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819 Intratumoral administration of high-concentration nitric oxide and anti PD-1 treatment leads to higher tumor regression rates and prolonged survival in CT26 tumor-bearing mice
  1. Hila Confino1,
  2. David Greenberg1,
  3. Selena Chaisson1,
  4. Jedidiah Mercer Monson1,
  5. Steve Lisi2,
  6. Amir Avniel1,
  7. Ido Wolf3 and
  8. Yana Epshtein1
  1. 1Beyond Cancer, Beyond Air Inc., Rehovot, Israel
  2. 2Beyond Cancer, Beyond Air Inc., Garden City, NY, USA
  3. 3Tel Aviv Sourasky Medical Center, Tel Aviv Yafo, Israel


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.


  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.


  1. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012; 12(4):252–264.

  2. Schoenfeld AJ, Hellmann MD. Acquired resistance to immune checkpoint inhibitors. Cancer Cell 2020; 37(4):443–455.

  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.

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