Exploratory Study Of Using Low-dose Radiotherapy,STAT6 ASO To Improve The Efficacy Of Radioimmunotherapy

BackgroundImmunotherapy,represented by immune checkpoint inhibitors,has changed the traditional treatment of cancers.However,there are a lot of patients resistant to the monotherapy of immune checkpoint inhibitor in the clinical practice.Studies have shown that the addition of radiotherapy can significantly enhance the efficacy of immune checkpoint inhibitors.Radiation therapy is able to convert tumors at the irradiated site into an in-situ tumor vaccine and further enhance the immune response through inflammatory cytokines directly stimulated by radiation.The combination of radiotherapy and immunotherapy,known as radioimmunotherapy(iRT),can not only enhance the regression of tumors at the irradiated site,but also improve the efficacy of the distant,unirradiated tumors,that is,the "Abscopal effect".However,more than half of patients can not get benefit from iRT.Therefore,it is urgent to explore new combination treatment to overcome immunotherapy resistance and further improve the anti-tumor efficacy of iRT.Mechanically,the formation of immunosuppressive tumor stroma(such as tumorassociated fibroblasts,etc.)and the recruitment of immunosuppressive cells(such as M2 macrophages,etc.)into tumor microenvironment after RT are the main reasons for the inhibition of effective immune cells entering the tumor,which leading to iRT treatment resistance.Preclinical studies have shown that low-dose radiotherapy modulates the immunosuppressive stroma by downregulating TGF-β,increasing the M1/M2 macrophage ratio,and significantly enhancing the infiltration of effector CD4+T cells and NK cells,improving the systemic antitumor effect of iRT.The efficacy of artificially administered low-dose radiation to abscopal lesions has not been validated in clinical trials,although retrospective analyses have shown improved local response to low-dose scatter lesions near areas of high-dose radiation.In addition,M2 tumor-associated macrophages activated by the STAT6 signaling pathway are closely related to radiotherapy resistance and immunotherapy progression,but traditional STAT6 small molecule drugs are difficult to achieve the desired inhibitory effect.Therefore,from two perspectives of enhancing anti-tumor immune cell infiltration and inhibiting intratumor M2 macrophages,this study used low-dose radiotherapy and STAT6 inhibitor(STAT6 ASO)respectively to improve the efficacy of iRT.In addition,this study initially explored strategies for improving the efficacy of iRT in specific tumor backgrounds,such as liver metastases and multiple metastases.Part Ⅰ:Clinical study of using low-dose RT to improve the efficacy of iRTObjectivesBased on the rationale that low-dose radiotherapy can enhance the effect of anti-tumor immune cell infiltration,we firstly used the strategy of combining low-dose and high-dose radiotherapy and conducted a prospective phase Ⅱ trial(NCT02710253)in immunotherapyresistant advanced patients at MD Anderson Cancer Center to investigate whether low-dose radiotherapy can improve the efficacy of iRT in immunotherapy-resistant patients.MethodsEligible patients had metastatic disease that progressed on immunotherapy within 6 months.Patients were given either high-dose radiotherapy(20-70 Gy total;3-12.5 Gy/fraction),or high-dose+low-dose radiotherapy(0.5-2 Gy/fraction 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 complete/partial response or stable disease)or an overall response(complete/partial response)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.ResultsSeventy-four patients(NSCLC,n=38;melanoma n=21)were analyzed(39 high-dose radiotherapy and 35 high-dose+low-dose radiotherapy).The median follow-up time was 13.6 months.The primary endpoint was met for 72 evaluable patients,with a 4-month disease control rate of 42%(47%[16/34]vs 37%[14/38]in high-dose+low-dose radiotherapy vs high-dose radiotherapy,P=0.38),and 19%overall response rate at any time(26%[9/34]vs 13%[5/38]in high-dose+low-dose radiotherapy vs high-dose radiotherapy,P=0.27).Three patients had toxicity≥grade 3.Low-dose radiotherapy lesion response(53%)was improved compared to nonirradiated lesions in high-dose+ low-dose radiotherapy(23%,P=0.002)and high-dose radiotherapy(11%,P<0.001).The infiltration of T cells and NK cells was enhanced in lesions treated with Low-dose radiotherapy.ConclusionFrom the prospective phase Ⅱ trial,we confirmed that low-dose radiotherapy can improve the infiltration of anti-tumor immune cells,and the addition of low-dose radiotherapy can safely and effectively improve the efficacy of iRT in patients with immunotherapy-resistant advanced tumors.Part Ⅱ:Preclinical study of using SATA6 ASO to improve the efficacy of iRTObjectivesTaking advantage of the inhibitory effect of STAT6 inhibitor on M2 macrophage polarization,we used antisense oligonucleotide based STAT6 inhibitor(STAT6 ASO)in combination with radiotherapy and immunotherapy for the first time to explore the effect of STAT6 ASO in improving the efficacy of iRT.MethodsWe used STAT6 ASO along with hypofractionated RT(hRT)(3 fractions of 12 Gy each)to primary tumors(irradiated)in three bilateral murine NSCLC models(Lewis lung carcinoma,344SQ-parental and 344SQ-anti-PD1-resistant lung adenocarcinomas).Methods used in this study mainly include RT-qPCR,flow cytometry,immunofluorescence,ELISA and NanoString.ResultsSTAT6 ASO plus hRT slowed growth of both primary and abscopal tumors,decreased lung metastases,and extended survival.Interrogating the mechanism of action showed increased M2 macrophage within tumor after hRT,but the combination of hRT and STAT6 ASO significantly reduced M2 macrophage tumor infiltration,as well as enhanced TH1 polarization,improved T cell and macrophage function,and decreased TGF-β levels.The addition of anti-PD1 further enhanced the systemic antitumor response.ConclusionsSTAT6 ASO improved the efficacy of iRT.These results provide a pre-clinical rationale for the pursuit of an alternative therapeutic approach for patients with immune-resistant NSCLC.Part Ⅲ:Preliminary exploration of other strategies to improve the efficacy of iRTIn addition to the above-mentioned two parts that use low-dose radiotherapy and STAT6 ASO to improve the efficacy of iRT,for iRT treatment in specific tumor backgrounds,such as liver metastases and multiple metastases,preliminary explorations were made on the use of low-dose radiotherapy to improve the efficacy of iRT for liver metastases and use of PulsedRT to improve the efficacy of iRT in poly-metastatic cancer.1.Using low-dose radiotherapy to improve the efficacy of iRT for liver metastasesLiver 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,which can reprogram the tumor microenvironment from an immunosuppressive to an immunostimulatory one.Likewise,low-dose radiotherapy 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 low-dose radiotherapy in the context of cancer immunotherapy.Finally,we will discuss the rationale of combining liver-directed low-dose radiotherapy 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 low-dose radiotherapy to the liver.2.Ues of Pulsed-RT to improve the efficacy of iRT in poly-metastatic cancerRadiotherapy,particularly in combination with immunotherapy,can enhance immune memory effects and shape the tumor-directed T cell populations.However,a single cycle of radiotherapy delivered to a limited number of polymetastatic lesions is rarely sufficient to achieve systemic control.We hypothesize that several rounds of radiotherapy,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.