, Kivin Jacobsen, Liselotte Brix, Thomas Holberg Blicher. Identification of functional MAIT-specific TCRs using MR1 dCODE Dextramer® reagents and evaluating antigen presentation [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 2062.

Deciphering Antigen Specificity of Protective Regulatory T Cell Clones in Patients after Allogeneic Stem Cell Transplantation

Introduction: Clonal expansion of donor regulatory T cells (Tregs) in recipients of allogeneic stem cell transplantations (allo-SCT) is associated with the absence of acute graft-versus-host disease (aGVHD). Therefore, we hypothesize that antigen-specific donor Tregs prevent aGVHD. In this project, we aim to identify the cognate antigen of expanded Treg clones in patients after allo-SCT. Methods: We recruited 177 consecutive patients undergoing allo-SCT for acute myeloid leukemia without severe infection and CMV-reactivation for this study. Healthy adults matching patients' sex and age served as the control group. We sorted CD3 +CD4 +CD25 hiCD127 low Tregs from peripheral blood mononuclear cell samples collected early after transplantation (21 to 36 days, median 27 days), and we performed single-cell RNA sequencing (scRNAseq) to profile Treg transcriptome including both TCR alpha and beta chain. To determine the antigen specificity of a given TCR, we adapted a cytokine-capturing antigen-screening system (Lee and Meyerson, Sci Immunol, 2021). We built the system by 1) expressing designated TCRαβ chains in Jurkat cells, and 2) expressing cell-surface interleukin-2 capture mAb, corresponding MHC-II molecules, and CD74-fused epitope libraries in HEK293T cells serving as antigen-presenting cells (APCs). By co-culturing both modified cell lines, we can perform high-throughput screening of pooled candidates. To obtain epitope-encoding minigenes, we either used RNA fragmentation to generate candidates randomly or customized oligonucleotide synthesis to encode the tiled human proteome. To scale down the diversity of the synthetic library, we utilized NetMHCIIpan (DTU Health Tech) for MHC-II binding affinity prediction. By sequencing the CD74-fused minigene after screening out APCs with the highest cytokine intensity on the cell surface, we can decode the peptide sequence recognized by the given TCR. We have validated the cytokine-capturing antigen-screening system using a CMV-specific TCR and its cognate antigen. Results: In total, we analyzed six patients with and six patients without aGVHD. ScRNAseq showed that the FOXP3 + Treg population in patients without aGVHD shared more transcriptional similarities with healthy control as compared to patients with aGVHD. Due to lymphocytopenia, Tregs are extremely low in numbers early after allo-SCT, but clustering analysis revealed highly clonal expanded populations of Tregs with various degrees of activation. Notably, one patient without aGVHD was found to have a distinct Treg-dominant CD4 population, and a markedly abundant Treg clone (>15% of the entire Treg repertoire) was found to be parts of both FOXP3 + and FOXP3 - clusters. We then transduced the most abundant TCR into JM-T, the TCR-deficient Jurkat cell. We used an RNA-fragmented randomized library for the first screening, in which we identified one shared epitope in three independent screenings of high frequency. We then moved on to perform screenings for synthesized candidate pools predicted by NetMHCIIpan, representing human proteome and common post-allo-SCT infections. Currently, we are analyzing sequencing reads from parallel screenings and attempting direct expansion of IL-2 + sort-outs. Conclusion: By expanding our established screening system, we aim to elucidate the antigens of multiple clonal expanded TCR candidates from the same patient. Identification of specific antigens leading to clonal Treg expansion and protection from GVHD holds the potential for the development of targeted cell therapies.

CTIM-16. DEVELOPING PATIENT SPECIFIC TUMOR INFILTRATING LYMPHOCYTES (TILS) WITH INCREASED TUMOR SPECIFICITY AS A THERAPY FOR GLIOBLASTOMA

Abstract Immunotherapy failure in glioblastoma results from three main hurdles; limited numbers of T cells within the tumor, T cell exhaustion and significant intra-tumoral and intra-patient heterogeneity leading to lack of T cell tumor recognition. Our group has developed an expansion protocol to isolate and expand less exhausted tumor infiltrating lymphocytes (TILs) from the tumor periphery, selecting for TILs that recognize tumor antigen ex vivo. This process will increase the number of T-cells specific for an individual patient’s diverse tumor neoantigens. We have successfully expanded glioblastoma associated TILs from 66% of the novel Lukens trap substrate, which collects TILs and tumor from the tumor periphery. Cells are expanded in two stages, preREP and REP, through novel methods based on a protocol with clinical success in lung cancer and modified to increase the percent of tumor specific T cells. Through co-culture with patient matched glioma neurospheres we have identified activated T cells from each stage of culture (initial product, preREP, REP) based on CD137+ expression and tumor specific cytokine release (IFNy+TNFa+IL2+IL17a+) by flow cytometry. Through 5’scRNA sequencing paired with TCR repertoire analysis we can identify activation specific TCRs. Overrepresented TCR alpha and beta chains will be linked by a P2A site, cloned into a lentiviral vector, and transduced into TCR negative Jurkat cells to confirm tumor reactivity. This strategy will allow us to enrich and assess tumor specific TCR activity compared to bystander activation in our TIL product. Following additional preclinical assessment of cytotoxicity against autologous tumor, we will evaluate safety and feasibility of by administering our novel TIL product intrathecally in patients with glioblastoma.

P06.04.B NEW APPROACHES FOR EXPANDING GLIOBLASTOMA INFILTRATING LYMPHOCYTES

Abstract BACKGROUND Adoptive cell therapy using tumor infiltrating lymphocytes (TILs) has shown promise in melanoma and Non-Small Cell Lung Cancer. TILs culture and expansion from GBM remains challenging due to paucity of infiltrated T-cells that are also exhausted. This hinders creation of potent TILs. We have developed a new efficient method to manufacture potent TILs from GBM samples. METHODS We have developed efficient TILs manufacturing methods using both solid and aspirated liquid samples collected during vacuum assisted excision containing tumor fragments that may stem predominantly from the tumor margins and provide a source for TILs. We followed the traditional two steps: initial expansion (preREP) where T-cells are expanded with tumor cells to selectively expand tumor antigen-reactive TILs, followed by a large-scale expansion (REP) after further activation. For both steps we optimized media and reagents. RESULTS TILs cultures from solid tumors were established from enzymatically digested tissue and by fragment plating. Successful TILs yield from liquid samples required enzymatic digestion, debris removal and RBC lysis prior to culture initiation. PreREP TILs yields ranged from a few million to 2 billion viable cells for solid and liquid samples. REP of TILs from liquid samples expanded similarly from solid samples. REP of TILs reached up to 556-fold expansion (mean: 346, range: 95-556) providing promising numbers of activated TILs. The potency of the TILs was demonstrated with tumor specific Interferon-gamma production when cultured with matched tumor digest or neurospheres. CONCLUSIONS We successfully expanded TILs that are rich in cytotoxic T-cells, demonstrated their tumor reactivity and further expanded the preREP TILs to reach therapeutic doses. We have characterized the TILs by scRNA and TCRseq and analyze paired TCR reads using scRNA sequencing. Overrepresented TCR alpha and beta chains will be linked by a P2A site and will be cloned into a lentiviral vector to establish a TCR library which will be transduced into novel Jurkat Triple Parameter Reporter Cell Lines under the control of different T cell activation programs. TCR Jurkat Libraries will be cocultured with original tumor material and sorted to identify tumor reactive TCRs; enabling us to define the percentage of TCRs that are enriched by TIL isolation that are specifically reactive to tumor vs bystander expanded TCR cells. Ultimately, we aim to bring this therapy to patients in a clinical trial.

The redirected killing of PD-L1 positive tumor cells by the expanded mucosa-associated invariant T (MAIT) cells is mediated with a bispecific antibody targeting TCR Vα7.2 and PD-L1

Pan-T cell targeting by CD3-based T cell engagers has brought program-shift treatment and management of blood tumors. However, these modalities have been shown to provoke all types of T cells leading to cytokine storm syndrome, and activate Treg cells. Thus, modulating and potentiating the antitumor responses of a specific T cell subset was encouraged. We initially found that high purity of mucosa-associated invariant T (MAIT) cells could be expanded by the combination of plate-immobilized Vα7.2 mAb (Clone 3C10) and IL2 plus IL15. Then, we generated a novel anti-Vα7.2 TCR bsAb, Vα7.2 x PD-L1, to invoke the anti-tumor potency of these expanded MAIT cells. Furthermore, our data have demonstrated that Vα7.2 x PD-L1 could mediate the cell-to-cell conjunction between MAIT cell and tumor cell line, selectively elicit the activation, cytokine production, degranulation, and cytotoxicity of the expanded MAIT cells in the presence of target cell only. Collectively, this proof-of-concept study provides a new tool to explore the clinical potential of MAIT cells in fighting against PD-L1 positive solid tumors and suggests additional encouragement in designing novel T cell engagers targeting TCR alpha chain specific innate-like T cells subsets, other than pan CD3+ T cells.

Non Classical MHC presentation to Non-classical lymphocytesMR1-restricted T cell activation in physiopathology

Similarly to iNKT cells, mucosal associated invariant T (MAIT) cells express an invariant TCRalpha chain with a limited number of beta chains. These innate-like T cells recognize bacterial ligands presented by the non-polymorphic MR1 molecule. Metabolites derived from bacterial riboflavin synthesis pathway activate MAIT cells to produce granzyme B, Th1 and Th17 cytokines. The recognition of bacterial ligands by MAIT cells is further illustrated by the near absence of MAIT cells in germ-free mice. Most interestingly the riboflavin precursor needs to be modified by small molecules such as methylglyoxal, which level is elevated in obesity and diabetes. Moreover gut micro- biota alterations have been described in diabetes and obesity, both in humans and mouse models. Our recent studies have shown major alterations of MAIT cells in the blood of obese and diabetic patients as compared to healthy lean control individuals, suggesting their potential role in these pathologies. On going studies on the role and activation of MAIT cells in these pathologies will be presented.

Structural insights into engineering a T-cell receptor targeting MAGE-A10 with higher affinity and specificity for cancer immunotherapy

BackgroundT-cell receptor (TCR) immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials. The safety of patients is paramount, so innovative cell engineering methods are being...

Abstract LB094: First of its kind T cell receptors and functional proteomics detected from the same single cells to advance cancer immunology discovery

Abstract T cell receptor repertoire diversity and T cell cytokine production are the important attributes for the assessment of effective T cell immunity against cancer and pathogens, yet existing single-cell genomics platforms cannot interrogate TCR sequences and proteomic cytokines simultaneously. IsoPlexis’ functional proteomics has demonstrated the values in cell therapy and cancer immunology against blood cancers and solid tumors. The ability to connect the T cell receptor diversity to the most potent single cells enables a wide variety of applications for tumor infiltrating lymphocytes, personalized neoantigen TCR’s, cancer immunology in terms of understanding antigen specificity, as well as self-potency. IsoPlexis single-cell TCR Duomic platform provide a unique measurement for connecting T cell receptor sequences to those T cell cytokine profiles, for the first time. Human PBMC-derived T cell line established from healthy donor was stimulated with PMA/ionomycin (2ul/ml, Biolegend) for 6hrs, stained and resuspended in Reverse transcription premix containing reverse transcriptase with buffer and protein phosphatase inhibitor at 1 x 106/ml and then loaded on Duomic chips. Ran on IsoLight overnight, cDNA was collected from the chip followed by cleanup and amplification, proceeded with TCR amplification and sequencing on NextSeq 1000/2000. Proteomic data from single cells was automatically generated and analyzed by IsoSpeak software. The single-cell TCR sequencing data on the IsoPlexis TCR Duomics platform shows that 78.9% cells have TCR alpha chain recombination and 94.4% cells have TCR beta chain recombination. Totally 77.7% cells have combination on both alpha and beta chain. VDJ Usage Plot further displays the top10 most common V (D) J combination flow between T cell receptor alpha chain and beta chain among the single cells, revealing the diversity of TCR repertoire at the single-cell level. In addition, the proteomics data identifies the dominant proteins among the single cells are Granzyme B, GM-CSF, IFN-g and TNF-a. Furthermore, we define 3 distinct cell clusters based on functional proteins and an association of VDJ distribution with these cell clusters, suggesting the occurrence of V(D)J sequences in the most common reconstructed alpha and beta sequence. IsoPlexis single-cell TCR Duomics platform, for the first time, enables simultaneous profiling of functional proteomics and TCR repertoires/TCR clonotypes as well as identification of specific TCR V(D)J combination with functional cell clusters across the same individual T cells. The combination of these two analytes at the single cells provides a key new modality of tracking the most potent and also the antigen specific immune cells together to fight cancer and infectious disease, for the first time. Citation Format: Jennifer Yang, Jing Zhou, Sean Mackay. First of its kind T cell receptors and functional proteomics detected from the same single cells to advance cancer immunology discovery [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 LB094.

Single-Cell Clonal Tracking in Allogeneic Hematopoietic Stem Cell Transplantation Reveals Time Dependent and Distinct Functional Patterns in Traceable Donor T Cell Clones

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only curative treatment option for various malignant hematological diseases. The therapeutic effect of alloHSCT is a long-lasting graft-versus-leukemia (GvL) effect of the transferred graft. T cells are important mediators of GvL and the longitudinal tracking of T-cell clones from donor to recipient is of particular interest in the setting of alloHSCT as this might provide further insight into mechanisms leading to survival and expansion of particular clones. In a broader sense, we used the unique setting of alloHSCT to study survival and expansion of mature T-cell clones after transfer into an immune cell depleted and allogeneic patient.We used single-cell RNA sequencing (scRNAseq) to integrate immune subset delineation, clone identification and transcriptome information of about 35500 single T cells in peripheral blood of 14 paired donor-recipient samples in four alloHSCT pairs. Donor samples were collected before and after treatment with Granulocyte-Colony Stimulating Factor (GCSF), and recipient samples were collected on days +90 and +180 post-transplant.Looking at common diversity scores of pooled donor versus pooled recipient time points we observe an expected decrease of TCR diversity after transplantation (median inv. Simpson's 379 in donor vs. 20 in recipient samples, p=0.011, Figure 1A). On single cell level, we observe a substantial decrease of unique T-cell clones after transplantation compared to donor samples, which in return means that certain TCR clones markedly expand, contributing to a skewing of the TCR repertoire in the post-transplant course. The majority of these cells represent CD8 effector memory T cells.Our main interest was a better understanding of traceable and persisting T-cell clones. In a first step, we looked at the overall clonal overlap between time points of the different donor-recipient pairs, using only combined TCR alpha and beta chain information to define specific T-cell clones. We find the highest overlaps of T-cell clones between time points within individuals (e.g., Morisita score 0.91 between preGCSF and postGCSF of donor 16 and Morisita score 0.65 between days +90 and +180 of recipient 16, Figure 1B). Additionally, we demonstrate an inter-individual overlap between donors and their respective recipients in all pairs on single cell level (Figure 1C).Next, we compared the differential gene expression of traceable and non-traceable T cell clones and found that the traceable T cell clones exhibit a distinct transcriptional program, characterized by upregulation of genes related to T cell proliferation and chemotaxis as well as antigen presentation, while housekeeping functions such as translation are downregulated.In order to examine the dynamic changes of the T-cell transcriptome, we looked at the differential gene expression at the consecutive time points of pooled traceable clones in all pairs. This shows an induction of an activation pattern during the donor-recipient transfer and post-transplant phase involving genes related to the cell cycle and graft-versus-host disease (Figure 1D). Phenotype analysis via antibody-derived tags accordingly revealed an upregulation of activation markers in the recipients.To our knowledge, this is the first time that longitudinal inter-individual (donor-to-recipient) overlap of single-cell TCR alpha/beta clones is demonstrated in the setting of alloHSCT revealing time point-dependent and distinct functional patterns in traceable donor T cell clones. [Display omitted] DisclosuresPenack: Neovii: Honoraria; MSD: Honoraria; Incyte: Research Funding; Priothera: Consultancy; Therakos: Honoraria; Gilead: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Takeda: Research Funding; Astellas: Honoraria; Jazz: Honoraria; Omeros: Consultancy; Shionogi: Consultancy. Bullinger: Amgen: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Hexal: Consultancy; Abbvie: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Astellas: Honoraria; Pfizer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bayer: Research Funding; Seattle Genetics: Honoraria; Novartis: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Menarini: Consultancy; Gilead: Consultancy; Celgene: Consultancy, Honoraria; Sanofi: Honoraria. Na: Bristol Myers Squibb: Research Funding; Shire/Takeda: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding.

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.

Abstract PS17-22: Intratumoral delivery of tavokinogene telseplasmid (plasmid IL-12) and electroporation induces an immune signature that predicts successful combination in patients

Abstract Interleukin-12 (IL-12) is a pro-inflammatory cytokine involved in the generation of an inflammatory tumor microenvironment and is critical in eliciting a productive anti-tumor immune response. It has been investigated as an anti-cancer therapeutic using various delivery routes, but intratumoral injection of plasmid IL-12 (tavokinogene telseplasmid; TAVO) followed by electroporation is a gene therapy approach that results in more sustained production of IL-12 locally with minimal systemic immune-related toxicity. Here we show that TAVO not only provides protection in the treated triple-negative breast cancer (TNBC) lesion, but also induces a systemic, abscopal effect. Single cell RNAsequencing (scRNAseq) of infiltrating immune cells shows a significant increase in both CD4 and CD8 T cells as well as dendritic cells within the treated lesions, while simultaneously decreasing a granulocytic myeloid derived suppressor population. scRNAseq allows for a detailed look into not only the overall pathway enrichment caused by TAVO treatment, but also the specific receptor-ligand interactions occurring between cell types. A combination of these analyses revealed an enrichment in the IFN-gamma induced PDL1 pathway by TAVO, typified by an increase in the interaction between PDL1 on dendritic cells and PD1 on CD8 T cells. Further, dramatic enrichment of the CXCL9/10/11/CXCR3 axis was observed, consistent with previous studies in melanoma. Analysis of paired TCR alpha and beta chains on T cells additionally demonstrated a dramatic shift in tumor infiltrating T cell (TIL) clonality and frequency. In sum, these preclinical studies identify a signature of increased antigen presentation, T cell infiltration and expansion, and a decrease in the number of granulocytes but also a particular enhancement of the PDL1 immunosuppressive pathway following TAVO treatment. Using this signature, we focus on an in-depth analysis of 2 patients from a single arm, prospective clinical trial of TAVO monotherapy (OMS-I140) in pre-treated advanced TNBC that went on to receive anti-PD-1 as their immediate next therapy with clinical anti-tumor response. Together these data support the combination of TAVO with PD1/PDL1 inhibitors while also identifying other key pathways that may enhance responsiveness in TNBC patients for whom treatment options remain limited. Citation Format: Erika J Crosby, Hiroshi Nagata, Melinda L Telli, Chaitanya R Acharya, Irene Wapnir, Kaitlin Zablotsky, Erica Browning, Reneta Hermiz, Lauren Svenson, Donna Bannavong, Kellie Malloy, David A Canton, Chris G Twitty, Takuya Osada, Herbert Kim Lyerly. Intratumoral delivery of tavokinogene telseplasmid (plasmid IL-12) and electroporation induces an immune signature that predicts successful combination in patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-22.

Exploiting CD1-restricted T cells for clinical benefit

CD1-restricted T cells were first described over 30 years ago along with the cloning of the CD1 family. Around the same time, invariant Natural Killer cells (iNKT) were identified based on invariant TCR-alpha chains with additional expression of natural killer (NK) cell markers. About 5 years later, iNKT were shown to react with CD1d. Since then, iNKT have been shown to be a major population of CD1d-restricted T cells in humans and many animals. Like NK cells, iNKT are innate lymphocytes with rapid and wide-ranging effector potential. These activities include cytotoxicity and an unusually broad and high-level cytokine production. The development of highly-specific methods of isolating, stimulating, expanding or depleting these relatively rare cells and controlling their potent activities has stimulated considerable interest in therapeutic targeting of iNKT cells. Potential applications include cancers, inflammatory, infectious and autoimmune among other diseases. To date, most trials have targeted various cancers, there are 2 published trials in viral hepatitis and one in sickle cell lung disease. Uniform safety, evidence of immunologic activity and increasingly clinical efficacy have been seen. Approaches to targeting iNKT cells in clinical development include highly specific natural glycolipid ligands presented by CD1d and chemical analogues thereof and monoclonal antibody-based targeting of iNKT cells. In the case of iNKT cell-based therapies, novel approaches include arming them with Chimeric Antigen Receptors (CARs) and recombinant TCRs (rTCR), gene editing and allogeneic use. Controlling the iTCR:CD1d molecular interaction and consequences is a unique and promising therapeutic technology.

T cell receptor diversity, specificity and promiscuity of functionally heterogeneous human MR1-restricted T cells

The monomorphic MHC-class I-like molecule, MR1, presents small metabolites to T cells. MR1 is the restriction element for microbe-reactive mucosal-associated invariant T (MAIT) cells. MAIT cells have limited TCR usage, including a semi-invariant TCR alpha chain and express high levels of CD161 and CD26. In addition to microbial lumazine metabolites, recent studies have demonstrated that MR1 is able to capture a variety of diverse chemical entities including folate-derivatives, a number of drug-like and other synthetic small molecules, and as yet undefined compounds of self-origin. This capacity of MR1 to bind distinct ligands likely accounts for the recent identification of additional, non-canonical, subsets of MR1-restricted T (MR1T) cells. These subsets can be defined based on their ability to recognize diverse microbes as well as their reactivity to non-microbial cell-endogenous ligands, including tumor-associated antigens. Herein, we will discuss our current understanding of MR1T cell diversity in terms of TCR usage, ligand recognition and functional attributes.

Abstract NG11: Landscape analysis of neoepitope-specific T-cell responses to immunotherapy

Abstract NG11: Landscape analysis of neoepitope-specific T-cell responses to immunotherapy

Establishing cGMP manufacturing of CRISPR/Cas9-edited human CAR T cells

Background & Aim Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumor rejection. CD19 targeted CAR T cells have showed remarkable effacy for chemorefractory/relapsed B cell malignancies. Recombinant retroviral vectors mediated- CAR gene transfer is currently the standard method to generate cGMP grade CAR T cells. However, CAR gene expression is highly variable, owing to position effects and vector incorporation variations. CAR T cells engineered in this manner are capable of tumor eradication, but are prone to tonic signaling and accelerated exhaustion. We have developed a novel genetic engineering strategy to insert CAR genes into a precise genomic location in human peripheral blood T cells. Methods, Results & Conclusion T cells are electroporated with Cas9 mRNA or Cas9 protein and a guide RNA, followed by transduction with a recombinant Adeno-Associated Virus encoding the CAR sequence to facilitate the insertion of the CAR gene upstream of the constant region of TCR alpha chain. This results in the endogenous TCR promoter (TRAC) controlled CAR expression and abrogation of TCR surface expression. This strategy not only allows uniform CAR expression, but also delays T-cell differentiation and exhaustion, leading to enhanced T cell function and anti-tumor efficacy. The edited cells vastly outperformes retrovirally modified CAR T cells in a pre-B ALL NALM6 mouse model. The targeting of CARs to the TCR locus also provides a safer therapeutic T cell by minimizing the risks of insertional oncogenesis, and TCR-induced autoimmunity and alloreactivity (thus spanning both autologous and allogeneic T cell applications). In addition, we have incorporated in the CAR a mutant CD3z chain encoding a single immunoreceptor tyrosine-based activation motif that improves the balance between effector and memory T cells composition. We are in the process of translating this novel approach into the clinical setting by establishing cGMP conditions and protocols for the manufacturing of TRAC-CAR T cells. Using the 4D-LV electroporator, we have demonstrated that we can knockout the TCR at large scale (100 E06 CD3+ T cells) with high efficiency (70-80%) as that obtained at small scale. We have evaluated AAV6 to deliver the CAR transgene in the TCR locus. Data will be presented on optimizing the manufacturing of the TRAC-CAR T cells and on evaluating their anti-tumor efficacy in vivo.

Abstract 1211: Identification, retrieval, and RNA sequencing of single rare antigen-specific T cells from circulation using the RareCyte® platform

Abstract Background. The immune system provides antigen-specific protection against pathogens as well as malignancies, both of which evolve strategies to evade immune surveillance and containment. Effective immune response often depends on activation of rare antigen-specific immune cell sub-types. The RareCyte platform provides integrated multi-parameter imaging and retrieval capabilities that allow phenotypic identification and isolation of rare cells for sequence and transcript level analyses in order to study the complexity of host defense. Methods. Antigen-specific T cells were identified by immunofluorescent staining of live purified T cells from normal donors with the following panel: CD3; CD8; exclusionary cocktail (CD4, CD14, CD15, CD20, and DAPI); and HLA-A2 restricted tetramer (MBL). Tetramers had specificity against influenza-M1 (GILGFVFTL) and the melanoma antigen Mart-1 (ELAGIGILTV). For differential expression experiments, cells were stimulated overnight with specific target peptide or control irrelevant peptide, stained with the multiparameter panel containing the relevant tetramer, then imaged using the CyteFinder® instrument. Automated image analysis identified candidate cells with positive signal in the tetramer and CD8 channels and negative signal in the exclusion channel. Single cells displaying membrane ring tetramer distributions were retrieved using the CytePicker® module, followed by single cell RNA sequencing using the SMART-Seq® v4 kit (Takara Bio) and the MiSeq® instrument (Illumina). Differential gene expression of stimulated versus unstimulated cells was performed using DESeq2 software (BioConductor). Single-cell RNA seq FASTQ files were analyzed with the TraCeR computational method to identify TCR alpha and beta chains in both stimulated and unstimulated cells. Results. Single influenza and Mart-1 antigen-specific T cells in donor blood samples were detected and retrieved using the CyteFinder® instrument. Anti-influenza specificity was confirmed by RNA sequencing, which revealed a majority of alpha/beta TCR pairings with identical match to literature reports. Comparative gene expression analysis of activated and control flu tetramer-positive T cells revealed 73 significantly down-regulated and 687 significantly up-regulated genes. Pathway analysis of differential expression revealed involvement of TCR signaling and inflammatory response/cytokine signaling. Conclusions. The RareCyte platform can be used to visually identify and retrieve rare antigen-specific T cells from a bulk population by using tetramers against influenza-specific T cell receptors. T cell receptor sequencing confirmed the flu-specific identity of the tetramer-positive cells. RNA signatures of activation were identified at the single T cell level after peptide stimulation. Citation Format: Nolan Ericson, Eric Kaldjian, Tad George, Lance U'Ren. Identification, retrieval, and RNA sequencing of single rare antigen-specific T cells from circulation using the RareCyte® platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1211.

Abstract 3377: T-cell receptor repertoire profiling using an augmented transcriptome

Abstract Immunotherapy is growing as one of the most promising therapeutic approaches in clinical oncology practice. This brings with it an increasing need for comprehensive immuno-genomic profiling of tumors to better understand the interaction with the immune system. This includes profiling of the T and B-cell receptor repertoires (TCR/BCR), which has traditionally not been feasible with an exome/transcriptome platform. To address these challenges, we developed ImmunoID NeXT, an augmented, immuno-oncology optimized exome/transcriptome platform designed to provide comprehensive information regarding the tumor and tumor microenvironment (TME) from limited FFPE tumor biopsies, including the TCR alpha, beta, gamma and delta chains and BCR heavy and light chains. We show how this platform accurately profiles abundant clones, and can be applied to understand the diversity and activity of the adaptive immune system. We characterize the performance of ImmunoID NeXT at profiling TCR beta from RNA. We analyze the reproducibility of clones identified using replicates of PBMC and FFPE samples, and assess the concordance of top clones from a standalone TCR sequencing approaches to ours. Then, we test LOD by diluting well-characterized clonal T-cell line samples into PBMCs. We also analyze patient-derived FFPE tumors to understand the profiles of tumor-infiltrating immune repertoires. First, we compare TCR beta profiling with IHC quantification of CD3+ cells in both tumor and adjacent normal tissues. Finally, to better understand both B and T cell infiltration, we profile the intra-sample heterogeneity of BCR and TCR in a set of tumors. Between replicates of PBMC samples, abundances for shared clones have high concordance (R2>0.98). We observe strong concordance of the abundances for shared clones between adjacent curls of a tumor FFPE sample (R2>0.87), showing that our approach is robust to degraded FFPE samples. Compared to a standalone approach, we identify over 93% of the top 1000 clones, with highly concordant abundances (R2>0.94). Assessing LOD in dilutions of T cell lines into a PBMC sample, we are able to identify clones present at over 0.00032% RNA by mass. In our analysis of T-cell infiltration, we find a significant T-cell population in normal tissues. We also compare TCR read counts detected by ImmunoID NeXT in tumor and normal tissues with IHC results. Finally, we find substantial inter-sample variations in the number of TCR and BCR clones in tumors. ImmunoID NeXT has been designed to enable sensitive detection of abundant TCR and BCR clones in addition to comprehensive biomarkers from exome/transcriptome data. We demonstrate that our platform is reproducible, sensitive, and concordant with the top-abundance clones derived from targeted TCR methods, as well as feasible with FFPE samples. Finally, we highlight how immune repertoire results from ImmunoID NeXT can be used to gain understanding about the immunological composition of the TME. Citation Format: Eric Levy, Pamela Milani, Sean M. Boyle, Shujun Luo, Gabor Bartha, Charles Abbott, Rena McClory, Robin Li, John West, Richard Chen. T-cell receptor repertoire profiling using an augmented transcriptome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3377.

Physical cognate pairing of mouseTCR alpha-beta VDJ-rearrangements with phenotype directly from FACS sorted cells

Abstract Phenotypic characterization of single cells paired with immune receptor information can provide valuable insight into T cell function, which may be beneficial for the development of personalized therapies. In order to comprehensively analyze T cell function and specificity, it is necessary to integrate antigen receptor sequencing with phenotypic markers at the single cell level. We therefore developed a sensitive technology called iPair+™ that enables the simultaneous amplification and sequencing of paired mouse TCR alpha and beta chains with functional gene markers in a single, sorted immune cell via a combination of flow cytometry, multiplex PCR, and next generation sequencing. We utilized iPair+™ to study CD4 and CD8 T cells isolated from treated and non-treated mice from a breast cancer model. CDR3 diversity, clonality, and frequency were analyzed together with 80+ phenotypic genes. When cells isolated from both treated and non-treated mice were compared, we found gene expression levels changed significantly with treatment. Our data demonstrates that genes that were upregulated prior to treatment were associated with cancer growth, while genes upregulated after treatment were associated with T cell activation and function in cancer repression or growth inhibition. Our study indicates that iPair+ technology could reveal important insights into T cell function, clonal development, and immunotherapy response during treatment.

A novel method for high throughput TCR single cell VDJ-pairing with phenotypic analysis

Abstract Single cell transcriptional analysis reveals a wealth of information that is obscured when analyzing the RNA of a sample en masse. Furthermore, it is important to know the identity of the TCR chain VDJ-rearrangement associated with the phenotypic state. This information can provide direct calculations of clonal frequency in various cell subsets, tracking of specific lymphocytes with treatment, and reveal paired information for both chains of the receptor for downstream Car-T development. Here, we discuss a novel method that identifies paired VDJ-rearrangements for both TCR alpha and beta chains from single cells barcoded via the BD Rhapsody Express single cell system. The phenotype of the cell is obtained using the BD Rhapsody RNA-seq kit, while the receptor information is amplified from the same cDNA using an iRepertoire-derived method incorporating a multiplex mix of primers associated with both the TCR alpha and beta loci. This targeted-seq strategy requires less read depth when compared to template-switch approaches. In one experiment, 3,000 TCR-beta unique CDR3 (uCDR3) were identified from ~7,000 barcoded cells using 1.5 million NGS reads with a 0.83 Pearson correlation upon repeat. Approximately 1,000 TCR alpha-beta paired uCDR3 were also called from the data. Additionally, the BD phenotyping results independently verify the immune repertoire data since the TCR beta chain constant gene overlays with the TCR-beta uCDR3 clonotypes for CD4+ and CD8+ subsets but is largely absent in the CD14+ monocyte population for both analyses. Thus, the developed technology provides the ability to simultaneously pair both alpha and beta chains for thousands of T-cells while obtaining information related to the functional status of each cell.