Chimeric antigen receptor (CAR)-T cell therapy is one of the most promising advances in cancer treatment. It is based on genetically modified T cells to express a CAR, which enables the recognition of the specific tumour antigen of interest. To date, CAR-T cell therapies approved for commercialisation are designed to treat haematological malignancies, showing impressive clinical efficacy in patients with relapsed or refractory advanced-stage tumours. However, since they all use the patient´s own T cells as starting material (i.e. autologous use), they have important limitations, including manufacturing delays, high production costs, difficulties in standardising the preparation process, and production failures due to patient T cell dysfunction. Therefore, many efforts are currently being devoted to contribute to the development of safe and effective therapies for allogeneic use, which should be designed to overcome the most important risks they entail: immune rejection and graft-versus-host disease (GvHD). This systematic review brings together the wide range of different approaches that have been studied to achieve the production of allogeneic CAR-T cell therapies and discuss the advantages and disadvantages of every strategy. The methods were classified in two major categories: those involving extra genetic modifications, in addition to CAR integration, and those relying on the selection of alternative cell sources/subpopulations for allogeneic CAR-T cell production (i.e. γδ T cells, induced pluripotent stem cells (iPSCs), umbilical cord blood T cells, memory T cells subpopulations, virus-specific T cells and cytokine-induced killer cells). We have observed that, although genetic modification of T cells is the most widely used approach, new approaches combining both methods have emerged. However, more preclinical and clinical research is needed to determine the most appropriate strategy to bring this promising antitumour therapy to the clinical setting.

Background/Objectives: Chimeric Antigen Receptor (CAR)-T cell therapy has demonstrated impressive clinical results against hematological malignancies. However, currently commercialized CAR-T therapies are designed for autologous use, which entails some disadvantages, including high costs, manufacturing delays, complex standardization, and frequent production failures due to patient T cell dysfunction. Moreover, their CARs target one specific antigen, increasing the probability of antigen-negative tumor relapses. To overcome these limitations, we developed a novel NKG2D CAR-T cell therapy for allogeneic use with broad target specificity, as this CAR targets eight different ligands commonly upregulated in both solid and hematological tumors. Additionally, the manufacturing process was optimized to improve the phenotypic characteristics of the final product. Methods: Multiplex CRISPR/Cas9 technology was applied to eliminate the expression of TCR and HLA class I complexes in healthy donor T cells to reduce the risk of graft-versus-host disease and immune rejection, respectively, as well as lentiviral transduction for introducing the second-generation NKG2D-CAR. Moreover, we sought to optimize this manufacturing process by comparing the effect of different culture interleukin supplementations (IL-2, IL-7/IL-15 or IL-7/IL-15/IL-21) on the phenotypic and functional characteristics of the product obtained. Results: Our results showed that the novel CAR-T cells effectively targeted cervicouterine and colorectal cancer cells, and that those manufactured with IL-7/IL-15/IL-21 supplementation showed the most suitable characteristics among the conditions tested, considering genetic modification efficiency, cell proliferation, antitumor activity and proportion of the stem cell memory T cell subset, which is associated with enhanced in vivo CAR-T cell survival, expansion and long-term persistence. Conclusions: In summary, this new prototype of NKG2D CAR-T cell therapy for allogeneic use represents a promising universal treatment for a wide range of tumor types.