Adoptive immunotherapy using autologous T cells endowed with chimeric antigen receptors (CAR) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator-like effector nuclease (TALEN) mRNA allows highly efficient multiplex gene editing in primary human T cells. We use this TALEN-mediated editing approach to develop a process for the large-scale manufacturing of T cells deficient in expression of both their ab T-cell receptor (TCR) and CD52, a protein targeted by alemtuzumab, a chemotherapeutic agent. Functionally, T cells manufactured with this process do not mediate graft-versus-host reactions and are rendered resistant to destruction by alemtuzumab. These characteristics enable the administration of alemtuzumab concurrently or prior to engineered T cells, supporting their engraftment. Furthermore, endowing the TALEN-engineered cells with a CD19 CAR led to efficient destruction of CD19 þ tumor targets even in the presence of the chemotherapeutic agent. These results demonstrate the applicability of TALEN-mediated genome editing to a scalable process, which enables the manufacturing of third-party CAR T-cell immunotherapies against arbitrary targets. As such, CAR T-cell immunotherapies can therefore be used in an "off-the-shelf" manner akin to other biologic immunopharmaceuticals. Cancer Res; 75(18); 3853-64. Ó2015 AACR.
Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. A limitation of the current autologous approach is that CAR T-cells must be manufactured on a "per patient basis". We have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" engineered CD19-CAR+ T-cell–based frozen products. Our platform involves the use of transcription activator-like effector nucleases (TALEN™), which mediate the simultaneous inactivation of two genes through genome editing. The knockout of the TCR alpha gene eliminates TCR expression and is intended to abrogate the donor T-cell’s potential for graft-versus-host disease (GvHD), while knocking out the CD52 gene makes donor T-cells resistant to the lymphodepleting agent alemtuzumab. In addition, our T-cells are engineered to coexpress the RQR8 gene as a safety feature, with the aim of rendering them sensitive to the monoclonal antibody rituximab. We previously provided proof-of-concept for the application of this approach by manufacturing TCR/CD52-deficient RQR8+ and CD19-CAR+ T-cells (UCART19) using a good manufacturing practice–compatible process, and we also demonstrated that the resulting UCART19 cells were functional using in vitro assays. Here we report the ability of UCART19 cells to engraft into an orthotopic human CD19+ lymphoma xenograft immunodeficient mouse model. UCART19 cells exhibited antitumor activity equivalent to that of standard CD19 CAR T-cells. We also demonstrated that UCART19 cells did not mediate alloreactivity in a xeno-GvHD mouse model. Furthermore, the effectiveness of the rituximab-induced depletion mechanism of RQR8+ cells was shown in an immunocompetent mouse model. In conclusion, our work significantly enlarges upon previous results by showing in vivo that (1) concomitant inactivation of a second gene has no deleterious effects on T-cells, (2) the antitumor potency of manufactured TCR/CD52-deficient CD19–CAR+ T-cells is similar to that of standard CD19-CAR+ T-cells, (3) TCR gene inactivation is efficient at preventing potential graft-versus-host reaction, and (4) allogeneic T-cells can be depleted by the use of rituximab. This valuable dataset supports the development of allogeneic CAR T-cells, and UCART19 will be investigated in an exploratory, first-in-human, clinical trial where refractory/relapsed CD19+ B-cell leukemia patients are to be enrolled. Disclosures Gouble: Cellectis SA: Employment. Poirot:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Galetto:Cellectis SA: Employment. Derniame:Cellectis SA: Employment. Arnould:Cellectis SA: Employment. Desseaux:Cellectis SA: Employment. Smith:Cellectis SA: Employment.
Adoptive T-cell therapies, where exogenous expression of a chimeric antigen receptor (CAR) confers cancer recognition, have shown significant promise in initial clinical trials. However, present adoptive immunotherapy Methods are limited by the need for manipulation of autologous patient T-cells. To permit such an approach in an allogeneic context, Transcription Activator-Like Effector Nucleases (TALENTM) have been used to simultaneously inactivate the endogenous T cell receptor and CD52, a cellular target for a lymphodepleting treatment. This approach reduces the risk of GVHD while permitting proliferation and activity of the introduced T lymphocytes in the presence of the immunosuppressive drug alemtuzumab. Electroporation of primary T cells with mRNA coding for the appropriate TALENTM result in double knock-out (dKO) frequencies of up to 70%. Furthermore, functional characterization demonstrates that the dKO cells are resistant to complement dependent lysis or in vivo depletion by alemtuzumab, and show no apparent potential for TCR-mediated activation. Finally, endowing the dKO cells with a CD19 CAR supports their capacity to kill CD19+ tumor targets as efficiently as unedited T-cells both in vitro and in vivo. Disclosures: Poirot: CELLECTIS THERAPEUTICS: Employment. Schiffer-Mannioui:CELLECTIS THERAPEUTICS: Employment. Philip:UCL Cancer Institute, London, United Kingdom: Employment. Derniame:CELLECTIS THERAPEUTICS: Employment. Gouble:CELLECTIS THERAPEUTICS: Employment. Chion-Sotinel:CELLECTIS THERAPEUTICS: Employment. Le Clerre:CELLECTIS THERAPEUTICS: Employment. Lemaire:CELLECTIS THERAPEUTICS: Employment. Grosse:CELLECTIS THERAPEUTICS: Employment. Cheung:UCL Cancer Institute, London, United Kingdom: Employment. Arnould:CELLECTIS THERAPEUTICS: Employment. Smith:CELLECTIS THERAPEUTICS: Employment. Pule:UCL Cancer Institute, London, United Kingdom: Employment. Scharenberg:CELLECTIS THERAPEUTICS: Employment.
Adoptive immunotherapy using autologous T-cells endowed with chimeric antigen receptors (CARs) has given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. However, a limitation of the current autologous approach is that CAR T-cells must be manufactured on a "per patient basis". To overcome this limitation, we have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" engineered CAR+ T-cell-based frozen products. Our allogenic platform utilizes the Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology to inactivate the TCRα constant (TRAC) gene, significantly reducing the potential for T-cells bearing alloreactive TCR’s to mediate Graft-versus-Host Disease (GvHD). We have previously demonstrated the precise and efficient disruption of the the TRAC gene by gene editing, yielding up to 85% of TCRαβ-negative cells. This allows the production of TCRαβ-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. In the clinic, the proof of concept of the applicability of our allogeneic platform was achieved with early compassionate use for patients treated with UCART19, an allogeneic engineered CAR T-cell product directed against CD19. UCART19 clinical trials are currently ongoing. Here, we have developed T-cells targeting CD22 which is expressed on tumor cells from the majority of patients with B-cells leukemia. In a first step, we have screened multiple antigen recognition domains in the context of different CAR architectures to identify effective CAR candidates displaying activity against cells expressing variable levels of the CD22 antigen. As a safety feature, T-cells are engineered to co-express a depletion gene, rendering them sensitive to the monoclonal antibody rituximab. Several constructs of depletion genes have been evaluated in the context of the CD22 CAR. In addition, experiments in an orthotopic ALL mouse model using UCART22 cells demonstrated important anti-tumor activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non-alloreactive CD22 specific T-cells from a single healthy donor can offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of leukemic patients. UCART22 could also offer an alternative to patients who may relapse with CD19-negative tumors after CD19 CAR T-cell treatment. Citation Format: Agnès Gouble, Cécile Schiffer-Mannioui, Severine Thomas, Anne-Sophie Gautron, Laurent Poirot, Julianne Smith. UCART22: allogenic adoptive immunotherapy of leukemia by targeting CD22 with CAR T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3763. doi:10.1158/1538-7445.AM2017-3763
Autologous T-cells engineered to express chimeric antigen receptors (CARs) that target specific tumor antigens are known to be of high potential in treating different kinds of cancer. However, they must be generated on a “per patient” basis, thereby limiting the population of patients that could benefit from this approach. In particular, immune homeostasis may be affected in heavily pre-treated patients, such that autologous T-cells may be low in number, not fully functional, or unable to expand, thereby restricting the amount of cells that could be manufactured. The use of allogeneic T-cells isolated from healthy third party donors could constitute an easy-to-scale-up alternative, producible in advance, with potential for standardized quality controls, better batch consistency, and immediate availability for administration to a larger number of patients. In this context, we have established a highly efficient, 18-day, good manufacturing practice (GMP)–compatible process to produce CAR T-cells from healthy donor peripheral blood mononuclear cells (PBMCs). To circumvent the potential of allogeneic T-cells inducing graft-versus-host disease (GvHD) in recipient patients, the TCR alpha constant (TRAC) gene was inactivated using a proprietary transcription activator-like effector nuclease (TALEN™)-mediated gene editing technology. The CD52 gene was also disrupted using another specific TALEN™ to allow the administration of engineered T-cells following an alemtuzumab-based lymphodepleting therapy. The antitumor activity of these double-knockout CAR T-cells was shown to be as potent as non-nuclease-edited cells expressing the same CAR in vitro. The current manufacturing process is highly reproducible, making it suitable for use in a larger scale manufacturing platform for administration as “off-the-shelf” immunopharmaceuticals. We estimate that a single production run, starting from a healthy volunteer leukapheresis product containing 109 PBMCs, would allow the production of up to 500 doses of CAR double-knockout T-cells, at 2x107 cells per dose, allowing the extension of CAR therapies to a larger number of patients. Our results provide the proof of concept for the general applicability of this approach as a platform for large-scale GMP–compliant manufacturing of allogeneic, off-the-shelf, non-alloreactive, frozen CAR T-cells. From this manufacturing platform, we produced UCART19 cells, which are TCR/CD52-deficient, RQR8+ (as a safety attribute), and anti-CD19 CAR+, to investigate their potential in the treatment of CD19+ B cell leukemias. We believe this adaptable manufacturing platform offers multiple opportunities to improve CAR T-cell therapies through multiplex genome editing, such as rendering UCART cells resistant to standard chemotherapy or to tumor evasion mechanisms. Disclosures Derniame: Cellectis SA: Employment. Poirot:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Galetto:Cellectis SA: Employment. Beurdeley:Cellectis SA: Employment. Reynier:Cellectis SA: Employment. Arnould:Cellectis SA: Employment. Smith:Cellectis SA: Employment.
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