An animal model of HLA-independent, chimeric antigen receptor-mediated adoptive immunotherapy against cytomegaloviruses
While an infection with the human cytomegalovirus (CMV) causes only mild symptoms in immunocompetent individuals, this virus is the most frequent cause of congenital infection and represents a serious health risk for immunocompromised patients. Especially in the context of an allogenic stem cell transplantation (SCT), CMV infection or reactivation remains a life‑threatening complication. Based on a strategy of adoptive T cell therapy, this project investigates CMV‑specific chimeric antigen receptor (CAR) expressing T cells for their potential to restrict a CMV infection in vitro and in the relevant murine CMV (MCMV) model. In detail, CAR T cells are equipped with a chimeric receptor that is composed of an extracellular single-chain variable fragment (scFv) redirecting the immune cell to a specific target structure. This scFv is structurally linked to an intracellular CD3ζ signal transduction domain derived from the T cell receptor complex and, characteristic for second generation CAR constructs, to an additional co‑stimulatory domain (4-1BB or CD28). Because of this structure, binding of CAR T cells to their target cells directly induces intracellular signalling and results in T cell activation, secretion of effector cytokines and finally lysis of the antigen‑expressing target cell. Multiple CAR constructs targeting different MCMV glycoproteins have previously been designed in our research group. In this project, murine T cells will be equipped with these CARs by retroviral transduction or mRNA based approaches. The CAR expression will be validated by flow cytometry (Aim 1). For each CAR construct, recognition and cytotoxicity will be analysed in vitro using MCMV glycoprotein expressing target cell lines (Aim 2). The killing assays will additionally be conducted using cells infected with a luciferase encoding MCMV reporter strain, MCMVm157luc. Here, different MCMVm157luc knock out variants of genes interfering with Perforin/Granzyme mediated lysis will be engineered and the influence on the CAR-mediated killing will be investigated (Aim 3). Finally, the therapeutic effect of the most promising CAR candidates will be examined in vivo using MCMVm157luc infected immune deficient RAG-/- mice(Aim 4). Once a therapeutic effect is demonstrated in the mouse model, this study can provide strong rationales to further investigate antiviral CAR‑mediated adoptive immunotherapies in order to combat CMV infections in SCT recipients.
