FAP-targeting Small molecule-immunostimulator conjugate bearing a novel TLR7 agonist payload induces tumor eradication and long-lasting antitumor immunity in vivo

While antibody-based immune checkpoint inhibitors have shown remarkable success in cancer therapy, their efficacy is often limited by poor T-cell infiltration in many tumors. Activation of pattern recognition receptors such as toll-like receptors (TLRs) can trigger cytokine production and amplify antitumor immune responses. However, systemic administration of TLR agonists frequently induces severe adverse effects through uncontrolled immune activation. To overcome this issue, targeting approaches such as immunostimulating antibody conjugates (ISACs) have been developed to selectively deliver the payload to the tumor, thereby potentiating efficacy and reducing systemic toxicities. The replacement of the tumor-homing antibody with a small molecule possessing a high affinity towards a tumor-associated antigen affords small molecule-immunostimulator conjugates (SMICs), offering several advantages, including a more rapid extravasation, an efficient tissue penetration, leading to a homogeneous and selective uptake within antigen-positive tumor masses. In this work, we developed a SMIC bearing the OncoFAP moiety targeting Fibroblast Activation Protein (FAP), an endopeptidase abundant in the stroma of most solid tumors, and featuring a novel proprietary TLR agonist as a payload. Through a screening of different TLR1/2 and TLR7 agonists, we identified MB357, a novel potent TLR7 agonist with a single-digit nanomolar activity in vitro. To enhance tumor specificity and improve tumor exposure to the drug, we employed the FAP-cleavable Glycine-Proline linker, which rapidly and selectively releases high concentrations of the active TLR agonist to the FAP-positive lesions. The SMIC was tested for its activity on immunocompetent mice bearing FAP positive tumors, showing complete tumor eradication in all treated animals and long-lasting antitumor immunity, without any sign of toxicity. The promising preclinical results provided the rationale to start a clinical trial to investigate the safety and efficacy of OncoFAP-GlyPro-MB357 in canine patients with spontaneous solid tumors.