Abstract 3645: Preclinical characterization of a multi-specific DARPin molecule targeting PD1 and VEGF
Authors: Krieg Jennifer , Dieter Schiegg , Joanna Taylor , Herzog Christel , Juglair Laurent , Rigamonti Nicolo , Mueller Mischa , Fiedler Ulrike , Stumpp Michael Tobias , Snell Dan 
Published: July 2017

Abstract
Background: Combination therapy of immune-modulating drugs with modalities targeting tumor cells directly or the tumor stroma are increasing in clinical investigation and practice. In particular, the combination of immune checkpoint modulators targeting PD1 and molecules targeting the tumor stroma, like anti-VEGF therapeutics, are being utilized in multiple permutations and have shown promising results. We have developed a new class of protein therapeutics DARPin® molecules for simultaneous targeting of PD1 and VEGF. The multi-specific PD1 and VEGF targeting molecules were tested for inhibition of PD1 and VEGF mediated functions in cellular functional assays and in syngeneic mouse models.
Methods: Inhibition of human PD1 functions were tested in cell binding assays, a reporter cell assay (Promega) and in human primary T-cell assays like mixed lymphocyte reaction (MLR). VEGF-A inhibition was tested in functional assays showing inhibition of VEGFR2 signaling. Molecules targeting murine PD1 were analyzed in cell binding assays and splenocyte assays as well as in syngeneic mouse models. The anti-tumoral-effect was monitored by caliper measurement and on T-cell infiltration and angiogenesis by immunohistochemistry.
Results: The multi-specific human PD1-VEGF targeting DARPin® molecule inhibits PD1 mediated downregulation of T-cell receptor signaling in a reporter cell assay in the range of the benchmark antibody Nivolumab. Potent picomolar inhibition of VEGF mediated VEGFR2 signaling was also demonstrated for the molecule. Moreover the molecule showed potent inhibition of PD1 in an MLR assay to a similar level as compared to Nivolumab. A murine surrogate molecule was also produced and showed potent PD1 inhibition in vitro. Syngeneic mouse models demonstrate a strong anti-tumoral effect of the surrogate molecule and an increase in the infiltration of T-cells into the tumor was observed upon treatment. The anti-tumoral effect was strongest by combining the anti-PD1 function with the anti-VEGF function. This indicates that the multi-specific DARPin® molecule is functional, shows potency beyond blocking of only PD1 or VEGF and has a strong potential for therapeutic benefit in the clinic.
Conclusions: We have generated a multi-specific PD1 and VEGF targeting DARPin® molecule which is potently active in blocking PD1 and VEGF mediated functions in tumor growth and immunity. The potency of the molecule goes beyond targeting PD1 or VEGF alone and has a strong potential for therapeutic benefit in the clinic.
* DARPins are small repeat proteins, designed to bind targets with high affinity and specificity, and which can be combined in a modular fashion to produce multi-functional agents.
Citation Format: Jennifer Krieg, Schiegg Dieter, Taylor Joanna, Christel Herzog, Laurent Juglair, Nicolo Rigamonti, Mischa Mueller, Ulrike Fiedler, Michael Tobias Stumpp, Dan Snell. Preclinical characterization of a multi-specific DARPin molecule targeting PD1 and VEGF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3645. doi:10.1158/1538-7445.AM2017-3645