Abstract P2-04-17: Directing NK cells to Trop-2-expressing breast and other cancers, with chimeric antigen receptors
Authors: C-H Chang, D Liu, DM Goldenberg
Journal: Cancer Research (2017)

Abstract Adoptive immunotherapy with chimeric antigen receptor (CAR)-engineered T or NK cells is in active clinical and preclinical development, with recent interest increasingly focused on advancing the availability of off-the-shelf allogeneic clonal NK cell lines capable of targeting tumors with appropriately designed CARs. Among the human NK cell lines established, the IL-2-dependent NK-92 is the most studied and has been transduced with human IL-2 to generate NK-92MI, which exhibits cytotoxicity similar to NK-92, and is independent of IL-2 for its cytotoxicity, viability and proliferation. We report the assembly of E1.BB.3z-92MI, a novel CAR-NK for potential therapy of diverse Trop-2-expressing breast, lung, bladder, ovarian, and other cancers. The Trop-2-specific CAR, referred to as hRS7-CAR, consists of the CD8α signal peptide, the VK and VH of hRS7 (a humanized anti-human Trop-2 mAb used in the antibody-drug conjugate, IMMU-132), the hinge region and transmembrane domain of CD8α, the intracellular domains (ICD) of 4-1BB, and the ICD of CD3ξ. The construct of hRS7-CAR is introduced into NK-92MI either by electroporation of the mRNA synthesized in vitro, or by inoculation with the lentiviral particles harvested from the 48-h supernatants of Lenti-X 293T cells transduced with pLVX-puro-hRS7-CAR. The presence of hRS7-CAR in E1.BB.3z-92MI was probed by Western blot using an HRP conjugate of a rat anti-id antibody against hRS7, which detected a distinct band of about 50 kDa from the cell lysates of NK-92MI transfected with hRS7-CAR mRNA, but not from the mock-transfected NK-92MI. As the calculated molecular weight of hRS7-CAR is about 51 kDa, these results support that hRS7-CAR is produced in NK-92MI cells transfected with hRS7-CAR mRNA. Additional evidence by flow cytometry shows about 41% of NK-92MI cells transfected with hRS7-CAR mRNA are alive at the time of analysis and 25% of this subpopulation expresses hRS7. The cytotoxicity of E1.BB.3z-92MI was evaluated against the Trop-2-expressing, human breast cancer cell line, HCC1806, in 96-well plates (4,500 cells/well) at 3 different effector-to-target (E/T) ratios (1:1, 2:1, or 4:1). As shown in the Table, the results of the MTS assay indicate that NK-92MI cells transfected with hRS7-CAR mRNA significantly killed more HCC1806 cells at the E/T ratio of 2:1 or 4:1, in comparison to mock-transfected NK-92MI. Another study performed by flow cytometry using dye-labeled HCC1806 at the E/T ratio of 3:1 indicates that the specific lysis of HCC1806 cells by NK-92MI cells transfected with hRS7-CAR mRNA was about 2-fold higher than that observed for mock-transfected NK-92MI. Finally, the initial results obtained for NK-92MI cells transduced with the lentiviral pLVX-puro-hRS7-CAR show about 70% viability and greater than 30-fold higher MFI in the live population than the control transduced with pLVX-puro or not transduced. Together, these results demonstrate the feasibility of developing E1.BB.3z-92MI as a novel CAR-NK for targeting Trop-2-expressing cancers, including breast cancer. Table. Cytotoxicity of Trop-2-targeting NKcellsE/TViability (mean ± SD)Mock vs. hRS7-CAR MockhRS7-CARΔP-value1:188.5 ± 9.980.3 ± 11.18.20.392:189.9 ± 10.667.4 ± 4.422.50.0274:184.8 ± 4.072.4± 2.512.40.010 Citation Format: Chang C-H, Liu D, Goldenberg DM. Directing NK cells to Trop-2-expressing breast and other cancers, with chimeric antigen receptors [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-17.