Summary

Purpose: Despite tremendous results achieved by immune checkpoint inhibitors, most patients are not responders, mainly because of the lack of a pre-existing anti-tumor immune response. Thus, solutions to efficiently prime this immune response are currently under intensive investigations. Radiotherapy elicits cancer cell death, generating an antitumor-specific T cell response, turning tumors in personalized in situ vaccines, with potentially systemic effects (abscopal effect). Nonetheless, clinical evidence of sustained anti-tumor immunity as abscopal effect are rare.

Methods: Hafnium oxide nanoparticles (NBTXR3) have been designed to increase energy dose deposit within cancer cells. We examined the effect of radiotherapy-activated NBTXR3 on anti-tumor immune response activation and abscopal effect production using a mouse colorectal cancer model.

Results: We demonstrate that radiotherapy-activated NBTXR3 kill more cancer cells than radiotherapy alone, significantly increase immune cell infiltrates both in treated and in untreated distant tumors, generating an abscopal effect dependent on CD8+ lymphocyte T cells.

Conclusion: These data show that radiotherapy-activated NBTXR3 could increase local and distant tumor control through immune system priming. Our results may have important implications for immunotherapeutic agent combination with radiotherapy.

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.

Summary

For decades, radiotherapy (RT) has been a cornerstone of cancer treatment. Currently, approximately 50% of cancer patients will be treated with RT. Beyond the ability of RT to produce free radicals and to generate single and double-strand breaks in DNA, triggering cell death, preclinical and clinical studies have demonstrated that RT can have immunomodulatory effects. For example, RT can stimulate MHC class I expression on cancer cells, induce immunogenic cell death (ICD), and activate expression of various pro- and anti-inflammatory cytokines and adhesion molecules, allowing recruitment and activation of both innate and adaptive immune cells into the tumor. Unfortunately, RT rarely produces a sustained anti-tumor response as immune escape frequently occurs with tumor recurrence. Moreover, the so-called ‘abscopal effect’ which corresponds to reduction of metastatic burden outside the irradiated area is rarely observed after RT. Finally, the maximum dose of irradiation is limited because of toxicity to surrounding healthy tissues.

The high electron density of functionalized hafnium oxide nanoparticles (NBTXR3) allows a high probability of interaction with incoming ionizing radiation, increasing energy dose deposit within cells. We have previously reported in nonclinical studies the ability of RT-activated NBTXR3 (NBTXR3+RT) to increase cancer cell destruction as well as better control of treated tumor growth through this physical mode of action leading, compared to RT alone. Furthermore, NBTXR3+RT demonstrated clinically meaningful benefit for patients with locally advanced Soft Tissue Sarcoma compared to RT alone, in the randomized controlled phase II/III Act.in.Sarc study (NCT02379845).

To explore the impact of NBTXR3+RT on the anti-tumor immune response, we used CT26 mouse colorectal cancer cells to perform a series of abscopal assays in immunocompetent mice. We showed that NBTXR3+RT can generate a significant abscopal effect along with a substantial increase of CD8+ T cell infiltrates both in treated and untreated tumors, compared to RT alone. We showed that this distant effect was fully dependent on CD8+ T cells, as their depletion completely abolished the abscopal effect. To better understand how NBTXR3+RT treatment could generate this abscopal effect, we compared the TCR repertoire of treated and untreated tumors for the different conditions. This analysis revealed that NBTXR3+RT was able to broaden clonal diversity in both treated and untreated tumors, compared to RT alone. This indicates that NBTXR3+RT has the ability to transform the tumor into a in situ vaccine more efficiently than RT alone and could have important implications for the use of NBTXR3+RT in combination with immunotherapy.

Summary

Recent results of a phase II/III in locally advanced Soft Tissue Sarcoma patients demonstrated clinical benefits of intratumorally injected HfO2-NP activated by radiotherapy compared to radiotherapy alone, validating their first-in-class mode of action. In addition, animal studies have reported that HfO2-NP+RT can induce an abscopal effect, where RT alone cannot. Here, using a mouse abscopal assay, we measured T cells infiltrates in treated and untreated tumors after HfO2-NP intratumor injection and activation with RT, and their role in the abscopal effect.

These data indicate that the immunogenic conversion of the tumor microenvironment triggered by HfO2-NP+RT generates the abscopal effect by activation of CD8+ T cells. HfO2-NP+RT may potentiate a pro-inflammatory microenvironment appropriate for enabling an anti-tumor immune response. It may act as effective in-situ cancer vaccine and be combined with immunotherapeutic agents across oncology.