Development of a bispecific DARPin® molecule for tumor-restricted CD40 activation
 
Nicolo' Rigamonti^1*, Anja Schlegel¹, Sophie Barsin¹, Jennifer Krieg¹, Susanne Mangold¹, Maria Paladino¹, Sandra Bruckmaier¹, Simon Fontaine¹, Valérie Calabro¹, Victor Levitsky¹, Dan Snell¹, Michael Stumpp¹ and Clara Metz^1
1Molecular Partners AG, Schlieren, Switzerland.
^*Corresponding Author: nicolo.rigamonti@molecularpartners.com
 
CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily which can activate both innate and adaptive tumor immunity, making it an attractive target for cancer immunotherapy. Systemic administration of agonistic CD40 antibodies (Ab) has shown signs of activity in cancer patients, but dose-limiting toxicity impaired the clinical efficacy. New therapeutic approaches to achieve tumor-restricted activation through intratumoral administration of CD40 Ab are now in early clinical development, aiming to reduce systemic toxicity and increase efficacy, but with clear limitations, such as tumor accessibility. Here, we report an alternative approach based on systemic administration of a bispecific DARPin® molecule targeting human CD40 and a tumor antigen (TA), enabling CD40 pathway activation exclusively in the presence of TA-expressing cells.
Using fibroblast activation protein (FAP) alpha, a glycoprotein abundantly expressed in many solid tumors, as a TA, we generated a panel of bispecific CD40/FAP DARPin® molecules intended to induce immune activation only when clustered by binding to FAP-expressing cells. In a reporter cell assay, bispecific CD40/FAP DARPin® molecules were able to trigger CD40 and activate the NF-κB pathway only in the presence of FAP-transfected CHO cells, but not in the presence of parental CHO cells.  These DARPin® molecules were then tested in different and more meaningful cell assays using primary human immune cell populations and showed a CD40-mediated activation in the presence of FAP-positive, but not with FAP-negative cells, again confirming a mechanism of action strictly dependent on FAP-mediated cross-linking.
In conclusion, we have generated bispecific agonist CD40/FAP DARPin® molecules able to activate the CD40 pathway in cellular assays with a targeting-dependent mechanism of action, supporting the hypothesis that these DARPin® molecules could lead to a tumor-localized immune activation in vivo. Selected bispecific DARPin® molecules will next be assessed in mouse tumor models to test this hypothesis.