Background Immune checkpoint inhibitors have shown dramatic activity transforming clinical oncology.¹ Yet, their activity is limited to a subset of highly sensitive tumors (e.g., melanoma). Even then response is observed in only 50% of patients.²

Nitric Oxide (NO) is a signaling molecule in multiple diseases, including cancer and has been shown to activate anti-tumor immune responses.³ Previously, we reported that treatment of CT26 tumor-bearing mice with high-concentration NO (UNO) stimulated anti-tumor immune responses leading to the rejection of a secondarily-induced tumor. More specifically, a significant increase of tumor-infiltrating T-cells and blood and spleen B and T-cells were observed 14-21 days post-UNO treatment.

In this study we investigated the ability of UNO to improve the efficacy of anti-PD-1 antibody.

Methods Day zero, 5.0 X 10⁶ CT26 cells were injected to the right flank of Balb/c mice (n=15-16 per group). Day six, CT26 cells were injected to the contralateral flank and anti-PD1 injections (5mg/kg mouse, q2d, x 5) commenced.

Day eight, tumors (average size 71.9±37.2mm³) were treated intratumorally with UNO (50,000 ppm, 5 or 10 minutes, flow rate ~0.2 liter per minute). Post-treatment tumor volume and survival were monitored thereafter.

Results

1. Complete regression of the primary tumor occurred in 9/15 (60%) of mice treated with combination 10-minute NO and anti-PD-1, post-treatment day 26. This compared to 4/16 (25%) of controls treated with anti-PD-1 alone (p=0.13) and 0/15 (0%) treated with UNO alone (p=0.0027).

2. Survival was drastically increased in the 10-minute UNO/anti-PD-1 combination arm compared to anti-PD-1 alone (p<0.05), post-treatment day 32.

3. Secondary, contralateral flank tumor take in the anti-PD-1 alone arm was 21.4% but reduced by 38% to 13.3% in the 10-min combination arm and reduced by 67% to 7.14% in the 5-min combination arm, post-treatment day 19.

4. Survival was significantly improved for both the 5- and 10-minute combination arms compared to the 5- and 10-minute UNO controls (p=0.02 and p<0.0001, respectively).

Conclusions Combination of UNO with anti-PD-1 significantly improved outcomes compared with UNO or anti-PD-1 alone. A strong possibility is that high-concentration NO assists the immune system in overcoming anti-PD-1 resistance. Thus, the combination of high-concentration NO and immune checkpoint inhibitors such as anti-PD-1 can be a breakthrough therapy with important clinical implications.

References

1. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012; 12(4):252–264. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856023/

2. Schoenfeld AJ, Hellmann MD. Acquired resistance to immune checkpoint inhibitors. Cancer Cell 2020; 37(4):443–455. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7182070/

3. Khan FH, Dervan E, Bhattacharyya DD, McAuliffe JD, Miranda KM, Glynn SA. The role of nitric oxide in cancer: master regulator or not? Int J Mol Sci 2020; 21(24):9393–9423. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763974/