EGFRvIII is the most prevalent tumor-specific variant of the wild-type EGFR and represents an attractive tumor target in various solid tumors such as GMB, HNSCC or NSCLC. Despite the clinical successes achieved with EGFR targeting antibodies or small molecule inhibitors little therapeutic progress has been made with EGFRvIII. There is still a high medical need in such cancers and several agents are in development to address this notoriously difficult target: Celldex′ vaccine rindopepimut in combination with bevacizumab in late-stage development, or ADCs in early development by Amgen or Abbvie. However, despite the high tumor specificity of EGFRvIII expression, no potent T- or NK-cell engaging are currently in clinical development.

It has been recognized that there may be more therapeutic potential with bi-specific antibodies recruiting immune effectors for targeted destruction of antigen-positive tumor cells. We developed tetravalent, bi-specific TandAbs that recognize EGFRvIII, and recruiting either T-cells or NK-cells by binding to the activating receptors CD3 and CD16A, respectively. This targeted antibody approach allows the selective destruction of EGFRvIII+ tumor cells employing highly potent and efficacious immune effector cells whilst sparing normal cells that are EGFRvIII- or cells presenting the ubiquitously expressed EGFR.

The selected EGFRvIII/CD3 and EGFRvIII/CD16A TandAbs exhibited exquisite specificity towards EGFRvIII in Western Blot, SPR, ELISA, and flow cytometric assays. No binding was observed to recombinant EGFR or to EGFR+ cells. They also displayed potent cytotoxicity towards EGFRvIII+ cell lines with EC50 values in the low picomolar range. No cytotoxicity was observed on EGFRvIII- target cells or cells expressing EGFR demonstrating the high selectivity of anti-EGFRvIII TandAbs. Importantly, in the absence of EGFRvIII+ target cells our TandAbs did not elicit T- or NK-cell activation and activation-induced immune cell proliferation suggesting an excellent safety profile. In vivo pharmacodynamics for anti-EGFRvIII TandAbs was demonstrated in a mouse xenograft model.

The clinical relevance of EGFRvIII as a tumor marker and the binding of our anti-EGFRvIII variable domains were evaluated by immunohistochemistry. Binding was shown in glioblastoma and other solid tumors, but no expression was detectable on healthy tissue.

In summary, the presented in vitro and in vivo studies qualify EGFRvIII/CD3 and EGFRvIII/CD16A TandAbs as highly attractive therapeutic antibody candidates and provides us with the possibility of employing both T-cell and NK-cell recruiting strategies for selective immunotherapy of EGFRvIII+ tumors. Due to tumor specific expression of EGFRvIII and the absence of off-target activity our TandAbs display an excellent safety profile reducing the risks of side effects associated with other anti-EGFRvIII therapies.

Citation Format: Kristina Ellwanger, Uwe Reusch, Ivica Fucek, Michael Weichel, Thorsten Gantke, Stefan Knackmuss, Martin Treder. Anti-EGFRvIII TandAbs recruiting either T or NK cells are highly specific and potent therapeutic antibody candidates for the treatment of EGFRvIII+ tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 580.