A Novel Platform to Manufacture High-Efficient "Off-the-Shelf" CAR-NK Products from Peripheral Blood Mononuclear Cells (PBMC)

Nature killer (NK) cells, which don't require the human leukocyte antigen (HLA) matching, were considered as a promising immune cell for adoptive immunotherapy. Chimeric antigen receptor (CAR) has been proved as a powerful weapon to enhance antitumor ability of NK cell, which makes CAR-NK therapy an attractive candidate for "off-the-shelf" products. However, to date, CAR-NK development still faces several issues including low transduction efficiency, large-scale preparation, and short-time persistence in vivo.

NK cells express a variety of signal-activating molecules, such as NKp44, NKp46, NKG2D, DAP10 and DAP12, which are the significant features of NK distinguish them from T cells. Thus, we speculated that CAR structure may be a critical factor in both tumoricidal function and persistence of CAR-NK. Therefore, we designed multiple CAR structures based on the signal pathways of NK (Figure 1A top). We engaged the transmembrane domain of NKp44, which could make CAR form a tripolymer with NK active molecule DAP12, and intracellular domains of 4-1BB and DAP10, which could either activate NF-κB or MAPK pathway, respectively (Figure 1A bottom). The antitumor efficacy of CAR-NK cells with the different CAR were evaluated in NCG model. Interestingly, CD19 scfv-8h-p44TM-BB-DAP10 (The scfv targeting CD19 was attached to the hinge of CD8, and fused to the NKp44 (transmembrane domain), 4-1BB and DAP10 (cytoplasmic) domains, CAR-4) showed the superior tumoricidal function (Figure 1B, C) and exhibited an outstanding persistence in NCG model without exogenous cytokines (Figure 1D). Our data suggested that natural immune receptor-like CAR structure provides a new perspective for CAR-NK optimization.

To improve the transduction efficiency of CAR gene in PBMC derived NK cells (PBNK), we developed a lentiviral based NK.affi-LV vector. Our data showed that the positive rate of CAR could reach above 80% when transduced with NK.affi-LV (MOI=3) (Figure 1E) and kept stable in vitro and in vivo. Moreover, we confirmed that the vector could efficiently delivery a large exogenous gene (up to 4, 300 bp, a polycistron linked by 2A self-cleaving peptides). Apart from PBNK cells, highly efficient transduction of other lymphocytes, including γδ-T and B cells, was also achieved. Over 50% of cells were transduced at MOI of 3. These results indicate that the NK.affi-LV vectors have a broader application potency for adoptive immunotherapy. Additionally, a feeders-free culture procedure was established for producing CAR-NK from PBNK. The proliferation curve of CAR-NK from 3 health donors were concluded at Figure 1F, and the average amplification was 8250 (range, 3,000-15,000) after 21 days of culture. Thus, with this procedure, we are able to produce over 8e12 cells per batch when initialed from 1e9 PBNK, which should be sufficient for hundreds of patients.

Together, our data give some new solutions to overcome the barriers of the clinical application of CAR-NK, and bring a new hope for the future "Off-the-shelf" CAR-NK production.

Huang:Chongqing Precision Biotech Co., Ltd: Current Employment. Chen:Chongqing Precision Biotech Co., Ltd: Current Employment. Shen:Chongqing Precision Biotech Co., Ltd: Current Employment. Xu:Chongqing Precision Biotech Co., Ltd: Current Employment. Qi:Chongqing Precision Biotech Co., Ltd: Current Employment. Yang:Chongqing Precision Biotech Co., Ltd: Current Employment, Research Funding. Zhang:Chongqing Precision Biotech Co., Ltd: Current Employment. Chen:Chongqing Precision Biotech Co., Ltd: Current Employment. Zhao:Chongqing Precision Biotech Co., Ltd: Current Employment. Hong:Chongqing Precision Biotech Co., Ltd: Current Employment. Hu:Chongqing Precision Biotech Co., Ltd: Current Employment. Jiao:Chongqing Precision Biotech Co., Ltd: Current Employment. Zhu:Chongqing Precision Biotech Co., Ltd: Current Employment. Wang:Chongqing Precision Biotech Co., Ltd: Current Employment. Zhao:Chongqing Precision Biotech Co., Ltd: Current Employment. Huang:Chongqing Precision Biotech Co., Ltd: Current Employment. Qin:Chongqing Precision Biotech Co., Ltd: Current Employment. Yao:Chongqing Precision Biotech Co., Ltd: Current Employment. Dai:Chongqing Precision Biotech Co., Ltd: Current Employment. Li:Chongqing Precision Biotech Co., Ltd: Current Employment. Wang:Chongqing Precision Biotech Co., Ltd: Current Employment. Qian:Chongqing Precision Biotech Co., Ltd: Current Employment.