Specific T-cell Receptor Research Articles

First-in-human phase 1/2 study of autologous T cells engineered using the Sleeping Beauty System transposon/transposase to express T-cell receptors (TCRs) reactive against cancer-specific mutations in patients with advanced solid tumors.

TPS2679 Background: In 2022 approximately 1.7 million Americans will die from solid cancers. Recently there have been significant advances in the genetic engineering of T lymphocytes to recognize neoantigens on tumors in vivo, resulting in remarkable cases of tumor regression and remission. Cancer cells frequently harbor KRAS, TP53, and EGFR somatic hotspot mutations that can be processed and presented by tumor HLA as neoantigens to T cells through their T-cell receptor (TCR). These neoantigens are not present in the normal tissues; thus, they are attractive targets for adoptive T cell therapy. Given the number and complexity of different neoantigen/HLA combinations on solid tumors, a TCR library approach is warranted. Therefore, we have developed a library of TCR-T cell therapies including those targeting shared KRAS, TP53 and EGFR mutations. Methods: Patients for whom a TCR matching the subject’s somatic mutation(s) and HLA type is available in our TCR library, and have progressive or recurrent disease following standard therapy are eligible for enrollment on this protocol. Patients with the following tumor types will be enrolled: ovarian, endometrial, colorectal, pancreatic, cholangiocarcinoma, and non-small cell lung cancer. This first-in-human study includes Screening, Pre-Treatment, Treatment and Follow-up Periods. During the Pre-Treatment Period, subjects will undergo apheresis for PBMCs isolation. The PBMCs will be transposed using the Sleeping Beauty system to express the subject’s mutation specific TCR. Bridging therapy after apheresis is allowed once the apheresis product has been accepted. During the Treatment Period, patients will undergo lymphodepletion with cyclophosphamide and fludarabine. After which, the TCR-T cell drug product will be administered to the subject by infusion at the assigned dose level. The starting dose level of Arm A (monotherapy) will be DL1 (5 x10 9 TCR+ Cells) administered on Day 0. Dose escalation will continue utilizing the accelerated BOIN design (planned escalation dose levels: 5 x10 9, 4 x10 10 and 1 x10 11 TCR+ Cells). In Arm B, if initiated by protocol, subjects will also receive aldesleukin (interleukin-2) infusion starting on Day 0 (within 24 hours of TCR-T cell product infusion) at 720K IU/kg, every eight hours for up to 4 days. The Follow-up Period will begin after the subject completes their Day 28 visit. Clinical and radiologic response will be assessed at 6 and 12 weeks after TCR-T cell drug product infusion and every 12 weeks thereafter until up to 2 years or study discontinuation (e.g., disease progression, initiation of new anti-cancer therapy, consent withdrawn), whichever occurs first. All subjects will continue to be followed in the Long-Term Follow-up Protocol for up to 15 years post-TCR-T cell drug product infusion. Clinical trial information: NCT05194735.

Phase I clinical trial to assess safety, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of NY-ESO-1–specific TCR T-cells (TAEST16001) in HLA-A*02:01 patients with advanced soft tissue sarcoma.

11502 Background: NY-ESO-1 is a cancer/testis antigen with expression in a wide range of tumor types. TAEST16001 cells are genetically engineered autologous T cells that express high-affinity NY-ESO-1-specific T-cell receptor (TCR) targeting NY-ESO-1+ soft tissue sarcoma in the context of HLA-A*02:01. Here, we report preliminary results of TAEST16001 cells from an ongoing, dose-escalation and expansion study in HLA-A*02:01 positive patients with advanced soft tissue sarcoma expressing NY-ESO-1 antigen. Methods: This is an open, single arm, dose-escalation and expansion study to evaluate safety, tolerability, PK, PD and preliminary efficacy of TAEST16001 cells in patients with soft tissue sarcoma. Enrolled patients underwent apheresis, and their isolated T cells were expanded in vitro after transducing with a lentiviral vector containing NY-ESO-1 TCR. Prior to TAEST16001 cells infusion, patients were to receive lymphodepleting chemotherapy consisting of cyclophosphamide (15 mg/kg/day ´ 3 days) and fludarabine (20 mg/m2/day ´ 3 days). TAEST16001 cells were administered at 5 × 108 ± 30% (dose level 1), 2 × 109 ± 30% (dose level 2), 5 × 109 ± 30% (dose level 3) and 1.2 × 1010 ± 30% (dose level 4/expansion) transduced cells. After TAEST16001 cells infusion, patients were to receive interleukin-2 subcutaneous injection for 14 days. Tumor responses were assessed by RECIST/iRECIST. Results: As of 31 December 2021, 12 patients with advanced soft tissue sarcoma were enrolled (M:F 7:5; mean age = 37.9; median prior regimens = 2 (range 1-3)). TAEST16001 cells were well-tolerated with no dose limiting toxicity. The most frequently reported grade ³ 3 adverse events were lymphopenia (n = 12), leukopenia (n = 10), neutropenia (n = 11), anemia (n = 4), thrombocytopenia (n = 1), hypokalemia (n = 1), and fever (n = 1). Two patients presented with cytokine release syndrome (grades 2) and resolved after symptomatic treatment. None of the patients had neurotoxicity, or serious adverse events related to cell infusion. The maximum tolerated dose (MTD) was not reached. PK modelling indicated that Tmax of TAEST16001 cells were 6.23 days after cells infusion, and there were no relationship between clinical response and Cmax/AUC0-28. Among 12 efficacy-evaluable patients, 5 patients had a partial response, 5 patients had stable disease, and 2 patients had progressive disease. The overall response rate was 41.7%. The median time to an initial response was 1.9 month (range, 0.9 to 3.0), and the median duration of response was 14.1 months (range, 5.0 to 14.2). Conclusions: TAEST16001 cells showed an acceptable tolerability profile overall. MTD was not reached. Emerging efficacy data encouraged the continued expansion study of TAEST16001 cells in advanced soft tissue sarcoma. Clinical trial information: NCT04318964.

Merkel polyoma virus specific T-cell receptor transgenic T-cell therapy in PD-1 inhibitor refractory Merkel cell carcinoma.

9549 Background: Merkel cell carcinoma is an aggressive neuroendocrine tumor of skin origin with most cases caused by the Merkel polyoma virus (MCPyV). While many patients benefit from PD-1/PD-L1 axis blockade, most patients do not respond or develop resistance. We sought to ask whether adoptive transfer of autologous T cells transduced with MCPyV specific T cells could lead to clinical responses in PD-1 inhibitor refractory patients. Methods: Five MCPyV positive, HLA-A02 patients with PD-1 inhibitor refractory metastatic Merkel cell carcinoma were treated with adoptive transfer of CD62L+ CD8+ and CD4+ autologous T cells transduced with a T cell receptor (TCR) targeting an HLA-A0201 restricted MCPyV epitope. Two different strategies were used to facilitate T cell expansion: In 3 patients, single fraction radiation was administered to a subset of lesions prior to T cell transfer. In 2 patients, lymphodepleting chemotherapy with cyclophosphamide and fludarabine was administered prior to T cell transfer. Anti PD-1/PD-L1 therapy was given 14 days after T cell infusion. Transgenic T cells were visualized in tumor biopsies by multiplex immunohistochemistry. Results: 5 patients were treated, with 4 patients receiving 100 million tetramer positive CD8+ T cells and one patient receiving 500 million cells. 3 patients received second infusions with between 300 million and 900 million tetramer positive cells. No dose limiting toxicities or cytokine release syndrome were observed. T cell persistence was lower in the 2 patients treated with lymphodepleting chemotherapy relative to the 3 patients treated with single fraction radiation. Transgenic T cells persisted at tumor sites greater than 1 month following transfer. 4 patients experienced progressive disease, and a single patient had a mixed response and greater than 1 year disease free interval following local therapy of an isolated site of progression. The responding patient was the only patient with class I MHC staining on tumor cells prior to treatment, and the site of local progression in that patient showed the presence of TCR transgenic T cells but loss of class I MHC expression. Conclusions: MCPyV specific transgenic T cells are safe, traffic to tumor sites, and can result in a clinical response, but their clinical activity may be limited by down-regulation of class I MHC expression on tumors. A future trial will address strategies to increase class I MHC expression on Merkel tumors. Clinical trial information: NCT03747484.

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.

Autoantigen specific T-cell receptor induces organ-specific autoimmunity by escaping T cell negative selection

Abstract Previously we reported a new model of spontaneous autoimmune uveitis in which mice with a hypomorphic mutation of Aire (AireGW/+) are combined with deletion of Lyn. The key autoantigen in this model is interphotoreceptor retinoid-binding protein (IRBP) as AireGW/+Lyn−/−IRBP−/− mice failed to develop uveitis. We performed single-cell RNA-seq with TCR sequencing for IRBP 271–290 (P2)-specific CD4+ T cells from eye-draining lymph nodes (LN) of these mice with and without uveitis. Both mice showed an expansion of these T cells in eye-draining LNs, but it was greater in mice with disease. A remarkable clonal diversity of TCRs was seen. Expansions of particular T cell clonotypes were more evident in LN of mice with disease, and even greater in the retinas of mice with disease. We reconstituted several P2–specific TCR clonotypes in a T cell hybridoma line and verified their specificity. One of the TCRs was expressed in thymocytes by retroviral transduction of bone marrow stem cells from Rag2−/− mice and transplanted into WT or AireGW/+ recipient mice. Remarkably, the small residual expression of IRBP in the thymus of AireGW/+ mice was sufficient to induce substantial negative selection of thymocytes with this TCR. In addition, we generated TCR transgenic mice with this TCR clonotype. The TCR transgenic mice spontaneously developed uveitis with expansion of P2-specific CD4+ T cells. Combined with AireGW/+ or IRBP−/−, there were more P2-specific CD4+ T cells in the thymus of TCR transgenic mice. Thus, suboptimal Aire function in AireGW/+Lyn−/− mice results in a partial defect in negative selection of T cells recognizing IRBP in the thymus, promoting development of uveitis. This disease is accelerated in mice expressing a TCR transgene recognizing IRBP. Supported by AI138479

Human Coronary Plaque T Cells Are Clonal and Cross-React to Virus and Self.

Coronary artery disease is an incurable, life-threatening disease that was once considered primarily a disorder of lipid deposition. Coronary artery disease is now also characterized by chronic inflammation' notable for the buildup of atherosclerotic plaques containing immune cells in various states of activation and differentiation. Understanding how these immune cells contribute to disease progression may lead to the development of novel therapeutic strategies. We used single-cell technology and in vitro assays to interrogate the immune microenvironment of human coronary atherosclerotic plaque at different stages of maturity. In addition to macrophages, we found a high proportion of αβ T cells in the coronary plaques. Most of these T cells lack high expression of CCR7 and L-selectin, indicating that they are primarily antigen-experienced memory cells. Notably, nearly one-third of these cells express the HLA-DRA surface marker, signifying activation through their TCRs (T-cell receptors). Consistent with this, TCR repertoire analysis confirmed the presence of activated αβ T cells (CD4<CD8), exhibiting clonal expansion of specific TCRs. Interestingly, we found that these plaque T cells had TCRs specific for influenza, coronavirus, and other viral epitopes, which share sequence homologies to proteins found on smooth muscle cells and endothelial cells, suggesting potential autoimmune-mediated T-cell activation in the absence of active infection. To better understand the potential function of these activated plaque T cells, we then interrogated their transcriptome at the single-cell level. Of the 3 T-cell phenotypic clusters with the highest expression of the activation marker HLA-DRA, 2 clusters expressed a proinflammatory and cytolytic signature characteristic of CD8 cells, while the other expressed AREG (amphiregulin), which promotes smooth muscle cell proliferation and fibrosis, and, thus, contributes to plaque progression. Taken together, these findings demonstrate that plaque T cells are clonally expanded potentially by antigen engagement, are potentially reactive to self-epitopes, and may interact with smooth muscle cells and macrophages in the plaque microenvironment.

Isolation of Neoantigen-Specific Human T Cell Receptors from Different Human and Murine Repertoires

Simple SummaryT cell-based immunotherapy has achieved remarkable clinical responses in patients with cancer. Neoepitope-specific T cells can specifically recognize mutated tumor cells and have led to tumor regression in mouse models and clinical studies. However, isolating neoepitope-specific T cell receptors (TCRs) from the patients’ own repertoire has shown limited success. Sourcing T cell repertoires, other than the patients’ own, has certain advantages: the availability of larger amounts of blood from healthy donors, circumventing tumor-related immunosuppression in patients, and including different donors to broaden the pool of specific T cells. Here, for the first time, a side-by-side comparison of three different TCR donor repertoires, including patients and HLA-matched allogenic healthy human repertoires, as well as repertoires of transgenic mice, is performed. Our results support recent studies that using not only healthy donor T cell repertoires, but also transgenic mice might be a viable strategy for isolating TCRs with known specificity directed against neoantigens for adoptive T cell therapy.(1) Background: Mutation-specific T cell receptor (TCR)-based adoptive T cell therapy represents a truly tumor-specific immunotherapeutic strategy. However, isolating neoepitope-specific TCRs remains a challenge. (2) Methods: We investigated, side by side, different TCR repertoires—patients’ peripheral lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs), PBLs of healthy donors, and a humanized mouse model—to isolate neoepitope-specific TCRs against eight neoepitope candidates from a colon cancer and an ovarian cancer patient. Neoepitope candidates were used to stimulate T cells from different repertoires in vitro to generate neoepitope-specific T cells and isolate the specific TCRs. (3) Results: We isolated six TCRs from healthy donors, directed against four neoepitope candidates and one TCR from the murine T cell repertoire. Endogenous processing of one neoepitope, for which we isolated one TCR from both human and mouse-derived repertoires, could be shown. No neoepitope-specific TCR could be generated from the patients’ own repertoire. (4) Conclusion: Our data indicate that successful isolation of neoepitope-specific TCRs depends on various factors such as the heathy donor’s TCR repertoire or the presence of a tumor microenvironment allowing neoepitope-specific immune responses of the host. We show the advantage and feasibility of using healthy donor repertoires and humanized mouse TCR repertoires to generate mutation-specific TCRs with different specificities, especially in a setting when the availability of patient material is limited.

Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information.

The study and manipulation of T cell receptors (TCRs) is central to multiple fields across basic and translational immunology research. Produced by V(D)J recombination, TCRs are often only recorded in the literature and data repositories as a combination of their V and J gene symbols, plus their hypervariable CDR3 amino acid sequence. However, numerous applications require full-length coding nucleotide sequences. Here we present Stitchr, a software tool developed to specifically address this limitation. Given minimal V/J/CDR3 information, Stitchr produces complete coding sequences representing a fully spliced TCR cDNA. Due to its modular design, Stitchr can be used for TCR engineering using either published germline or novel/modified variable and constant region sequences. Sequences produced by Stitchr were validated by synthesizing and transducing TCR sequences into Jurkat cells, recapitulating the expected antigen specificity of the parental TCR. Using a companion script, Thimble, we demonstrate that Stitchr can process a million TCRs in under ten minutes using a standard desktop personal computer. By systematizing the production and modification of TCR sequences, we propose that Stitchr will increase the speed, repeatability, and reproducibility of TCR research. Stitchr is available on GitHub.

Effects of Reduced-Dose Anti-Human T-Lymphocyte Globulin on Overall and Donor-Specific T-Cell Repertoire Reconstitution in Sensitized Kidney Transplant Recipients.

BackgroundPre-sensitized kidney transplant recipients have a higher risk for rejection following kidney transplantation and therefore receive lymphodepletional induction therapy with anti-human T-lymphocyte globulin (ATLG) whereas non-sensitized patients are induced in many centers with basiliximab. The time course of lymphocyte reconstitution with regard to the overall and donor-reactive T-cell receptor (TCR) specificity remains elusive.Methods/DesignFive kidney transplant recipients receiving a 1.5-mg/kg ATLG induction therapy over 7 days and five patients with 2 × 20 mg basiliximab induction therapy were longitudinally monitored. Peripheral mononuclear cells were sampled pre-transplant and within 1, 3, and 12 months after transplantation, and their overall and donor-reactive TCRs were determined by next-generation sequencing of the TCR beta CDR3 region. Overall TCR repertoire diversity, turnover, and donor specificity were assessed at all timepoints.ResultsWe observed an increase in the donor-reactive TCR repertoire after transplantation in patients, independent of lymphocyte counts or induction therapy. Donor-reactive CD4 T-cell frequency in the ATLG group increased from 1.14% + -0.63 to 2.03% + -1.09 and from 0.93% + -0.63 to 1.82% + -1.17 in the basiliximab group in the first month. Diversity measurements of the entire T-cell repertoire and repertoire turnover showed no statistical difference between the two induction therapies. The difference in mean clonality between groups was 0.03 and 0.07 pre-transplant in the CD4 and CD8 fractions, respectively, and was not different over time (CD4: F(1.45, 11.6) = 0.64 p = 0.496; CD8: F(3, 24) = 0.60 p = 0.620). The mean difference in R20, a metric for immune dominance, between groups was -0.006 in CD4 and 0.001 in CD8 T-cells and not statistically different between the groups and subsequent timepoints (CD4: F(3, 24) = 0.85 p = 0.479; CD8: F(1.19, 9.52) = 0.79 p = 0.418).ConclusionReduced-dose ATLG induction therapy led to an initial lymphodepletion followed by an increase in the percentage of donor-reactive T-cells after transplantation similar to basiliximab induction therapy. Furthermore, reduced-dose ATLG did not change the overall TCR repertoire in terms of a narrowed or skewed TCR repertoire after immune reconstitution, comparable to non-depletional induction therapy.

Immortalization and functional screening of natively paired human T cell receptor repertoires.

Functional analyses of the T cell receptor (TCR) landscape can reveal critical information about protection from disease and molecular responses to vaccines. However, it has proven difficult to combine advanced next-generation sequencing technologies with methods to decode the peptide-major histocompatibility complex (pMHC) specificity of individual TCRs. We developed a new high-throughput approach to enable repertoire-scale functional evaluations of natively paired TCRs. In particular, we leveraged the immortalized nature of physically linked TCRα:β amplicon libraries to analyze binding against multiple recombinant pMHCs on a repertoire scale, and to exemplify the utility of this approach, we also performed affinity-based functional mapping in conjunction with quantitative next-generation sequencing to track antigen-specific TCRs. These data successfully validated a new immortalization and screening platform to facilitate detailed molecular analyses of disease-relevant antigen interactions with human TCRs.

Abacavir inhibits but does not cause self-reactivity to HLA-B*57:01-restricted EBV specific T cell receptors

Pre-existing pathogen-specific memory T cell responses can contribute to multiple adverse outcomes including autoimmunity and drug hypersensitivity. How the specificity of the T cell receptor (TCR) is subverted or seconded in many of these diseases remains unclear. Here, we apply abacavir hypersensitivity (AHS) as a model to address this question because the disease is linked to memory T cell responses and the HLA risk allele, HLA-B*57:01, and the initiating insult, abacavir, are known. To investigate the role of pathogen-specific TCR specificity in mediating AHS we performed a genome-wide screen for HLA-B*57:01 restricted T cell responses to Epstein-Barr virus (EBV), one of the most prevalent human pathogens. T cell epitope mapping revealed HLA-B*57:01 restricted responses to 17 EBV open reading frames and identified an epitope encoded by EBNA3C. Using these data, we cloned the dominant TCR for EBNA3C and a previously defined epitope within EBNA3B. TCR specificity to each epitope was confirmed, however, cloned TCRs did not cross-react with abacavir plus self-peptide. Nevertheless, abacavir inhibited TCR interactions with their cognate ligands, demonstrating that TCR specificity may be subverted by a drug molecule. These results provide an experimental road map for future studies addressing the heterologous immune responses of TCRs including T cell mediated adverse drug reactions.

Identification and Tracking of Alloreactive T Cell Clones in Rhesus Macaques Through the RM-scTCR-Seq Platform.

T cell receptor (TCR) clonotype tracking is a powerful tool for interrogating T cell mediated immune processes. New methods to pair a single cell’s transcriptional program with its TCR identity allow monitoring of T cell clonotype-specific transcriptional dynamics. While these technologies have been available for human and mouse T cells studies, they have not been developed for Rhesus Macaques (RM), a critical translational organism for autoimmune diseases, vaccine development and transplantation. We describe a new pipeline, ‘RM-scTCR-Seq’, which, for the first time, enables RM specific single cell TCR amplification, reconstruction and pairing of RM TCR’s with their transcriptional profiles. We apply this method to a RM model of GVHD, and identify and track in vitro detected alloreactive clonotypes in GVHD target organs and explore their GVHD driven cytotoxic T cell signature. This novel, state-of-the-art platform fundamentally advances the utility of RM to study protective and pathogenic T cell responses.

Structural basis of T cell receptor specificity and cross-reactivity of two HLA-DQ2.5-restricted gluten epitopes in celiac disease

Celiac disease is a T cell-mediated chronic inflammatory condition often characterized by human leukocyte antigen (HLA)-DQ2.5 molecules presenting gluten epitopes derived from wheat, barley, and rye. Although some T cells exhibit cross-reactivity toward distinct gluten epitopes, the structural basis underpinning such cross-reactivity is unclear. Here, we investigated the T-cell receptor specificity and cross-reactivity of two immunodominant wheat gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). We show by surface plasmon resonance that a T-cell receptor alpha variable (TRAV) 4+-T-cell receptor beta variable (TRBV) 29-1+ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1 with similar affinity, whereas a TRAV4- (TRAV9-2+) TCR recognized HLA-DQ2.5-glia-ω1 only. We further determined the crystal structures of the TRAV4+-TRBV29-1+ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1, as well as the structure of an epitope-specific TRAV9-2+-TRBV7-3+ TCR-HLA-DQ2.5-glia-ω1 complex. We found that position 7 (p7) of the DQ2.5-glia-α1a and DQ2.5-glia-ω1 epitopes made very limited contacts with the TRAV4+ TCR, thereby explaining the TCR cross-reactivity across these two epitopes. In contrast, within the TRAV9-2+ TCR-HLA-DQ2.5-glia-ω1 ternary complex, the p7-Gln was situated in an electrostatic pocket formed by the hypervariable CDR3β loop of the TCR and Arg70β from HLA-DQ2.5, a polar network which would not be supported by the p7-Leu residue of DQ2.5-glia-α1a. In conclusion, we provide additional insights into the molecular determinants of TCR specificity and cross-reactivity to two closely-related epitopes in celiac disease.

Specific TCR V–J gene segment recombinations leading to the identification pan-V–J CDR3s associated with survival distinctions: diffuse large B-cell lymphoma

In the diffuse large B-cell lymphoma (DLBCL) setting, we examined lymph node biopsy, T-cell receptor features, and the DLBLC patient human leukocyte antigen (HLA) alleles, to provide a basis for assessing survival distinctions represented by the National Cancer Institute Center for Cancer Research (NCICCR) dataset. While previous analyses of other cancer datasets have indicated that specific T-cell receptor (TCR) V or J gene segments, independently, can be associated with a survival distinction, we have here identified V–J recombinations, representing specific V and J gene segments associated with survival distinctions. As specific V–J recombinations represent relatively conserved complementarity determining region-3 (CDR3) amino acid sequences, we assessed the entire DLBCL NCICCR dataset for such conserved CDR3 features. Overall, this approach indicated the opportunity of identifying DLBCL patient subpopulations with TCR CDR3 features, and HLA alleles, with significant survival distinctions, possibly identifying cohorts more likely to benefit from a given immunotherapy.

Identification of Claudin 6-specific HLA class I- and HLA class II-restricted T cell receptors for cellular immunotherapy in ovarian cancer

ABSTRACT Adoptive cell therapy (ACT) is one of promising immunotherapies for cancer patients by providing a large amount of cancer antigen-specific effector T cells that can be manufactured rapidly by ex vivo gene engineering. To provide antigen-specificity to patients’ autologous T cells in a short-term culture, T-cell receptors (TCRs) or chimeric antigen receptors (CARs) are transduced to bulk T cells. Because of intra- and inter-tumoral heterogeneity in tumor antigen expression, a repertoire of TCR or CAR genes targeting a wide range of tumor antigens are required for a broad and effective treatment by ACT. Here, we characterized immunogenicity of claudin 6 (CLDN6) in ovarian cancer patients and identified specific TCR genes from CD8+ and CD4+ T cells. CLDN6 protein was frequently expressed on EpCAM+ ovarian cancer cells but not CD45+ lymphocytes in tumor ascites of ovarian cancer patients. Spontaneous CLDN6-specific CD4+ and CD8+ T-cell response was detected in peripheral blood mononuclear cells (PBMCs) from 1 out of 17 ovarian cancer patients. HLA-A*02:01 (A2) and DR*04:04 (DR4)-restricted TCR genes were isolated from CLDN6-specific CD8+ and CD4+ T cells, respectively. T cells that were engineered with A2-restricted TCR gene recognized and killed A2+CLDN6+ cancer cells. DR4-restricted TCR-transduced T cells directly recognized DR4+CLDN6+-overexpressed cancer cells. Our results demonstrate that these CLDN6-specific TCR genes are useful as therapeutic genes for ACT to patients with ovarian and other solid tumors expressing CLDN6.

Phase I clinical trial evaluating the safety and efficacy of ADP-A2M10 SPEAR T cells in patients with MAGE-A10+ advanced non-small cell lung cancer

BackgroundADP-A2M10 specific peptide enhanced affinity receptor (SPEAR) T cells (ADP-A2M10) are genetically engineered autologous T cells that express a high-affinity melanoma-associated antigen A10 (MAGE-A10)-specific T-cell receptor (TCR) targeting MAGE-A10+ tumors...

A High-Throughput Strategy for T-Cell Receptor Cloning and Expression.

Expression of T-cell receptor (TCR) genes is a critical step for TCR characterization and epitope identification. The recent interest in using specific TCRs for cancer immunotherapy has further increased the demand for practical and robust methods to rapidly clone and express TCRs. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TCR transgene into different expression systems. In this protocol, we first constructed all the human TRAV and TRBV genes into individual plasmid. To clone any TCR, we only need to ligate a short CDR3 fragment to its corresponding V gene plasmid using Golden Gate cloning. This strategy significantly improves the efficiency of individual TCR cloning and mutagenesis, providing a flexible high-throughput method for TCR analysis and TCR-mediated therapeutics.

Smart Lipid-Based Nanosystems for Therapeutic Immune Induction against Cancers: Perspectives and Outlooks.

Cancer immunotherapy, a promising and widely applied mode of oncotherapy, makes use of immune stimulants and modulators to overcome the immune dysregulation present in cancer, and leverage the host’s immune capacity to eliminate tumors. Although some success has been seen in this field, toxicity and weak immune induction remain challenges. Liposomal nanosystems, previously used as targeting agents, are increasingly functioning as immunotherapeutic vehicles, with potential for delivery of contents, immune induction, and synergistic drug packaging. These systems are tailorable, multifunctional, and smart. Liposomes may deliver various immune reagents including cytokines, specific T-cell receptors, antibody fragments, and immune checkpoint inhibitors, and also present a promising platform upon which personalized medicine approaches can be built, especially with preclinical and clinical potentials of liposomes often being frustrated by inter- and intrapatient variation. In this review, we show the potential of liposomes in cancer immunotherapy, as well as the methods for synthesis and in vivo progression thereof. Both preclinical and clinical studies are included to comprehensively illuminate prospects and challenges for future research and application.

LB17. Immunosequencing of the T-Cell Receptor Repertoire Reveals Signatures Specific for Diagnosis and Characterization of Early Lyme Disease

BackgroundChanging climate and demographic trends have led to recent increases in the incidence of tick-borne illnesses. Early diagnosis of Lyme disease (LD) is critical for initiation of antibiotics to mitigate symptoms and prevent late manifestations. In patients not presenting with a typical erythema migrans rash, 2-tiered serologic testing is recommended to support a diagnosis of LD. However, 2-tiered testing is limited by ambiguity in interpretation and low sensitivity in early disease, highlighting an unmet clinical need for alternative diagnostic approaches. We identified a clinical signal for early LD based on evaluation of the T-cell response to B. burgdorferi infection.MethodsWe immunosequenced T-cell receptor (TCR) repertoires in blood samples from 3 independent cohorts of patients with laboratory-confirmed or clinically diagnosed early LD and endemic/non-endemic controls to identify 251 public, LD-associated TCRs. These TCRs were used to train a classifier that identified early LD with 99% specificity. Classifier sensitivity was evaluated in 211 LD cases and 2631 endemic controls and compared to that of standard 2-tiered testing (STTT). Biologic specificity was assessed by correlating TCR assay scores with clinical measures and by mapping the antigen specificity of Lyme-associated TCRs to B. burgdorferi antigens.Figure 1. LD-associated TCRs distinguish cases (orange) from controls (blue) in training cohorts. (A) Logistic-growth curve used to define a scoring function. (B) Positive-call threshold (99th percentile in endemic controls). ResultsIn early LD, TCR testing demonstrated a 1.9-fold increase in sensitivity compared to STTT (56% vs 30%), with a 3.1-fold increase ≤4 days from the onset of symptoms (44% vs 14%). TCR positivity predicted subsequent seroconversion in 37% of initially STTT-negative patients, suggesting the T-cell response is detectable before the humoral response. While positivity for both tests declined following treatment, greater declines in posttreatment sensitivity were observed for STTT compared to TCR testing. Higher TCR scores were associated with measures of disease severity, including abnormal liver function tests, disseminated rash, and number of symptoms. A subset of LD-associated TCRs mapped to B. burgdorferi antigens, demonstrating the high specificity of a TCR immunosequencing approach. Figure 2. Validation of the TCR classifier in the JHU cohort and other holdout endemic controls. Distribution of model scores (A) and assay sensitivity (B). Model scores (C) and ROC (D) curves by serostatus. Figure 3. Clinical correlates of TCR scoring. (A) Liver function test; (B) lymphocyte count, (C) rash presentation, (D) number of symptoms. ConclusionT-cell-based testing has potential clinical utility as a sensitive and specific diagnostic for early LD, particularly in the initial days of illness.Disclosures Sudeb C. Dalai, MD, PhD, Adaptive Biotechnologies (Employee, Shareholder) Julia Greissl, PhD, Microsoft (Employee, Shareholder) Mitch Pesesky, PhD, Adaptive Biotechnologies (Employee, Shareholder) Allison W. Rebman, MPH, Global Lyme Alliance (Research Grant or Support)Steven and Alexandra Cohen Foundation (Research Grant or Support) Mark J. Soloski, PhD, NIH grant P30 AR070254 (Grant/Research Support)Steven and Alexandra Cohen Foundation (Research Grant or Support) Elizabeth J. Horn, PhD, Adaptive Biotechnologies (Research Grant or Support)Bay Area Lyme Foundation (Research Grant or Support)Lyme Disease Biobank (Employee)Steven and Alexandra Cohen Foundation (Research Grant or Support) Jennifer N. Dines, MD, Adaptive Biotechnologies (Employee, Shareholder) Rachel M. Gittelman, PhD, Adaptive Biotechnologies (Employee, Shareholder) Thomas M. Snyder, PhD, Adaptive Biotechnologies (Employee, Shareholder) Ryan O. Emerson, PhD, Adaptive Biotechnologies (Other Financial or Material Support, Employment with Adaptive Biotechnologies during the time of this study) Edward Meeds, PhD, Microsoft (Employee, Shareholder) Thomas Manley, MD, Adaptive Biotechnologies (Other Financial or Material Support, Declares employment with Adaptive Biotechnologies during the time of this study) Ian M. Kaplan, PhD, Adaptive Biotechnologies (Employee, Shareholder) Lance Baldo, MD, Adaptive Biotechnologies (Employee, Shareholder, Leadership Interest) Jonathan M. Carlson, PhD, Microsoft (Employee, Shareholder) Harlan S. Robins, PhD, Adaptive Biotechnologies (Board Member, Employee, Shareholder) John Aucott, MD, Adaptive Biotechnologies (Advisor or Review Panel member)Bay Area Lyme Foundation (Other Financial or Material Support, Scientific Advisory Board member)Department of Health and Human Services (Other Financial or Material Support, Past Chair, 2018, HHS Tick-borne Disease Working Group, Office of HIV/AIDS and Infectious Disease Policy, Office of the Assistant Secretary of Health)Expert testimony (Other Financial or Material Support, Expert testimony)Global Lyme Alliance (Research Grant or Support)Pfizer (Consultant)Steven and Alexandra Cohen Foundation (Research Grant or Support)Tarsus Pharmaceuticals (Consultant)

Empirical identification and validation of tumor-targeting T cell receptors from circulation using autologous pancreatic tumor organoids

BackgroundTumor-specific cytotoxic T cells and T cell receptors are effective tools for cancer immunotherapy. Most efforts to identify them rely on known antigens or lymphocytes that have infiltrated into the...