From ex vivo to in vivo chimeric antigen T cells manufacturing: new horizons for CAR T-cell based therapy

Abstract

In the past decades, Chimeric Antigen Receptor (CAR)-T cell therapy has achieved remarkable success, leading to the approval of six therapeutic products for haematological malignancies. Recently, the therapeutic potential of this therapy has also been demonstrated in non-tumoral diseases. Currently, the manufacturing process to produce clinical-grade CAR-T cells is complex, time-consuming, and highly expensive. It involves multiple steps, including the collection of T cells from patients or healthy donors, in vitro engineering and expansion, and finally reinfusion into patients. Therefore, despite the impressive clinical outcomes, ex vivo manufacturing process makes CAR-T cells out of reach for many cancer patients. Direct in vivo engineering of T cells could be a more rapid solution able to circumvent both the complexity and the costs associated with ex vivo manufactured CAR-T cells. This novel approach allows to completely eliminate ex vivo cell manipulation and expansion while producing therapeutic cell populations directly in vivo. To date, several studies have demonstrated the feasibility of in vivo T cell reprogramming, by employing injectable viral- or nanocarrier-based delivery platforms in tumour animal models. Additionally, in vivo production of CAR-T cells might reduce the incidence, or at least the severity, of systemic toxicities frequently occurring with ex vivo produced CAR-T cells, such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. In this review, we highlight the challenges associated with the current ex vivo manufacturing protocols and review the latest progresses in the emerging field of in vivo CAR-T therapy, by comparing the various platforms so far investigated. Moreover, we offer an overview of the advantages deriving from in vivo reprogramming of other immune cell types, such as Natural Killer and macrophages, with CAR constructs.

Keywords: CAR T cell; Manufacturing; T cell engineering.

Fig. 1

A Schematic representation of CAR-T cell structure: intracellular, transmembrane, extracellular domain. B Evolution of the five generation of CARs: from the first generation, containing only one activation domain, to the last next generation CARs, aiming to improve their safety and efficacy

Fig. 2

Ex vivo vs In vivo CAR-T cell production. In this illustration, ex vivo CARs generation passages are compared with in vivo CARs production by intravenous injection of viral vectors or nanocarriers

Fig.3

Comparison of CAR-NK and CAR-M cell mechanism of action. A CAR-dependent tumor killing pathway of CAR-NK cells involves the binding of a specific tumor antigen with CAR, and the following secretion of perforin and granzyme B, to kill tumor cells, and pro-inflammatory cytokines (TNF-α and INF-γ), to promote CAR-NKs activation and stimulate antitumor response of other T-cells. B CAR-NK cells can mediate a direct killing of tumor cells through the following CAR-independent mechanisms: signaling of activating receptors expressed on NK cells surface, that lead to secretion of TNF-α, INF-γ, perforin and granzyme B; induction of cell apoptosis through FasL/Fas and TRAIL/TRAIL-R pathways; triggering of ADCC via the CD16 Fc receptor; secretion of chemokines and cytokines that recruit and activate other immune cells. C CAR-dependent tumor killing pathway of CAR-M cells involves the binding of a specific tumor antigen with CAR and the subsequent antigen-specific tumor phagocytosis and release of pro-inflammatory cytokines, which stimulate antitumor response of other T cells. D CAR-M cells can mediate a direct killing of tumor cells through the following CAR-independent mechanisms: direct phagocytosis of tumour cells and subsequent presentation of processed tumor antigens to T cells trough MHC-I molecules; secretion of pro-inflammatory cytokines; triggering of ADCC via the CD16 Fc receptor; expression of “killing molecules”, such as ROS and iNOS, which mediate cytotoxic effects on tumor cells