Abstract B43: Combining SQ3370, a doxorubicin-based therapeutic, with TLR and STING agonists enhances antitumor effects in murine tumor models

SQ3370, the lead investigational asset of the CAPACTM (Click Activated Protodrugs Against Cancer) platform, is a therapeutic based on doxorubicin which can induce immunogenic cell death in mouse tumors making it a suitable candidate for combination approaches with immunotherapies that target antigen-presenting cells. Here, we show the effects of SQ3370 combined with either TLR9 or STING agonists. Immunocompetent mice were inoculated with MC38 tumors. Once tumors reached ~100mm3, SQ3370 was administered in two parts: 1) a tetrazine-modified biopolymer was injected into the tumor and 2) a protodrug of doxorubicin modified with a trans-cyclooctene (TCO) was given intravenously as five daily doses. Efficient reaction between the biopolymer and protodrug releases the active drug in situ, delivering higher drug concentrations specifically to the tumor site compared to conventional doxorubicin, which leads to increased efficacy and reduced systemic toxicity. TLR9 and STING agonists were administered intratumorally either as monotherapies or in combination with SQ3370 by co-injection with the biopolymer. Tumor immune composition was assessed by flow cytometry and multiplex immunofluorescent analysis. Combining SQ3370 with either TLR9 or STING agonists led to a significant reduction in tumor growth and prolonged survival of MC38 tumor-bearing mice compared to any monotherapy. Higher doses of either immune adjuvant increased the effect on tumor growth inhibition, suggesting a dose-dependent effect. Complete regression of tumors was observed occasionally in mice treated with a single agent, most commonly with the TLR9 agonist. However, both combination treatments with SQ3370 increased the number of complete responses. Assessment of immune cell infiltration of tumors treated with SQ3370 suggested an increase in antitumor immune responses that were consistent with a doxorubicin-driven induction of immunogenic cell death, including an increase in tumor-infiltrating T-cells. The addition of the TLR9 agonist to SQ3370 further elevated this antitumor immune response. In line with these results, we observed regression of distal, non-injected tumors using a MC38 dual tumor model. A higher fraction of animals treated with SQ3370 + TLR9 agonist (5 out of 10) showed complete tumor regression (absence of both tumors) compared to animals receiving the SQ3370 monotherapy (1 out of 10). Tumor rechallenge of animals with complete regressions revealed a sustained response in line wit