NBTXR3 generates long term immune memory

Summary

Purpose/Objectives: Although treatment of high-dose (HD) radiation (XRT) and NBTXR3 (R3) on primary tumors in combination with systemic anti-PD1 was able to significantly improve abscopal effect in 344SQR murine metastatic lung cancer, most of the mice eventually expired due to the growth of metastatic tumors. Therefore, we studied the effects of R3 injection into primary tumors plus high-dose radiation on primary tumor and low-dose raditionon metastatic tumor plus dual-agent immunotherapy (IT) of anti-PD1 and anti-CTLA-4 to achive complete control of tumor growth at both the primary and the metastatic tumors in mice.

Materials/Methods: Five groups of 8 mice each were inoculated subcutaneously with 5×104 anti-PD1-resistant 344SQR murine lung cancer cells in each hind leg, 3 days apart, to establish ‘primary’ (right) and ‘metastatic’ (left) tumors. All mice in treatment groups received intraperitoneal anti-PD1 and anti-CTLA-4 on days 4, 7, 10, and 13, and contining anti-PD1 treatment on days 20, 27, 34, 41, and 49 and 12-Gy high-dose (HD) XRT to the primary tumors on days 7, 8 and 9. Primary tumors in groups 3 and 5 also received intratumoral R3 on day 6. Metastatic tumors in groups 4 and 5 were also irradiated with 1-Gy low-dose (LD) XRT on days 12 and 13 (Radscopal™ approach). Experimetal groups were designated as 1=Control, 2=HD+IT, 3=R3+HD+IT, 4=HD+LD+IT, and 5=R3+HD+LD+IT. On day 178, the right flank of the survived mice in group 5 was rechallenged with 5×104 344SQR cells and the tumor growth was monitored.

Results: All the mice in groups 1, 2, 3, 4 expired due to the growth of either the primary tumor or the metastatic tumor by day 36. Both the primary and the metastatic tumors in 4 mice of group 5 were completely eliminated. No tumor growth was observed in the 4 survived mice which was rechallenged with 344SQR cells. It was also found that the mice in group 5 had significantly fewer spontaneous lung metastases than the mice in any other groups. Nanostring data for the metastatic tumor collected on day 19 demonstrated that all the treatments group had significant upregulation of major anti-tumor immune pathways than the control. In addition, R3 nanoparticle exhibited stronger immune activation in mice than the ones without it. The flow cytometry data for the metastatic tumors collected on day 16 demonstrated that only the mice in group 3 and 5 showed significantly more CD8+ T cell infiltration in the metastatic tumor than the control group. Both flow cytometry and Nanostring data showed that only the mice received R3+HD+LD+IT treatment had significantly higher CD8+ Tcell/Treg cell ratio than the control group.

Conclusions: The combination of R3+HD+LD+IT could effectively eliminate the growth of both the primary and the metastasized tumors, significantly extend the survival of the treated mice, and create long-term immune memory against tumor cells. The combination therapy was able to contribute the immune-mediated control of the metastasized tumor at both genetic and cellular levels.