Building and Optimizing a Virus-specific T Cell Receptor Library for Targeted Immunotherapy in Viral Infections

Banu, Nasirah; Chia, Adeline; Ho, Zi Zong; Garcia, Alfonso Tan; Paravasivam, Komathi; Grotenbreg, Gijsbert M.; Bertoletti, Antonio; Gehring, Adam J.

Restoration of antigen-specific T cell immunity has the potential to clear persistent viral infection. T cell receptor (TCR) gene therapy can reconstitute CD8 T cell immunity in chronic patients. We cloned 10 virus-specific TCRs targeting 5 different viruses, causing chronic and acute infection. All 10 TCR genetic constructs were optimized for expression using a P2A sequence, codon optimization and the addition of a non-native disulfide bond. However, maximum TCR expression was only achieved after establishing the optimal orientation of the alpha and beta chains in the expression cassette; 9/10 TCRs favored the beta-P2A-alpha orientation over alpha-P2A-beta. Optimal TCR expression was associated with a significant increase in the frequency of IFN-gamma1 T cells. In addition, activating cells for transduction in the presence of Toll-like receptor ligands further enhanced IFN-gamma production. Thus, we have built a virus-specific TCR library that has potential for therapeutic intervention in chronic viral infection or virus-related cancers. C D8 T cells are a critical component to clearing or controlling viral infections. Lack of a virus-specific T cell response is associated with failure to control chronic Hepatitis B virus (HBV) infection 1 and loss of virusspecific T cells due to immune suppression during hematopoietic stemcell or solid organ transplant can lead to life-threatening Epstein-Barr Virus (EBV) and human cytomegalovirus (hCMV) infections 2 . Reconstitution of virus-specific immunity, either through bone marrow transplant [3] [4] [5] or adoptive transfer of virus-specific T cells 6, 7 , can control these persistent infections. In addition, data from influenza has demonstrated that pre-existing virus-specific T cell immunity can protect against lethal infection 8, 9 . Therefore, strategies to manipulate the virus-specific T cell response could lead to clinical therapies to treat chronic infections or prevent mortality related to severe acute infections.