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311 PD-1 blockade protein coupled in 4th generation armored CAR-T cells enhances cytotoxicity effect with in vitro re-challenge system
  1. Zhifeng Zhao,
  2. Jinyan Ma,
  3. Tiantian Wang,
  4. Ka Fung Noi,
  5. Jie Xu and
  6. Qingyang Gu
  1. WXi ApTec, Nantong, China


Background Chimeric antigen receptor (CAR)-T cells are genetically engineered T cells expressing a receptor on their surface to recognize tumor-associated antigens (TAA). CAR-T cell therapy has been coined as a ‘living drug’ and have demonstrated remarkable success with hematological malignancies. However, limited headway has been made with solid tumors due to various challenges, including recognition of tumor-specific antigens, trafficking and penetration, and survival within an immunosuppressive tumor microenvironment (TME). Some overexpression of immunosuppressive cytokines/proteins, such as TGF-β and PD-L1, downregulates cytotoxic CD8+ T cells and reduces the efficacy of CAR-T cell therapy. Immune checkpoint inhibitors (ICIs), such as anti-PD-1/PD-L1 and anti-CTLA-4 antibodies have gathered immense attention due to their efficacy across multiple solid malignancies. Therefore, a combination of CAR-T and ICIs could be a viable strategy to overcome the unfavorable TME. Here, we have established a 4th generation, armored CAR-T coupled with a PD-1 blockade protein to enhance the anti-exhaustion effect of CAR-T cells.

Methods CAR-T cells were generated from primary human T cells which were isolated from human PBMC using a pan T cell isolation kit. Lentiviral particles containing the MSLN CAR gene with/without PD-1 transgene were generated in 293T cells. T cells were stimulated by CD3/CD28 beads for 2 days, followed by the addition of CAR lentiviral particles for an additional 24 hours, then further proliferated for 6 days. Cells were collected for surface marker analysis after the second stimulation or co-cultured with luciferase-transduced HCT-116 cells for re-challenge cytotoxicity assay.

Results • 4th generation CAR-T cells have reduced surface PD-1 available for binding, compared to 3rd generation CAR-T cells and untransduced T cells.

• CAR-negative 4th generation T cells also have reduced available PD-1 binding sites.

• 4th generation CAR-T cell retains >70% cytotoxicity effect, while 3rd and 2nd generation CAR-T cells have markedly reduced cytotoxicity effect after two re-challenges with HCT-116-luc cells.

Conclusions PD-1 immune checkpoint blockade enhances and prolongs CAR-T cells cytotoxic effect in an in vitro re-challenge assay. This research indicates the possibility of adding an immune checkpoint inhibitor transgene to the existing CAR gene to enhance CAR-T cytotoxicity.

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