Increasing apparent affinity of EGFR-directed target modules results in enhanced anti-tumor and diagnostic properties of the UniCAR system

Immunotherapy with CAR-modified T cells has recently entered into the clinical routine. Nonetheless, until now most concerning side effects associated with CAR T cell therapies are cytokine release syndrome and “on-target, off-tumor” reactions. In order to improve CAR technology regarding safety, we developed a novel switchable platform termed UniCAR. It relies on the separation of the functional domains of conventional CARs. Thus, the UniCAR system is composed of (I) T cells modified to express an universal CAR (UniCAR) and (II) tumor-specific target modules (TM). UniCAR T cell activity can be easily controlled: While they are inert in the absence of TMs, their anti-tumor reactivity can be only switched on in the presence of TMs. For redirection of UniCAR T cells to EGFR+ epithelial tumors, we recently established a monovalent nanobody-based α-EGFR TM, either expressed in bacterial or eukaryotic cells. In spite of the identical primary sequence the eukaryotic α-EGFR TM showed a reduced killing capability and affinity. This observation encouraged us, to elucidate whether TM functionality can be further improved by an increase in affinity. Consequently, we here constructed a novel bivalent α-EGFR-EGFR TM, expressed it in eukaryotic cells and compared its anti-tumor reactivity and pharmacokinetic properties with the monovalent α-EGFR TM. As expected, the avidity of the bivalent TM is higher than that of its monovalent counterpart. By raising the number of binding sites, the resulting bivalent α-EGFR-EGFR TM shows also an improved killing efficacy and capability in vitro and in vivo. 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 also redirect UniCAR T cells to tumor cells expressing lower levels of EGFR. According to in vivo PET experiments, the increased molecular weight of the bivalent α-EGFR-EGFR TM delays its elimination and thereby improves the enrichment at the tumor site. Consequently, the bivalent TM seems to be more suitable for PET imaging approaches and tumor eradication.