Beringer, D. | Search bioRxiv

bioRxiv2021

Challenges of γ9δ2TCR affinity maturation when using phage display

BackgroundOver the past years we showed that the efficacy of {beta}T cells engineered to express a defined {gamma}{delta}TCR (TEG) depends on the functional avidity of the {gamma}9{delta}2TCR. We hypothesized that functional avidity mediated through {gamma}9{delta}2TCR in the TEG format could be further enhanced by increasing affinity of the {gamma}9{delta}2TCR. MethodsWe attempted to overcome limited affinity of natural occurring {gamma}9{delta}2TCRs through affinity maturation by phage display using a library containing mutations in CDR1 and CDR2 of both TCR chains. ConclusionAffinity maturation of {gamma}9{delta}2TCR by using phage display was not successful. The largest hurdle was the periplasmic expression of {gamma}9{delta}2TCR 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.

immunology↗

bioRxiv2021

Tumor-reactivity of d2 negative gdT cells and the role of the gdT cell receptor

{beta}T cells engineered to express a defined {gamma}{delta}TCR (TEG) to attack cancer cells have shown great promise when using a {gamma}9{delta}2TCR to redirect {beta}T cells (1-9). Reports by us (1-9) and recent reports by others (10, 11) support the key role of the {gamma}9{delta}2TCR in cancer recognition. We further emphasized the crucial role of the {delta}TCR chain and that differences in CDR3 sequences of the {delta}TCR chain modulates functional avidity of TEGs (2, 8). We and others demonstrated that also {delta}2 negative {gamma}{delta}TCRs are able to redirect {beta}T cells towards different tumor cell lines (12-15). However, some studies suggest that {delta}2 negative {gamma}{delta}TCRs play a minor role in the tumor recognition by {delta}2 negative {gamma}{delta}T cells (16, 17). In addition for both modes of action for tumor-recognition, {delta}2 negative {gamma}{delta}TCR-dependent and -independent, it has been suggested that CMV infection is not only a major driver of {delta}2 negative {gamma}{delta}T cell expansion (18) but also induces tumor-cross reactive {delta}2 negative {gamma}{delta}T cells (19-21). Therefore, we aimed to systematically explore frequencies of tumor reactive {delta}2 negative {gamma}{delta}T cells in naive repertoires (cord blood) and patients with or without CMV infection and examined the potential role the parental {delta}2 negative {gamma}{delta}TCR in anti-tumor reactivity of selected clones. We observed that approximately 30% of all tested clones were tumor-reactive, though no differences were observed between different sources. Surprisingly, none of the so far tested {gamma}{delta}TCR did mediate strong anti-tumor reactivity of the parental clones. Though numbers of tested TCR sequences are still low, our data imply that tumor-reactivity of {delta}2 negative {gamma}{delta}T cells is frequently not mediated by the {delta}2 negative {gamma}{delta}TCR alone.

immunology↗