Challenges of γ9δ2TCR affinity maturation when using phage display

Background Over the past years we showed that the efficacy of αβT cells engineered to express a defined γδTCR (TEG) depends on the functional avidity of the γ9δ2TCR. We hypothesized that functional avidity mediated through γ9δ2TCR in the TEG format could be further enhanced by increasing affinity of the γ9δ2TCR. Methods We attempted to overcome limited affinity of natural occurring γ9δ2TCRs through affinity maturation by phage display using a library containing mutations in CDR1 and CDR2 of both TCR chains. Conclusion Affinity maturation of γ9δ2TCR by using phage display was not successful. The largest hurdle was the periplasmic expression of γ9δ2TCR constructs in E.coli which is a prerequisite for successful phage display. The underlying reason for this lack of expression was the instability of the single chain (sc)TCR format. Expression of scTCR formats in HEK293F cells yielded only 15-20% correctly folded scTCR. ### Competing Interest Statement JK is shareholder of Gadeta. JK, EvD, and DXB are inventors on patents with γδTCR related topics.