STING Agonist Therapy In Combination With CAR-NK Cells For The Treatment Of Pancreatic Cancer

ObjectivePancreatic cancer is one of the malignant tumors with the highest mortality in the world due to its late stage at diagnosis and poor prognosis.However,so far,the clinical therapeutic strategies for pancreatic cancer are mainly limited to traditional methods such as surgical resection,chemotherapy and radiotherapy.The 5-year survival rate of patients is less than 7%.Therefore,finding a more effective therapeutic strategy is essential for the pancreatic cancer.In the past few years,immunotherapy has been developed into a new cancer therapeutic strategy.For example,FDA has approved for three important immune checkpoint inhibitors,programmed cell death protein-1(PD-1),programmed death ligand 1(PD-L1)and cytotoxic T lymphpcyte associated antigen 4(CTLA-4),are used in the treatment of various cancers.Unfortunately,these immune checkpoint inhibitors have not been found to be effective for patients with pancreatic cancer.In addition,anti-mesothelin monoclonal antibodies(mAb),including the anti-mesothelin immunotoxin SSlP which recognizes MSLN N-terminal 296-359 monoclonal antibodies and MORAb-009(also known as ametuximab)are still under clinical or preclinical trials.In recent years,immune activators targeting pattern recognition receptors have become a hot spot in tumor immunotherapy.Innate immune cells can recognize the pathogen-associated molecular patterns(PAMPs)and dangerous-associated molecular patterns(DAMPs)signals through pattern recognition receptors(PRRs)to initiate different immune responses,and play a key role in regulating body homeostasis and host defense.In recent years,PRRs have gradually become a target of clinical disease treatment,and has good therapeutic potential in anti-tumor and anti-infection.Among them,STING protein agonists have shown ideal therapeutic effects in preclinical studies and clinical experimental treatments of various tumors,and may become potential therapeutic methods for cancer.When exogenous DNA viruses,bacteria,and DNA from damaged cells enter the cytoplasm,they are easily recognized by the DNA receptor cGAS in APCs and catalyze the production of cyclic cGAMP.As a second messenger,cGAMP can bind to the STING protein anchored on the endoplasmic reticulum,inducing the conformation of the STING molecule from monomer to dimer,and transported to the perinuclear microsome through the Golgi apparatus.STING protein can recruit and activate TBK1,further activate IRF3,finally induce the expression of type I IFN.Type I IFN produced by DCs can promote the maturation of DCs,and enhance the anti-tumor response of CD8+T cells.Studies have confirmed that cyclic dinucleotide(CDN)can not only inhibit mouse B16 melanoma,4T1 breast cancer,CT26 colon cancer,pancreatic cancer,and skin cancer,as well as inhibit the growth of tumors at distant untreated sites.In addition,clinical trials of ML RR-S2 CDA(also known as ADU-S100 or MIW815)have showed a total objective effective rate of 5%and a disease control rate of 32.5%.This means that STING agonists are expected to become a new immunotherapy drugs for the treatment of pancreatic cancer.For the anti-tumor mechanism of STING agonists,previous studies thought that STING agonists mainly activate the STING signaling pathway in APCs,and promote the anti-tumor response mediated by CD8+T cells.However,in recent years,there are also literatures showing that,in addition to APCs,STING protein is also widely expressed in natural killer cells(NK)and tumor cells.However,whether STING agonists directly activate NK cells and enhance the anti-tumor activity of NK cells?There is a lack of meticulous and definite research.In order to explore the therapeutic effect and detailed mechanism of STING agonists in the treatment of pancreatic cancer,we explored from the following aspects:First,to investigate whether STING agonists have direct activation on NK cells,NK-92 cells were stimulated with STING agonist 2’-3’-cGAMP,we found that 2’-3’-cGAMP can directly activite cGAS-STIING signaling pathway in NK-92 cells,up-regulate the expression of activating receptors and down-regulate the expression of inhibitory receptors,2’-3’-cGAMP also can improve the killing ability of NK-92 cells.Secondly,2’-3’-cGAMP can directly activate the cGAS-STING signaling pathway in pancreatic cancer cells,and improve the killing sensitivity of pancreatic cancer cells to NK cells.Further,we established a xenogradft model of pancreatic cancer in NOD-SCID mice to verify the therapeutic effect of STING agonists.We found that STING agonists can induce apoptosis in tumor tissues and induce the production of IFN-β,CCL5 and CXCL10 in tumor tissues.Finally,we combined STING agonists with anti-MSLN-CAR-NK cells established by our group for treating pancreatic cancer in NOD-SCID mice,we found that treatment with STING agonist and anti-MSLN-CAR-NK cells obviously reduced tumour burden,and prolonged survival of tumor-bearing mice.Our results showed that STING agonists can directly activate NK cells and enhance the anti-tumor activity of NK cells.We also confirmed that STING agonists combined with anti-MSLN-CAR-NK cells can significantly improve the therapeutic effect of NK cells on pancreatic cancer.This immunotherapy strategy provided new ideas and basis for immunotherapy of pancreatic cancer.Methods1.The expression levels of STING gene in NK-92 cells and pancreatic cancer cell lines were detected by RT-PCR.2.The phosphorylation levels of IRF3 were detected by Western blot.3.The expression levels of IFN-β,activating receptors and inhibitory receptors in NK-92 cells,and the expression levels of IFN-β,NKG2DL and chemokines in AsPC-1 and Capan-2 cells,were detected by Q-PCR method.4.The killing ability of NK-92 cells against pancreatic cancer cells was detected by lactate dehydrogenase(LDH)cytotoxicity detection method.5.The proliferation of pancreatic cancer cells was detected by MTT method.6.The apoptosis of pancreatic cancer cells was deteceted by Annexin V-PI method.7.The levels of chemokines CCL5 and CXCL10 in AsPC-1 and Capan-2 cells were detected by ELISA method.8.The expressions of mesothelin in AsPC-1 and Capan-2 cells were detected by flow cytometry and immunohistochemistry.9.NOD-SCID mice were administrated with AsPC-1 cells via subcutaneous injection to construct the pancreatic cancer model.10.The apoptosis of tumor tissues was detected by TUNEL method.Results1.STING agonist 2’-3’-cGAMP can directly activate the cGAS-STING signaling pathway in the NK-92 cells.2.2’-3’-cGAMP promoted the phosphorylation of IRF3 in a time-dependent manner,and induced the production of IFN-β in NK-92 cells.3.2’-3’-cGAMP upregulated the expression of activting receptors and down-regulated the expression of inhibitory receptors in NK-92 cells.4.2’-3’-cGAMP enhanced the killing activity of NK-92 cells against AsPC-1 cells and Capan-2 cells.5.2’-3’-cGAMP can directly activate the STING signaling pathway in AsPC-1 and Capan-2 cells.6.2’-3’-cGAMP stimulation promoted the phosphorylation of IRP3 in a time-dependent manner and up-regulated the expression of IFN-β in AsPC-1 and Capan-2 cells.7.2’-3’-cGAMP stimulation significantly inhibited the proliferation and promoted the apoptosis of AsPC-1 and Capan-2 cells in a dose-dependent manner.8.2’-3’-cGAMP stimulation up-regulated the expression of NKG2DL and promoted the production of CCL5 and CXCL10 in AsPC-1 and Capan-2 cells.9.2’-3’-cGAMP pre-stimulated AsPC-1 cells and Capan-2 cells can enhance their killing sensitivity to NK-92 cells.10.Mesothelin is highly expressed in AsPC-1 and Capan-2 cells.11.Anti-MSLN-CAR-NK cells were successfully constructed.Anti-MSLN-CAR-NK cells had a higher killing capacity for AsPC-1 cells and Capan-2 cells than NK-92 cells.12.Intratumoral injection 2’-3’-cGAMP can significantly inhibit tumor growth.13.STING agonists combined with anti-MSLN-CAR-NK cells can significantly improve the therapeutic effect of NK cells on pancreatic cancer.Conclusions1.STING agonist 2’-3’-cGAMP has a direct activation effect on NK cells,and enhance the killing activity of NK-92 cells aganist pancreatic cancer cells.2.2’-3’-cGAMP can activate the cGAS-STING signaling pathway in pancreatic cancer cells,enhance the killing sensitivity of pancreatic cancer cells to NK cells.3.STING agonists can directly inhibit proliferation and induce apoptosis of pancreatic cancer cells.4.STING agonist combined with anti-MSLN-CAR-NK cells can significantly improve the therapeutic effects of NK cells on pancreatic cancer.