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Abstract Background The combination of radiotherapy and immunotherapy (immunoradiotherapy) has been increasingly used for treating a wide range of cancers. However, some tumors are resistant to immunoradiotherapy. We have previously shown that MER proto-oncogene tyrosine kinase (MerTK) expressed on macrophages mediates resistance to immunoradiotherapy. We therefore sought to develop therapeutics that can mitigate the negative impact of MerTK. We designed and developed a MerTK specific antisense oligonucleotide (ASO) and characterized its effects on eliciting an anti-tumor immune response in mice. Methods 344SQR cells were injected into the right legs on day 0 and the left legs on day 4 of 8-12 weeks old female 129sv/ev mice to establish primary and secondary tumors, respectively. Radiation at a dose of 12 Gy was given to the primary tumors on days 8, 9, and 10. Mice received either anti-PD-1, anti-CTLA-4 or/and MerTK ASO starting from day 1 post tumor implantation. The composition of the tumor microenvironment and the level of MerTK on macrophages in the tumor were evaluted by flow cytometry. The expression of immune-related genes was investigated with NanoString. Lastly, the impact of MerTK ASO on the structure of the eye was histologically evaluated. Results Remarkably, the addition of MerTK ASO to XRT+anti-PD1 and XRT+anti-CTLA4 profoundly slowed the growth of both primary and secondary tumors and significantly extended survival. The ASO significantly reduced the expression of MerTK in tumor-associated macrophages (TAMs), reprograming their phenotype from M2 to M1. In addition, MerTK ASO increased the percentage of Granzyme B+ CD8+ T cells in the secondary tumors when combined with XRT+anti-CTLA4. NanoString results demonstrated that the MerTK ASO favorably modulated immune-related genes for promoting antitumor immune response in secondary tumors. Importantly, histological analysis of eye tissues demonstrated that unlike small molecules, the MerTK ASO did not produce any detectable pathology in the eyes. Conclusions The MerTK ASO can significantly downregulate the expression of MerTK on TAMs, thereby promoting antitumor immune response. The combination of MerTK ASO with immunoradiotherapy can safely and significantly slow tumor growth and improve survival. Graphical Abstract

Abstract Background While improvements in immunoradiotherapy have significantly improved outcomes for cancer patients, this treatment approach has nevertheless proven ineffective at controlling the majority of malignancies. One of the mechanisms of resistance to immunoradiotherapy is that immune cells may be suppressed via the myriad of different immune checkpoint receptors. Therefore, simultaneous blockade of multiple immune checkpoint receptors may enhance the treatment efficacy of immunoradiotherapy. Methods We combined NBTXR3-enhanced localized radiation with the simultaneous blockade of three different checkpoint receptors: PD1, LAG3, and TIGIT, and tested the treatment efficacy in an anti-PD1-resistant lung cancer model in mice. 129 Sv/Ev mice were inoculated with fifty thousand αPD1-resistant 344SQR cells in the right leg on day 0 to establish primary tumors and with the same number of cells in the left leg on day 4 to establish the secondary tumors. NBTXR3 was intratumorally injected into the primary tumors on day 7, which were irradiated with 12 Gy on days 8, 9, and 10. Anti-PD1 (200 µg), αLAG3 (200 µg), and αTIGIT (200 µg) were given to mice by intraperitoneal injections on days 5, 8, 11, 14, 21, 28, 35, and 42. Results This nanoparticle-mediated combination therapy is effective at controlling the growth of irradiated and distant unirradiated tumors, enhancing animal survival, and is the only one that led to the destruction of both tumors in approximately 30% of the treated mice. Corresponding with this improved response is robust activation of the immune response, as manifested by increased numbers of immune cells along with a transcriptional signature of both innate and adaptive immunity within the tumor. Furthermore, mice treated with this combinatorial therapy display immunological memory response when rechallenged by the same cancer cells, preventing tumor engraftment. Conclusion Our results strongly attest to the efficacy and validity of combining nanoparticle-enhanced radiotherapy and simultaneous blockade of multiple immune checkpoint receptors and provide a pre-clinical rationale for investigating its translation into human patients. Graphical Abstract

The efficacy of immunoradiotherapy consisting of radiation therapy and immune checkpoint blockade relies on effectively promoting the systemic antitumor immune response’s activation while simultaneously reducing local factors favoring immune suppression. We previously demonstrated that NBTXR3, a nanoparticle radioenhancer, significantly improved immune responses in a murine anti-PD1-resistant metastatic lung cancer model. We hypothesize that radioactivated-NBTXR3 addition to anti-PD1 and a second-generation anti-CTLA4 could improve treatment effectiveness. To test this hypothesis, we inoculated mice with 344SQR cells in the right and left legs to establish primary and secondary tumors. The primary tumors were intratumorally injected with NBTXR3 nanoparticles on day 7, followed by three fractions of 12 Gy radiation on days 8, 9, and 10. The secondary tumors received two fractions of 1Gy radiation on days 13 and 14. Multiple rounds of anti-PD1, anti-CTLA4 or nonfucosylated anti-CTLA4 were given to the mice. Immune profiling of the tumors revealed that the combination of NBTXR3 with immunoradiotherapy significantly upregulated the activities of a wide range of antitumor immune pathways and reduced the abundance of regulatory suppressor T cells. This combination effectively eradicated the primary and secondary tumors and increased animal survival to 75%. Remarkably, previously treated with NBTXR3-containing treatment, the survivor mice exhibited a long-lasting antitumor memory immune response. This data provides compelling evidence of the efficacy of NBTXR3 to synergize with the immunoradiotherapy approach when combined with an anti-PD1 and multiple checkpoints such as a second generation anti-CTLA4 and show the potential for clinical uses of antitumor immunomodulatory effects of NBTXR3.