Targeting CD70 using CAR NK cells to enhance NK cells cytolytic effect against osteosarcoma
Introduction
Osteosarcoma (OS) is the most common primary malignant bone tumor in pediatric patients primarily affecting adolescents. Cure rates for OS have not improved in over thirty years, and no new, effective therapies have been identified for metastatic disease. Natural killer (NK) cells have become an attractive tool for cancer immunotherapy. However, therapeutic efficacy has been limited in solid tumors. Thus, direct tumor targeting introducing a specific chimeric antigen receptor (CAR) may improve efficacy. Emerging knowledge suggests CD70 to be a viable target candidate in OS as expression has been correlated with tumor escape from immune surveillance and was shown to be higher in OS lung metastases. Therefore, the primary objective of this study is to assess whether using CD70 CAR-NK cells we can enhance trafficking, homing, and proliferation of NK cells and thus therapeutic efficacy against OS.
Methods
Surface expression of CD70 on human OS cell lines and normal osteoblasts was determined by flow cytometry. Western blotting was used to confirm CD70 expression by OS cell lines. Immunohistochemistry was used to analyze CD70 expression on OS patient derived xenograft tumor samples and tumor tissues from human OS mouse models. Publicly available databases were analyzed to determine CD70 mRNA expression on OS patient samples and OS cell lines compared to normal tissues. RNAseq was used to analyze CD70 mRNA expression of multiple OS cell lines. CD70 CAR constructs were sequenced, verified, and transduced into NK cells. CD70 CAR NK and control NK cells cytolytic activity against OS cells was evaluated by IncuCyte. Trafficking and cytotoxicity of CD70 CAR NK cells was analyzed in a 3D OS spheroid model. In vitro cytokine release was measured upon CD70 CAR NK/NK cells exposure to OS cells (IsoPlexis).
Results
Flow cytometry showed variable expression of CD70 on OS cells and no expression on human osteoblasts. Western blot confirmed these results on the OS cell lines tested. CD70 expression as determined by IHC, was present on tissues from OS PDX models and human OS mouse models but was found to be heterogeneous. RNAseq consistently showed increased CD70 expression in patient samples and OS cell lines compared to normal bone. There was an increase in CD70 CAR NK percent lysis against OS cells at the highest effector:target ratios compared to control, non-transfected (NT) NK cells. CD70 CAR NK cells were able to effectively traffic to, penetrate, and exhibit cytotoxicity in a 3D OS spheroid model. We found a predominant release in the chemoattractive/stimulatory cytokines upon OS cells exposure to CD70 CAR NK cells.
Conclusion
CD70 has immunotherapy potential against OS. CD70 CAR NK cells have increased cytolytic activity against OS cells in vitro. The cytolytic activity may be influenced by the release of specific cytokines.