Activity of a Sting and a TLR9 Agonist As an “in Situ Therapeutic Vaccination” Against Lymphomas
Authors: Sallets Adrienne , Sagiv Barfi Idit , Levy Ron 
Published: October 2019

Abstract
We have developed a therapeutic “in situ vaccination” strategy against lymphomas involving the intratumoral injection of a STING- or a TLR9 agonist. The advantage of this approach is that it does not require the isolation of tumor specific antigens since the immune system is already primed at the tumor site, but inhibited for acting.
The Stimulator of Interferon Gene (STING) is part of the innate immunity involved in the intracellular pathogen recognition. More recently, it was shown to be a crucial player in the spontaneous immune response against immunogenic tumors. STING agonists are able to induce an immune response through activating dendritic cells that then produce cytokines and chemokines including type 1 IFN. Modified cyclic dinucleotides (CDNs) have enhanced ability to activate both mouse and human STING variants. When injected intratumorally, these CDNs were able to induce a tumor-specific CD8 T cell response in various tumor models. A Phase 1 clinical trial is evaluating the safety and the efficacy of intratumoral injection of CDNs in patients with advanced/metastatic solid tumors or lymphomas [NCT02675439]. Toll-like receptor 9 (TLR9) is another receptor of the innate immune system. Oligodeoxynucleotides containing unmethylated CpG motifs (CpG) are well known TLR9 agonists. Upon TLR9 stimulation, CpG can activate dendritic cells and B cells, and induce the release of multiple cytokines, such as interferons (IFNs). Intratumoral injection of CpG results in a CD8 T cell-dependent immune response against tumors. Multiple phase 1 / 2 clinical trials are evaluating the efficacy of CpG in cancer treatments. Among them, some are investigating intratumoral injection to patients with lymphomas [NCT01745354, NCT02254772, NCT00185965, and NCT00880581].
STING or TLR9 activations, both result in similar immune outcomes. However, they work by independent intracellular signal transduction pathways and their expression pattern is different. TLR9 is expressed on immune cells whereas STING is expressed on a wider variety of cells among immune- and somatic cells, including endothelial cells. Therefore, we studied the properties of both, CpG and CDNs, in the context of lymphomas. We examined the direct effect of these molecules on lymphoma cells in vitro and their therapeutic effects in vivo.
In vitro, both agonists were able to induce the expression of costimulatory molecules on lymphoma B cells. Interestingly, the STING agonist showed toxicity on mouse lymphoma cell lines whereas CpG showed increased proliferation on some of these same B cell lines.
We studied the in vivo effects of both agonists using the A20 lymphoma injected into two different bilateral subcutaneous sites. Either the TLR9- or the STING agonist was injected in one tumor site, and both, the injected- and the uninjected tumor sites were monitored to assess the local- and the systemic effects of the treatment. As single agents, both agonists showed a similar local and systemic outcome. The treated tumor disappeared, and the growth of the distant tumor was retarded. We assumed that the failure to cure the second tumor was due to the dampening of the immune response by local immunosuppressive mechanisms. Therefore, we investigated the combination of each agonist with different checkpoint blockade strategies, with the checkpoint blocking agent being given intratumorally or systemically. Combining the TLR9- or the STING agonist with a checkpoint blocking agent induced the regression of the distant tumor. Interestingly, the preferred route of administration of the checkpoint blocking agents was different depending on TLR9 or STING stimulation. TLR9 stimulation required intratumoral checkpoint blocking, whereas STING stimulation worked better with systemic checkpoint blocking.
Our results indicate that in situ stimulation of the immune system, using either STING or TLR9, combined with checkpoint blockade is a promising approach for the therapy of lymphoma.

Disclosures
No relevant conflicts of interest to declare.