Summary

Background: When exposed to radiotherapy (RT), NBTXR3 nanoparticles increase radiation dose deposition from within the cancer cells. NBTXR3 is intended for a single intratumor injection. Results from a phase II/III clinical trial in patients with locally advanced Soft Tissue Sarcoma demonstrated significant superiority and clinical benefits of NBTXR3 activated by RT compared to RT alone, and was well tolerated. NBTXR3 is currently being evaluated in several other tumors including head and neck, liver, and pancreatic cancer as a single agent or in combination with anti-PD1. Moreover, preclinical studies have demonstrated that NBTXR3 can produce a significant abscopal effect, whereas RT alone cannot. Here, we explored the impact of NBTXR3 activated by RT on CD8+ infiltrates and TcR repertoire diversity change, and the effect on the immunopeptidome of cancer cells.

Methods: CT26 (murine colorectal cancer cells) were subcutaneously injected in BALB/c mice in one flank. Then, tumors were intratumorally injected with NBTXR3 (or vehicle) and irradiated 24 hours later with 4Gy per fraction for 3 consecutive days. Tumors were collected 3 days after the last RT fraction and immune cell infiltrates were measured using immunohistochemistry (IHC) and digital pathology. For TcR repertoire sequencing, the same workflow was followed, except cells were injected in both flanks. Only right tumors received treatment, while left tumors remained untreated. For immunopeptidome analysis, in vitro cells were irradiated by 4Gy. After one day, cells were collected for isolation and sequencing of MHC I-loaded peptides.

Results: IHC analyses showed a significant increase of CD8+ T cell infiltrates in tumors of mice treated with NBTXR3+RT, while RT alone had no significant effect. In addition, NBTXR3+RT treatment was able to increase TcR repertoire diversity, both in treated and untreated tumors, compared to RT alone. Finally, analysis of immunopeptidome showed that NBTXR3+RT changed the profile of MHC-I-loaded peptides.

Conclusions: Our in vivo data indicate that NBTXR3+RT can modulate the microenvironment of treated tumors, leading to enhanced CD8+ T cell infiltration as well as modification of the TcR repertoire, both in treated and distant untreated tumors. These NBTXR3+RT-induced responses may be related to changes in the immunopeptidome of cancer cells triggered by this treatment.

Summary

Background: TIGIT and LAG3 are inhibitory receptors expressed on cytotoxic CD8+ T cells and NK cells and directly inhibit the activation and proliferation of these cells. We proposed that blockade of TIGIT and LAG3 could improve antitumor immune response in a mouse model of anti-PD1 (aPD1)-resistant mice.

Methods: 129Sv/Ev mice were inoculated with 50,000 aPD1-resistant 344SQR cells in the right leg on day 0 (primary tumor) and with 50,000 cells in the left leg on day 4 (secondary tumor). Primary tumors were injected with NBTXR3 radioenhancer nanoparticles on day 7 and irradiated with 12 Gy on days 8, 9, and 10. Anti-PD1, aLAG3, and aTIGIT were given to mice by intraperitoneal injections on days 5, 8, 11, 14, 21, 28, 35, and 42. On day 21, primary tumors, secondary tumors, and blood samples were harvested and analyzed with flow cytometry to evaluate changes in immune cell populations. The RNA extracted from the tumors were also analyzed by Nanostring. Mice in which tumors were completely eradicated were re-challenged with another 50,000 344SQR cells in the right flank at least two months post radiation; no further treatment was given to these mice, and tumor growth was monitored.

Results: The addition of aTIGIT, aLAG3, or aTIGIT+aLAG3 to NBTXR3+XRT+aPD1 therapy significantly improved control of tumors, and the addition of aTIGIT+aLAG3 also led to fewer spontaneous lung metastases. The addition of either aTIGIT or aLAG3 to NBTXR3+XRT+aPD1 extended mouse survival time relative to NBTXR3+XRT+aPD1. None of the 8 mice in either the NBTXR3+XRT+aPD1+aTIGIT group or the NBTXR3+XRT+aPD1+aLAG3 group survived more than 32 days; in contrast, 3 of the 8 mice that received NBTXR3+XRT+aPD1+aTIGIT+aLAG3 survived until the end of the experiment. These surviving mice were found to have developed memory against 344SQR cells, and no further tumor growth was observed after re-challenge. Flow cytometry analysis showed that adding aTIGIT+aLAG3 to NBTXR3+XRT+aPD1 increased the percentages of proliferating CD8+ T cells in primary tumors, secondary tumors, and blood. Furthermore, Nanostring transcriptomic analysis of cells isolated from the tumors of mice thus treated showed evidence of classical two-step immunological priming, with an elevation of innate immune genes at the primary tumor and full-blown activation of the immune system within the secondary tumor.

Conclusions: Blockade of TIGIT and LAG3 with NBTXR3+XRT+aPD1 improved CD8+ T-cell proliferation, augmented the antitumor response at both irradiated and unirradiated (abscopal) tumors, and induced potent long-term antitumor memory in mice.

Summary

Purpose/Objective(s): Immune checkpoint inhibitors (ICI) can improve outcomes in patients who respond to treatment, however most patients exhibit resistance. Overcoming this resistance is the main challenge in immune-oncology and recent studies suggest radiotherapy (RT) may improve ICI response rates. NBTXR3, composed of functionalized hafnium oxide nanoparticles, is injected intratumorally and activated by RT. NBTXR3 increases RT energy deposit inside tumor cells and subsequent tumor cell death, without adding toxicity to healthy tissues. Here we present evidence that NBTXR3 activated by RT primes the immune system, producing an anti-tumor immune response, including activation of the cGAS-STING pathway, that overcomes anti-PD-1 resistance both in murine models and patients.

/Methods: Abscopal assays were conducted in immunocompetent mice. Anti-PD-1 sensitive or resistant lung tumor cell lines were injected in both flanks. Intratumoral injection of NBTXR3 (or vehicle) followed by RT was performed in right flank (primary) tumors only. Some mice also received anti-PD-1 injections. Tumor growth was monitored, and tumor immune cell infiltrates analyzed by immunohistochemistry (IHC). Separately, in the phase II/III randomized Act.in.Sarc [NCT02379845] trial patients with locally advanced soft tissue sarcoma (STS) received either NBTXR3+RT or RT alone followed by tumor resection. Pre- and post-treatment tumor samples from patients in both groups were analyzed by IHC and Digital Pathology for immune biomarkers. The safety and efficacy of NBTXR3 plus stereotactic body radiotherapy (SBRT) in combination with anti-PD-1 is being evaluated in three cohorts of patients with advanced cancers in the Phase I 1100 [NCT03589339] trial.

Results: Pre-clinical studies demonstrated that NBTXR3+RT induces an immune response not observed with RT alone and enhances systemic control. IHC showed significant increase of CD8+ T-cell infiltrates in both NBTXR3+RT treated and untreated tumors compared to RT alone. Increased CD8+ T-cell and decreased FOXP3+ Treg density (pre- vs post-treatment) was also observed in tumors from STS patients treated with NBTXR3+RT. Furthermore, NBTXR3+RT in combination with anti-PD-1 improved local and systemic control in mice bearing anti-PD-1 resistant lung tumors, produced long-term memory, and reduced spontaneous lung metastases. Preliminary efficacy data from the 1100 trial showed tumor regression in 8/9 patients. Of note, tumor regression was observed in 6/7 patients who had progressed on prior anti-PD-1.

Conclusion: The clinical efficacy of NBTXR3+RT has been demonstrated as a single agent in STS. Here we demonstrate that it overcomes resistance to anti-PD-1 treatment mechanisms in mice and led to tumor regression in patients having progressed on anti-PD-1 therapy. These results highlight the potential of NBTXR3+RT to positively impact the immuno-oncology field.

Summary

Background: Although treatment with high-dose (HD) radiation (XRT) and NBTXR3 on primary tumors in combination with systemic anti-PD1 was able to significantly improve abscopal effect in 344SQR murine metastatic lung cancer model, most of the mice eventually died due to the growth of secondary tumors. Therefore, we intended to use HD-XRT plus NBTXR3 injection into primary tumors and low-dose (LD) radiation on secondary tumors plus dual-agent immunotherapy (IT) of anti-PD1 and anti-CTLA-4 to acheive complete control of both the primary and secondary tumors in mice.

Methods: Five groups of 8 mice each were inoculated subcutaneously with 5 × 104 anti-PD1-resistant 344SQR cells in each hind leg, 3 days apart, to establish ‘primary’ (right) and ‘secondary’ (left) tumors. All mice in treatment groups received intraperitoneal anti-PD1 and anti-CTLA-4 on days 4, 7, 10, and 13, and continuing anti-PD1 treatment on days 20, 27, 34, 41, and 49 and 12Gy x3 (HD-XRT) to the primary tumors on days 7, 8 and 9. Primary tumors in groups 3 and 5 also received intratumoral NBTXR3 on day 6. Secondary tumors in groups 4 and 5 were also irradiated with 1Gyx2 (LD-XRT) on days 12 and 13. Experimental groups were designated as 1=Control, 2=HD+IT, 3=NBTXR3+HD+IT, 4=HD+LD+IT, and 5=NBTXR3+HD+LD+IT. The secondary tumors were analyzed by flow cytometry and Nanostring. On day 178, the survivor mice were rechallenged with 5 × 104 344SQR cells on the right flank and the tumor growth was monitored for an additional 36 days.

Results: All mice in all the groups except NBTXR3+HD+LD+IT died due to the growth of either the primary tumor or the secondary tumor by day 36. Both the primary and the secondary tumors in 4 mice of NBTXR3+HD+LD+IT group were completely eliminated. No tumor growth was observed in these mice after rechallenged with 344SQR cells. Flow cytometry data demonstrated that only the mice in the groups with NBTXR3 had significantly more CD8+ T cell infiltration in the secondary tumor collected on day 16 than the control. Both flow cytometry and Nanostring data showed that only the mice in NBTXR3+HD+LD+IT had a significantly higher CD8+ Tcell/Treg cell ratio than the control.

Conclusions: The combination of NBTXR3 plus high and low dose radiation with immunotherapy effectively controlled the growth of both primary and secondary tumors, significantly extended the survival, generating long-term antitumor memory. This combination therapy induced immune-mediated control of the secondary tumor at both genetic and cellular levels.

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

Background: Despite recent advances, resistance to immune checkpoint inhibitors (ICI), observed in over 80% of treated patients, is currently the main challenge immuno-oncology is facing. Intense efforts are being made to identify combination therapies that could improve ICI response rates. Administered intratumorally, NBTXR3 enhances the energy dose deposited by ionizing radiation within tumor cells, increasing the anti-tumor efficacy of radiation therapy (XRT) without adding toxicity to surrounding tissues. Here we present evidence that NBTXR3 activated by XRT primes the immune system, producing an anti-tumor response, including activation of the cGAS-STING pathway, that overcomes anti-PD-1 resistance both in mice models and patients.

Methods: Abscopal assays were conducted in immunocompetent mice. Tumor cell lines, sensitive or resistant to anti-PD-1, were injected in both flanks of mice. Intratumoral injection of NBTXR3 (or vehicle) followed by XRT was performed in right flank (primary) tumors only. Some mice also received anti-PD-1 injections. Tumor growth was monitored, and tumor immune cell infiltrates were analyzed by immunohistochemistry (IHC).
Separately, in the phase II/III randomized trial Act.in.Sarc [NCT02379845] patients with locally advanced soft tissue sarcoma (STS) received either NBTXR3+XRT or XRT alone followed by wide tumor resection. Pre- and post-treatment tumor samples from patients in both groups were analyzed by IHC and Digital Pathology for immune biomarkers.
The safety and efficacy (RECIST 1.1/iRECIST) of NBTXR3 plus stereotactic ablative radiotherapy (SABR) in combination with anti-PD-1 is being evaluated in three cohorts of patients with advanced cancers [NCT03589339].

Results: Pre-clinical studies demonstrated that NBTXR3+XRT induces an immune response a not observed with XRT alone and enhances systemic control. IHC showed significant increase of CD8+ T-cell infiltrates in both NBTXR3+XRT treated and untreated tumors compared to XRT alone. Similarly, increased CD8+ T-cell density (pre- vs post-treatment) was observed in tumor tissues from STS patients treated with NBTXR3+XRT. Tumor samples from the NBTXR3+XRT group also displayed increased PD-1+ cell density. Furthermore, in combination with anti-PD-1, NBTXR3+XRT improved local and systemic control in mice bearing anti-PD-1 resistant lung tumors, as well as resulted in reduced number of spontaneous lung metastases. Preliminary efficacy data from the first in human trial of NBTXR3+XRT in combination with anti-PD-1 showed tumor response in patients who progressed on prior anti-PD-1.

Conclusions: The clinical efficacy of NBTXR3+XRT has been demonstrated as a single agent. We now demonstrate that it potentiates anti-PD-1 treatment to overcome resistance mechanisms. These results highlight the potential of NBTXR3+XRT to positively impact the immuno-oncology field.

Summary

Purpose/Objective(s): Hafnium oxide nanoparticles (NBTXR3) activated by radiotherapy (RT) increase radiation dose deposit within cancer cells compared to RT alone. Currently 7 clinical trials are underway to evaluate NBTXR3+RT. To date, no dose limiting toxicities (DLTs) have been observed. Given that RT can prime an anti-tumor immune response we hypothesized that this response could be enhanced by NBTXR3+RT in both animals and humans.

Materials/Methods: Immunocompetent mice were injected in both flanks with CT26 cells. An intratumoral injection of NBTXR3 (or vehicle) was performed in right flank tumors, followed by RT (3x4Gy). Tumor growth was followed, and animals sacrificed when tumors reached 800mm3. Alternatively, tumors were collected 3 days after last RT fraction and immune cell infiltrates analyzed by immunohistochemistry (IHC). Pts with locally advanced soft tissue sarcoma (STS) [NCT02379845] received either NBTXR3+RT or RT alone. Pre- and post-treatment tumor tissues (biopsy and tumor resection respectively) from pts were analyzed by IHC and Digital Pathology for immune biomarkers (>16 pts per arm).

Results: Animal studies demonstrated that NBTXR3+RT can induce an immune response which was not observed with RT alone. IHC analyses showed that significantly more CD8+ cells were present in NBTXR3+RT treated and untreated tumors, compared to tumors from mice treated with RT alone. Similarly, increased CD8+ T cell infiltration pre- vs post-treatment was observed in tumor tissues from STS pts treated with NBTXR3+RT. An increase in biomarkers, including CD8 and PD1, following NBTXR3 +RT was also observed by IHC in tumor samples from STS pts compared to RT alone.

Conclusion: These results demonstrate that NBTXR3+RT induces a specific adaptive immune profile in both mice and STS pts. As such, it may convert immunologically “cold” tumors into “hot” tumors, opening the potential for combination with immunotherapeutic agents. We have therefore sought to investigate the safety and systemic effect of NBTXR3 activated by stereotactic ablative radiotherapy (SABR) in combination with anti-PD-1 antibody in pts with locoregionally recurrent or metastatic (to lung or liver) head and neck squamous cell carcinoma (HNSCC), as well as in metastatic non-small cell lung cancer (NSCLC) and liver metastasis patients [NCT03589339].

Summary

Purpose: First in class hafnium oxide nanoparticles (NBTXR3) activated by radiotherapy (RT) increase radiation dose deposit within cancer cells compared to RT alone. Given that RT can prime an anti-tumor immune response we hypothesized that this response could be enhanced by NBTXR3+RT in both animals and humans.

Method & Materials: Different abscopal assays in mice were conducted. Immunocompetent mice were injected in both flanks with murine tumor cells. Intratumoral injection of NBTXR3 (or vehicle) was performed in right flank tumors, followed by RT of right flank tumors only. Tumor growth was followed and immune cell infiltrates were analyzed by immunohistochemistry (IHC). Some mice received anti-PD-1 injections and tumor growth was monitored. Pts with locally advanced soft tissue sarcoma (STS) [NCT02379845] received either NBTXR3+RT or RT alone. Pts pre- and post-treatment tumor tissues were analyzed by IHC and Digital Pathology for immune biomarkers.

Results: Animal studies demonstrated that NBTXR3+RT induces an immune response which was not observed with RT alone. IHC showed significantly more CD8+ cells present in NBTXR3+RT treated and untreated tumors. Furthermore, NBTXR3+RT improved the effect of anti-PD-1. Similarly, increased CD8+ T cell infiltration pre- vs post-treatment was observed in tumor tissues from STS pts treated with NBTXR3+RT. An increase in biomarkers, including CD8, following NBTXR3+RT was also observed by IHC in tumor samples from STS pts compared to RT alone.

Conclusion: These results demonstrate that NBTXR3+RT induces a specific adaptive immune profile in both mice and STS pts. NBTXR3+RT also improved response to anti-PD-1 in mice, opening the potential for combination with immunotherapeutic agents in humans. We have therefore sought to investigate the safety and systemic effect of NBTXR3 activated by stereotactic ablative radiotherapy (SABR) in combination with anti-PD-1 in pts with locoregionally recurrent or metastatic (lung or liver) head and neck squamous cell carcinoma, as well as in metastatic non-small cell lung cancer and liver metastasis pts [NCT03589339].

Clinical Relevance & Application: The results of this study highlight the potential of NBTXR3 to be used in combination with immune checkpoint inhibitors in order to improve patient outcomes.

Summary

Purpose/Objective(s): Hafnium oxide nanoparticles (NBTXR3) activated by radiotherapy (RT) increase radiation dose deposit within cancer cells compared to RT alone. Currently 7 clinical trials are underway to evaluate NBTXR3+RT. To date, no dose limiting toxicities (DLTs) have been observed. Given that RT can prime an anti-tumor immune response we hypothesized that this response could be enhanced by NBTXR3+RT in both animals and humans.

Materials/Methods: Immunocompetent mice were injected in both flanks with CT26 cells. An intratumoral injection of NBTXR3 (or vehicle) was performed in right flank tumors, followed by RT (3x4Gy). Tumor growth was followed, and animals sacrificed when tumors reached 800mm3. Alternatively, tumors were collected 3 days after last RT fraction and immune cell infiltrates analyzed by immunohistochemistry (IHC). Pts with locally advanced soft tissue sarcoma (STS) [NCT02379845] received
either NBTXR3+RT or RT alone. Pre- and post-treatment tumor tissues (biopsy and tumor resection respectively) from pts were analyzed by IHC
and Digital Pathology for immune biomarkers (>16 pts per arm).

Results: Animal studies demonstrated that NBTXR3+RT can induce an immune response which was not observed with RT alone. IHC analyses showed that significantly more CD8+ cells were present in NBTXR3+RT treated and untreated tumors, compared to tumors from mice treated with RT alone. Similarly, increased CD8+ T cell infiltration pre- vs post-treatment was observed in tumor tissues from STS pts treated with NBTXR3+RT. An increase in biomarkers, including CD8 and PD1, following NBTXR3 +RT was also observed by IHC in tumor samples from STS pts compared to RT alone.

Conclusion: These results demonstrate that NBTXR3+RT induces a specific adaptive immune profile in both mice and STS pts. As such, it may convert immunologically “cold” tumors into “hot” tumors, opening the potential for combination with immunotherapeutic agents. We have therefore sought to investigate the safety and systemic effect of NBTXR3 activated by stereotactic ablative radiotherapy (SABR) in combination with anti-PD-1 antibody in pts with locoregionally recurrent or metastatic (to lung or liver) head and neck squamous cell carcinoma (HNSCC), as well as in metastatic non-small cell lung cancer (NSCLC) and liver metastasis patients [NCT03589339].