Redirection of UniCAR T cell against EGFR+ tumor cells by using different αEGFR targeting module formats

Since epithelial growth factor receptor (EGFR) mutations or overexpression is linked with variety of malignancies, including lung, breast, stomach, colorectal, head and neck carcinomas as well as glioblastomas, it is an attractive target for tailored treatment. As chimeric antigen receptor (CAR) engineered T cells highly effectively eliminate hematological malignancies already in the clinics, one idea is to redirect CAR T cells also against EGFR expressing solid cancers. However, CAR T cell therapy can lead to severe, even life‐threatening side effects with high risk of on‐target/off‐tumor activity. To overcome these challenges our UniCAR technology might be an appropriate answer combining high anti‐tumor effectiveness, tumor specificity, flexibility, and safety control mechanisms. In contrast to conventional CARs, UniCAR T cells are per se inert because UniCARs are directed against a small peptide epitope, which is not present on living cells. The redirection of UniCAR T cells toward target cells occurs only in the presence of a tumor specific targeting molecule (TM) – bifunctional moieties carrying the specificity for a certain tumor antigen and contain the UniCAR peptide epitope recognized by UniCARs. TMs can be made of different molecules showing various structures. Here we are presenting the comparison in functionality, in vitro and in vivo, of two TM formats: nanobody based αEGFR TM derived from the camelid αEGFR antibody 7C12 and scFv derived TM from the clinically used chimeric monoclonal antibody Cetuximab (IMC C‐225). In principle, we observed that both TM formats are able to redirect UniCAR T cells to eliminate EGFR‐expressing tumor cells in an antigen‐specific and TM‐dependent manner. Most interestingly, the tumor killing efficiency of the αEGFR scFv TM was significantly superior in comparison to the nanobody based TM, what might decide whether UniCAR T cells attack target cells showing different EGFR density level.