Aim for a million targets, Strike with a giant hammer
Naval Medical University CHINA

Experiments

Experiment

Abstract

We firstly verified the specific killing effect of CAR-NK92 cells. Through modeling we rejected the plan to express diverse CARs on a single NK92 cell, which led us to express a CAR library on a group of NK92 cells to make up a CAR-NK92 library. We then verified the feasibility of the kill switch circuit and the growth dynamics of logic-gated NK-92 cells according to antigens. Together with statistics we gathered from modeling, we can preliminarily demonstrate that our project is feasible and promising in tumor therapy, especially in addressing tumor heterogeneity.

Our modeling work is detailed in MODELS.

Experiments

1.verification of the specific killing effect of CAR-NK92 cells

Material：

①Engineered cell: NK-92-CTX and NK-92-TTZ

②Target cell: MCF-7 cells (EGFR-HER2-), MCF-7 EGFR+ cells and MCF-7 HER2+ cells

Method:

fluorescent target array killing assays

Result:

Figure 1 The specific killing effect of CAR-NK92 cells in response to tumour cells. The killing effect of CAR-NK92 and control cells against MCF-7 cells was assessed by fluorescent target array killing assays at the indicated E:T ratios. Data are presented as mean ± s.d. of five independent biological replicates.

NK-92 cells with no CAR performed no killing effect and NK-92-CTX or NK-92-TTZ had specific killing effect only when they encountered corresponding antigens, which verified the specific killing effect of CAR-NK92 cells.

2.verification of the feasibility of the kill switch circuit

Material:

①Engineered cell: NK-92-CTX and NK-92-TTZ, engineered with kill switch circuit

②Target cell: MCF-7 cells (EGFR-HER2-), MCF-7 EGFR+ cells and MCF-7 HER2+ cells

Method:

co-culture experiment

Result:

Figure 2 Cells were first stimulated with different MCF-7 cells or unstimulated for 24 or 72 h. The addition of 10 nM AP1903 to the cultures induced apoptosis/necrosis of the CAR-NK92 cells, as assessed by annexin-V staining in four independent experiments. Data are presented as mean ± s.d. *P< 0.0001 compared with no AP1903, from two-way ANOVA followed by Bonferroni post-test.

Our data showed that AP1903 had no effect on the viability of NK-92 cells but caused apoptosis/necrosis of the engineered NK92 cells with no stimulatory cells. However, after 24h of stimulation with specific antigens, the engineered NK92 cells were notably affected by the reduced pro-apoptotic effect of AP1903, and both the EGFR-specific and HER2-specific CAR-NK92 cells were resistant to AP1903 after antigen stimulation for 72h.

3.verification of the growth dynamics of logic-gated NK-92 cells according to antigens

While AP1093 extensively induces apoptosis, we want to ensure that CAR-NK92 cells that recognize the corresponding antigen, could continue to proliferate and enrich without interference. To achieve this goal, we introduced the suicide repressor section.

Material:

①Engineered cell: a group of NK-92 cells engineered with 10 different kinds of CARs and the kill switch circuit, making up a 10-CAR-NK92 library.

The origin of 10 CARs: cetuximab, trastuzumab, CH65, 9.8B, 2F5, 7D11, 8D6, omalizumab, TE33, and R10

②Target cell: MCF-7 cells (EGFR-HER2-), MCF-7 EGFR+ cells and MCF-7 HER2+ cells

Method:

co-culture experiment

Result:

Figure 3 Growth dynamics of the CAR-NK92 cell library in the different co-culture methods. Frequencies of αEGFR and αHER2 CAR-NK92 cells were analysed before the addition of fresh target cells.

Repeat stimulations with MCF-7 EGFR cells or MCF-7 HER2 cells resulted in no significant change in the CAR composition of the library co-cultured with either MCF-7 cells or antigen-positive MCF-7 derivatives without AP1903 treatment, while enrichment of NK-92 CTX and NK-92 TTZ cells was observed after AP1903 treatment in the presence of specific MCF-7 derivatives. These data suggest that the gene circuit was effective in the cell-based CAR screening method and adding AP1903 was essential for the enrichment of CAR NK-92 cells.

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