Protective T cell receptor identification for orthotopic reprogramming of immunity in refractory virus infections

Abstract

Viral infections cause life-threatening disease in immunocompromised patients and especially following transplantation. Tcell receptor (TCR) engineering redirects specificity and can bring significant progress to emerging adoptive Tcell transfer (ACT) approaches. Tcell epitopes are well described, although knowledge is limited on which TCRs mediate protective immunity. In this study, refractory adenovirus (AdV) infection after hematopoietic stem cell transplantation (HSCT) was treated with ACT of highly purified Hexon5-specific Tcells using peptide major histocompatibility complex (pMHC)-Streptamers against the immunodominant human leukocyte antigen (HLA)-A∗0101-restricted peptide LTDLGQNLLY. AdV was successfully controlled through this oligoclonal ACT. Novel protective TCRs were isolated exvivo and preclinically engineered into the TCR locus of allogeneic third-party primary Tcells by CRISPR-Cas9-mediated orthotopic TCR replacement. Both TCR knockout and targeted integration of the new TCR in one single engineering step led to physiological expression of the transgenic TCR. Reprogrammed TCR-edited Tcells showed strong virus-specific functionality such as cytokine release, effector marker upregulation, and proliferation capacity, as well as cytotoxicity against LTDLGQNLLY-presenting and AdV-infected targets. In conclusion, exvivo isolated TCRs with clinical proven protection through ACT could be redirected into Tcells from naive third-party donors. This approach ensures that transgenic TCRs are protective with potential off-the-shelf use and widened applicability of ACT to various refractory emerging viral infections.