From mono- to bivalent: improving theranostic properties of target modules for redirection of UniCAR T cells against EGFR-expressing tumor cells in vitro and in vivo

// Susann Albert 1, * , Claudia Arndt 2, * , Stefanie Koristka 2 , Nicole Berndt 3 , Ralf Bergmann 2 , Anja Feldmann 2 , Marc Schmitz 3, 4, 5 , Jens Pietzsch 2, 6 , Jörg Steinbach 2, 5, 6 and Michael Bachmann 1, 2, 3, 5 1 UniversityCancerCenter (UCC) Dresden, Tumor Immunology, ‘Carl Gustav Carus’ Technische Universität Dresden, Dresden, Germany 2 Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Institute of Radiopharmaceutical Cancer Research, Dresden, Germany 3 German Cancer Consortium (DKTK), part site Dresden and German Cancer Research Center (DKFZ), Heidelberg, Germany 4 Institute of Immunology, Medical Faculty, ‘Carl Gustav Carus’ Technische Universität Dresden, Dresden, Germany 5 National Center for Tumor Diseases (NCT), partner site Dresden, Dresden, Germany 6 Faculty of Chemistry and Food Chemistry, School of Science, Technische Universität Dresden, Germany * These authors contributed equally to this work Correspondence to: Michael Bachmann, email: m.bachmann@hzdr.de Keywords: CAR T cell immunotherapy; solid epithelial tumor; nanobody; affinity; PET imaging Received: January 22, 2018 Accepted: April 28, 2018 Published: May 22, 2018 ABSTRACT CAR-modified T cells show impressive results in clinical trials. However, cytokine release syndrome and “on-target, off-tumor” reactions represent most concerning side effects. To improve the safety of CAR-T cell therapy, we established a switchable CAR platform termed UniCAR system consisting of two components: UniCAR-modified T cells and tumor-specific target modules (TM). For treatment of EGFR + epithelial tumors, we recently described a monovalent nanobody-based α-EGFR TM, either expressed in bacteria or eukaryotic cells. In spite of the identical primary sequence the eukaryotic TM showed a reduced killing capability and affinity. Here we describe a novel bivalent α-EGFR-EGFR TM. As expected, the avidity of the bivalent TM is higher than that of its monovalent counterpart. Binding of neither the monovalent α-EGFR TM nor the bivalent α-EGFR-EGFR TM to EGFR effected the EGF-mediated signaling. While the monovalent α-EGFR TM could only mediate the killing of tumor cells expressing high levels of EGFR, the bivalent α-EGFR-EGFR TM could redirect UniCAR T cells to tumor cells expressing low levels of EGFR. According to PET experiments in vivo , the increased avidity of the bivalent α-EGFR-EGFR TM improves the enrichment at the tumor site and its use for PET imaging.