STG-968 (azido) - VacciGrade™ - Conjugatable STING Ligand

STING as a therapeutic target

Given its critical implication in NF-κB- and IRF-mediated production of type I IFNs (IFN-α/β) and pro-inflammatory cytokines in response to cytosolic DNA, STING has been a major target for the development of immunomodulatory therapeutics [1, 2]. InvivoGen offers conjugatable STING agonists in multiple functional formats to support diverse bioconjugation strategies. STG-968 and its derivatives STG-982 and STG-1151 are based on the same proprietary STING agonist, CL845, and differ by their reactive functional handle, enabling azido-, maleimide-, or DBCO-based coupling, respectively.

STG-968 azido functional handle for click-chemistry

STG-968 features an azido functional handle coupled to the phosphorothioate group of CL845 via a “built-in” linker (see below). It is compatible with commonly used click-chemistry linkers (e.g., those bearing a DBCO functional group), enabling evaluation of multiple bioconjugate architectures.

The bioconjugation to a protein of interest (POI) occurs in two steps:
– Step 1: Linker attachment to solvent-accessible cysteine or lysine residues on the POI
– Step 2: Click reaction between the azido group of STG-968 and the complementary linker functional group

Common features of conjugatable STING ligands

InvivoGen STG-968 was engineered from CL845 (cAIM(PS) Difluor (Rp)), an analog of the clinical STING agonist CL656 (cAIM(PS)₂ Difluor (Rp/Sp)) [2-6]. STING signaling is triggered upon ligand binding to a small pocket that cannot accommodate large molecules [1]. Therefore, once the bioconjugate has entered the target cell, CL845 must dissociate from the rest of the molecule. To secure this step, STING conjugatable ligands were designed with a “cleavable linker”, composed of: 

- a spacer: polyethylene glycol (PEG) to improve flexibility and reduce steric hindrance [7, 8],

- a cleavage site: Val-Ala dipeptide, a substrate of the lysosomal protease cathepsin B [8],

- a connector: para-aminobenzoic (PAB) moiety that supports protease accessibility and self-immolative release of CL845 [8].

Applications for conjugatable STING ligands

Conjugatable STING agonists are designed for the development of targeted immunotherapeutic bioconjugates. Their controlled conjugation to antibodies or antigenic proteins enables localized activation of the STING pathway, enhancing innate and adaptive immune responses in cancer and vaccination models.

• Antibody-drug conjugates (ADCs / ISACs)

STING agonist conjugation to monoclonal antibodies (mAbs) generates immunostimulatory ADCs. Such constructs are promising cancer immunotherapeutic strategies due to their complementary mechanisms of action [9, 10] (see illustration):
– Antibody-mediated tumor cell killing upon recognition of specific tumor antigen by the mAb variable region.
– mAb binding to Fcγ receptor (FcγR)-expressing cells, such as antigen-presenting cells (APCs). The engagement of  FcγR may induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).
– STING activation in the tumor target cells (not shown) or neighboring myeloid cells after ADC internalization. This results in the production of type I interferons and pro-inflammatory cytokines.

• Antigen-adjuvant conjugates (AACs)

Conjugation of STING agonists to antigenic peptides/proteins generates AACs for vaccination and immune priming. These AACs enable (see illustration):
– Co-delivery of antigen and STING agonist to APCs
– Antigen processing and STING activation in the same APC
The conjugation strategy helps overcome the dissociation of the antigen and STING agonist upon mixture administration and allows a prolonged antigen presentation for the induction of adaptive immune responses, probably through an intracellular storage/depot effect [11-12].

References:

1. Zhang H., et al. 2020. Targeting Stimulator of Interferon Genes (STING): a medicinal chemistry perspective. Journal of Medicinal Chemistry. 63(8):3785.
2. Jang S., et al. 2021. ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance. Commun. Biol. 4:497.
3. Pro-cyclic dinucleotides and pro-cyclic dinucleotide conjugates for cytokine induction. WO 2019/129880A1 and foreign equivalents.
4. Cyclic dinucleotides for cytokine induction, patent US10011630B2 and foreign equivalents.
5. Lioux T., et al. 2016. Design, synthesis, and biological evaluation of novel cyclic adenosine-inosine monophosphate (cAIMP) analogs that activate stimulator of interferon genes (STING). J. Med. Chem. 59:10253.
6. https://clinicaltrials.gov/ct2/show/NCT04592484
7. Drago J.Z, et al. 2021. Unlocking the potential of antibody-drug conjugates for cancer therapy. Nat Rev Clin Oncol. 18(6):327.
8. Poreba M., 2020. Protease-activated prodrugs strategies: challenges, and future directions. The FEBS Journal. 287(10):1936.
9. Wu TY, et al., 2022. Tumor-targeted delivery of a STING agonist improves cancer immunotherapy. PNAS, 119(49):e2214278119.
10.  Cetinbas, MN, et al., 2024. Tumor cell-directed STING agonist antibody-drug conjugates induce type III interferons and anti-tumor innate immune responses. Nat comm., 15(1):5842.
11. Van Montfoort N. et al., 2009. Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity. PNAS. 106(16):6730.
12. Ho N.I. et al., 2021. Distinct antigen uptake receptors route to the same storage compartments for cross-presentation in dendritic cells. Immunology. 164(3):494.