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Since the momentous discovery of X-rays, high-dose radiotherapy (H-XRT) has been a cornerstone for combating cancer. The high-energy electromagnetic waves induce direct damage to tumor-cells’ DNA, thereby halting cell growth and proliferation, and eventually leading to tumor eradication. Furthermore, recent evidence suggests that H-XRT may have immunomodulatory properties which arise from its ability to induce the release of neoantigens, which in turn prime T-cells and contribute to T-cell repertoire diversity. Throughout the years, there have been different treatment modalities introduced as complements to H-XRT that have yielded greater results than monotherapy alone. In this review, we will discuss preclinical and clinical data related to the recently introduced low-dose radiotherapy (L-XRT) modality. We will also explore the justification for combining L-XRT and H-XRT, which became known as the “RadScopal Technique”, as a novel immune adjuvant to treat cancer. In this analysis, we detail and dissect the physiological mechanisms of action of each modality and describe the synergistic amalgamation effect observed on primary and metastatic tumors. Finally, we will explore the impetus for further studies to investigate combinations of the “RadScopal Technique” with various immune-oncology drug candidates.

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.

Immunotherapy has revolutionized cancer treatment and revitalized efforts to harness the power of the immune system to combat a variety of cancer types more effectively. However, low clinical response rates and differences in outcomes due to variations in the immune landscape among patients with cancer continue to be major limitations to immunotherapy. Recent efforts to improve responses to immunotherapy have focused on targeting cellular metabolism, as the metabolic characteristics of cancer cells can directly influence the activity and metabolism of immune cells, particularly T cells. Although the metabolic pathways of various cancer cells and T cells have been extensively reviewed, the intersections among these pathways, and their potential use as targets for improving responses to immune-checkpoint blockade therapies, are not completely understood. This review focuses on the interplay between tumor metabolites and T-cell dysfunction as well as the relationship between several T-cell metabolic patterns and T-cell activity/function in tumor immunology. Understanding these relationships could offer new avenues for improving responses to immunotherapy on a metabolic basis.

Supplementary Figure S1. CONSORT flow diagram for patient selection

Patient characteristics

Ipilimumab is effective for patients with melanoma, but not for those with less immunogenic tumors. We report a phase II trial of ipilimumab with concurrent or sequential stereotactic ablative radiotherapy to metastatic lesions in the liver or lung (NCT02239900). Ipilimumab (every 3 weeks for 4 doses) was given with radiotherapy begun during the first dose (concurrent) or 1 week after the second dose (sequential) and delivered as 50 Gy in 4 fractions or 60 Gy in 10 fractions to metastatic liver or lung lesions. In total, 106 patients received ≥1 cycle of ipilimumab with radiation. Median follow-up was 10.5 months. Median progression-free survival time was 2.9 months (95% confidence interval, 2.45–3.40), and median overall survival time was not reached. Rates of clinical benefit of nonirradiated tumor volume were 26% overall, 28% for sequential versus 20% for concurrent therapy (P = 0.250), and 31% for lung versus 14% for liver metastases (P = 0.061). The sequential lung group had the highest rate of clinical benefit at 42%. There were no differences in treatment-related adverse events between groups. Exploratory analysis of nontargeted lesions revealed that lesions receiving low-dose radiation were more likely to respond than those that received no radiation (31% vs. 5%, P = 0.0091). This phase II trial of ipilimumab with stereotactic radiotherapy describes satisfactory outcomes and low toxicities, lending support to further investigation of combined-modality therapy for metastatic cancers.

Supplementary Figures

Response Rates of Unirradiated Tumors by Target site and Histology

Supplementary Figures and Tables

Immune checkpoint inhibitors, such as anti–PD-1/PD-L1, have emerged as promising therapies for advanced non–small cell lung cancer (NSCLC). However, approximately 80% of patients do not respond to immunotherapy given alone because of intrinsic or acquired resistance. Radiotherapy (XRT) can overcome PD-1 resistance and improve treatment outcomes, but its efficacy remains suboptimal. The tyrosine phosphatase SHP-2, expressed in some cancers and in immune cells, has been shown to negatively affect antitumor immunity. Our hypothesis was that SHP-2 inhibition in combination with anti–PD-L1 would enhance immune-mediated responses to XRT and synergistically boost antitumor effects in an anti–PD-1–resistant mouse model. We treated 129Sv/Ev mice with anti–PD-1–resistant 344SQ NSCLC adenocarcinoma with oral SHP099 (a SHP-2 inhibitor) combined with XRT and intraperitoneal anti–PD-L1. Primary tumors were treated with XRT (three fractions of 12 Gy each), whereas abscopal (out-of-field) tumors were observed but not treated. XRT in combination with SHP099 and anti–PD-L1 promoted local and abscopal responses, reduced lung metastases, and improved mouse survival. XRT also increased SHP-2+ M1 tumor-associated macrophages in abscopal tumors (P = 0.019). The addition of SHP099 also associated with a higher M1/M2 ratio, greater numbers of CD8+ T cells, and fewer regulatory T cells. This triple-combination therapy had strong antitumor effects in a mouse model of anti–PD-1–resistant NSCLC and may be a novel therapeutic approach for anti–PD-1–resistant NSCLC in patients.

Hepatic lesions measured at baseline for patients with evaluable imaging

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.

We have previously demonstrated that PD-1 blockade decreased the incidence of high-grade dysplasia in a carcinogen-induced murine model of oral squamous cell carcinoma (OSCC). It remains unknown, however, whether there are additional factors involved in escape from immune surveillance that could serve as additional targets for immunoprevention. We performed this study to further characterize the immune landscape of oral premalignant lesions (OPL) and determine the impact of targeting of the PD-1, CTLA-4, CD40, or OX40 pathways on the development of OPLs and oral carcinomas in the 4-nitroquinoline 1-oxide model. The immune pathways were targeted using mAbs or, in the case of the PD-1/PD-L1 pathway, using PD-L1–knockout (PD-L1ko) mice. After intervention, tongues and cervical lymph nodes were harvested and analyzed for malignant progression and modulation of the immune milieu, respectively. Targeting of CD40 with an agonist mAb was the most effective treatment to reduce transition of OPLs to OSCC; PD-1 alone or in combination with CTLA-4 inhibition, or PD-L1ko, also reduced progression of OPLs to OSCC, albeit to a lesser extent. Distinct patterns of immune system modulation were observed for the CD40 agonists compared with blockade of the PD-1/PD-L1 axis with or without CTLA-4 blockade; CD40 agonist generated a lasting expansion of experienced/memory cytotoxic T lymphocytes and M1 macrophages, whereas PD-1/CTLA-4 blockade resulted in a pronounced depletion of regulatory T cells among other changes. These data suggest that distinct approaches may be used for targeting different steps in the development of OSCC, and that CD40 agonists merit investigation as potential immunoprevention agents in this setting.Prevention Relevance:PD-1/PD-L1 pathway blockade, as well as activation of the CD40 pathway, were able to prevent OPL progression into invasive OSCC in a murine model. A distinct pattern of immune modulation was observed when either the CD40 or the PD-1/PD-L1 pathways were targeted.

Percentage of CD4+CD44hi

Table 1: Pathology classification criteria. Table 2: Flow cytometry antibodies

Purpose: Radiation is used extensively to treat localized cancer, but improved understanding of its effects on the immune system has increased interest in its potential systemic (abscopal) effects, particularly in combination with checkpoint inhibitors such as anti-PD1. The majority of patients either do not respond or develop resistance to monotherapy over time. Here, we investigated the efficacy of OX40 (CD134) stimulation as an alternative immunotherapeutic approach in combination with radiotherapy (XRT) in a murine model of anti–PD1-resistant lung tumors.Experimental Design: We established a bilateral tumor model in 129Sv/Ev mice using an anti–PD1-resistant lung tumor cell line. Primary tumors were treated with intratumoral injection of an OX40 agonist antibody, given as adjuvant therapy after XRT (36 Gy in three 12-Gy fractions), whereas secondary tumors were left untreated to investigate abscopal outcomes.Results: The combination of XRT followed by OX40 stimulation effectively inhibited local and systemic antitumor growth, limited lung metastases, and improved survival rates. This treatment regimen augmented CD4+ and CD8+ T-cell expansion. XRT induced the expression of OX40 on T cells in tumors and spleens and increased the percentages of splenic CD103+ dendritic cells.Conclusions: Our data extend the benefits of radiation to systemic disease control, especially when combined with anti-OX40 agonist to promote immunologically mediated abscopal effects. Moreover, this study provides a rational treatment approach and sequence to overcome anti–PD1-resistant poorly immunogenic tumors. Clin Cancer Res; 24(22); 5735–43. ©2018 AACR.

CD8+ CD103+ TRM cells express high levels of PD1. Flow cytometric analysis for TILs on day 29 from the triple therapy group (RT + α-PD1 + α-MerTK). Cells were gated on CD45 population then on CD4 and CD8, followed by CD8 and CD103 to check for PD1 expression in the final gate.

Purpose:Radiotherapy (RT) traditionally has been used for local tumor control in the treatment of cancer. The recent discovery that radiotherapy can have anticancer effects on the immune system has led to recognition of its ability to sensitize the tumor microenvironment to immunotherapy. However, radiation can also prompt adverse immunosuppressive effects that block aspects of systemic response at other tumor sites. Our hypothesis was that inhibition of the MER proto-oncogene tyrosine kinase (MerTK) in combination with anti-programmed cell death-1 (α-PD1) checkpoint blockade will enhance immune-mediated responses to radiotherapy.Experimental Design:We tested the efficacy of this triple therapy (Radiation + α-PD1 + α-MerTK mAbs) in 129Sv/Ev mice with bilateral lung adenocarcinoma xenografts. Primary tumors were treated with stereotactic radiotherapy (36 Gy in 3 12-Gy fractions), and tumors were monitored for response.Results:The triple therapy significantly delayed abscopal tumor growth, improved survival rates, and reduced numbers of lung metastases. We further found that the triple therapy increased the activated CD8+ and NK cells populations measured by granzyme B expression with upregulation of CD8+CD103+ tissue-resident memory cells (TRM) within the abscopal tumor microenvironment relative to radiation only.Conclusions:The addition of α-PD1 + α-MerTK mAbs to radiotherapy could alter the cell death to be more immunogenic and generate adaptive immune response via increasing the retention of TRM cells in the tumor islets of the abscopal tumors which was proven to play a major role in survival of non-small cell lung cancer patients.

High dose RT induced higher apoptosis. Mice were inoculated in the right leg with 344SQ for establishment of primary tumor. Tumors were treated locally by intermediate dose RT (15 Gy in three 5-Gy fractions) or High dose RT (36 Gy in three 12-Gy fractions). Tumors were harvested 7 days after the last fraction of RT then sectioned for Immunohistochemistry using Caspase-3 staining.

The triple therapy expresses high levels of TNF-α and IL-12 β cytokines. The NanoString results showed significant increase in IL-12 and TNF-α with the triple therapy group compared to no treatment. On the other hand, IL-6 was not significantly modulated.

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.

To report early findings from a phase II trial of high-dose radiotherapy (HD-RT) with or without low-dose RT (LD-RT) for metastatic cancer.Eligible patients had metastatic disease that progressed on immunotherapy within 6 months. Patients were given either HD-RT (20-70 Gy total; 3-12.5 Gy/f), or HD-RT + LD-RT (0.5-2 Gy/f up to 1-10 Gy total) to separate lesions, with continued immunotherapy. Radiographic response was assessed per RECIST 1.1 and Immune-Related Response Criteria (irRC). Primary endpoints: (1) 4-month disease control (DCR, complete/partial response [CR/PR] or stable disease [SD]) or an overall response (ORR, CR/PR) at any point in ≥10% of patients, per RECIST 1.1; (2) dose-limiting toxicity within 3 months not exceeding 30%. Secondary endpoint was lesion-specific response.Seventy-four patients (NSCLC, n = 38; melanoma n = 21) were analyzed (39 HD-RT and 35 HD-RT + LD-RT). The median follow-up time was 13.6 months. The primary endpoint was met for 72 evaluable patients, with a 4-month DCR of 42% (47% [16/34] vs. 37% [14/38] in HD-RT + LD-RT vs. HD-RT, P = 0.38), and 19% ORR at any time (26% [9/34] vs. 13% [5/38] in HD-RT + LD-RT vs. HD-RT, P = 0.27). Three patients had toxicity ≥grade 3. LD-RT lesion response (53%) was improved compared to nonirradiated lesions in HD-RT + LD-RT (23%, P = 0.002) and HD-RT (11%, P 0.001). T- and NK cell infiltration was enhanced in lesions treated with LD-RT.HD-RT plus LD-RT safely improved lesion-specific response in patients with immune resistant solid tumors by promoting infiltration of effector immune cells into the tumor microenvironment.

Background: while improvements in immunoradiotherapy have significantly improved outcomes for cancer patients, this treatment approach has nevertheless proven ineffective at controlling the majority of malignancies. One of the mechanisms of resistance to immunoradiotherapy 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 immunoradiotherapy. 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. 129 Sv/Ev mice were inoculated with fifty thousand alpha PD1-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 into the primary tumors on day 7, which were irradiated with 12 Gy on days 8, 9, and 10. Anti-PD1 (200 mu g), alpha LAG3 (200 mu g), and alpha TIGIT (200 mu g) 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 the growth of irradiated and distant unirradiated tumors, enhancing animal survival, and is the only one that led to the destruction of both tumors in approximately 30% of the treated mice. Corresponding with this improved response is 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. Conclusion: our results strongly attest to the efficacy and validity of combining nanoparticle-enhanced radio-therapy and simultaneous blockade of multiple immune checkpoint receptors and provide a pre-clinical rationale for investigating its translation into human patients., This 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.

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.

Radiation therapy (XRT) has a well-established role in cancer treatment. Given the encouraging results on immunostimulatory effects, radiation has been increasingly used with immune-check-point inhibitors in metastatic disease, especially when immunotherapy fails due to tumor immune evasion. We hypothesized that using high-dose stereotactic radiation in cycles (pulses) would increase T-cell priming and repertoire with each pulse and build immune memory in an incremental manner. To prove this hypothesis, we studied the combination of anti-CTLA-4 and Pulsed radiation therapy in our 344SQ non-small cell lung adenocarcinoma murine model. Primary and secondary tumors were bilaterally implanted in 129Sv/Ev mice. In the Pulsed XRT group, both primary and secondary tumors received 12Gyx2 radiation one week apart, and blood was collected seven days afterwards for TCR repertoire analysis. As for the delayed-Pulse group, primary tumors received 12Gyx2, and after a window of two weeks, the secondary tumors received 12Gyx2. Blood was collected seven days after the second cycle of radiation. The immunotherapy backbone for both groups was anti-CTLA-4 antibody to help with priming. Treatment with Pulsed XRT + anti-CTLA-4 led to significantly improved survival and resulted in a delayed tumor growth, where we observed enhanced antitumor efficacy at primary tumor sites beyond XRT + anti-CTLA-4 treatment group. More importantly, Pulsed XRT treatment led to increased CD4+ effector memory compared to single-cycle XRT. Pulsed XRT demonstrated superior efficacy to XRT in driving antitumor effects that were largely dependent on CD4+ T cells and partially dependent on CD8+ T cells. These results suggest that combinatorial strategies targeting multiple points of tumor immune evasion may lead to a robust and sustained antitumor response., This work was supported in part by Cancer Center Support (Core) Grant P30 CA016672 from the National Cancer Institute, National Institutes of Health, to The University of Texas MD Anderson Cancer Center.

We have previously demonstrated that PD-1 blockade decreased the incidence of high-grade dysplasia in a carcinogen-induced murine model of oral squamous cell carcinoma (OSCC). It remains unknown, however, whether there are additional factors involved in escape from immune surveillance that could serve as additional targets for immunoprevention. We performed this study to further characterize the immune landscape of oral premalignant lesions (OPL) and determine the impact of targeting of the PD-1, CTLA-4, CD40, or OX40 pathways on the development of OPLs and oral carcinomas in the 4-nitroquinoline 1-oxide model. The immune pathways were targeted using mAbs or, in the case of the PD-1/PD-L1 pathway, using PD-L1–knockout (PD-L1ko) mice. After intervention, tongues and cervical lymph nodes were harvested and analyzed for malignant progression and modulation of the immune milieu, respectively. Targeting of CD40 with an agonist mAb was the most effective treatment to reduce transition of OPLs to OSCC; PD-1 alone or in combination with CTLA-4 inhibition, or PD-L1ko, also reduced progression of OPLs to OSCC, albeit to a lesser extent. Distinct patterns of immune system modulation were observed for the CD40 agonists compared with blockade of the PD-1/PD-L1 axis with or without CTLA-4 blockade; CD40 agonist generated a lasting expansion of experienced/memory cytotoxic T lymphocytes and M1 macrophages, whereas PD-1/CTLA-4 blockade resulted in a pronounced depletion of regulatory T cells among other changes. These data suggest that distinct approaches may be used for targeting different steps in the development of OSCC, and that CD40 agonists merit investigation as potential immunoprevention agents in this setting.Prevention Relevance:PD-1/PD-L1 pathway blockade, as well as activation of the CD40 pathway, were able to prevent OPL progression into invasive OSCC in a murine model. A distinct pattern of immune modulation was observed when either the CD40 or the PD-1/PD-L1 pathways were targeted.

Introduction: Immunoradiotherapy combining radiotherapy and immune checkpoint blockade, has achieved impressive treatment outcomes. We have previously demonstrated that photon (XRT) and proton (PRT) localized radiotherapy in combination with immune checkpoint blockade and NBTXR3, a radioenhancing nanoparticle, could induce a systemic antitumor immune response. However, no studies have compared the treatment efficacy of PRT and XRT immunoradiotherapy. Here, we compared the capabilities of PRT and XRT when combined with NBTXR3 and PD1 blockade for inducing an antitumor immune response. Methods: 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 five treatment groups: 1) untreated control; 2) XRT+αPD1; 3) NBTXR3+XRT+αPD1; 4) PRT+αPD1; 5) NBTXR3+PRT+αPD1. The primary tumors were intratumorally injected with NBTXR3 nanoparticles on day 7, followed by two fractions of 12 Gy XRT or PRT on days 8 and 9 (total dose of 24 Gy). Two hundred μg of αPD1 was given to the mice on days 7, 10, 14, 21, 28, 35, and 42 through intraperitoneal injection. We assayed whole tumor RNAs with a Nanostring pan-cancer immune panel to profile the tumor immune microenvironment changes. In addition, immune cells extracted from the tumors were analyzed with single-cell RNA sequencing (scRNAseq). The survivor mice treated with the NBTXR3+PRT+αPD1 were rechallenged with 344SQR and 344SQP cells. Results: PRT+ αPD1 resulted in significantly better control of the primary tumors than XRT+ αPD1. No significant difference was observed in the volume of the secondary tumors treated with XRT+ αPD1 and the control. In contrast, PRT+ αPD1 resulted in a pronounced abscopal effect. Adding NBTXR3 to XRT+ αPD1 and PRT+ αPD1 improved the control of both the primary and secondary tumors. In addition, NBTXR3+PRT+αPD1 achieved significantly slower growth of the two tumors and more prolonged survival than NBTXR3+XRT+αPD1. Remarkably, NBTXR3+PRT+αPD1 eradicated both primary and secondary tumors in 40% of the treated mice, while no mice were cured by NBTXR3+XRT+αPD1. Nanostring analysis and scRNAseq revealed better activation and infiltration of antitumoral immune cells to the two tumors when treated with PRT than XRT. The survivor mice treated with NBTXR3+PRT+αPD1 effectively rejected tumor growth after rechallenge with 344SQR and 344SQP cells. Conclusions: When combined with NBTXR3, PRT with αPD1 exhibited significantly better treatment efficacy than XRT with αPD1. The superior efficacy of NBTXR3+PRT+αPD1 is accompanied by a more robust immune activation and immune cell infiltration in irradiated and unirradiated tumors. Citation Format: Yun Hu, Sébastien Paris, Narayan Sahoo, Qi Wang, Qianxia Wang, Hampartsoum B. Barsoumian, Jordan Da Silva, Célia Bienassis, Ailing Huang, Nahum Puebla-Osorio, Saumil Gandhi, Quynh-Nhu Nguyen, Jing Wang, Maria A. Cortez, James W. Welsh. NBTXR3 radio-enhancing nanoparticle achieves a more robust antitumor immune response when combined with proton radiotherapy than photon radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2415.

Metastatic cancer is a heterogeneous entity, some of which could benefit from local consolidative radiation therapy (RT). Although randomized evidence is growing in support of using RT for oligometastatic disease, a highly active area of investigation relates to whether RT could benefit patients with polymetastatic disease. This article highlights the preclinical and clinical rationale for using RT for polymetastatic disease, proposes an exploratory framework for selecting patients best suited for these types of treatments, and briefly reviews potential challenges. The goal of this hypothesis-generating review is to address personalized multimodality systemic treatment for patients with metastatic cancer. The rationale for using high-dose RT is primarily for local control and immune activation in either oligometastatic or polymetastatic disease. However, the primary application of low-dose RT is to activate distinct antitumor immune pathways and modulate the tumor stroma in efforts to better facilitate T cell infiltration. We explore clinical cases involving high- and low-dose RT to demonstrate the potential efficacy of such treatment. We then group patients by extent of disease burden to implement high- and/or low-dose RT. Patients with low-volume disease may receive high-dose RT to all sites as part of an oligometastatic paradigm. Subjects with high-volume disease (for whom standard of care remains palliative RT only) could be treated with a combination of high-dose RT to a few sites for immune activation, while receiving low-dose RT to several remaining lesions to enhance systemic responses from high-dose RT and immunotherapy. We further discuss how emerging but speculative concepts such as immune function may be integrated into this approach and examine therapies currently under investigation that may help address immune deficiencies. The review concludes by addressing challenges in using RT for polymetastatic disease, such as concerns about treatment planning workflows, treatment times, dose constraints for multiple-isocenter treatments, and economic considerations.

Immune function (e.g. absolute lymphocyte count (ALC)) and modifiable predictors thereof (e.g. volume of the heart/lungs receiving low-dose radiation) impact outcomes of cancer patients, but this has not been well-studied in the immunotherapy era. This investigation of metastatic lung cancer assessed the interaction of dosimetric parameters (e.g. lung/heart V5), radiotherapy technique (e.g. stereotactic (SBRT) or traditional radiotherapy), lymphopenia, and survival outcomes.Patients were collected from three institutional phase I/II trials of combined immunotherapy and lung irradiation. SBRT referred to 50 Gy/4 fractions or 60 Gy/10 fractions, and traditional RT as 45 Gy/15 fractions. Blood collections were standardized on the first and last day of radiotherapy and each cycle of immunotherapy. Statistics included multivariable linear regression to identify variables associated with ALC decline, Kaplan-Meier analysis of overall and progression-free survival (PFS), and Cox multivariate analysis.The median follow-up of the 165 patients was 21 months. The only factor independently predictive of ALC decline was traditional RT (p 0.001). Therefore, the analysis was repeated for traditional RT and SBRT separately; lung V5 was associated with lymphopenia for traditional RT (p 0.001) but not SBRT (p = 0.12). Pre-radiotherapy ALC was independently associated with PFS in both cohorts (p 0.05 for both); post-RT ALC predicted for PFS in the traditional RT (p = 0.048) but not the SBRT (p = 0.90) group. Neither heart nor lung V5 was independently associated with PFS.When combined with immunotherapy, SBRT may better preserve lymphocytes (and hence improve outcomes) than traditional RT. When administering traditional RT, constraining the lung V5 may indirectly impact outcomes by means of ALC preservation.

Immune checkpoint inhibitors, such as anti–PD-1/PD-L1, have emerged as promising therapies for advanced non–small cell lung cancer (NSCLC). However, approximately 80% of patients do not respond to immunotherapy given alone because of intrinsic or acquired resistance. Radiotherapy (XRT) can overcome PD-1 resistance and improve treatment outcomes, but its efficacy remains suboptimal. The tyrosine phosphatase SHP-2, expressed in some cancers and in immune cells, has been shown to negatively affect antitumor immunity. Our hypothesis was that SHP-2 inhibition in combination with anti–PD-L1 would enhance immune-mediated responses to XRT and synergistically boost antitumor effects in an anti–PD-1–resistant mouse model. We treated 129Sv/Ev mice with anti–PD-1–resistant 344SQ NSCLC adenocarcinoma with oral SHP099 (a SHP-2 inhibitor) combined with XRT and intraperitoneal anti–PD-L1. Primary tumors were treated with XRT (three fractions of 12 Gy each), whereas abscopal (out-of-field) tumors were observed but not treated. XRT in combination with SHP099 and anti–PD-L1 promoted local and abscopal responses, reduced lung metastases, and improved mouse survival. XRT also increased SHP-2+ M1 tumor-associated macrophages in abscopal tumors (P = 0.019). The addition of SHP099 also associated with a higher M1/M2 ratio, greater numbers of CD8+ T cells, and fewer regulatory T cells. This triple-combination therapy had strong antitumor effects in a mouse model of anti–PD-1–resistant NSCLC and may be a novel therapeutic approach for anti–PD-1–resistant NSCLC in patients.

Purpose: Radiotherapy (RT) traditionally has been used for local tumor control in the treatment of cancer. The recent discovery that radiotherapy can have anticancer effects on the immune system has led to recognition of its ability to sensitize the tumor microenvironment to immunotherapy. However, radiation can also prompt adverse immunosuppressive effects that block aspects of systemic response at other tumor sites. Our hypothesis was that inhibition of the MER proto-oncogene tyrosine kinase (MerTK) in combination with anti-programmed cell death-1 (α-PD1) checkpoint blockade will enhance immune-mediated responses to radiotherapy. Experimental Design: We tested the efficacy of this triple therapy (Radiation + α-PD1 + α-MerTK mAbs) in 129Sv/Ev mice with bilateral lung adenocarcinoma xenografts. Primary tumors were treated with stereotactic radiotherapy (36 Gy in 3 12-Gy fractions), and tumors were monitored for response. Results: The triple therapy significantly delayed abscopal tumor growth, improved survival rates, and reduced numbers of lung metastases. We further found that the triple therapy increased the activated CD8+ and NK cells populations measured by granzyme B expression with upregulation of CD8+CD103+ tissue-resident memory cells (TRM) within the abscopal tumor microenvironment relative to radiation only. Conclusions: The addition of α-PD1 + α-MerTK mAbs to radiotherapy could alter the cell death to be more immunogenic and generate adaptive immune response via increasing the retention of TRM cells in the tumor islets of the abscopal tumors which was proven to play a major role in survival of non-small cell lung cancer patients.

Ipilimumab is effective for patients with melanoma, but not for those with less immunogenic tumors. We report a phase II trial of ipilimumab with concurrent or sequential stereotactic ablative radiotherapy to metastatic lesions in the liver or lung (NCT02239900). Ipilimumab (every 3 weeks for 4 doses) was given with radiotherapy begun during the first dose (concurrent) or 1 week after the second dose (sequential) and delivered as 50 Gy in 4 fractions or 60 Gy in 10 fractions to metastatic liver or lung lesions. In total, 106 patients received ≥1 cycle of ipilimumab with radiation. Median follow-up was 10.5 months. Median progression-free survival time was 2.9 months (95% confidence interval, 2.45–3.40), and median overall survival time was not reached. Rates of clinical benefit of nonirradiated tumor volume were 26% overall, 28% for sequential versus 20% for concurrent therapy (P = 0.250), and 31% for lung versus 14% for liver metastases (P = 0.061). The sequential lung group had the highest rate of clinical benefit at 42%. There were no differences in treatment-related adverse events between groups. Exploratory analysis of nontargeted lesions revealed that lesions receiving low-dose radiation were more likely to respond than those that received no radiation (31% vs. 5%, P = 0.0091). This phase II trial of ipilimumab with stereotactic radiotherapy describes satisfactory outcomes and low toxicities, lending support to further investigation of combined-modality therapy for metastatic cancers.

Despite multiple therapeutic approaches, the presence of liver metastases carries a guarded prognosis, urgently necessitating further clinical and scientific research to develop curative interventions. The liver is an immunoprivileged organ that suppresses the effectiveness of immunotherapies in patients with hepatic metastases. Cancer immunotherapies have been successfully bolstered by low-dose radiotherapy (LDRT), which is capable of reprogramming the tumor microenvironment (TME) from an immunosuppressive to an immunostimulatory one. Likewise, LDRT may be able to revoke the immune privilege enjoyed by the liver, permitting successful immunotherapies there. Here, we first review challenges that face the treatment of liver metastases. We next outline emerging preclinical and clinical evidence supporting enhanced systemic tumor control of LDRT in the context of cancer immunotherapy. Finally, we will discuss the rationale of combining liver-directed LDRT with immunostimulatory strategies to overcome immune resistance and achieve better clinical response. This notion is supported by a recent case study in which a patient who had progressed following T cell therapy experienced a complete response after LDRT to the liver.

Purpose The limitation of hypofractionated radiation efficacy is due partly to the immunosuppressive tumor microenvironment. Indoleamine 2,3-dioxygenase 1 (IDO1) is an important regulator of tumor immune suppression. We evaluated the effects of IDO1 in hypofractionated radiation using a Lewis lung carcinoma (LLC) mouse model and tested whether IDO1 inhibition could sensitize those tumors to hypofractionated radiation. Methods and Materials Bilateral LLC tumors were established in C57BL/6 mice. Primary tumors were treated with 3 fractions of either 12 Gy or 6 Gy, and the IDO1 inhibitor INCB023843 was given starting on the first day of radiation. Plasma tryptophan and kynurenine levels were quantified by liquid chromatography and tandem mass spectrometry. Tumor-infiltrating immune cells were isolated from the tumors, stained, and quantified by flow cytometry. Results The combination of INCB023843 and three 12-Gy fractions led to better tumor control and survival than radiation alone; INCB023843 plus three 6-Gy fractions had no benefit. IDO1 expression by tumor-infiltrating immune cells was increased by three 12-Gy doses and inhibited by the addition of INCB023843. Nearly all IDO1+ immune cells were also F4/80+. Percentages of IDO1+F4/80+ immune cells were drastically increased by three 12-Gy fractions and by three 6-Gy fractions, but only INCB023843 combined with three 12-Gy fractions reduced those percentages. IDO1+F4/80+ immune cells were further found to be CD11b+, Gr1-intermediate-expressing, CD206–, and CD11c– (ie, myeloid-derived suppressor cells). Three 12-Gy fractions also increased the percentages of tumor-infiltrating T regulatory cells and CD8+ T cells, but adding INCB023843 did not affect those percentages. Conclusions In addition to its immune activation effects, hypofractionated radiation induced “rebound immune suppression” in the tumor microenvironment by activating and recruiting IDO1-expressing myeloid-derived suppressor cells in a dose-dependent manner. Adding an IDO1 inhibitor to hypofractionated radiation reduced the percentages of these cells, overcame the immune suppression, and sensitized LLC tumors to hypofractionated radiation.

Costimulation through 4-1BB (CD137) receptor generates robust CD8+ T-effector and memory responses. The only known ligand, 4-1BBL, is a trimeric transmembrane protein that has no costimulatory activity as a soluble molecule. Thus, agonistic antibodies to the receptor have been used for cancer immunotherapy in preclinical models and are currently being evaluated in the clinic. Here, we report that treatment with an oligomeric form of the ligand, SA-4-1BBL, as a single agent is able to protect mice against subsequent tumor challenge irrespective of the tumor type. Protection was long-lasting (>8 weeks) and a bona fide property of SA-4-1BBL, as treatment with an agonistic antibody to the 4-1BB receptor was ineffective in generating immune protection against tumor challenge. Mechanistically, SA-4-1BBL significantly expanded IFNγ-expressing, preexisting memory-like CD44+CD4+ T cells and NK cells in naïve mice as compared with the agonistic antibody. In vivo blockade of IFNγ or depletion of CD4+ T or NK cells, but not CD8+ T or B cells, abrogated the immunopreventive effects of SA-4-1BBL against cancer. SA-4-1BBL as a single agent also exhibited robust efficacy in controlling postsurgical recurrences. This work highlights unexpected features of SA-4-1BBL as a novel immunomodulator with implications for cancer immunoprevention and therapy. Significance: This study demonstrates the unique and unexpected immunomodulatory features of SA-4-1BBL that bridge innate and adaptive immune responses with both preventive and therapeutic efficacy against cancer.

Purpose/Objective(s) Despite advances in radiotherapy (RT) and immunotherapy, their antitumor activity remains suboptimal in metastatic non-small cell lung cancer (NSCLC). The tumor microenvironment after hypofractionated RT (hRT) is largely immunosuppressive and replete with M2 tumor-associated macrophages. STAT6 is a highly activated transcription factor that contributes to M2 macrophage polarization but has been difficult to target therapeutically. We selectively targeted STAT6 with antisense nucleotides (ASOs), hypothesizing that using ASOs to inhibit STAT6 in combination with hRT and anti-PD1 would inhibit M2 polarization and improve systemic antitumor effects in NSCLC. Materials/Methods We tested three bilaterally established lung cancer murine models: Lewis lung carcinoma in B6.Foxp3EGFP mice, and 344SQ-parental or 344SQ anti-PD1-resistant adenocarcinomas in 129Sv/Ev mice. The primary tumor was treated with hRT (3 fractions of 12 Gy each) and secondary tumors were observed for evidence of abscopal effects. STAT6 knockdown was determined by Western blotting and qRT-PCR. Tumor-infiltrating leukocytes were characterized by immunohistochemical tissue microarray-based immunofluorescence, flow cytometry, and 770 gene-Immune NanoString analyses. Also, serum TGF-β was measured by ELISA. Results The combination of hRT and STAT6 ASOs reduced the volumes of both primary and secondary tumors and extended survival (vs. either treatment alone). The addition of anti-PD1 enhanced the systemic antitumor effect (vs. STAT6 ASOs + hRT or hRT+anti-PD1) and led to decreased STAT6, increased M1 genes (IL1R, iNOS, CD80, CD86), and lowered M2 genes (Arg-1, Fizz-1, CD163, CCR2) in tumor-infiltrating leukocytes by qRT-PCR. Treatment with hRT+STAT6 ASOs ± anti-PD1 also enhanced tumor infiltration by macrophages and Th1 cells, improved T-cell and macrophage function, and slowed cancer progression. Flow cytometry and tissue microarray confirmed the hRT (vs. control) led to higher STAT6 and lower M1/M2 ratio, but hRT+ STAT6 ASOs ± anti-PD1 led to decreased STAT6, reduced Tregs, increased M1/M2 ratio, and lowered TGF-β levels. Depletion of macrophages by anti-F4/80 abrogated the observed antitumor effect. Conclusion The combination of STAT6 ASOs and hRT reduces immunosuppressive M2 macrophage infiltration into tumors and enhances the systemic antitumor effect of hRT; this radio-immunotherapy combination may provide an alternative therapeutic approach for NSCLC patients with PD1-resistance.

PURPOSE/OBJECTIVE(S) Radiation therapy (XRT) induces lymphocyte infiltration of primary tumors that is increased when using NBTXR3 radioenhancer nanoparticle, leading to improved response to combination immunotherapy (IT). However, treatment effects on the metastatic tumor microenvironment are poorly understood. We studied the effects of NBTXR3 injection into primary tumors plus XRT plus dual-agent immunotherapy on secondary tumor-infiltrating T cells in a mouse model of lung cancer. The hypothesis is that intratumoral primary tumor treatment with NBTXR3 nanoparticle enriches conserved T cell receptor repertoires among metastatic tumor-infiltrating lymphocytes. MATERIALS/METHODS Four groups of 4 mice each were inoculated subcutaneously with anti-PD1-resistant 344SQR murine lung cancer cells in each hind leg, 3 days apart, to establish 'primary' (right) and 'secondary' (left, which represented metastasis) tumors. All mice received intraperitoneal anti-PD1 and anti-CTLA-4 (IT) on days 4, 7, 10, and 13, and 12-Gy high-dose XRT (HD) to the primary tumors on days 7, 8 and 9. Primary tumors in groups 2 and 4 also received intratumoral NBTXR3 on day 6. Secondary tumors in groups 3 and 4 were irradiated with 1-Gy low-dose XRT (LD) on days 12 and 13. Treatment groups were designated as 1 = HD+IT, 2 = NBTXR3+HD+IT, 3 = HD+LD+IT, and 4 = NBTXR3+HD+LD+IT. All secondary tumors were harvested on day 19 for tumor-infiltrating T-cell RNA extraction and sequencing on an Illumina MiSeq. T-cell receptor (TCR) beta complementarity-determining region 3 (CDR3b) repertoires were analyzed with MiXCR v3.0.12 software, and CDR3b analyses and Circos plots were generated using software. P < 0.05 from Mann-Whitney U tests were considered statistically significant. RESULTS NBTXR3 significantly increased the proportion of conserved CDR3b clonotypes within the secondary tumors among mice that received only HD XRT to the primary tumors (group 2 vs 1, P = 0.0476). When the secondary tumors were exposed to LD XRT, NBTXR3 still increased the frequency of overlapping clonotypes although not significantly (group 4 vs 3, P = 0.3095). Most interestingly, there were significantly more conserved CDR3b repertoires from the secondary tumors that were shared between groups 2 and 4 that received NBTXR3 compared to those shared between groups 1 and 3 (P < 0.0001). Comparisons of CDR3b normalized Shannon clonality indices and Circos plots did not reveal any difference in repertoire diversity between NBTXR3-treated and non-NBTXR3-treated groups. CONCLUSION NBTXR3 radioenhancer nanoparticle, given with immunoradiation, might reshape the TCR repertoire of metastatic lung cancer tumor-infiltrating T cells by driving the selection of conserved CDR3b clonotypes.

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. Materials/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.

Purpose Radiation combined with PD1 blockade offers significant treatment benefits in several tumor types; however, anti-PD1 resistance precludes such benefits in many cases. Here we attempted to overcome anti-PD1 resistance by combining localized radiation with a radioenhancing nanoparticle (NBTXR3) and systemic anti-PD1 treatment to achieve abscopal effects in an anti-PD1–resistant mouse model of lung cancer. Methods and Materials Female 129Sv/Ev mice were inoculated with 344SQ anti-PD1–resistant (344SQR) or anti-PD1–sensitive (344SQP) metastatic lung cancer cells in the right leg on day 0 (“primary” tumor) and the left leg on day 4 (“secondary” tumor). Primary tumors were injected intratumorally with NBTXR3 on day 7 and were irradiated with 12 Gy on days 8, 9, and 10. Mice were given 6 intraperitoneal injections of anti-PD1. T cell receptor repertoire was analyzed in tumor samples with RNA sequencing, infiltration of CD8 T cells with immunohistochemical staining, and activities of various immune pathways with NanoString analysis. Results The triple combination of NBTXR3 with localized radiation and systemic anti-PD1 significantly delayed the growth of both irradiated and unirradiated tumors in both 344SQP and 344SQR tumor models. NBTXR3 remodeled the immune microenvironment of unirradiated tumors by triggering the activation of various immune pathways, increasing the number of CD8+ T cells, and modifying the T cell receptor repertoire in the 344SQR tumor model. Conclusions The ability of NBTXR3 to evoke significant abscopal effects in both anti-PD1–sensitive and anti-PD1–resistant lung cancers could open the possibility of its use for treating patients with metastatic lung cancer regardless of sensitivity (or resistance) to immunotherapies.

Highlights • High-dose RT upregulated pDCs within the tumor microenvironment. • The administration of intratumoral TLR9 agonist (CMP-001) after stereotactic RT significantly enhanced the anti-tumor immune response both locally and at secondary tumor site. • CMP-001 Post-RT delayed the abscopal tumor growth and extended the survival rate via increasing the percentages of activated CD4+ and CD8+ T-cells within the tumor microenvironment. • The treatment proved efficacious in both lung adenocarcinoma and colon carcinoma syngeneic models used., Radiotherapy (RT) has been used to control tumors by physically damaging DNA and inducing apoptosis; it also promotes antitumor immune responses via neoantigens release and augmenting immune-oncology agents to elicit systemic response. Tumor regression after RT can recruit inflammatory cells, such as tumor-associated macrophages and CD11b+ myeloid cell populations, a major subset of which may actually be immunosuppressive. However, these inflammatory cells also express Toll-like receptors (TLRs) that can be stimulated to reverse suppressive characteristics and promote systemic antitumor outcomes. Here, we investigated the effects of adding CMP-001, a CpG-A oligodeoxynucleotide TLR9 agonist delivered in a virus-like particle (VLP), to RT in two murine models (344SQ metastatic lung adenocarcinoma and CT26 colon carcinoma). High-dose RT (12Gy x 3 fractions) significantly increased the percentages of plasmacytoid dendritic cells within the tumor islets 3- and 5-days post-RT; adding CMP-001 after RT also enhanced adaptive immunity by increasing the proportion of CD4+ and CD8+ T cells. RT plus CMP-001-mediated activation of the immune system led to significant inhibition of tumor growth at both primary and abscopal tumor sites, thereby suggesting a new combinatorial treatment strategy for systemic disease., Graphical Abstract Image, graphical abstract

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. Acknowledgements This 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.

Indoleamine 2,3-dioxygenase 1 (IDO1), involved in the catabolism of tryptophan (Trp) to kynurenine (Kyn) is an important regulator of tumor-mediated immunosuppression implicated in resistance to anti-PD1 immunotherapy. We investigated the role of IDO1 in an anti-PD1-resistant lung cancer model (344SQ_R) compared to the parental 344SQ tumors (344SQ_P). IDO1 was overexpressed in tumor-infiltrating leukocytes, and plasma Kyn levels were increased, in 344SQ_R vs. 344SQ_P. The IDO1 inhibitor INCB023843 retarded tumor growth and reduced lung metastases in 344SQ_R. IDO1 was expressed at higher levels in F4/80+Gr1intCD11b+ myeloid-derived suppressor cells (MDSCs) that were prominent in 344SQ_R. The INCB023843 reduced IDO1 expression and percentages of these MDSCs while increasing CD8+ T cells infiltration, hence reactivating antitumor T-cell responses in 344SQ_R. Therefore, IDO1 inhibition holds promise for treating lung cancer that does not respond to anti-PD1 therapy.

BackgroundThis study compared response rates and outcomes of combined radiotherapy and immunotherapy (iRT) based on the type of checkpoint inhibitor (anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) vs antiprogrammed death-1 (PD1)) for metastatic non-small cell lung cancer (mNSCLC).MethodsWe retrospectively reviewed two prospective trials of radiation combined with anti-CTLA4 or anti-PD1 for patients with mNSCLC. Patients undergoing non-salvage stereotactic body radiation therapy (SBRT) to lung sites were selected from both trials and grouped by the immunotherapeutic compound received. Endpoints included in-field and out-of-field response rates, and overall response rate (complete or partial response) (all by response evaluation criteria in solid tumors). Progression-free survival (PFS) and overall survival (OS) were estimated with the Kaplan-Meier method.ResultsMedian follow-up times for the 33 patients (n=17 SBRT+anti-CTLA4, n=16 SBRT+anti-PD1) were 19.6 and 19.9 months. Response rates for out-of-field lesions were similar between anti-PD1 (37%) and anti-CTLA4 (24%) (p=0.054). However, global response rates for all lesions were 24% anti-CTLA4 vs 56% anti-PD1 (p=0.194). The PFS was 76% for anti-CTLA4 vs 94% anti-PD1 at 3 months, 52% vs 87% at 6 months, 31% vs 80% at 12 months, and 23% vs 63% at 18 months (p=0.02). Respective OS values were 76% vs 87% at 6 months, 47% vs 80% at 12 months, and 39% vs 66% at 18 months (p=0.08).ConclusionsBoth anti-CTLA4 and anti-PD1 agents prompt a similar degree of in-field and out-of-field responses after iRT, although the global response rate and PFS were statistically higher in the anti-PD1 cohort. Further dedicated study and biological mechanistic assessment is required.Trial registration numbersNCT02239900andNCT02444741.

Radiotherapy is a component of the standard of care for many patients with locally advanced nonmetastatic tumors and increasingly those with oligometastatic tumors. Despite encouraging advances in local control and progression-free and overall survival outcomes, continued manifestation of tumor progression or recurrence leaves room for improvement in therapeutic efficacy. Novel combinations of radiation with immunotherapy have shown promise in improving outcomes and reducing recurrences by overcoming tumor immune tolerance and evasion mechanisms via boosting the immune system's ability to recognize and eradicate tumor cells. In this review, we discuss preclinical and early clinical evidence that radiotherapy and immunotherapy can improve treatment outcomes for locally advanced and metastatic tumors, elucidate underlying molecular mechanisms and address strategies to optimize timing and sequencing of combination therapy for maximal synergy.

Purpose/Objective(s) Intratumoral pressure creates a major barrier for delivery and retention of immuno-oncology drugs, such as checkpoint inhibitors. A strong pressure differential between the center region and outer region of the tumor produces a convection force that prevents the diffusion of therapeutic agents into the tumor. Radiation (XRT), a cornerstone of cancer treatment and immune system activator, is an attractive strategy to reduce intratumoral pressure prior to drug delivery. Here, we explored the effects of XRT on intratumoral pressure in a mouse model for lung cancer. Materials/Methods 344SQ-parental lung adenocarcinoma tumors (0.5 × 106 cells) were subcutaneously implanted into the legs of 8- to 12-week-old 129Sv/Ev mice. When tumors reached 7-8 mm in diameter, they were irradiated locally with XRT (two fractions of 1 Gy, three fractions of 5 Gy, 8 Gy or 12 Gy, or one fraction of 20 Gy) from a Cesium source. The different fractions were scheduled such that all treatments were completed on Day 10 after tumor implantation. Intratumoral pressure was measured using a pressure transducer device at six different time points (Days 11, 13, 18, 22, 27, 33), following 24 hours after the last fraction of XRT. Results Low dose radiation (two fractions of 1 Gy) and higher XRT doses (three fractions of 12 Gy, one fraction of 20 Gy) reduced intratumoral pressure levels between days 11 and 13 and 13 and 22, respectively, compared to control tumors that were not irradiated. An unpaired t-test was used to compare the mean pressure between study groups. On day 13, the mean intratumoral pressure for tumors irradiated with two fractions of 1 Gy was 69.6 mmHg versus 111.4 mmHg in controls (P = 0.06). On day 22, the mean intratumoral pressure for tumors irradiated with three fractions of 12 Gy was 64.2 mmHg versus 119.2 mmHg in controls (P = 0.01); tumors irradiated with one fraction of 20 Gy had mean pressures of 39.3 mmHg on day 22 (P = 0.001). Intermediate doses of XRT (three fractions of 5 Gy or 8 Gy) failed to reduce intratumoral pressure in the measurement period. Conclusion XRT is widely being explored, pre-clinically and clinically, in combination with immuno-oncology agents due to synergistic effects of these therapies. Optimized XRT doses can help overcome the pressure barrier for successful intratumoral delivery of immuno-oncology agents. Further work will examine how the intratumoral pressure reduction improves retention of macromolecules injected directly into tumors.

PURPOSE/OBJECTIVE(S) To report updated findings from a prospective phase II trial of salvage high-dose radiotherapy (HD-RT) with or without low-dose RT (LD-RT) for metastatic cancer (NCT02710253). MATERIALS/METHODS Eligible patients had metastatic disease that progressed on immunotherapy within the prior 6 months. Patients were given either HD-RT (20-70Gy total; 3-12.5Gy/fraction), or HD-RT and LD-RT to separate lesions (LD-RT comprised of < 2Gy/fraction up to 10Gy total), with continued immunotherapy. Radiographic response was assessed per RECIST1.1 and Immune-Related Response Criteria (irRC). Primary endpoints were 1) 4-month disease control (complete/partial response [CR/PR] or stable disease [SD]) or an overall response (CR/PR) at any point in ≥10% of patients, and 2) dose-limiting toxicity within 3 months not exceeding 30%. Also, where applicable, biopsies pre- and post- low-dose RT were collected, FFPE slides were prepared and analyzed by Multiplex Immunofluorescence (Multi-IF) microscopy for immune-cell infiltration. RESULTS Seventy-four patients were analyzed (39 in HD-RT group and 35 in HD-RT+LD-RT group), mostly with non-small cell lung cancer (n = 38) or melanoma (n = 21). Patients received immunotherapy for a median of 7.1 months (3.7-10.6 months) before RT, and 95% maintained the same therapy after RT. The median follow-up time was 13.8 months. The site most commonly treated with HD- or LD-RT was lung/mediastinum. The primary endpoint was met for the 72 evaluable patients, with a 4-month overall disease control rate of 42% (RECIST1.1, 47% [16/34] for the HD-RT+LD-RT group and 37% [14/38] for the HD-RT only group). Three (4.2%) patients experienced grade ≥3 toxicity. LD-RT treated lesion response rates (irRC, 53%) was improved compared to nonirradiated lesions in the HD-RT+LD-RT (23%, P = 0.002) and HD-RT only (11%, P < 0.001) group. The median PFS was 7.0 months vs. 5.8 months, while median OS was 15.9 months vs. 10.5 months for the HD-RT+LD-RT group vs. HD-RT only group respectively; however, statistical significance was not reached. Furthermore, the addition of LD-RT did not significantly affect the absolute lymphocyte counts (ALC), with ALC reduction after treatment recorded as 420/μL vs. 340/μL in the HD-RT only vs. HD-RT+LD-RT groups (P = 0.44). The Multi-IF data for 3 analyzed biopsies of LD-RT lesions showed strong correlation between T cell and NK cell infiltration and clinical response rate. Lesion 1 for example (24% SD), had CD56+ NK cell numbers increase from 67 to 1837 n/mm2 post LD-RT. Lesion 2 (-80% PR) had its CD3+CD4+ T cells increase from 287 to 1233 n/mm2 and NK cells increase from 0 to 187 n/mm2; while lesion 3 (-8% SD) showed an increase from 79 to 131 n/mm2 and 3.6 to 7.3 n/mm2 in the T cell and NK cell compartments respectively. CONCLUSION Low-dose RT is an effective and safe way to enhance individual lesion-specific response rates among progressing patients with solid tumors, with the ability to improve the infiltration of effector immune cells into the tumor microenvironment.

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.