A Multispecific Anti-CD40 DARPin Construct Induces Tumor-Selective CD40 Activation and Tumor Regression

Cancer Immunol Res. 2022 May 3;10(5):626-640. doi: 10.1158/2326-6066.CIR-21-0553.


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 antitumor immunity. Importantly, in contrast to the mouse anti-CD40 tested in parallel, the in vivo antitumor activity of α-mFAPxCD40 was associated neither 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 suggests 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).

MeSH terms

  • Animals
  • Antibodies, Monoclonal / pharmacology
  • Antibodies, Monoclonal / therapeutic use
  • Antineoplastic Agents, Immunological* / therapeutic use
  • CD40 Antigens
  • Cell Line, Tumor
  • Designed Ankyrin Repeat Proteins
  • Humans
  • Immunotherapy
  • Lymphocyte Activation
  • Mice
  • Neoplasms* / drug therapy


  • Antibodies, Monoclonal
  • Antineoplastic Agents, Immunological
  • CD40 Antigens
  • Designed Ankyrin Repeat Proteins

Associated data

  • ClinicalTrials.gov/NCT05098405