NK-92MI Cells Engineered with Anti-claudin-6 Chimeric Antigen Receptors in Immunotherapy for Ovarian Cancer

Background: The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR-NK cell efficacy. Claudin-6 (CLDN6) has been reported to be overexpressed in ovarian cancer and may be an attractive target for CAR-NK cells immunotherapy. However, the feasibility of using anti-CLDN6 CAR-NK cells to treat ovarian cancer remains to be explored. Methods: CLDN6 expression in primary human ovarian cancer, normal tissues and cell lines were detected by immunohistochemistry and western blot. Two types of third-generation CAR NK-92MI cells targeting CLDN6, CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) and CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB) were constructed by lentivirus transfection, sorted by flow cytometry and verified by western blot and qPCR. OVCAR-3, SK-OV-3, A2780, Hey and PC-3 cells expressing the GFP and luciferase genes were transduced. Subcutaneous and intraperitoneal tumor models were established via NSG mice. The ability of CLDN6-CAR NK cells to kill CLDN6-positive ovarian cancer cells were evaluated in vitro and in vivo by live cell imaging and bioluminescence imaging. Results: Both CLDN6-CAR1 and CLDN6-CAR2 NK-92MI cells could specifically killed CLDN6-positive ovarian cancer cells (OVCAR-3, SK-OV-3, A2780 and Hey), rather than CLDN6 negative cell (PC-3), in vitro. CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) exhibited stronger cytotoxicity than CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB). Furthermore, CLDN6-CAR1 NK cells could effectively eliminate ovarian cancer cells in subcutaneous and intraperitoneal tumor models. More importantly, CAR-NK cells combined with immune checkpoint inhibitors, anti-PD-L1, could synergistically enhance the antitumor efficacy of CLDN6-targeted CAR-NK cells. Conclusions: These results indicate that CLDN6-CAR NK cells possess strong antitumor activity and represent a promising immunotherapeutic modality for ovarian cancer.


Figure S1 .
Figure S1.Construction and characterization of CLDN6-specific CARs.(A) Schematic representation of CD19-and CLDN6-targeted CARs.CD19-CAR cells were used as a control.

Figure S2 .
Figure S2.Structural verification of lentiviral vectors and flow cytometry sorting (FCMS).(A)After digestion of the CAR lentiviral vector plasmid by restriction endonucleases PmeI and SpeI, the CAR gene was detected by agarose gel electrophoresis (AGE).(B)Flow cytometry sorting (FCMS).eGFP served as a marker.

Figure S3 .
Figure S3.The morphology and GFP expression of CAR-NK92MI cells were observed by confocal microscopy.Scale bars, 10 μm.

Figure S4 .
Figure S4.CD107a detection on the CAR-NK92MI cell surface.Detection of the cell surface activation marker CD107a on CAR-NK cells after stimulation with SK-OV-3 and OVCAR-3 cells at an E:T ratio of 1:1 or no stimulation.Flow cytometry using Abs specific to human CD107a conjugated with APC fluorochromeswas performed.

Figure S5 .
Figure S5.Representative time-lapse images of CLDN6-CAR1 NK92MI serial killing (yellow lines) and SK-OV-3 (red lines).In the same field of view indicated in Figure 3, the CAR-NK cells killed one target cell and then moved on to kill another.See also Figure 3.

Figure S6 .
Figure S6.Analysis of the interaction between CAR-NK92MI cells and PC-3 (CLDN6-negative) cytotoxicity based on live-cell imaging (A) Representative time-lapse images of the interaction between different CAR-NK cells (yellow lines) and PC-3 cells (white lines).(B-C) Time for killing (B) and the number of NK cells engaging with a cancer cell (C).Error bars denote SD. ns, not significant, Mann-Whitney test adjusted p value.A time display on the left upper corner indicated in (A) represents the changed time post CAR-NK cell coculture with target cells.

Figure S7 .
Figure S7.PD-L1 expression in all ovarian cancer cell lines and NK-92MI cells was detected by western blot (A and B) or flow cytometry (C).A549 cells were used as a negative control, and H358 cells were used as a positive control.

Figure S8 .
Figure S8.PD-1/PD-L1 was detected by western blotting and flow cytometry.(A and B) SK-OV-3 and OVCAR-3 cells were treated with different concentrations of IFN-γ (0, 5, 10, 30, 50, and 100 ng/ml), and then the expression of PD-L1 was detected by western blot (A) and analyzed by ImageJ software (B).Error bars denote SD. (C) CD19-CAR, CLDN6-CAR1, and CLDN6-CAR2 NK cells treated with IFN-γ were examined by flow cytometry with Abs specific to human PD-L1 conjugated to APC fluorochromes.(D) PD-1 detection in the resting state of NK and CLDN6-CAR1 NK cells with flow cytometry.(E) The PD-1 expression level of CLDN6-CAR1 NK cells stimulated by Hey was detected by anti-PD-1 conjugated with APC fluorochromes.

Figure S9 .
Figure S9.Activation and cytokine secretion of CLDN6-CAR1 NK92MI cells combined with anti-PD-L1.(A) Detection of the cell surface activation marker CD107a on CLDN6-CAR1 NK cells combined with anti-PD-L1 via FACS.(B) The CD107a+ percentage of NK and CAR-NK cells and fluorescence intensity were analyzed.Error bars denote SD. *** p < 0.001, one-way ANOVA with Holm-Sidak test adjusted p value.(C) The secretion of IFN-γ, granzyme B, and perforin by CAR-NK cells combined with anti-PD-L1 therapy was determined by enzyme-linked immunosorbent assay (ELISA).Error bars denote SD. ns, not significant, * p < 0.05, two-tailed Student's t test adjusted p value.

Table 1 . Determination of lentivirus titers with different CAR molecules
. Unit (TU/ml).