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.

Numerous sophisticated approaches have been developed to optimize the TCR expression cassette 14, 19, 22, 23 , pairing of the introduced TCR 24-29 and function/maturation of the resulting T cell population 30, 31 . These approaches have largely been based on the modification of a single TCR in each study. We chose the simplest modifications previously reported in the literature and applied them to our library of 10 different virus-specific TCRs to optimize TCR expression and the antiviral potential of redirected primary human T cells. In line with previous reports, we observed that codon optimization, linking the alpha and beta chains with a P2A sequence, including a glycine-serine-glycine spacer, and the addition of a non-native cysteine disulfide bond in the TCR constructs led to increased expression and pairing. Surprisingly, when we tested whether simply inverting the orientation of the alpha and beta genes within the TCR expression cassette impacted TCR expression and function we found evidence for clear positive (high frequency) and negative (low frequency) orientation. In 9/10 of the TCRs, placing the beta chain upstream of the P2A sequence resulted in enhanced expression. This enhanced expression translated to increased HLA-multimer staining in the non-CD8 T cell population as well, comprised primarily of CD4 T cells, which provide help for virus specific CD8 T cells in vivo 32 .