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Diverse factors contribute to the limited clinical response to radiotherapy (RT) and immunotherapy in metastatic non–small cell lung cancer (NSCLC), among which is the ability of these tumors to recruit a retinue of suppressive immune cells—such as M2 tumor-associated macrophages (TAM)—thereby establishing an immunosuppressive tumor microenvironment that contributes to tumor progression and radio resistance. M2 TAMs are activated by the STAT6 signaling pathway. Therefore, we targeted STAT6 using an antisense oligonucleotide (ASO) along with hypofractionated RT (hRT; 3 fractions of 12 Gy each) to primary tumors in three bilateral murine NSCLC models (Lewis lung carcinoma, 344SQ-parental, and anti–PD-1–resistant 344SQ lung adenocarcinomas). We found that STAT6 ASO plus hRT slowed growth of both primary and abscopal tumors, decreased lung metastases, and extended survival. Interrogating the mechanism of action showed reduced M2 macrophage tumor infiltration, enhanced TH1 polarization, improved T-cell and macrophage function, and decreased TGFβ levels. The addition of anti–PD-1 further enhanced systemic antitumor responses. These results provide a preclinical rationale for the pursuit of an alternative therapeutic approach for patients with immune-resistant NSCLC.

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Diverse factors contribute to the limited clinical response to radiotherapy (RT) and immunotherapy in metastatic non–small cell lung cancer (NSCLC), among which is the ability of these tumors to recruit a retinue of suppressive immune cells—such as M2 tumor-associated macrophages (TAM)—thereby establishing an immunosuppressive tumor microenvironment that contributes to tumor progression and radio resistance. M2 TAMs are activated by the STAT6 signaling pathway. Therefore, we targeted STAT6 using an antisense oligonucleotide (ASO) along with hypofractionated RT (hRT; 3 fractions of 12 Gy each) to primary tumors in three bilateral murine NSCLC models (Lewis lung carcinoma, 344SQ-parental, and anti–PD-1–resistant 344SQ lung adenocarcinomas). We found that STAT6 ASO plus hRT slowed growth of both primary and abscopal tumors, decreased lung metastases, and extended survival. Interrogating the mechanism of action showed reduced M2 macrophage tumor infiltration, enhanced TH1 polarization, improved T-cell and macrophage function, and decreased TGFβ levels. The addition of anti–PD-1 further enhanced systemic antitumor responses. These results provide a preclinical rationale for the pursuit of an alternative therapeutic approach for patients with immune-resistant NSCLC.

Radiotherapy (XRT), a well-known activator of the inflammasome and immune priming, is in part capable of reversing resistance to anti-PD1 treatment. Although NLRP3 is typically observed for its role in exacerbating XRT-induced tissue damage, the NLRP3 inflammasome can also be protective and augment the effect of XRT when used in proper dosing and sequencing. However, whether NLRP3 agonist boosts radiation-induced immune priming and promote abscopal responses in anti-PD1 resistant model is still unknown. Therefore, in this study, we paired intratumoral injection of an NLRP3 agonist with XRT to stimulate the immune system in both wild type (344SQ-P) and anti-PD1 resistant (344SQ-R) murine-implanted lung adenocarcinoma models. We found that the combination of XRT + NLPR3 agonist enhanced control of implanted lung adenocarcinoma primary as well as secondary tumors in a radiological dose-dependent manner, in which 12Gy x 3 fractions of stereotactic XRT was better than 5Gy x 3, while 1Gy x 2 did not improve the NLRP3 effect. Survival and tumor growth data also showed significant abscopal response with the triple therapy (12Gyx3 + NLRP3 agonist + α-PD1) in both 344SQ-P and 344SQ-R aggressively growing models. Multiple pro-inflammatory cytokines (IL-1b, IL-4, IL-12, IL-17, IFN-γ and GM-CSF) were elevated in the serum of mice treated with XRT + NLRP3 or triple therapy. The Nanostring results showed that NLRP3 agonist is capable of increasing antigen presentation, innate function, and T-cell priming. This study can be of particular importance to treat patients with immunologically-cold solid tumors whom are also refractory to prior checkpoint treatments.

Background: While improvements in radioimmunotherapy have made significant headway in improving outcomes for cancer patients, this treatment approach has nevertheless proven ineffective at controlling the majority of malignancies. One of the mechanisms of resistance to radioimmunotherapy 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 radioimmunotherapy.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. 129Sv/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 to the primary tumors on day 7, which were irradiated with 12 Gy on days 8, 9, and 10. Anti-PD1 (200 mg), αLAG3 (200 mg), and αTIGIT (200 mg) 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 growth of treated and distant untreated tumors, enhancing animal survival and is the only one that led to complete destruction of both tumors in approximately 30% of treated mice. Corresponding with this improved response is a 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.Conclusions: Our results strongly attest to the efficacy and validity of combining nanoparticle-enhanced radiotherapy and simultaneous blockade of multiple immune check point receptors and provide pre-clinical rationale for investigation into its translation into human patients.

Introduction: Checkpoint inhibition (CPI) has been a game-changer for cancer therapy, but most cancers do not respond. Part of the problem is that cancers employ multiple checkpoints; thus, targeting multiple checkpoint receptors simultaneously may yield increased benefit. Previously, we found that radiotherapy (XRT) in concert with a radiation-enhancing nanoparticle (NBTXR3) and PD1 blockade significantly improved tumor control both at the irradiative site and in remote, unirradiated tumors in a mouse model of anti-PD1 (αPD1)-resistant lung cancer (344SQR). Here, we tested whether blockade of TIGIT, LAG3, and PD1 in combination with NBTXR3-enhanced XRT could improve antitumor immune responses in our 344SQR tumor model. Methods: Fifty thousand 344SQR cells were inoculated in the right and left hindlimbs of 129Sv/Ev mice on day 0 and day 4 to establish primary and secondary tumors, respectively. Mice were divided into six treatment groups: 1) untreated; 2) XRT+αPD1; 3) NBTXR3+XRT+αPD1; 4) NBTXR3+XRT+αPD1+αLAG3; 5) NBTXR3+XRT+αPD1+αTIGIT; and 6) NBTXR3+XRT+αPD1+αLAG3 +αTIGIT. Primary tumors were intratumorally injected with NBTXR3 nanoparticles on day 7 and received 3 fractions of 12 Gy X-ray radiation on days 8, 9, and 10. Immune checkpoint inhibitors, composed of αPD1 (200 μg), αLAG3 (200 μg), and αTIGIT (200 μg), were intraperitoneally administered to the mice on days 5, 8, 11, 14. The expression of 770 immune-related genes were measured via NanoString from the RNA extracted from the primary and secondary tumors on day 21. Results: CPIs+NBTXR3-enhanced radiation significantly promoted the upregulation of mRNA transcripts involved in innate immunity, the humoral response, B cell function, dendritic cell (DC) function, and antigen processing within primary, irradiated tumors relative to untreated controls. No additional increase in immune gene activity in the irradiated tumors was observed when LAG3, TIGIT, or both were blocked in addition to PD1. However, within the non-irradiated tumors, triple blockade of PD1, LAG3, and TIGIT in concert with NBTXR3+XRT produced elevations in multiple immune-related pathways that were significantly higher than those produced by other treatment combinations. These pathways included both adaptive and innate immunity; B, T, and natural killer (NK) cell, and DC function; and antigen processing. Conclusions: Simultaneous inhibition of LAG3 and TIGIT in tandem with PD1 blockade and NBTXR3-enhanced radiotherapy promotes immune activation at the irradiated site. In addition, abscopal immune responses are improved with blockade of LAG3 and TIGIT. These results suggest that blockade of multiple immune checkpoints in parallel with NBTXR3-mediated radiotherapy may be effective in metastatic cancers. [Y.H. and B.G. contributed equally to this work.] Citation Format: Yun Hu, Genevieve Bertolet, Sebastien Paris, Hampartsoum Barsoumian, Jordan DA SILVA, Saumil Gandhi, Quynh-Nhu Nguyen, Maria A. Cortez, James W. Welsh. Nanoparticle-enhanced radiotherapy combined with triple blockade of PD1, LAG3, and TIGIT enhances anti-tumor immune activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5516.

BackgroundTIGIT 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.Methods129Sv/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.ResultsThe 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.ConclusionsBlockade 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.AcknowledgementsThis work was supported by Cancer Center Support (Core) Grant CA016672 to The University of Texas MD Anderson Cancer Center; the Goodwin family research fund; the family of M. Adnan Hamed and the Orr Family Foundation to MD Anderson Cancer Center‘s Thoracic Radiation Oncology program; an MD Anderson Knowledge Gap award; Nanobiotix.