1 Nanomedicine, Department of Pharmacy, Faculty of Health and Medical Sciences, Københavns Universitet2 National Cell Bank of Iran, Pasteur Institute of Iran3 bDepartment of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University4 aNational Cell Bank of Iran, Pasteur Institute of Iran5 cSchool of Allied Medical Sciences, Tehran University of Medical Sciences6 d Biotechnology Department, Pasteur Institute of Iran7 Department of Pharmacy, Faculty of Pharmaceutical Sciences, Københavns Universitet8 Department of Pharmacy, Faculty of Pharmaceutical Sciences, Københavns Universitet
Despite the preclinical success of adoptive therapy with T cells bearing chimeric nanoconstructed antigen receptors (CARs), certain limitations of this therapeutic approach such as the immunogenicity of the antigen binding domain, the emergence of tumor cell escape variants and the blocking capacity of soluble antigen still remain. Here, we address these issues using a novel CAR binding moiety based on the oligoclonal camelid single domain antibodies. A unique set of 13 single domain antibodies were selected from an immunized camel phage library based on their target specificity and binding affinity. A combination of these single domain antibodies was used to generate four tumor associated glycoprotein (TAG-72)-specific CARs harboring an identical antigen binding site, but with different signaling and spacer domains. Although all four CARs were functionally active against the TAG-72 expressing tumor cells, the combination of CD3ζ, OX40, CD28 as well as the CH3-CH2-hinge-hinge domains most efficiently triggered T cell activation. Importantly, CAR mediated functions were not blocked by the soluble TAG-72 antigen at a supraphysiological concentration. Our approach may have the potential to reverse multiple tumor immune evasion mechanisms, avoid CAR immunogenicity, and overcome problems in cancer gene therapy with engineered nanoconstructs.