Design And Synthesis Of STING Agonists And Their Application In Antitumor Vaccines

STING protein is an emerging target in immunotherapy,as its activation can induce the expression of type I interferons and other proinflammatory cytokines,thus playing a critical role in immune responses.The cyclic dinucleotide molecule acts as a natural ligand for the STING protein and plays an important role in regulating the pathogenicity of infectious agents and suppressing tumor cells.However,the structural features of cyclic dinucleotide molecules,such as their negative charge,low transmembrane efficiency,and poor stability,limit their application in immunotherapy.This article is based on the structural characteristics of the natural cyclic dinucleotide ligands for STING,and focuses on two aspects,namely structural modification（fluorination）and cationic lipid carrier delivery,to address the limitations of poor stability and low transmembrane efficiency.The immunological activity of these modifications was evaluated in an anti-tumor vaccine.Chapter 1:A brief overview of the STING pathway is provided,followed by the development of STING agonists,including nucleotide and non-nucleotide agonists.The research status of nucleotide agonists and their derivatives is emphasized,and the application of STING agonists in vaccines is described.Chapter 2:The design,synthesis,and activity study of fluorinated cyclic dinucleotide molecules is presented.In this chapter,based on the structural characteristics of natural cyclic dinucleotide ligands of STING,the 2’-OH site of the ligand was modified by fluorination.Five related compounds,3,3-GG^FMP,3,3-AA^FMP,3,3-c GA^FMP,3,3-c G^FAMP,and 2,3-c GA^FMP,were synthesized by using a one-pot synthesis method and evaluated for their stimulatory effects at the cellular and mouse levels.The stimulatory effects of each derivative on RAW264.7（mouse macrophage cells）were tested in vitro,and the results showed that compared to the PBS group and the natural cyclic dinucleotide3,3-c GAMP group,the fluorinated cyclic dinucleotide molecules increased the expression of CD86 on the surface of RAW264.7 cells and produced more IL-6.In vivo stimulation results also demonstrated the feasibility of the fluorination strategy.In conclusion,fluorination of the 2’-OH site of natural cyclic dinucleotide ligands can promote the maturation and activation of macrophages,effectively activate the immune system.Chapter 3:Development of delivery carriers based on double adjuvants and cationic lipids.We adopted a dual adjuvant and cationic lipid delivery STING agonist strategy by first using electrostatic interactions to bind the positively charged Pam₃Cys Ser K₄（Pam₃CSK₄）to the negatively charged cyclic diguanylate（CDG）,and then the CDG and Pam₃CSK₄ agonists were co-encapsulated in cationic liposomes to prepare a liposome-formulated adjuvant that was evaluated for its immune effect using 4T1 cell lysate as an antigen.Analysis of the results showed that（1）the liposome-formulated vaccine lip（CDG+Pam₃CSK₄+4T1）and the vaccine combined with adjuvant（CDG+Pam₃CSK₄+4T1）both induced higher titers of specific Ig G antibodies;（2）the vaccine induced a strong cellular immune response.Therefore,utilizing double adjuvant and cationic lipid-based carrier for STING agonist delivery has great potential for enhancing the efficacy of vaccines.