A multispecific anti-CD40 DARPin® construct induces tumor-selective CD40 activation and tumor regression

Abstract The CD40 receptor is an attractive target for cancer immunotherapy. Although a modest pharmacodynamic effect is seen in patients following administration of CD40-targeting monoclonal antibodies (mAb), the doses that could be safely administered do not result in a meaningful clinical response, most likely due to the limited therapeutic window associated with systemic CD40 activation. To overcome this issue, we developed a multispecific DARPin® construct, α-FAPxCD40, which has conditional activity at the site of disease. α-FAPxCD40 activation of CD40 depends on binding to fibroblast activation protein (FAP), a cell surface protease overexpressed in the stroma of solid tumors. In vitro studies demonstrated that α-FAPxCD40 potently activates human antigen-presenting cells in the presence, but not in the absence, of FAP-positive cells. After intravenous injection, a murine surrogate construct (α-mFAPxCD40) accumulated in FAP-positive tumors, elicited rejection of 88% of these tumors and induced memory anti-tumor immunity. Importantly, in contrast to the mouse anti-CD40 tested in parallel, the in vivo anti-tumor activity of α-mFAPxCD40 was neither associated with elevated blood cytokines nor with hepatotoxicity, both of which contribute to the clinical dose-limiting toxicities of several CD40 mAb. This study demonstrates that α-(m)FAPxCD40 engages CD40 in an FAP-restricted manner leading to tumor eradication without signs of peripheral toxicity. This distinct preclinical profile indicates that a favorable therapeutic index may be achieved in humans. It further supports the development of α-FAPxCD40, currently tested in a first-in-human clinical study in patients with solid tumors (NCT05098405).