Endogenous T Cell Receptor Research Articles

The Generation of CAR-Transfected Natural Killer T Cells for the Immunotherapy of Melanoma

Natural killer T (NKT) cells represent a cell subpopulation that combines characteristics of natural killer (NK) cells and T cells. Through their endogenous T-cell receptors (TCRs), they reveal a pronounced intrinsic anti-tumor activity. Thus, a NKT cell transfected with a chimeric antigen receptor (CAR), which recognizes a tumor-specific surface antigen, could attack tumor cells antigen-specifically via the CAR and additionally through its endogenous TCR. NKT cells were isolated from peripheral blood mononuclear cells (PBMCs), expanded, and electroporated with mRNA encoding a chondroitin sulfate proteoglycan 4 (CSPG4)-specific CAR. The CAR expression on NKT cells and their in vitro functionality were analyzed. A transfection efficiency of more than 80% was achieved. Upon stimulation with melanoma cells, CAR-NKT cells produced cytokines antigen-specifically. Compared with conventional CAR-T cells, cytokine secretion of CAR-NKT cells was generally lower. Specific cytotoxicity, however, was similar with CAR-NKT cells showing a trend towards improved cytotoxicity. Additionally, CAR-NKT cells could kill target cells through their endogenous TCRs. In summary, it is feasible to generate CAR-NKT cells by using mRNA electroporation. Their CAR-mediated cytotoxicity is at least equal to that of conventional CAR-T cells, while their intrinsic cytotoxic activity is maintained. Thus, CAR-NKT cells may represent a valuable alternative to conventional CAR-T cells for cancer immunotherapy.

Abstract CT008: A pilot trial of the combination of transgenic NY-ESO-1-reactive adoptive cellular therapy with dendritic cell vaccination with or without ipilimumab in patients with sarcoma and melanoma

Abstract Background: Transgenic adoptive cell therapy (ACT) targeting the tumor antigen NY-ESO-1 can be effective for the treatment of sarcoma and melanoma. Preclinical models have shown that this therapy can be improved with the addition of dendritic cell (DC) vaccination and immune checkpoint blockade. In this study, we report the results of the safety, feasibility, and antitumor efficacy of this approach alone and in combination with the CTLA-4 blockade with ipilimumab. Methods: NY-ESO-1 specific T-cell receptor (TCR) transgenic lymphocytes were adoptively transferred together with NY-ESO-1 peptide-pulsed DC vaccination in HLA-A*0201-positive subjects with (ESO, NCT02070406) or without ipilimumab (INY, NCT01697527) in patients with advanced sarcoma and melanoma under Investigator New Drug IND#15167. Patients received autologous retrovirally TCR-transduced T cells following a lymphodepleting preparative chemotherapy regimen. NY-ESO-1157-165 peptide-pulsed autologous DCs were administered on Days 1, 14 and 30, and low-dose IL-2 was given twice daily for 7-14 days. In the INY cohort, ipilimumab (1mg/kg) was given on day 0 or 1, and every three weeks for a maximum of four doses. Response rates were evaluated by RECIST. Results: Four patients with synovial sarcoma, two patients with melanoma, one patient with osteosarcoma, one patient with liposarcoma, and one patient with malignant peripheral nerve sheath tumor were enrolled into these cohorts. Two patients (one treated per ESO, and another treated per INY) experienced cytokine release syndrome requiring hospitalization. Patients treated per INY had significantly higher serum levels of the cytokine FLT-3L. Two out of four patients treated per INY, and four out of six patients treated per ESO demonstrated an objective clinical response by day 30. One patient treated per ESO (17%) has had an ongoing complete response for 3 years. One patient was treated per ESO and, following disease progression after an objective clinical response, was subsequently enrolled in INY. TCR sequencing of dextramer-positive cells demonstrated that the NY-ESO TCR integrated across a highly polyclonal population of endogenous TCR clonotypes. Tracking of transgenic T cells to the tumor and acquisition of PD-1 expression was demonstrated by TCR sequencing and immunohistochemistry of on-treatment biospies, respectively. Conclusions: ACT of fresh NY-ESO-1 transgenic T cells prepared via a short ex vivo protocol and given with DC vaccination and low dose IL-2, with or without ipilimumab, is feasible and results in transient antitumor activity, with no apparent clinical benefit of the addition of ipilimumab. Improvements are needed to maintain tumor responses. Citation Format: Theodore S. Nowicki, Beata Berent-Maoz, Rong Rong Huang, Xiaoyan Wang, Gardenia Cheung-Lau, Paula Kaplan-Lefko, Paula Cabrera, Justin Tran, Ignacio Baselga Carretero, Catherine S. Grasso, Siwen Hu-Lieskovan, Bartosz Chmielowski, Begoña Comin-Anduix, Arun Singh, Antoni Ribas. A pilot trial of the combination of transgenic NY-ESO-1-reactive adoptive cellular therapy with dendritic cell vaccination with or without ipilimumab in patients with sarcoma and melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT008.

Efficient Non-Viral T-Cell Engineering bySleeping BeautyMinicircles Diminishing DNA Toxicity and miRNAs Silencing the Endogenous T-Cell Receptors

Transposon-based vectors have entered clinical trials as an alternative to viral vectors for genetic engineering of T cells. However, transposon vectors require DNA transfection into T cells, which were found to cause adverse effects. T-cell viability was decreased in a dose-dependent manner, and DNA-transfected T cells showed a delayed response upon T-cell receptor (TCR) stimulation with regard to blast formation, proliferation, and surface expression of CD25 and CD28. Gene expression analysis demonstrated a DNA-dependent induction of a type I interferon response and interferon-β upregulation. By combining Sleeping Beauty transposon minicircle vectors with SB100X transposase-encoding RNA, it was possible to reduce the amount of total DNA required, and stable expression of therapeutic TCRs was achieved in >50% of human T cells without enrichment. The TCR-engineered T cells mediated effective tumor cell killing and cytokine secretion upon antigen-specific stimulation. Additionally, the Sleeping Beauty transposon system was further improved by miRNAs silencing the endogenous TCR chains. These miRNAs increased the surface expression of the transgenic TCR, diminished mispairing with endogenous TCR chains, and enhanced antigen-specific T-cell functionality. This approach facilitates the rapid non-viral generation of highly functional, engineered T cells for immunotherapy.

TCR deep sequencing of transgenic RAG-1-deficient mice reveals endogenous TCR recombination: a cause for caution.

The utility of T-cell receptor (TCR) transgenic mice in medical research has been considerable, with applications ranging from basic biology all the way to translational and clinical investigations. Crossing of TCR transgenic mice with either recombination-activating gene (RAG)-1 or RAG-2 knockouts is frequently used to generate mice with a monoclonal T-cell repertoire. However, low level productive TCR rearrangement has been reported in RAG-deficient mice expressing transgenic TCRs. Using deep sequencing, we set out to directly examine and quantify the presence of these endogenous TCRs. Our demonstration that functional nontransgenic TCRs are present in nonmanipulated mice has wide reaching ramifications worthy of critical consideration.

A novel method to generate T-cell receptor–deficient chimeric antigen receptor T cells

AbstractPractical methods are needed to increase the applicability and efficacy of chimeric antigen receptor (CAR) T-cell therapies. Using donor-derived CAR-T cells is attractive, but expression of endogenous T-cell receptors (TCRs) carries the risk for graft-versus-host-disease (GVHD). To remove surface TCRαβ, we combined an antibody-derived single-chain variable fragment specific for CD3ε with 21 different amino acid sequences predicted to retain it intracellularly. After transduction in T cells, several of these protein expression blockers (PEBLs) colocalized intracellularly with CD3ε, blocking surface CD3 and TCRαβ expression. In 25 experiments, median TCRαβ expression in T lymphocytes was reduced from 95.7% to 25.0%; CD3/TCRαβ cell depletion yielded virtually pure TCRαβ-negative T cells. Anti-CD3ε PEBLs abrogated TCRαβ-mediated signaling, without affecting immunophenotype or proliferation. In anti-CD3ε PEBL-T cells, expression of an anti-CD19-41BB-CD3ζ CAR induced cytokine secretion, long-term proliferation, and CD19+ leukemia cell killing, at rates meeting or exceeding those of CAR-T cells with normal CD3/TCRαβ expression. In immunodeficient mice, anti-CD3ε PEBL-T cells had markedly reduced GVHD potential; when transduced with anti-CD19 CAR, these T cells killed engrafted leukemic cells. PEBL blockade of surface CD3/TCRαβ expression is an effective tool to prepare allogeneic CAR-T cells. Combined PEBL and CAR expression can be achieved in a single-step procedure, is easily adaptable to current cell manufacturing protocols, and can be used to target other T-cell molecules to further enhance CAR-T-cell therapies.

CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells

AbstractAdoptive transfer of T cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities. To circumvent these problems, we designed a system where the endogenous TCR-β is knocked out from the recipient cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic αβ and γδ TCRs, which in turn translated to a stronger, and more polyfunctional, response of engineered T cells to their target cancer cell lines. Additionally, the TCR-plus-CRISPR–modified T cells were up to a thousandfold more sensitive to antigen than standard TCR-transduced T cells or conventional model proxy systems used for studying TCR activity. Finally, transduction with a pan-cancer–reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αβ TCR resulted in more efficient redirection of CD4+ and CD8+ T cells against a panel of established blood cancers and primary, patient-derived B-cell acute lymphoblastic leukemia blasts compared with standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new, improved generations of cancer immunotherapies.

Safety and persistence of WT1-specific T-cell receptor gene−transduced lymphocytes in patients with AML and MDS

AbstractWilms' tumor 1 (WT1) is constantly expressed in leukemic cells of acute leukemia and myelodysplastic syndrome (MDS). A T-cell receptor (TCR) that specifically reacts with WT1 peptide in the context of HLA-A*24:02 has been identified. We conducted a first-in-human trial of TCR–gene transduced T-cell (TCR–T-cell) transfer in patients with refractory acute myeloblastic leukemia (AML) and high-risk MDS to investigate the safety and cell kinetics of the T cells. The WT1-specific TCR-gene was transduced to T cells using a retroviral vector encoding small interfering RNAs for endogenous TCR genes. The T cells were transferred twice with a 4-week interval in a dose-escalating design. After the second transfer, sequential WT1 peptide vaccines were given. Eight patients, divided into 2 dose cohorts, received cell transfer. No adverse events of normal tissue were seen. The TCR-T cells were detected in peripheral blood for 8 weeks at levels proportional to the dose administered, and in 5 patients, they persisted throughout the study period. The persisting cells maintained ex vivo peptide-specific immune reactivity. Two patients showed transient decreases in blast counts in bone marrow, which was associated with recovery of hematopoiesis. Four of 5 patients who had persistent T cells at the end of the study survived more than 12 months. These results suggest WT1-specific TCR-T cells manipulated by ex vivo culture of polyclonal peripheral lymphocytes survived in vivo and retained the capacity to mount an immune reaction to WT1. This trial was registered at www.umin.ac.jp as #UMIN000011519.

A TCR-based Chimeric Antigen Receptor

Effector T cells equipped with engineered antigen receptors specific for cancer targets have proven to be very efficient. Two methods have emerged: the Chimeric Antigen Receptors (CARs) and T-cell Receptor (TCR) redirection. Although very potent, CAR recognition is limited to membrane antigens which represent around 1% of the total proteins expressed, whereas TCRs have the advantage of targeting any peptide resulting from cellular protein degradation. However, TCRs depend on heavy signalling machinery only present in T cells which restricts the type of eligible therapeutic cells. Hence, an introduced therapeutic TCR will compete with the endogenous TCR for the signalling proteins and carries the potential risk of mixed dimer formation giving rise to a new TCR with unpredictable specificity. We have fused a soluble TCR construct to a CAR-signalling tail and named the final product TCR-CAR. We here show that, if expressed, the TCR-CAR conserved the specificity and the functionality of the original TCR. In addition, we demonstrate that TCR-CAR redirection was not restricted to T cells. Indeed, after transduction, the NK cell line NK-92 became TCR positive and reacted against pMHC target. This opens therapeutic avenues combing the killing efficiency of NK cells with the diversified target recognition of TCRs.

NY-ESO-1 TCR single edited stem and central memory T cells to treat multiple myeloma without graft-versus-host disease

AbstractTransfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and β genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR “single editing” (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1157-165–specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy.

Defining an allogeneic CAR-T approach by shRNA-mediated knockdown of the T-cell receptor.

e14551 Background: An allogeneic approach for CAR T-Cell therapy can significantly streamline manufacturing, provide more accessible options to patients as well as enhance safety by reducing the possibility of graft-versus-host-disease. The T-Cell Receptor (TCR) is comprised of multiple subunits and functions to activate T-cells by a signal transduction cascade that is initiated upon antigen binding. Eliminating expression of the endogenous TCR on modified CAR-T-Cells may eliminate the ability to recognize major and minor histocompatibility antigens in the recipient. This study assessed if simultaneous expression of multiple short hairpin RNAs that knockdown levels of individual TCR subunits could result in the loss of TCR expression and TCR-mediated T-Cell activation. Methods: Recombinant DNA producing short hairpin RNAs against the TCR complex was transfected into T-cells. Cell surface TCR was analyzed by FACS. Following CD3 activation or co-culture with B-cells, T-Cell activation was quantified by measuring the levels of IL-2 by ELISA and QPCR. Results: When expressed individually, each shRNA inhibited it’s cognate TCR subunit by up to 93% of the endogenous levels. However, upon simultaneous expression of the shRNAs against the different subunits from the same vector, we observed a near complete depletion of the TCR complex from the cell surface ( > 99%) as measured by FACS analyses. Furthermore, TCR functionality was inhibited when treated cells were stimulated with either CD3 or in B cell co-cultures with Staphylococcal enterotoxins. IL-2 secretion was inhibited to undetectable levels by ELISA by the multi-shRNA treatment and > 98% by qPCR. Conclusions: Though knockdown of any single component of TCR never exceeded 93%, simultaneous knockdown of several TCR subunits abrogated surface TCR and downstream activation suggesting that disruption of stoichiometric expression the subunits prevented TCR formation. The small size of the shRNA expression cassette ( < 2 Kb) permits co-expression from the same lentiviral vector as the CAR. Altogether, these data point to a viable strategy towards generating a single vector approach for the production of allogeneic T-Cells for immunotherapies against certain cancers.

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies.

Development of a novel T-cell immunotherapy targeting HEATR1.

149 Background: Allogeneic hematopoietic stem cell transplantation (HCT) often cures acute leukemia. However leukemic relapse remains a major cause of HCT failure, and patients with post-HCT relapse have a very poor prognosis. We are developing T cell immunotherapies targeting leukemia-associated minor histocompatibility (H) antigens to manage post-HCT relapse. Because the presentation of minor H antigens is HLA-restricted, a panel of hematopoietic-restricted, leukemia-associated minor H antigens is required to enable the development of broadly applicable minor H antigen targeted immunotherapy. We previously discovered a HLA- B*0801-restricted, leukemia-associated minor H antigen, HEATR1. We showed that HEATR1-specific T cells can specifically kill leukemic blasts and prevent engraftment in a murine model, implying that this minor H antigen is expressed in leukemic stem cells. We have now developed immunotherapy targeting HEATR1 using genetically modified T cells. Methods: We isolated a HEATR1-specific T cell clone from the peripheral blood of a normal donor and sequenced its T cell receptor (TCR). The TCR was codon-optimized and cysteine-modified to maximize expression in T cells and reduce the risk of mispairing with endogenous TCR chains, then cloned into a multicistronic lentiviral vector (LV). Primary CD8+T cells were transduced with the HEATR1 TCR LV and evaluated with a range of assays. Results: The HEATR1-specific T cell clone specifically killed B8+ HEATR1+ primary leukemia. Similarly, CD8+ HEATR1 TCR-transduced T cells killed target cells pulsed with HEATR1 peptide (ISKERAEAL), but not the allelic variant control peptide (ISKERAGAL), and specifically killed cell lines that endogenously present the HEATR1 minor H antigen. HEATR1-transduced T cells also secrete cytokines and proliferate in response to HEATR1+ but not HEATR1-cells. Conclusions: We have demonstrated that HEATR1-specific T cell clone and HEATR1 TCR-transduced T cells can kill HEATR1+ cells. If the safety and efficacy of HEATR1 TCR immunotherapy is confirmed in further preclinical studies, development of phase I clinical studies will be warranted and may ultimately provide a new option for management of relapse after HCT.

Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells

Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19+ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+ tumors in an in vivo mouse model.

Immunology: T-cell tweaks to target tumours.

Immune cells known as T cells can destroy tumour cells, but their clinical use requires complex preparation and the cells can lose effectiveness over time. A new approach might improve the efficiency of T-cell therapy. See Letter p.113 Using T cells transduced with synthetic chimeric antigen receptors (CARs) is a promising strategy for treating certain types of cancer. Here Michel Sadelain and colleagues provide evidence in a mouse tumour model that knocking the CAR into the endogenous T-cell receptor α constant locus reduces tonic signalling, avoids accelerated T-cell differentiation, and delays T-cell exhaustion. This results in enhanced function and anti-tumour efficacy compared with random integrations.

γδTCR immunoglobulin constant region domain exchange in human αβTCRs improves TCR pairing without altering TCR gene-modified T cell function.

The adoptive genetic transfer of T cell receptors (TCRs) has been shown to be overall feasible and offer clinical potential as a treatment for different types of cancer. However, this promising clinical approach is limited by the serious potential consequence that exogenous TCR mispairing with endogenous TCR chains may lead to the risk of self-reactivity. In the present study, domain-exchange and three-dimensional modeling strategies were used to create a set of chimeric TCR variants, which were used to exchange the partial or complete constant region of αβTCR with corresponding γδTCR domains. The expression, assembly and function of the chimeric TCR variants were examined in Jurkat T cells and peripheral mononuclear blood cells (PBMCs). Genetically-encoded chimeras were fused with a pair of fluorescent proteins (ECFP/EYFP) to monitor expression and the pairing between chimeric TCRα chains and TCRβ chains. The fluorescence energy transfer based on confocal laser scanning microscopy showed that the introduction of γδTCR constant sequences into the αβTCR did not result in a global reduction of mispairing with endogenous TCR. However, the TCR harboring the immunoglobulin-like domain of the γδTCR constant region (i.e., TCR∆IgC), showed a higher expression and preferential pairing, compared with wild-type (wt)TCR. The function analysis showed that TCR∆IgC exhibited the same levels of interferon-γ production and cytotoxic activity, compared with wtTCR. Furthermore, these modified TCR-transduced T cells retained the classic human leukocyte antigen restriction of the original TCR. The other two chimeric TCRs, had either exchange of the cp+tm+ic domain or exchange of the whole C domain (Fig. 1). Ultimately, exchange of these domains demonstrated defective function in the transduced T cells. Taken together, these findings may provide further understanding of the γδTCR constant domain with implications for the improvement of TCR gene transfer therapy.

Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification

In T cells with transgenic high-avidity T cell receptors (TCRs), endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially causing autoimmunity. To knock out endogenous TCR expression, we assembled 12 transcription activator-like effector nucleases (TALENs) and five guide RNAs (gRNAs) from the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Using TALEN mRNA, TCR knockout was successful in up to 81% of T cells. Additionally, we were able to verify targeted gene addition of a GFP gene by homology-directed repair at the TALEN target site, using a donor suitable for replacement of the reporter transgene with therapeutic TCR chains. Remarkably, analysis of TALEN and CRISPR/Cas9 specificity using integrase-defective lentiviral vector capture revealed only one off-target site for one of the gRNAs and three off-target sites for both of the TALENs, indicating a high level of specificity. Collectively, our work shows highly efficient and specific nucleases for T cell engineering.

Electroporation of Dicer-Substrate siRNA Duplexes Targeting Endogenous TCR Enhance Tumor Killing Activity of Wilms' Tumor 1 (WT1)-Specific TCR-Redirected Cytotoxic T Cells

In adoptive cellular immunotherapy, T cells can be genetically engineered to express a novel T-cell receptor (TCR) that recognizes a tumor-associated antigen. However, mispairing between transgene and endogenous TCR chains may result in a reduction of transgene TCR expression and potentially harmful off-target reactivities. Here, we sought to develop a novel clinically safe strategy to promote transgene expression of a Wilms' tumor 1 (WT1)-specific TCR by Dicer-substrate small interfering RNA (DsiRNA)-mediated silencing of the endogenous TCR, using a double electroporation protocol.First, we isolated and cloned an HLA-A*0201-restricted WT1 peptide-specific TCR derived from a leukemia patient who demonstrated clinical benefit after receiving a WT1-targeted DC vaccine. Next, we produced a codon-optimized TCR sequence from the wild-type TCR construct and both TCR mRNAs were generated by in vitro transcription. TCR expression levels were validated by electroporation of TCR-deficient Jurkat J76.7 cells stably transduced with CD8 and an NFAT-driven GFP reporter gene. TCR functionality was confirmed by high expression levels of GFP (70% GFP+cells) upon TCR signaling after co-culture with WT1 peptide-pulsed T2 cells.In order to suppress the translation of endogenous TCR mRNA in CD8+ T cells, DsiRNA duplexes were designed to specifically target the constant regions of wild-type TCR α- and β-chains, but not the codon-optimized TCR. We further developed a double electroporation protocol combining DsiRNA and TCR mRNA transfection in which DsiRNA electroporation was performed 24 hours prior to TCR mRNA electroporation. Our results show more than 2-fold increase in WT1-specific TCR expression by HLA-A2/WT1 tetramer staining after DsiRNA treatment as compared to TCR mRNA electroporation only. This specific TCR expression was maintained at least 5 days after TCR mRNA electroporation in resting peripheral blood CD8+ lymphocytes from healthy donors. The enhanced TCR expression in DsiRNA-transfected CD8+T cells was also correlated with an increase of epitope recognition as shown by interferon (IFN)-γ ELISpot.To determine the killing capacity of DsiRNA/TCR mRNA-transfected CD8+ T cells against epitope-bearing target cells, we performed a flow cytometry-based cytotoxicity assay using WT1 peptide-pulsed T2 cells. Specific cytotoxicity, which was already present in WT1 TCR-transfected cells, was significantly enhanced in TCR mRNA-electroporated T cells following suppression of the endogenous TCR expression by DsiRNA treatment. Accordingly, DsiRNA-treated TCR mRNA transfected CD8+T cells presented higher levels of CD137 and CD69 activation markers and secretion of cytokines (IFN-γ and tumor necrosis factor-α), granzyme B, and perforin upon TCR triggering as compared to the non-DsiRNA treated T cells.In summary, we show a marked enhancement of transgene WT1-specific TCR expression upon silencing of the endogenous TCR using DsiRNA electroporation prior to TCR mRNA electroporation. Importantly, this enhancement in TCR expression was correlated with a significant increase in WT1-specific CD8+ T-cell killing activity, expression of CD69 and CD137 activation markers and cytokine secretion after recognition of WT1 peptide-bearing target cells. These results pave the way for developing a clinically safer strategy for T cell-based adoptive immunotherapy of patients with WT1-expressing malignancies. DisclosuresNo relevant conflicts of interest to declare.

Phase 1 Clinical Trial of Adoptive Immunotherapy for Acute Myelogenous Leukemia and Myelodysplastic Syndrome, Using Gene-Modified Autologous Lymphocytes Expressing WT1-Specific T-Cell Receptor

Elderly patients (pts) with acute myelogenous leukemia (AML) and high-risk myelodysplastic syndrome (MDS) particularly who are ineligible for allogeneic hematopoietic stem cell transplantation (allo-HSCT) and have disease relapse after allo-HSCT still have a poor prognosis. Thus, the development of novel treatment option providing higher response rate and prolonged survival time for those patients still remains an unmet need. Because not a few clinical and preclinical studies have described a promising value of Wilms Tumor 1 (WT1), a leukemia-associated antigen as a therapeutic target of antileukemia immunotherapy, we conducted a clinical study of novel adoptive immunotherapy using gene-modified autologous lymphocytes expressing WT1-specific T-cell receptor (TCR) for the treatment of refractory AML and high-risk MDS.A multicenter phase 1 study was conducted to assess feasibility, safety and preliminary antileukemia reactivity of patient-derived gene-modified lymphocytes expressing WT1-specific TCR. Antigen-specific TCR-gene transfer may cause a serious autoimmune disease mediated by mispaired TCR between introduced and endogenous TCR α/β chains. To avoid that, we established a retroviral vector system encoding siRNAs for endogenous TCR genes (siTCR vector). We conducted a first-in-human clinical trial employing this siTCR vector. After given written informed consents, mononuclear cells were collected from at most 200ml of peripheral blood (PB) from each patient. Then, proliferating lymphocytes pre-cultured with IL-2, anti-CD3 antibody and RetroNectinTM were infected with a retroviral vector, MS3-WT1-siTCR composed of DNAs encoding WT1235-243/HLA-A*24:02 complex specific TCR-α/β chains and siRNAs against endogenous TCR genes. Expanded gene-modified lymphocytes (WT1-siTCR/T cells) in additional culture for 13-14 more days were harvested and frozen until use. Eligibility included HLA-A*24:02 positive pts with refractory AML or high-risk MDS, > 20 y.o, ineligible for allo-HSCT and performance status of 0 to 2. WT1-siTCR/T cells were intravenously infused twice on days 0 and 28. Heteroclitic WT1235-243 ninemer peptide (300mg) emulsified with MontanideTM was given subcutaneously on day 2 and 16 after the second infusion. Besides safety assays, kinetics of WT1-siTCR/T cells in PB, immunological responses and residual leukemia burden determined by qRT-PCR for WT1 mRNA were serially measured until day 58 since the first infusion.Among 12 pts enrolled, 8 pts (5 AML, 3 MDS) with a median age of 68.5 y. received study treatment. Three pts received 2x108 cells/ infusion (cohort 1), 3 received 1x109 cells/ infusion (cohort 2), and 2 received extra-cohort doses. Median follow-up time after the first infusion was 257 days (as June 13, 2016). At the first infusion, all pts contracted progressive disease. Circulatory WT1-siTCR/T cells retaining the target reactivity appeared immediately after each infusion, peaked between 1 to 3 days, and declined thereafter. WT1 peptide vaccination did not seem to affect the transition of infused cells. Values of WT1 mRNA in PB were transiently suppressed in all pts and declined in 4 pts thereafter. Clinical outcomes included one with stable disease and 2 pts with partial remission (PR). In one with PR, the epitope-spreading phenomenon was suggested. In all pts, no serious adverse events associated with infused WT1-siTCR/T cells were observed.Adoptive transfer of autologous WT1-siTCR/T cells was feasible and safe. Although the persistence of infused WT1-siTCR/T cells was limited, infused WT1-siTCR/T cells at least seemed to be involved in the antileukemia reactivity. DisclosuresTawara:Astellas: Honoraria. Akatsuka:Takara Bio Inc.: Consultancy. Nukaya:Takara Bio Inc.: Employment. Takesako:Takara Bio Inc.: Employment.

Tumor-Specific Donor Lymphocyte Infusion for Tumor Relapse after MHC-Haploidentical Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for refractory hematologic malignancies such as leukemia and myelodysplastic syndrome. Tumor relapse, graft-vs host disease (GVHD) and infections are, however, major obstacles to successful allo-HSCT. Donor lymphocyte infusion (DLI) is carried out in some cases of tumor relapse and infections after allo-HSCT. DLI potentially induces or aggravates GVHD due to allo-reactivity of the lymphocytes used for infusion and the efficacy of DLI for tumor relapse is actually limited. Therefore, development of the method of DLI with enhanced anti-tumor effects without induction or aggravation of GVHD is expected to improve the results of DLI in the context of tumor relapse. Tumor antigen-specific T cell receptor (TCR)-expressing cell infusion is one of the potentially effective immunotherapies for refractory tumors. We previously reported that tumor antigen-specific TCR-gene-transduced human lymphocytes engineered with a novel retrovirus vector silencing endogenous TCRs showed reduced allo-reactivity (ASH annual meeting 2014). Utilizing this technology, we may be able to enhance anti-tumor effects without induction or aggravation of GVHD. In this study, we conducted a mouse pre-clinical model that mimics tumor-specific TCR-engineered DLI for tumor relapse after MHC-haploidentical HSCT and explored the effect of DLI on tumor growth and GVHD.We performed experimental study utilizing the following model: MHC-haploidentical BALB/c(H-2d)→CB6F1(H-2b/d) HSCT, BALB/c-derived sarcoma CMS5a and T cells from TCR-transgenic mouse DUC18. CMS5a has a unique mutated form of a mitogen-activated kinase ERK2, and a nonamer peptide, called 9m, incorporating the resulting amino acid substitution is presented on H-2Kd and recognized by the CD8+ CTL clone C18. DUC18 is a BALB/c-background TCR transgenic line that expresses the rearranged Vα10.1 and Vβ8.3 genes of the C18. CD8+ T cells of DUC18 mice can suppress CMS5a tumor growth in the syngeneic BALB/c hosts. Allo-reactivity of DUC18 T cells was reduced as compared to that of wild type BALB/c T cells. Based on the results of preliminary experiments, we set standard experimental conditions as follows: X-ray irradiation (10 Gy) for pre-conditioning, 5x106 whole bone marrow and 0.25x106 T cell transplant (BMT) for mild GVHD induction, 1x106 CMS5a inoculation for progressive tumor growth in BMT recipients, and 4x106 CD8+ T cells for observation of the effect of DLI on tumor growth and GVHD. In some experiment, CD4+ T cells were co-infused with CD8+ T cells. DLI was carried out 3 days after CMS5a inoculation at 2, 4 or 8 weeks after BMT.We first used CD8+ T cells from naïve BALB/c or DUC18 mice for DLI at 8 weeks after BMT. No GVHD aggravation was observed in either BALB/c or DUC18 CD8+ T cell recipients and, as expected, suppression of tumor growth was observed only in the DUC18 CD8+ T cell recipients. Next, DLIs using CD8+ T cells only, or both CD8+ and CD4+ (CD8+/CD4+) T cells from naïve mice were carried out at 4 or 2 weeks after BMT. No apparent effect of CD4+ T cell co-infusion on tumor growth and GVHD was observed in both types of donor T cell recipients when DLI was carried out at 4 weeks after BMT. However, GVHD was aggravated in CD8+/CD4+ T cell recipients from both types of donors when DLI was carried out at 2 weeks after BMT, and no CD4-driven additional anti-tumor effect was observed in DUC18 T cell recipients. We then performed DLI using in vitro activated/expanded T cells including both CD8+ and CD4+ as a more clinically relevant model. BALB/c and DUC18 splenocytes were activated and expanded in vitro and infused (adjusted CD8+ T cell number to 4x106/mouse) into recipients at 2 or 8 weeks after BMT. Aggravation of GVHD was observed in either BALB/c or DUC18 activated/expanded T cell recipients when DLI was carried out at 2 weeks after BMT. On the other hand, it was not observed when DLI was carried out at 8 weeks after BMT. Anti-tumor effect of DUC18 T cells was observed regardless of timings of DLI.Taken together, infusion of tumor-specific donor lymphocytes with reduced allo-reactivity for tumor relapse after MHC-haploidentical HSCT will be promising strategy. Timing of DLI and condition of recipients such as inflammatory status may affect GVHD. Multiple model studies are required for development of more effective DLI strategies without aggravation of GVHD. DisclosuresTawara:Astellas: Honoraria. Katayama:Nippon Shinyaku: Honoraria; Chugai: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria; Dainippon Sumitomo Pharma: Honoraria; Pfizer: Honoraria; Celgene: Honoraria; Shionogi: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Taisho Toyama Pharma: Honoraria; Shire: Honoraria; Astellas: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Eisai: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb Japan: Honoraria. Ikeda:Ono Pharmaceutical Co., Ltd: Honoraria; Daiichi Sankyo Co., Ltd: Honoraria.

646. Development of a Novel Chimeric Antigen Receptor as a Therapy Against Solid Tumors

The use of chimeric antigen receptor (CAR) T cells as a therapy for cancer is gaining traction as they potentially target tumors with high specificity and minimal off-target toxicities. Current CAR designs consist of an antigen-specific single chain antibody fragment (scFv) fused to the transmembrane and signaling domains of receptors capable of activating T cells. In CAR T cell therapy, patients’ T cells are transduced with cDNA encoding the CAR allowing CAR expressing T cells to target tumor cells. Clinical trials using anti-CD19 CAR T cells to target B cell malignancy have demonstrated the potential of this therapy with numerous patients achieving partial or full remission. In the treatment of solid tumors, however, CAR T cell therapy has not proven as effective. Evidence supporting T cell persistence is sparse, suggesting that currently used CARs may not provide optimal signals for memory cell generation. In addition, some patients have experienced life-threating side effects including cytokine release syndrome and on-target off-tumor activity, indicating a need for better modulation of CAR T cell activity. In line with these clinical observations, data from our laboratory and others demonstrate that currently used CARs can activate T cells in the absence of antigen, which promotes toxicity and T cell exhaustion while preventing differentiation into memory T cells. We propose that this antigen-independent activation is a result of high expression of CAR molecules that lack the regulatory context of the endogenous T cell receptor (TCR) complex. To more effectively mimic the temporal regulation of endogenous TCR signaling pathways, we developed a novel TCR-based CAR in which a scFv is fused to the constant region of TCRβ, thereby becoming incorporated into the native complex. We hypothesize that incorporation of our CAR into the TCR complex will mimic endogenous T cell activation and regulation by including contributions from all CD3 members (CD3Δ, CD3e, CD3γ, and CD3ζ). Furthermore, we propose that endogenous regulation of CAR signaling will prevent the constitutive T cell activation observed with currently used CAR constructs and allow for the formation of memory T cells. We have demonstrated that surface expression of our TCR CAR is modulated by endogenous CD3 expression levels and mirrors normal TCR surface expression. In contrast to both first and second generation CARs, T cells expressing our novel CAR lack constitutive activation, are specific for tumor cells expressing high levels of antigen, and retain higher proliferative potential in culture. Moreover, TCR CAR T cells are capable of signaling through the TCR complex and activating T cells in an antigen specific manner. Currently, we are examining the effector potential of our TCR CAR T cells compared to first and second generation CARs. Additionally, we are developing TCR CARs capable of signaling not only through the TCR complex but also through co-stimulatory receptors. In summary, we are designing the next generation of CARs by incorporating the tumor recognition component of a scFv into the endogenous TCR complex.