Novel Anti-tumor Drug Development And Mechanism Study By Targeting STING

Today,cancer is the second leading cause of death in the world,placing a heavy burden on society.Cancer treatment that has been a medical problem,but with the progress of the life science and technology in recent decades,cancer surgery and drug treatment has made significant progress,especially in terms of drug therapies,from traditional cytotoxic drugs,gradually to the molecular targeted drugs,immune therapy and cell therapy,the patient’s treatment effect is promoted,extended survival.It is worth mentioning that the rapid development of tumor immunotherapy in the past decade has brought new hope to patients.However,tumor immunotherapy,such as immune checkpoint inhibitors,is only effective in about 20% of patients,and the majority of patients still do not receive effective treatment.Therefore,the development of new tumor immunotherapy methods to benefit more patients has become the focus of current drug research and development.As the body’s first line of defense,natural immunity plays a key role in immune surveillance of tumors and resistance to pathogenic microorganisms.Among them,the c GAS-STING pathway is an important pathway in the cytoplasm of cells to feel its own broken double-stranded DNA(ds DNA)or viral DNA.When c GAS feels ds DNA,it generates 2’3’-c GAMP(c GAMP).As the natural ligand of STING,c GAMP can activates STING.STING activated can transport from endoplasmic reticulum to Golgi apparatus and lysosome,recruitment of TBK1 and IRF3,activation of type I interferon pathway,activation of NF-κB and STAT6 and other pathways,and mobilization of antiviral and anti-tumor immune responses.STING can also sense the natural ligands of cyclic dinucleotide produced by bacteria,such as cyclic-di-GMP,cyclic-di-AMP,3’,3’-c GAMP,etc.,and then activate the downstream signaling pathway.Therefore,the c GAS-STING pathway plays an important role in anti-tumor and anti-pathogen immune surveillance.In recent years,targeted STING of agonist become the new hot spots for antitumor drugs at home and abroad,the early stage of the research results shown that the STING agonists showed potent antitumor activity in melanoma,liver cancer,colon cancer,breast cancer and lymphoma and other tumor models,especially for tumor "cold" also have a good antitumor effect,which makes up for the deficiency of immune checkpoint inhibitors.Therefore,it has very important scientific value and transformation potential to develop new STING agonists for the research and development of anti-tumor drugs,explore the mechanism of its anti-tumor action and explore its indications.This paper is mainly divided into two parts: The first part was mainly based on the problems of poor stability and difficult chemical synthesis of cyclic dinucleotide STING agonists.We jointly developed genetically engineered bacteria that could produce natural STING ligand(cyclic-di-AMP).This kind of candidate living biological drugs had the ability to produce STING agonists stably and efficiently,overcoming the difficulties of chemical synthesis.We have achieved the goal of killing tumor efficiently in vivo,and the mechanism of anti-tumor has been deeply explored.In the second part,aiming at the restriction of only local injection of cyclic dinucleotide compounds,we cooperated with the drug chemistry research group to develop a small molecule STING agonist that could be systematically administered.We obtained a lead compound SOMCL-18-277 and conducted systematic pharmacodynamics evaluation.Both local injection and oral administration showed significant anti-tumor activity.SOMCL-18-277 provides the possibility of STING agonists in the treatment of deep tumors,and these works provide a good research basis for further development.Details are as follows:In the first part of the study,we selected Escherichia coli Nissle 1917 as the carrier,and transformed it through synthetic biology with its advantages of mainly colonizing tumor tissues,stable genetic material,no hemolysin,no antibiotic resistance and serum sensitivity.Through the construction,we obtain the genetically engineered bacterium CIBT4503 which only survive in tumors but cannot proliferate in vivo,and on this basis,we obtain candidate strains,CIBT4523 and CIBT4712,which express cyclic-di-AMP(CDA)by anhydrotetracyclin(ATC)induced.Subsequently,ATC-induced CIBT4523 and CIBT4712 were added into human THP1-Dual reporter cells,mouse RAW Lucia,BMDM and BMDC,and it was found that the interferon stimulated gene(ISG)pathway downstream of STING was significantly activated,and the expression of cytokines IFN-β and CXCL10 increased significantly,these results indicated that CIBT4523 and CIBT4712 realized targeted activation of STING pathway in immune cell.At the cellular level,CIBT4712 had a stronger ability to activate the STING pathway than CIBT4523.Furthermore,we explored the pathway which CDA enters cells,we inhibit phagocytosis by immune cells through cytochalasin D,but does not affect cell uptake of small molecules.It was found that CIBT4523 and CIBT4712 lost the activation of STING pathway in immune cell after using cytochalasin D.These results indicated that CDA was incorporated into immune cells through endocytosis rather than secrete and uptake by immune cells.In THP1-Dual cell line,we found that the activation of ISG pathway of immune cell depended on STING,TBK1,IRF3 and MYD88,and partly on c GAS,after CIBT4523 and CIBT4712 treatment.In mouse tumor models,we tested the antitumor activity of CIBT4523 and CIBT4712 against B-cell lymphoma A20,breast cancer 4T1,and melanoma B16F10.In all three animal tumor models,we found that CIBT4523 exhibited stronger antitumor activity than CIBT4712 and the control strain CIBT4503.Since CIBT4712 has a higher CDA expression ability than CIBT4523,but its anti-tumor activity is not as good as CIBT4523 in vivo,we analyzed the reasons.By detecting the number of live strains in tumor tissues at 24 hours and 72 hours,we found that the number of CIBT4712 decreased significantly after 72 hours compared with 24 hours.However,CIBT4523 can still maintain the number of strains for 24 hours,indicating that CIBT4712 is not stable in vivo,suggesting that the high expression of CDA in the bacteria will cause the lysis of the bacteria itself.Therefore,the appropriate expression of CDA in bacteria is crucial to balance their stability and antitumor activity.To elucidate the antitumor immunological mechanism of CIBT4523 in vivo,we analyzed the changes in tumor immune microenvironment and cytokine secretion in blood induced by CIBT4523 treatment in B16F10 tumor-bearing mice.We found that both CIBT4523 and CIBT4503 had an immune-activating effect on the tumor microenvironment,represented by an increase in myeloid cells,neutrophils and granzyme B+ CD8+T cells.However,there are also many immune subsets that decline,such as P-MDSC,macrophages,DC,CD3+T and CD4+T cells,which may be associated with overactivation-induced anti-bacterial cell death.Importantly,we found that CIBT4523 significantly increased the proportion of active DC,Th1,and Th17,suggesting that the engineered strain CIBT4523 effectively enhanced antigenpresenting cell activity and anti-tumor function of CD4+T subsets,which may be the main reason for its higher anti-tumor activity.Correspondingly,CIBT4523 induced higher production of immune-activated plasma cytokines,which were not found in the CIBT4503-treated group,especially IFN-β,IL-3,G-CSF,TNF-α,IL-1β,IL-12,and IFN-γ,further confirming the stronger immune-activated effect of CIBT4523.For example,IL-3 and G-CSF are granulocyte/macrophage colony stimulating factors that promote the production,differentiation and function of various immune cells,while TNF-α,IL-1β,IL-12 and IFN-γ are effective immune-activating cytokines against tumor,and their simultaneous increase will promote anti-tumor immune response.The second part is the pharmacodynamics study of the small molecule STING agonist SOMCL-18-277.In cooperation with the chemistry research group,we obtained a highly active STING small molecule agonist SOMCL-18-277,whose have faster and better effect than ML RR-S2-CDA,the current STING agonist with the fastest clinical progress,in human cells.SOMCL-18-277 specifically targets human and mouse STING molecules and strongly activates STING signaling pathways,promoting the phosphorylation of TBK1/IRF3 and the expression and secretion of IFN-β and CXCL10 in downstream.In mouse tumor model,intra-tumoral injection of SOMCL-18-277 significantly inhibited the growth of B16F10 tumor in wild-type mice,but did not inhibit the growth of STING knockout B16F10 tumor in vivo,suggesting that SOMCL-18-277 specifically targeted STING molecules to play an anti-tumor role in vivo.In a CT26 colon cancer model,intra-tumoral injection of SOMCL-18-277 showed potent antitumor activity,clearing tumor growth in all mice without tumor recurrence.Two months later,CT26 tumors were then inoculated into the mice with tumor regression.It was found that the tumors of these mice did not grow,while the control mice grew completely,suggesting that SOMCL-18-277 treated mice developed immune memory and prevented the recurrence of the same tumor.In addition,SOMCL-18-277 was also found to significantly inhibit the growth of B16F10 and 4T1 tumors in vivo by oral administration,suggesting that SOMCL-18-277 is a small molecule STING agonist that can be systematically administered and deserves further research and development.To sum up,this paper carried out two parts of research on the problems existing in the development process of STING agonists.In the first part of the work,we clarified the importance of the exquisite regulation of the production of STING agonists by genetically engineered bacteria for optimal anti-tumor efficacy,and explored in depth the immunological mechanism of anti-tumor.Relevant candidate products have been transferred.In the second part of the work,we jointly developed an oral potent smallmolecule STING agonist,which has important potential for the treatment of deep tumors in vivo.Therefore,the study of this paper provides an important reference for the development and mechanism research of STING agonists,and provides a good research basis for subsequent studies,which has important scientific significance.