Cancer immunotherapy has gained attention as the supreme therapeutic modality for the treatment of various malignancies. Adoptive T-cell therapy (ACT) is one of the most distinctive modalities of this therapeutic approach, which seeks to harness the potential of combating cancer cells by using autologous or allogenic tumor-specific T-cells. However, a plethora of circumstances must be optimized to produce functional, durable, and efficient T-cells. Recently, the potential of ACT has been further realized by the introduction of novel gene-editing platforms such as the CRISPR/Cas9 system; this technique has been utilized to create T-cells furnished with recombinant T-cell receptor (TCR) or chimeric antigen receptor (CAR) that have precise tumor antigen recognition, minimal side effects and treatment-related toxicities, robust proliferation and cytotoxicity, and nominal exhaustion. Here, we aim to review and categorize the recent breakthroughs of genetically modified TCR/CAR T-cells through CRISPR/Cas9 technology and address the pearls and pitfalls of each method. In addition, we investigate the latest ongoing clinical trials that are applying CRISPR-associated TCR/CAR T-cells for the treatment of cancers.

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Background The successful ex vivo expansion of T-cells in great numbers is the cornerstone of adoptive cell therapy. We aimed to achieve the most optimal T-cell expansion condition by comparing the expansion of T-cells at various seeding densities, IL-2 concentrations, and bead-to-cell ratios. we first expanded the peripheral blood mononuclear cells (PBMCs) of a healthy donor at a range of 20 to 500 IU/mL IL-2 concentrations, 125 × 103 to 1.5 × 106 cell/mL, and 1:10 to 10:1 B:C (Bead-to-cell) ratios and compared the results. We then expanded the PBMC of three healthy donors using the optimized conditions and examined the growth kinetics. On day 28, CD3, CD4, and CD8 expression of the cell populations were analyzed by flow cytometry. Results T-cells of the first donor showed greater expansion results in IL-2 concentrations higher than 50 IU/mL compared to 20 IU/mL (P = 0.02). A seeding density of 250 × 103 cell/mL was superior to higher or lower densities in expanding T-cells (P = 0.025). Also, we witnessed a direct correlation between the B:C ratio and T-cell expansion, in which, in 5:1 and 10:1 B:C ratios T-cell significantly expanded more than lower B:C ratios. The results of PBMC expansions of three healthy donors were similar in growth kinetics. In the optimized condition, 96–98% of the lymphocyte population expressed CD3. While the majority of these cells expressed CD8, the mean expression of CD4 in the donors was 19.3, 16.5, and 20.4%. Conclusions Our methodology demonstrates an optimized culture condition for the production of large quantities of polyclonal T-cells, which could be useful for future clinical and research studies.

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