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.

As previously demonstrated, codon optimization and incorporating the alpha and beta genes into a single cassette increased TCR expression in all three TCRs compared to wild type TCRs on separate vectors (Fig. 1A) . We did not see substantial differences in the frequency of HLA-multimer positive cells between single cassette constructs with and without the additional disulfide bond ( Fig. 1A ; a-b-Opt vs a-bcys-Opt). However, there was a clear advantage with the inclusion of an additional disulfide bond. The HLA-multimer positive CD8 T cell population showed much more defined staining and a higher mean fluorescence intensity in 2 out of 3 TCRs (Fig. 1B) . We also observed that the additional disulfide bond increased fluorescence intensity in the non-CD8 T cell population. Therefore, all subsequent TCR expression cassettes employed the 2A cleavage site with codon optimization and the additional disulfide bond to maximize pairing.