A novel non-viral PDCD1 site-integrated CAR design: killing 2 birds with 1 stone Article Swipe

Although chimeric antigen receptor T-cell (CAR-T-cell) therapy has shown excellent efficacy against refractory/relapsed B-cell lymphoma, B-cell acute lymphoblastic leukemia and multiple myeloma,1,2 the complete response rate of patients with refractory/relapsed B-cell lymphoma receiving conventional CAR-T-cell therapy is approximately 40% to 50%.3–5 There are 3 drawbacks to viral CAR-T-cell therapy. First, random integration of the CAR cassette and lentivirus replication are potential risks for viral CAR-T-cell therapy (Fig. 1A).6–8 Second, the expression levels of CAR vary in different CAR-T-cells, depending on their integration sites, which may affect the efficacy and toxicity of this therapy. In addition, the complex manufacturing process and long preparation time of viral-derived CAR-T-cells needs improvement. To overcome these hurdles, several teams have used CRISPR−Cas9 to integrate the CAR cassette into specific genome loci, including TRAC and AAVS loci.9,10Figure 1.: Schematic representation of the viral CAR-T-cell and non-viral PDCD1 site-integrated CAR-T-cells production. (A) Lentiviruses deliver CAR cassette by randomly integrating it to T cell chromosome. Immune checkpoint molecule PD-1 is still expressed on CAR-T-cell surface and make it more prone to exhaustion. (B) By using CRISPR/Cas9 technology, CAR cassette specifically inserts into the PDCD1 locus in non-viral PDCD1 site-integrated CAR-T-cell. By knocking out the PDCD1, non-viral PDCD1 site-integrated CAR-T-cell product has higher memory T cell proportion with long lasting antitumor effect. CAR-T-cell = chimeric antigen receptor T-cell.The programmed cell death receptor-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) axis is considered one of the most important immunosuppressive signaling pathways in T-cells. Undoubtedly, the anti-tumor efficacy of CAR-T-cells is also hindered by the PD-1/PD-L1 axis. To date, various types of immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have been approved for the treatment of a number of tumors.11,12 However, owing to potential safety concerns and the difficulty of co-localization of CAR-T-cells and PD-1/PD-L1 inhibitors,13 the combination of CAR-T therapy and PD-1 inhibition needs to be further optimized.13–15 Previous studies have demonstrated that disrupting PD-1 in CAR-T-cells by CRISPR−Cas916 or converting immunosuppressive PD-L1 signaling into stimulatory signaling by chimeric switch receptor T (CSR-T) cells can augment the anti-tumor effects of CAR-T-cells.17,18 Recently, the teams of Mingyao Liu and He Huang (et al) developed a two-in-one approach to generate non-viral CAR-T-cells by inserting an anti-CD19 CAR cassette into the specific locus through CRISPR/Cas9.19 First, they inserted an anti-CD19 CAR cassette containing 4-1BB and CD3ζ into the AAVS1 safe-harbor locus to demonstrate the feasibility of non-viral CAR-T-cells. Then, an innovative CAR-T-cell was developed by integrating the anti-CD19 CAR cassette into the PDCD1 locus (Fig. 1B). These non-viral anti-CD19 CAR-T-cells exhibited a superior ability to eradicate cancer cells both in vitro and in xenograft models. Moreover, a phase 1 clinical trial was performed to evaluate the safety and efficacy of non-viral PDCD1-integrated anti-CD19 CAR-T-cells in treating patients with relapsed/refractory aggressive B-cell non-Hodgkin lymphoma. In this study, 87.5% (7 out of 8) of patients achieved complete remission after 12 months. In addition, durable responses without serious adverse events were observed in all 8 patients. Interestingly, the results of single-cell sequencing in this study show that a high proportion of memory T-cells are present in the non-viral PDCD1 site-integrated anti-CD19 CAR-T-cell products. These analyses are consistent with the robust and long-lasting anti-tumor effect in xenografts. By analyzing the single-cell sequencing data, the superior clinical efficacy of non-viral PDCD1-integrated anti-CD19 CAR-T-cell products is due to the higher proportion of memory T-cells. This inspiring study shows that non-viral site-directed integrated CAR-T-cell products elicit excellent clinical safety and anti-tumor efficacy. The feasibility of non-viral site-directed integrated T-cell therapy in clinical applications was also demonstrated. This technological innovation lays a solid foundation for the development of more site-directed modified CAR-T therapies in the future. The success of this study sheds light on the establishment of a new CAR-T-cell technology platform with better clinical outcomes and fewer toxic side effects in the treatment of refractory relapsed B-cell lymphoma. Clinical results from more patients with longer follow-up will be necessary to reveal the long-term efficacy and persistence of the non-viral PDCD1 site-specific integration anti-CD19 CAR-T-cells. Further investigations are warranted to uncover the mechanisms underlying the formation of memory T-cells in non-viral PDCD1 site-integrated CAR-T-cells compared with viral CAR-T-cells. It will be interesting to examine downstream pathways in T-cells that are activated post viral transduction and CRISPR−Cas9 plus DNA electroporation. In addition, a recent study revealed that PD-1 checkpoint inhibition promotes the proliferation of CD62L+ precursors of exhausted T-cells (Tpex) in an MYB-dependent manner and maintains long-term responsiveness to immunotherapy.20 It is important to explore the MYB expression and proportion of CD62L+Tpex in PDCD1 site-integrated CAR-T-cells to further explain the mechanism of the higher memory T-cell proportion in PDCD1 site-integrated CAR-T-cells. In conclusion, this study demonstrates the safety and efficacy of non-viral PDCD1 site-integrated anti-CD19 CAR-T-cells and allows for the "killing of two birds with one stone" strategy by integrating a CAR cassette into a targeted gene locus for CAR expression and gene ablation. In the past few years, many researchers, including our team, have attempted to improve the efficacy of CAR-T-cells against solid tumors. To achieve this goal, it is pivotal to improve the persistence and maintain the cytotoxicity of CAR-T-cells in the immunosuppressive tumor microenvironment. Recently, we demonstrated that overexpression of constitutively active GP130 in CAR-T-cells results in a more robust anti-tumor capacity, better persistence and less GVHD (graft-versus-host disease) in solid tumor-derived xenograft models.21 We also showed that the expression of the Toll/interleukin-1 receptor (TIR) domain of Toll-like receptor 2 (TLR2)22 or the DAP10 cytoplasmic domain23 augments the expansion and anti-tumor effects of CAR-T-cells. Similar strategies can be used to develop integrated CAR cassettes against GPC3, MSLN, and PSCA to further improve the effector functions and persistence of CAR-T-cell products for treating solid tumors. This is the first article published by scientists from China in the field of CAR-T-cell therapy in Nature, reporting the first CAR-T-cell product with CAR cassette integration into the PDCD1 locus with robust preclinical and clinical results. This study presents a paradigm for collaboration between scientists from translational and clinical research in the field of immunocellular therapy. The future of CAR-T-cell technology holds bright prospects due to the fast-paced development of gene-editing techniques and the evolving understanding of the mechanisms regulating T-cell memory and exhaustion formation.