Review: Sustainable Clinical Development of CAR-T Cells – Switching From Viral Transduction Towards CRISPR-Cas Gene Editing

Abstract

T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first gene-modified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as “off-the-shelf” products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.

GMP development and preclinical validation of CAR-T cells targeting a lytic EBV antigen for therapy of EBV-associated malignancies

“…We had previously produced gp350CAR-T cells using retroviral vectors ( 9 ) but since third generation self-inactivating (SIN) LVs remains currently as the leading the gene transfer tool for CAR-T cell manufacturing ( 11 ), we designed and tested four LVs containing the CD8 transmembrane domain (TM) and incorporating the 7A1 or 6G4 gp350-specific scFvs, the human IgG Fc or CD8 hinges, and the 4-1BB.CD3ζ or CD28.CD3ζ chimeric signaling domains ( Figure 1A ). One day after activation with cytokines, PBMCs were transduced with the LVs and further expanded ex vivo .…”

Chimeric Antigen Receptor Immunotherapy for Solid Tumors: Choosing the Right Ingredients for the Perfect Recipe

“…One of the most debated aspects of CAR-based therapies is the choice of the cell population to be genetically engineered for the expression of CAR. Since CAR-engineered cells will represent the active substance of final product, the choice of cell population has a deep impact on large-scale manufacturing and “off-the-shelf” potential of the CAR therapy: Two critical points that will strongly dictate the future of CAR-based therapies for solid tumors in terms of clinical availability and economic sustainability ( Figure 1 ) [ 49 , 50 ]. Moreover, since different immune cells exhibit different mechanisms of action and subsequent antitumor activity, each carrying advantages and challenges, the choice of the cell type will affect the in vivo clinical efficacy of CAR-based immunotherapy.…”

Non-viral TRAC-knocked-in CD19KICAR-T and gp350KICAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency

“…The currently marketed CD19 CAR-T cell products are CTL019 (10), KTE-C19 (11), brexucabtagene autoleucel (12), and liso-cell (13). Generation of these CAR-T cells relies heavily on the transduction of T cells with lentiviral vectors (LVs) or retroviral vectors (RVs), which are costly and impose biosafety and gene technology level 2 restrictions (14). As an alternative to lymphoma antigens such as CD19 that are also expressed on normal B cells, resulting in their depletion, we explored viral antigens expressed on lymphoma cells for CAR engineering.…”