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

Purpose/Objective(s): Elderly and/or frail patients (pts) with head and neck squamous cell carcinoma (HSNCC) remain a challenging to manage and neglected population regarding clinical trials and data generation to support treatment choices. Despite representing 20% of the HNSCC population no consensus exists on what is the optimal treatment for these pts with locally advanced (LA) disease, vulnerable to treatment-induced toxicities with the current standard of care. New approaches are needed to improve clinical outcomes without adding toxicity. NBTXR3 hafnium oxide nanoparticles injected intratumorally may represent such an option. Otherwise inert; this first-in-class radioenhancer, augments the radiotherapy (RT) dose within tumor cells when activated by RT, increasing tumor cell death compared to RT alone. The results presented here demonstrate the feasibility and safety of NBTXR3 activated by RT in elderly/frail patients, a population with few therapeutic options.

Materials/Methods: Elderly/frail pts received a single intratumoral injection of NBTXR3 and intensity modulated radiation therapy (IMRT; 70 Gy/35 fractions/7 weeks). The study was a 3 + 3 dose escalation to test the NBTXR3 dose equivalent to 5, 10, 15, and 22% of baseline theoretical tumor volume, followed by a dose expansion. Primary endpoints include Recommended Phase 2 Dose (RP2D) determination and early dose limiting toxicities (DLT). NBTXR3 presence in surrounding healthy tissues and anti-tumor activity (RECIST 1.1) were also evaluated.

Results: Enrollment was completed at all dose levels: 5% (3 pts), 10% (3 pts), 15% (5 pts), and 22% (8 pts). No early DLT or SAE related to NBTXR3 or injection were observed. One G1 AE (asthenia; 22%) related to NBTXR3 and four AEs (G2 oral pain, G1 tumor hemorrhage, asthenia, and injection site hemorrhage) related to injection were observed. RT-related toxicity was as expected with IMRT. The RP2D was determined to be 22%. CT-scan assessment demonstrated localization of NBTXR3 intratumorally without presence in surrounding healthy tissues. At a median follow-up of 231 days, 9/13 (2 unconfirmed) evaluable pts receiving doses ≥10%, achieved a complete response of the treated tumors. The final dose escalation safety and efficacy results will be presented herein.

Conclusion: NBTXR3 was well tolerated at all tested doses and demonstrated preliminary anti-tumor activity. A dose expansion phase at the RP2D is ongoing. These results highlight the potential of NBTXR3 as a novel treatment option for elderly/frail pts with LA HNSCC and address an unmet medical need.

Summary

Background: Radiation therapy (RT) has demonstrated ability to augment antitumor immunity, promoting immunogenic cell death (ICD) and stimulating immune adjuvant effects. On the other hand, RT has also been reported to induce immunosuppressive responses. A new class of material with high electron density, hafnium oxide, was designed at the nanoscale (HfO2-NP) to efficiently absorb ionizing radiation and augment the radiation dose
deposited from within the tumor cells. […]

Methods: The potential ability of HfO2-NP exposed to RT to transform tumors into immunologically active lesions was tested in vitro and in vivo. In vitro, the level of ICD markers was evaluated in a panel of human cancer cell lines, following cells treated or not with HfO2-NP and irradiated. In vivo, a vaccination assay was performed to evaluate the host immune responses in immunocompetent mice inoculated with murine CT26 cancer cells treated or not with HfO2-NP and irradiated with 6 Gy. […]

Results: Higher DAMPs levels (cell surface expression of calreticulin, extracellular adenosine triphosphate level and extracellular high-mobility group box 1 level) were observed in the tested cancer cells treated with HfO2-NP + RT when compared to cancer cells exposed to RT. […]

Conclusions: These results suggest an efficient cell killing (ability to generate ICD) with superior potential of HfO2-NP + RT to transform the tumor into an effective in situ vaccine when compared to RT. Moreover, HfO2-NP treatment generates a marked increase of immune cells infiltration in the tumors suggesting that it may convert immunologically “cold” tumor into “hot” tumor and could be combined with immunotherapeutic agents across oncology.

Summary

Today, more than half of all cancer patients receive radiotherapy as part of their treatment. However, radiotherapy efficacy is often limited by healthy tissues toxicity and needs to be optimized. One relevant solution is to increase the radiation dose deposition from within the tumor cells. The presence of high atomic number (high-Z) elements within the X-ray pathway increases the probability of interaction with ionizing radiation as compared with tissues (composed of low-Z elements). Likewise, mammalian cells can handle materials at the nanoscale. Therefore, materials made of high-Z elements designed at the nanoscale can enhance the deposit of the radiation dose at the cancer cell level.

Still, the most relevant design of these nano-objects has been scarcely explored. Here, we hypothesize that the packing of high-Z elements within the nano-object is a key parameter when considering its design. We used gold and probe how its packing at the nanoscale can achieve the best probability of interaction with ionizing radiation. […]

Summary

Background: Between 70 to 90% of patient have “cold” tumors, i.e. devoid or poorly infiltrated by immune cells, rendering inoperative their treatment by immune checkpoint inhibitors. To allow these patients to benefit from these therapies, it is fundamental to prime an antitumor immune response. Radiotherapy (RT) has demonstrated its ability to induce the immunogenic cell death (ICD), a crucial event allowing the priming of the antitumor immune response. Meanwhile, a new class of material with high electron density, hafnium oxide, was designed at the nanoscale (HfO2-NP) to efficiently absorb ionizing radiation and increase the radiation dose deposition from within the tumor cells and increase killing of cancer cells. Here, we compared the ability of HfO2-NP and RT to RT alone to kill cancer cells and induce immunogenic cell death.

Methods: A panel of human and mouse cancer cell lines (mesenchymal and epithelial origin, radiosensitive and radioresistant) were treated or not with HfO2-NP, then irradiated by X-rays. Impact of the treatments on apoptosis and necrosis was assessed by FACS analysis (Annexin V/Propidium iodide). […]

Results: For all the tested cell lines treated with HfO2-NP and RT, a marked increase of apoptosis and necrosis was demonstrated, compared to cells treated with RT alone. In addition, higher levels of DAMPs (ecto-CRT, ecto-HSP70, ecto-HSP90, secreted ATP and extracellular HMGB1) were measured in the cancer cells treated with HfO2-NP and RT when compared to cancer cells exposed to RT.

Conclusions: HfO2-NP has demonstrated its capacity to kill cancer cells more efficiently than radiotherapy alone. HfO2-NP, administered via a single intratumor injection, is currently evaluated in clinical trials including soft tissue sarcoma (phase II/III), head and neck, prostate, liver and rectum cancers (phase I) and would permit to improve the local control of tumors, a crucial parameter for the cure and survival of patients. […]

Summary

Immunotherapy Workshop: Bethesda, MD, June 15-16, 2017

Radiotherapy (RT) has proven its ability to function like an in-situ vaccine, showing potential for successful combination with immunotherapeutic agents. Hafnium oxide nanoparticle (HfO2-NP), undergoing clinical trials for enhancing RT, was designed as high electron density material at the nanoscale. HfO2-NPs are taken up by cancer cells and, when exposed to RT, locally increase the radiation dose deposit, triggering more cancer cells death when compared to RT. We hypothesized that HfO2-NP+RT could trigger an enhanced immune response when compared to RT, both in preclinical and clinical settings.

Summary

Combination of NBTXR3 and cisplatin has been evaluated in vitro and in vivo. No specific toxicity was observed for the cells exposed only to NBTXR3. For the combined treatment, a marked and enhanced cell destruction when compared to the single agent. In vivo, NBTXR3 combined with low dose of cisplatin delayed tumor growth when compared to single agent cisplatin in combination with radiotherapy.

Summary

Background: NBTXR3 are functionalized hafnium oxide nanoparticles, undergoing seven clinical trials for enhancing radiation therapy (RT). The high electron density of the nanoparticles, when exposed to radiotherapy (NBTXR3 + RT), allow absorption/deposition of a high radiation dose within the cancer cells to physically kill the cells, and possibly improve outcome. Besides, NBTXR3 + RT has shown subsequent ability to enhance immunogenic cell death and immune response in preclinics. We hypothesized that NBTXR3 + RT could trigger an enhanced immune response when compared to RT in patients with STS.

Methods: Tumor tissues pre- (biopsy) and/or post-treatment (resection) were collected from patients (pts) with locally advanced STS, who received either NBTXR3 as intratumor injection and RT (14 pts) or RT (12 pts), as preoperative treatment (NCT02379845). Immunohistochemistry and Digital Pathology for immune biomarkers and for Immunoscore (CD3/CD8) were analyzed. Gene expression profiling and pre-optimized immune-gene signatures called Immunosign were also used.

Results: A significant increase of T cells (CD3+, CD8+) and a marked increase of CD103+ immune cell infiltration post- vs pre-treatment were observed for NBTXR3 + RT (P< 0.01), while no differences were seen for RT. Post-treatment, an increased Immunoscore (CD3 + CD8 cell densities) was observed for NBTXR3 + RT compared to RT (P < 0.07). Consistently, the up-regulation of pan immune genes expression and specifically expression of adaptive immunity genes between pre- and post-treatment, was pronounced for NBTXR3 + RT when compared to RT. Functional analysis of genes up-regulated in NBTXR3 + RT showed an enrichment of cytokine activity (IL7, IFNA, IL16, IL11, IFNG), adaptive immunity (RAG1, GZMA, TAP1, TAP2, TBX21, STAT4, IFNG, LCK, LTK, CD37, CD22) and T cell receptor signaling pathway (CD28, CTLA4, CD274, BTLA, TIGIT, CD40LG, CD5, CD3E, ZAP70).

Conclusions: NBTXR3 + RT induces a specific adaptive immune pattern. As such, it may contribute to convert “cold” tumor into “hot” tumor and be effectively combined with immunotherapeutic agents across oncology. These data warrant more tissue samples evaluation to reinforce these findings.

Summary

NBTXR3 exposed to irradiation enhanced cancer cells destruction and immunogenic cell death compared to irradiation alone, suggesting a strong potential for transforming tumor into an effective in situ vaccine. This may contribute to transform “cold” tumor into “hot” tumor and effectively be combined with most of the immunotherapeutic agents across oncology.

Summary

Since the discovery of cisplatin about 40 years ago, the design of innovative metal-based anticancer drugs is a growing area of research. Metal elements offer specific characteristics due to their intrinsic properties and could be used in relation to their final state: a metal complex, a radionuclide, a metal-based nanoparticle product. Transition metal coordination complexes interact with cell molecular targets, affecting biochemical functions resulting in cancer cell destruction. Radionuclides are another way to use metals as anticancer therapy. The metal nucleus of the unstable radionuclide becomes stable by emitting energy. The biological effect in different tissues is obtained by the absorption of this energy from the radiation emitted by the radionuclide, the principal target generally agreed for ionizing radiations being DNA. A new area of clinical research is now emerging using the same experimental metal elements, but in a radically different manner: metals and metal oxides used as crystalline nanosized radiation enhancers particles. The use of metals as a high electron density material tailored at the nanoscale when exposed to radiotherapy is a unique approach that can allow entry to the cell and make feasible the absorption/deposition of a high-energy dose within the tumor cell (on/off activity). Therefore, high electron density metal or metal oxide nanoparticles may bring well known physical mode of action, that of radiotherapy, within malignant cells and achieve the paradigm of local cancer treatment.