Superior antitumor immune response achieved with proton over photon immunoradiotherapy is amplified by the nanoradioenhancer NBTXR3.

Recent findings suggest that immunoradiotherapy (IRT), combining photon radiotherapy (XRT) or proton radiotherapy (PRT) with immune checkpoint blockade, can enhance systemic tumor control. However, the comparative efficacy of XRT and PRT in IRT remains understudied. To address this, we compared outcomes between XRT + αPD1 and PRT + αPD1 in murine αPD1-resistant lung cancer (344SQR). We also assessed the impact of the nanoparticle radioenhancer NBTXR3 on both XRT + αPD1 and PRT + αPD1 for tumor control and examined the tumor immune microenvironment using single-cell RNA sequencing (scRNAseq). Additionally, mice cured by NBTXR3 + PRT + αPD1 were rechallenged with three lung cancer cell lines to evaluate memory antitumor immunity. PRT + αPD1 showed superior local tumor control and abscopal effects compared to XRT + αPD1. NBTXR3 + PRT + αPD1 significantly outperformed NBTXR3 + XRT + αPD1 in tumor control, promoting greater infiltration of antitumor lymphocytes into irradiated tumors. Unirradiated tumors treated with NBTXR3 + PRT + αPD1 had more NKT cells, CD4 T cells, and B cells, with fewer Tregs, than those treated with NBTXR3 + XRT + αPD1. NBTXR3 + PRT + αPD1 also stimulated higher expression of IFN-γ, GzmB, and Nkg7 in lymphocytes, reduced the TGF-ß pathway, and increased tumor necrosis factor alpha expression compared to NBTXR3 + XRT + αPD1. Moreover, NBTXR3 + PRT + αPD1 resulted in greater M1 macrophage polarization in both irradiated and unirradiated tumors. Mice achieving remission through NBTXR3 + PRT + αPD1 exhibited a robust memory immune response, effectively inhibiting growth of subsequent tumors from three distinct lung cancer cell lines. Proton IRT combined with NBTXR3 offers enhanced tumor control and survival rates over photon-based treatments in managing αPD1-resistant lung cancer, indicating its potential as a potent systemic therapy.

Novel engineered IL-2 Nemvaleukin alfa combined with PD1 checkpoint blockade enhances the systemic anti-tumor responses of radiation therapy.

BACKGROUND: Combining interleukin-2 (IL-2) with radiotherapy (RT) and immune checkpoint blockade (ICB) has emerged as a promising approach to address ICB resistance. However, conventional IL-2 cytokine therapy faces constraints owing to its brief half-life and adverse effects. RDB 1462, the mouse ortholog of Nemvaleukin alfa, is an engineered IL-2 with an intermediate affinity that selectively stimulates antitumor CD8 T and NK cells while limiting regulatory T cell expansion. This study aimed to evaluate the antitumor activity and mechanism of action of the combination of RDB 1462, RT, and anti-PD1 in mouse tumor models. METHODS: Two bilateral lung adenocarcinoma murine models were established using 344SQ-Parental and 344SQ anti-PD1-resistant cell lines. Primary tumors were treated with RT, and secondary tumors were observed for evidence of abscopal effects. We performed immune phenotyping by flow cytometry, analyzed 770 immune-related genes using NanoString, and performed T cell receptor (TCR) repertoire analysis. Serum pro-inflammatory cytokine markers were analyzed by 23-plex kit. RESULTS: Compared to native IL-2 (RDB 1475), RDB 1462 demonstrated superior systemic antitumoral responses, attributable, at least in part, to augmented levels of CD4 and CD8 T cells with the latter. Our findings reveal substantial reductions in primary and secondary tumor volumes compared to monotherapy controls, with some variability observed among different dosing schedules of RDB 1462 combined with RT. Blood and tumor tissue-based flow cytometric phenotyping reveals an increase in effector memory CD8 and CD4 T cells and a decrease in immunosuppressive cells accompanied by a significant increase in IL-2, IFN-γ, and GM-CSF levels in the combination group. Transcriptomic profiling and TCR sequencing reveal favorable gene expression and T cell repertoire patterns with the dual combination. Furthermore, integrating anti-PD1 therapy with RT and RDB 1462 further reduced primary and secondary tumor volumes, prolonged survival, and decreased lung metastasis. Observations of immune cell profiles indicated that RT with escalating doses of RDB 1462 significantly reduced tumor growth and increased tumor-specific immune cell populations. CONCLUSION: The addition of Nemvaleukin therapy may enhance responses to RT alone and in combination with anti-PD1.

Inhibition of MER proto-oncogene tyrosine kinase by an antisense oligonucleotide enhances treatment efficacy of immunoradiotherapy.

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.

Nanoparticle-enhanced proton beam immunoradiotherapy drives immune activation and durable tumor rejection.

The combination of radiation therapy (RT) and immunotherapy has emerged as a promising treatment option in oncology. Historically, x-ray radiation (XRT) has been the most commonly used form of RT. However, proton beam therapy (PBT) is gaining recognition as a viable alternative, as it has been shown to produce similar outcomes to XRT while minimizing off-target effects. The effects of PBT on the antitumor immune response have only just begun to be described, and to our knowledge no studies to date have examined the effect of PBT as part of a combinatorial immunoradiotherapeutic strategy. Here, using a 2-tumor model of lung cancer in mice, we show that PBT in tandem with an anti-PD1 antibody substantially reduced growth in both irradiated and unirradiated tumors. This was accompanied by robust activation of the immune response, as evidenced by whole-tumor and single-cell RNA sequencing showing upregulation of a multitude of immune-related transcripts. This response was further significantly enhanced by the injection of the tumor to be irradiated with NBTXR3 nanoparticles. Tumors of mice treated with the triple combination exhibited increased infiltration and activation of cytotoxic immune cells. This triple combination eradicated both tumors in 37.5% of the treated mice and showed robust long-term immunity to cancer.

NBTXR3 improves the efficacy of immunoradiotherapy combining nonfucosylated anti-CTLA4 in an anti-PD1 resistant lung cancer model.

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.