Enhanced tumor control and survival in preclinical models with adoptive cell therapy preceded by low-dose radiotherapy.

Introduction: Effective infiltration of chimeric antigen receptor T (CAR-T) cells into solid tumors is critical for achieving a robust antitumor response and improving therapeutic outcomes. While CAR-T cell therapies have succeeded in hematologic malignancies, their efficacy in solid tumors remains limited due to poor tumor penetration and an immunosuppressive tumor microenvironment. This study aimed to evaluate the potential of low-dose radiotherapy (LDRT) administered before T-cell therapy to enhance the antitumor effect by promoting CAR-T cell infiltration. We hypothesized that combining LDRT with T-cell therapy would improve tumor control and survival compared to either treatment alone. Methods: We investigated this hypothesis using two NSG mouse models bearing GSU or CAPAN-2 solid tumors. The mice were treated with engineered CAR-T cells targeting guanyl cyclase-C (GCC) or mesothelin as monotherapy or in combination with LDRT. Additionally, we extended this approach to a C57BL/6 mouse model implanted with MC38-gp100+ cells, followed by adoptive transfer of pmel+ T cells before and after LDRT. Tumor growth and survival outcomes were monitored in all models. Furthermore, we employed atomic force microscopy (AFM) in a small cohort to assess the effects of radiotherapy on tumor stiffness and plasticity, exploring the role of tumor nanomechanics as a potential biomarker for treatment efficacy. Results: Our results demonstrated enhanced tumor control and prolonged survival in mice treated with LDRT followed by T-cell therapy across all models. The combination of LDRT with CAR-T or pmel+ T-cell therapy led to superior tumor suppression and survival compared to monotherapy, highlighting the synergistic impact of the combined approach. Additionally, AFM analysis revealed significant changes in tumor stiffness and plasticity in response to LDRT, suggesting that the nanomechanical properties of the tumor may be predictive of therapeutic response. Discussion: The findings of this study highlight the transformative potential of incorporating LDRT as a precursor to adoptive T-cell therapy in solid tumors. By promoting CAR-T and pmel+ T-cell infiltration into the tumor microenvironment, LDRT enhanced tumor control and improved survival outcomes, offering a promising strategy to overcome the challenges associated with CAR-T therapy in solid tumors. Additionally, the changes in tumor nanomechanics observed through AFM suggest that tumor stiffness and plasticity could be biomarkers for predicting treatment outcomes. These results support further investigation into the clinical application of this combined approach to improve the efficacy of cell-based therapies in patients with solid tumors.

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.

Is Single-Site Radiation Therapy Enough to Augment the Immune System and Enhance Immunotherapy for Metastatic Disease?

Despite the promise of combining immunotherapy and radiotherapy (RT) for metastatic cancers, existing randomized data have not been consistent on whether RT to a single irradiated site improves clinical outcomes. Mechanistically, this could result from a low quantity/diversity of tumor antigens released for immune detection, immunosuppressive molecules released by tumor masses, and the lack of immune infiltration into tumor bulk. Herein, multi-site RT is discussed as a potential solution, given that it can directly improve upon each of the mechanistic issues. Just as it is illogical to use systemic therapy alone in place of a dedicated local therapeutic option (e.g., RT) for most stage II-III malignancies, so too is illogical to irradiate one site only in case of metastatic neoplasms instead of implementing systemic therapy and/or multi-site RT. Although it may theoretically be possible to address all systemic disease with systemic therapy, that notion assumes that all areas of systemic disease will be responsive to systemic therapy in the first place. However, in reality, certain sites may develop innate or acquired resistance to systemic therapy, hence opening the door to multi-site localized treatment strategies. Further investigation is required to address whether multi-site RT would be effective in the setting of suboptimal immune function and/or resistance/refractoriness to multiple prior systemic therapies. Methods to improve the effectiveness of multi-site RT are also discussed, such as ablatively-/definitively-dosed RT, along with staggered timing of RT administration (pulsed RT).

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.

NLRP3 agonist enhances radiation-induced immune priming and promotes abscopal responses in anti-PD1 resistant model.

Radiotherapy (XRT), a well-known activator of the inflammasome and immune priming, is in part capable of reversing resistance to anti-PD1 treatment. The NLRP3 inflammasome is a pattern recognition receptor which is activated by both exogenous and endogenous stimuli, leading to a downstream inflammatory response. Although NLRP3 is typically recognized for its role in exacerbating XRT-induced tissue damage, the NLRP3 inflammasome can also yield an effective antitumor response when used in proper dosing and sequencing with XRT. 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 the control of implanted lung adenocarcinoma primary as well as secondary tumors in a radiological dose-dependent manner, in which 12Gyx3 fractions of stereotactic XRT was better than 5Gyx3, while 1Gyx2 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.

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.

The role of tumor metabolism in modulating T-Cell activity and in optimizing immunotherapy.

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.

NLRP3 agonist enhances radiation-induced immune priming and promotes abscopal responses in anti-PD1 resistant model.

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.

Radiation Therapy Modulates Tumor Physical Characteristics to Reduce Intratumoral Pressure and Enhance Intratumoral Drug Delivery and Retention.

Purpose: High intratumoral pressure, caused by tumor cell-to-cell interactions, interstitial fluid pressure, and surrounding stromal composition, plays a substantial role in resistance to intratumoral drug delivery and distribution. Radiation therapy (XRT) is commonly administered in conjunction with different intratumoral drugs, but assessing how radiation can reduce pressure locally and help intratumoral drug administration and retention is important. Methods and Materials: 344SQ-parental or 344SQ-anti-programmed cell death protein 1-resistant lung adenocarcinoma cells were established in 129Sv/Ev mice, and irradiated with either 1 Gy × 2, 5 Gy × 3, 8 Gy × 3, 12 Gy × 3, or 20 Gy × 1. Intratumoral pressure was measured every 3 to 4 days after XRT. Contrast dye was injected into the tumors 3- and 6-days after XRT, and imaged to measure drug retention. Results: In the 344SQ-parental model, low-dose radiation (1 Gy × 2) created an early window of reduced intratumoral pressure 1 to 3 days after XRT compared with untreated control. High-dose stereotactic radiation (12 Gy × 3) reduced intratumoral pressure 3 to 12 days after XRT, and 20 Gy × 1 showed a delayed pressure reduction on day 12. Intermediate doses of radiation did not significantly affect intratumoral pressure. In the more aggressive 344SQ-anti-programmed cell death protein 1-resistant model, low-dose radiation reduced pressure 1 to 5 days after XRT, and 12 Gy × 3 reduced pressure 1 to 3 days after XRT. Moreover, both 1 Gy × 2 and 12 Gy × 3 significantly improved drug retention 3 days after XRT; however, there was no significance detected 6 days after XRT. Lastly, a histopathologic evaluation showed that 1 Gy × 2 reduced collagen deposition within the tumor, and 12 Gy × 3 led to more necrotic core and higher extracellular matrix formation in the tumor periphery. Conclusions: Optimized low-dose XRT, as well as higher stereotactic XRT regimen led to a reduction in intratumoral pressure and increased drug retention. The findings from this work can be readily translated into the clinic to enhance intratumoral injections of various anticancer agents.

Inhibition of STAT6 with Antisense Oligonucleotides Enhances the Systemic Antitumor Effects of Radiotherapy and Anti-PD-1 in Metastatic Non-Small Cell Lung Cancer.

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 TGFß 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.

Corrigendum: Lipid metabolism in tumor immunology and immunotherapy.

[This corrects the article DOI: 10.3389/fonc.2023.1187279.].

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.

Bone morphogenetic proteins, activins, and growth and differentiation factors in tumor immunology and immunotherapy resistance.

The TGF-ß superfamily is a group of secreted polypeptides with key roles in exerting and regulating a variety of physiologic effects, especially those related to cell signaling, growth, development, and differentiation. Although its central member, TGF-ß, has been extensively reviewed, other members of the family-namely bone morphogenetic proteins (BMPs), activins, and growth and differentiation factors (GDFs)-have not been as thoroughly investigated. Moreover, although the specific roles of TGF-ß signaling in cancer immunology and immunotherapy resistance have been extensively reported, little is known of the roles of BMPs, activins, and GDFs in these domains. This review focuses on how these superfamily members influence key immune cells in cancer progression and resistance to treatment.

Pulsed radiotherapy to mitigate high tumor burden and generate immune memory.

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.

Combining a nanoparticle-mediated immunoradiotherapy with dual blockade of LAG3 and TIGIT improves the treatment efficacy in anti-PD1 resistant lung cancer.

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 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 into the primary tumors on day 7, which were irradiated with 12 Gy on days 8, 9, and 10. Anti-PD1 (200 µg), αLAG3 (200 µg), and αTIGIT (200 µg) 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 radiotherapy and simultaneous blockade of multiple immune checkpoint receptors and provide a pre-clinical rationale for investigating its translation into human patients.

High Plus Low Dose Radiation Strategy in Combination with TIGIT and PD1 Blockade to Promote Systemic Antitumor Responses.

Tumors deploy various immune-evasion mechanisms that create a suppressive environment and render effector T-cells exhausted and inactive. Therefore, a rational utilization of checkpoint inhibitors may alleviate exhaustion and may partially restore antitumor functions. However, in high-tumor-burden models, the checkpoint blockade fails to maintain optimal efficacy, and other interventions are necessary to overcome the inhibitory tumor stroma. One such strategy is the use of radiotherapy to reset the tumor microenvironment and maximize systemic antitumor outcomes. In this study, we propose the use of anti-PD1 and anti-TIGIT checkpoint inhibitors in conjunction with our novel RadScopal technique to battle highly metastatic lung adenocarcinoma tumors, bilaterally established in 129Sv/Ev mice, to mimic high-tumor-burden settings. The RadScopal approach is comprised of high-dose radiation directed at primary tumors with low-dose radiation delivered to secondary tumors to improve the outcomes of systemic immunotherapy. Indeed, the triple therapy with RadScopal + anti-TIGIT + anti-PD1 was able to prolong the survival of treated mice and halted the growth of both primary and secondary tumors. Lung metastasis counts were also significantly reduced. In addition, the low-dose radiation component reduced TIGIT receptor (PVR) expression by tumor-associated macrophages and dendritic cells in secondary tumors. Finally, low-dose radiation within triple therapy decreased the percentages of TIGIT+ exhausted T-cells and TIGIT+ regulatory T-cells. Together, our translational approach provides a new treatment alternative for cases refractory to other checkpoints and may bring immunotherapy into a new realm of systemic disease control.

A radioenhancing nanoparticle mediated immunoradiation improves survival and generates long-term antitumor immune memory in an anti-PD1-resistant murine lung cancer model.

BACKGROUND: Combining radiotherapy with PD1 blockade has had impressive antitumor effects in preclinical models of metastatic lung cancer, although anti-PD1 resistance remains problematic. Here, we report results from a triple-combination therapy in which NBTXR3, a clinically approved nanoparticle radioenhancer, is combined with high-dose radiation (HDXRT) to a primary tumor plus low-dose radiation (LDXRT) to a secondary tumor along with checkpoint blockade in a mouse model of anti-PD1-resistant metastatic lung cancer. METHODS: Mice were inoculated with 344SQR cells in the right legs on day 0 (primary tumor) and the left legs on day 3 (secondary tumor). Immune checkpoint inhibitors (ICIs), including anti-PD1 (200 µg) and anti-CTLA4 (100 µg) were given intraperitoneally. Primary tumors were injected with NBTXR3 on day 6 and irradiated with 12-Gy (HDXRT) on days 7, 8, and 9; secondary tumors were irradiated with 1-Gy (LDXRT) on days 12 and 13. The survivor mice at day 178 were rechallenged with 344SQR cells and tumor growth monitored thereafter. RESULTS: NBTXR3 + HDXRT + LDXRT + ICIs had significant antitumor effects against both primary and secondary tumors, improving the survival rate from 0 to 50%. Immune profiling of the secondary tumors revealed that NBTXR3 + HDXRT + LDXRT increased CD8 T-cell infiltration and decreased the number of regulatory T (Treg) cells. Finally, none of the re-challenged mice developed tumors, and they had higher percentages of CD4 memory T cells and CD4 and CD8 T cells in both blood and spleen relative to untreated mice. CONCLUSIONS: NBTXR3 nanoparticle in combination with radioimmunotherapy significantly improves anti-PD1 resistant lung tumor control via promoting antitumor immune response.

Pulsed Radiation Therapy to Improve Systemic Control of Metastatic Cancer.

Radiation therapy (RT) is emerging as an interventional modality in the cancer-immunity cycle, augmenting the activation of an adaptive immune response against tumors. RT, particularly in combination with immunotherapy, can enhance immune memory effects and shape the tumor-directed T-cell populations. However, a single cycle of RT delivered to a limited number of polymetastatic lesions is rarely sufficient to achieve systemic control. We hypothesize that several rounds of RT, akin to several rounds of immunotherapeutic drugs, is likely to provide greater clinical benefit to patients with metastatic disease. We propose that the repeated exposure to tumor antigens released by "pulsed-RT" (i.e., treating 2-4 tumor lesions with 3 irradiation cycles given one month apart) may amplify the adaptive immune response by expanding the tumor-specific T-cell receptor repertoire, the production of high-affinity tumor antibodies, and the generation of memory lymphocytes and thereby improve immune control of systemic disease.