Automated generation of gene-edited CAR T cells at clinical scale

Alzubi, Jamal; Lock, Dominik; Rhiel, Manuel; Schmitz, Sarah; Wild, Stefan; Mussolino, Claudio; Hildenbeutel, Markus; Brandes, Caroline; Rositzka, Julia; Lennartz, Simon; Haas, Simone; Chmielewski, Kay O.; Schaser, Thomas; Kaiser, Andrew; Cathomen, Toni; Cornu, Tatjana I.

doi:10.1016/j.omtm.2020.12.008

Cited by 36 publications

(37 citation statements)

References 35 publications

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“…There are currently two approaches to automation: fully automated closed systems or partially automated systems. Fully automated closed processes eliminate any handling of the product during manufacturing but are restricted to a single product in each production run (77)(78)(79). Fully automated systems such as the CliniMACS Prodigy (Miltenyi Biotec) and the Cocoon (Lonza) are capable of isolating T cells (either CD3 + or CD4/ CD8 enriched) from an apheresis product and moving them into a functionally closed culture and transduction process (80)(81)(82).…”

Section: Replacing Manual Processing With Automationmentioning

confidence: 99%

Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy

Abou‐El‐Enein

Elsallab

Feldman³

et al. 2021

Blood Cancer Discovery

102

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As of April 2021, there are five commercially available chimeric antigen receptor (CAR) T cell therapies for hematologic malignancies. With the current transition of CAR T cell manufacturing from academia to industry, there is a shift toward Good Manufacturing Practice (GMP)-compliant closed and automated systems to ensure reproducibility and to meet the increased demand for patients with cancer. In this review, we describe current CAR T cell clinical manufacturing models and discuss emerging technologic advances that embrace scaling and production optimization. We summarize measures being used to shorten CAR T cell manufacturing times and highlight regulatory challenges to scaling production for clinical use. Significance:As the demand for CAR T cell cancer therapy increases, several closed and automated production platforms are being deployed, and others are in development. This review provides a critical appraisal of these technologies, which can be leveraged to scale and optimize the production of nextgeneration CAR T cells. iNtRODUctiONChimeric antigen receptor (CAR)-modified T cells have emerged as an efficacious treatment for patients with certain hematologic malignancies (1). Currently, five CAR T cell products are approved for commercial use and available on the U.S. market: three for B-cell leukemia and lymphoma (tisagenlecleucel, axicabtagene ciloleucel, lisocabtagene maraleucel), one for mantle cell lymphoma (brexucabtagene autoleucel), and one for multiple myeloma (idecabtagene vicleucel; refs. 2-8). These therapies involve genetically modifying patient-derived (autologous) peripheral blood T cells to express a CAR directed against antigens present on the surface of targeted tumor cells, such as the CD19 molecule, or, in the case of idecabtagene vicleucel, B-cell maturation antigen (BCMA;. After antigen recognition, the intracellular signaling domains activate the immune effector and memory functions of the CAR T cells. Once activated, these T cells proliferate, infiltrate tumor sites, secrete cytokines, and release cytolytic granules to eliminate targeted cells in an antigen-dependent manner (1). All approved CAR T cell products are second-generation CARs that incorporate CD28 or CD137 (4-1BB) costimulatory signals, which are essential for eliciting a clinically relevant immune response (9-15). These CAR T cells were able to elicit complete responses (CR) in 32% to 67% of patients with lymphoma and showed better CR rates in patients with leukemia (16)(17)(18)(19).The increasing success of CAR T immunotherapies in relapsed/refractory hematologic malignancies sparked the interest of pharmaceutical companies, and several products targeting a variety of cancers are currently in the pipeline (20). However, many limitations to current CAR T cell manufacturing must be overcome before this modality can be fully integrated into routine clinical practice. First, most CAR T cell trials to date have used autologous peripheral blood and apheresis as the main cell sources for manufacturing

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Section: Replacing Manual Processing With Automationmentioning

confidence: 99%

Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy

Abou‐El‐Enein

Elsallab

Feldman³

et al. 2021

Blood Cancer Discovery

102

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“…As described, three main technologies, containing ZFNs, TALEN, and CRISPR/Cas9, facilitate gene disruption in the human cell. Remarkably, the ablation of endogenous TCR expression largely obtained through utilizing genome-editing technologies abrogate the continuous districts of TRAC genes, and thereby offer the chance for manufacturing universal CAR-T cells [7,42].…”

Section: Car-t Cells Generation From Autologous and Allogeneic T Cellsmentioning

confidence: 99%

A deep insight into CRISPR/Cas9 application in CAR-T cell-based tumor immunotherapies

et al. 2021

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To date, two chimeric antigen receptors (CAR)-T cell products from autologous T cells have been approved by The United States Food and Drug Administration (FDA). The case-by-case autologous T cell generation setting is largely considered as a pivotal restraining cause for its large-scale clinical use because of the costly and prolonged manufacturing procedure. Further, activated CAR-T cells mainly express immune checkpoint molecules, including CTLA4, PD1, LAG3, abrogating CAR-T anti-tumor activity. In addition, CAR-T cell therapy potently results in some toxicity, such as cytokine releases syndrome (CRS). Therefore, the development of the universal allogeneic T cells with higher anti-tumor effects is of paramount importance. Thus, genome-editing technologies, in particular, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 are currently being used to establish “off-the-shelf” CAR-T cells with robust resistance to immune cell-suppressive molecules. In fact, that simultaneous ablation of PD-1, T cell receptor alpha constant (TRAC or TCR), and also β-2 microglobulin (B2M) by CRISPR-Cas9 technique can support the manufacture of universal CAR-T cells with robust resistance to PD-L1. . Indeed, the ablation of β2M or TARC can severely hinder swift elimination of allogeneic T cells those express foreign HLA-I molecules, and thereby enables the generation of CAR-T cells from allogeneic healthy donors T cells with higher persistence in vivo. Herein, we will deliver a brief overview of the CAR-T cell application in the context of tumor immunotherapy. More importantly, we will discuss recent finding concerning the application of genome editing technologies for preparing universal CAR-T cells or cells that can effectively counter tumor escape, with a special focus on CRISPR-Cas9 technology.

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“…The precision afforded by CRISPR, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) is useful to not only knock-out endogenous genes but also to knockin transgenes [137]. T cells edited to remove their endogenous TCR or inhibitory receptors, such as PD-1, have already been tested in preclinical and clinical trials [68,[138][139][140]. The abrogation of HLA class I expression via the ablation of ß 2 -microglobulin is being actively pursued to build allogeneic universal CAR T cells [141].…”

Section: Genetic Engineering and Genome Editingmentioning

confidence: 99%

CAR T cells: Building on the CD19 paradigm

et al. 2021

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Spearheaded by the therapeutic use of chimeric antigen receptors (CARs) targeting CD19, synthetic immunology has entered the clinical arena. CARs are recombinant receptors for antigen that engage cell surface molecules through the variable region of an antibody and signal through arrayed T-cell activating and costimulatory domains. CARs allow redirection of T-cell cytotoxicity against any antigen of choice, independent of MHC expression. Patient T cells engineered to express CARs specific for CD19 have yielded remarkable outcomes in subjects with relapsed/refractory B-cell malignancies, setting off unprecedented interest in T-cell engineering and cell-based cancer immunotherapy. In this review, we present the challenges to extend the use of CAR T cells to solid tumors and other pathologies. We further highlight progress in CAR design, cell manufacturing, and genome editing, which in aggregate hold the promise of generating safer and more effective genetically instructed immunity. Novel engineered cell types, including innate T-cell types, natural killer (NK) cells, macrophages, and induced pluripotent stem cell-derived immune cells, are on the horizon, as are applications of CAR T cells to treat autoimmunity, severe infections, and senescence-associated pathologies.

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Automated generation of gene-edited CAR T cells at clinical scale

Cited by 36 publications

References 35 publications

Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy

Scalable Manufacturing of CAR T Cells for Cancer Immunotherapy

A deep insight into CRISPR/Cas9 application in CAR-T cell-based tumor immunotherapies

CAR T cells: Building on the CD19 paradigm

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