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,Jörg Steinbach,Stefanie Koristka,Michael Bachmann,Nicole Berndt,Jens Pietzsch,Marc Schmitz,Ralf Bergmann,Claudia Arndt,Anja Feldmann

doi:10.18632/oncotarget.25390

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.

Highlights

The epidermal growth factor receptor (EGFR) is a ubiquitously expressed transmembrane protein of the HER family of tyrosine kinase receptors that plays a central role for normal cell and organ development [1,2,3]
As proofof-concept, we recently reported on universal CAR (UniCAR)-armed T cells successfully redirected to EGFR+ tumors in vitro and in vivo via a novel nanobody (Nb)-based α-EGFR target modules (TM) expressed in E. coli (termed α-EGFR TM) or eukaryotic CHO cells [23]
To elucidate whether TM functionality can be further improved by an increase in affinity, we here performed comparative analyses between monovalent and bivalent EGFR-specific TMs both expressed in CHO cells

Summary

Introduction

The epidermal growth factor receptor (EGFR) is a ubiquitously expressed transmembrane protein of the HER family of tyrosine kinase receptors that plays a central role for normal cell and organ development [1,2,3]. Within the last two decades, many EGFR-targeted therapies have emerged These include tyrosine kinase inhibitors (Erlotinib, Gefitinib, Afatinib) and α-EGFR monoclonal antibodies (Cetuximab, Panitumumab, Necitumumab) approved for treatment of metastatic colorectal cancer, head and neck cancer, pancreatic cancer and lung cancer [3, 16,17,18]. As learned from both preclinical and clinical studies therapeutic approaches are often accompanied by mild to severe side effects due to the widespread EGFR expression on healthy tissues [19, 20] This clearly emphasizes the urgent need for a more precise control of highly effective EGFR directed cancer treatments

Methods

Results

Conclusion