Design Of Polymeric STING Agonists For Cancer Immunotherapy

Immunotherapy is playing an increasingly crucial role in the field of cancer treatment.In cancer treatment,the activation of innate immunity can not only directly mobilize innate immune cells to exert the immune killing effect,but also play an important role in activating the adaptive immune response.Stimulator of interferon genes(STING),a pattern recognition receptor(PRR)located on the endoplasmic reticulum(ER),is involved in innate immune activation.When activated,the STING immune pathway can induce the release of type Ⅰ interferon(IFN-I)from immune cells to mobilize innate and adaptive immune cells to eliminate pathogens or tumors.There are generally two ways to activate the STING pathway:one is that double-stranded DNA(dsDNA)present in cytosol is sensed by the cyclic GMP-AMP(cGAMP)synthase(cGAS)to induce the production of cGAMP—the natural ligand STING;the other is delivery small-molecular STING agonists from outside,which can directly bind to STING protein to activate downstream signals.However,the current development of STING agonists is still limited,with unsatisfied clinical feedback.Besides,small-molecular agonists usually have toxic side effects such as insolubility,high clearance,and poor targeting.Since polymers have abundant architectures and modifiable functional groups,they are often used to solve the delivery defects of small-molecular drugs and improve pharmaceutical activity,or to achieve special physiological activities by themselves through structural modification.In this paper,based on polymers as the main framework,we explored the effect of small-molecular drug-conjugated polymers and drug-free polymers on stimulating STING pathway.Three works were carried out gradually:First,from the perspective of increasing cytoplasmic dsDNA,we revealed a new manner to in situ activate STING pathway in tumor microenvironment by applying DNA-targeted drugs modified with polymers;Second,from the point of STING pathway can be activated by directly binding with ligands,we designed a novel drug-free polymeric STING agonists only through the terminal modification of polymers;Third,we systematically investigated the correlation between macromolecular architectures or physicochemical properties and immunostimulatory activity of polymeric STING agonists,and summarized some guiding rules for the design of polymeric STING agonists.The specific research content and main conclusions are as follows:(1)Polymeric 7-ethyl-10-hydroxycamptothecin(SN38)stimulates STING pathway in immune cells by damaging DNA of tumor cells and transferring DNA fragments by exosomes.Given that cytoplasmic dsDNA is the initial inducer of STING immune pathway,we considered that small molecule chemotherapeutic drugs with DNA-targeting damage capabilities,such as SN38,topotecan(TPT),doxorubicin(DOX),and cisplatin(CDDP),could induce DNA damage and release into the cytoplasm,thereby activating the STING pathway.After screening,it was found that the supernatant from SN38-treated mouse breast cancer tumor cell line E0771 had a significant advantage in inducing mouse bone marrow-derived dendritic cells(BMDCs)to release interferon β(IFN-β).After mechanism investigation,we found that SN38 can induce DNA damage and DNA fragments released in the form of exosomes,which are subsequently taken up by innate immune cells such as DCs and activate the STING immune pathway.however,considering the drawbacks of SN38 being insoluble in water,high clearance rate,and acute physiological toxicity,we modified SN38 on polyglutamic acid-grafted polyethylene glycol(PLG-g-mPEG/SN38)to obtain a SN38 nano-formulation(SN38-NPs).In vivo,SN38-NPs significantly reduced the toxicity of free SN38,greatly prolonged the half-life,and effectively induced the activation of the STING pathway.As results,in the E0771 tumor microenvironment after SN38-NPs treatment,cytokine(IL-6,TNF-α,IFN-y,IFN-β)levels were markedly increased,DCs and NK cells were activated,and finally the treatment of SN38-NPs achieved a tumor suppression rate(TSR)of 82.6%.This study revealed a novel mechanism by which SN38 can activate the STING immune pathway in cancer therapy,and provided a new idea for the application of nanoformulation-based DNA-targeting damaging drugs in cancer immunotherapy.(2)Design of a new class of polymeric STING agonists—azole molecules end-capped branched polyethyleneimine(bPEI)derivatives(bPEI-M).Given that the nitrogenous heterocyclic structures are essential in a variety of small molecular STING agonists(e.g.,cGAMP,diABZIs,SR717)and play a vital role in the docking of ligands with the STING protein-binding domain.We modified the terminal amino groups of bPEI with five-membered nitrogenous cyclic small molecules,i.e.,azole molecules,to prepare a new class of bPEI derivatives(bPEI-M)without physiological active small agents.Interactions between effector groups and STING protein domain would be enhanced by modulation effects and multivalent effects of bPEI backbone architecture,reducing the requirement for complex small molecule agonist structural design.Among the 15 bPEI-M polymers we designed,more than 60%exhibited immunostimulatory activity of IFN-β-related pathways.In the investigation experiments on the immune activation mechanism of several representative bPEI-M materials,bPEI-5Thi,-4Imi,and-4BImi could all induce a significant increase in the phosphorylation level of STING pathway-related proteins.In the human monocyte cell line THP-1,distinct STING protein clusters were induced after incubation with bPEI-4BImi,which represents possible direct interactions between bPEI-4BImi and STING proteins and indicates the following signal transduction.In further molecular docking simulation calculations,we found that the docking ability of 4BImi small molecules to the STING protein pocket-binding domain was markedly increased after modification by bPEI,illustrating the direct interactions between bPEI-4BImi polymers and STING proteins.We then prepared a minimalist binary nanovaccine(BiVax)by simply mixing bPEI-4BImi with antigen proteins,which integrated innate immune stimulatory activity into the carrier.The BiVax achieved good spatiotemporal orchestration of antigen cross-presentation with innate stimulation in APCs,strongly induced the activation of antigen-specific T cells and achieved tumor-specific killing.In personalized postoperative tumor model therapy,BiVax in combination with the immune checkpoint inhibitor aPD-Ll achieved a 60%cure rate.This study revealed the structure of a new class of macromolecular STING stimulators and opened new horizons for the design of novel cancer vaccine carriers.(3)Study on the correlation between polymer architectures and STING immunostimulatory capacities of nitrogenous cyclic molecules-modified cationic polymers.In the previous work,a series of bPEI-M generally exhibited STING signaling pathway immunostimulatory activity.However,due to the random hyperbranched structure of bPEI,the instruction significance for the design of polymeric STING agonists is relatively unclear.Therefore,we systematically explored the correlation of nitrogenous cyclic molecules,polymer architectures,and side chains with the STING immunostimulatory activity,which provided certain guidance for the design of polymeric STING agonists.First,after extending azole molecules of bPEI-M to six-or seven-membered nitrogenous cyclic molecules,we still observed a general immunostimulatory effect.And the molecular weight(Mw)of bPEI(within a certain range)had less effect on the immunostimulatory effect,even though the Mw of bPEI decreased from 10 kDa to 0.6 kDa.However,we found that the topology of polymer backbone is an important influencing factor.Both linear PEI(1PEI)and dendrimeric PAMAM exhibited significantly reduced immunostimulatory activity after modification with azole molecules compared with hyperbranched bPEI-4BImi,which exemplifies the advantages of branched structures in achieving immunostimulation.To further explore the importance of branched structures,we prepared monovalent,bivalent and tetravalent side chains with well-defined branched structures based on the cationic polymer poly-L-lysine(PLL)(PLL,PLL-2V,PLL-4V),whose terminal amino groups were subsequently modified with 4BImi.We successfully validated PLL-4V-4BImi with multivalent branched side chains was superior to PLL-2V-4BImi and PLL-4BImi in achieving STING pathway immunostimulation.And we also observed that the binding interaction between polymer and STING protein was in a multivalent structure-dependent manner.This study focuses on revealing the importance of polymer architecture in the design of STING pathway-dependent immunoreactive reagents,and provides certain references for the design of polymeric STING agonists.Through the studies of this paper,it is hoped that it can provide references for the study of polymers-based STING pathway-dependent immunostimulatory nanomedicines and the design of polymeric STING agonists,and provides impetus for the application of STING pathway-dependent antitumor immunotherapy.