Fusion of an F c chain to a VHH boosts the accumulation levels in A rabidopsis seeds

Summary Nanobodies ® ( VHH s) provide powerful tools in therapeutic and biotechnological applications. Nevertheless, for some applications, bivalent antibodies perform much better, and for this, an F c chain can be fused to the VHH domain, resulting in a bivalent homodimeric VHH ‐ F c complex. However, the production of bivalent antibodies in Escherichia coli is rather inefficient. Therefore, we compared the production of VHH 7 and VHH 7‐ F c as antibodies of interest in A rabidopsis seeds for detecting prostate‐specific antigen ( PSA ), a well‐known biomarker for prostate cancer in the early stages of tumour development. The influence of the signal sequence (camel versus plant) and that of the F c chain origin (human, mouse or pig) were evaluated. The accumulation levels of VHH s were very low, with a maximum of 0.13% VHH of total soluble protein ( TSP ) in homozygous T 3 seeds, while VHH ‐ F c accumulation levels were at least 10‐ to 100‐fold higher, with a maximum of 16.25% VHH ‐ F c of TSP . Both the camel and plant signal peptides were efficiently cleaved off and did not affect the accumulation levels. However, the F c chain origin strongly affected the degree of proteolysis, but only had a slight influence on the accumulation level. Analysis of the m RNA levels suggested that the low amount of VHH s produced in A rabidopsis seeds was not due to a failure in transcription, but rather to translation inefficiency, protein instability and/or degradation. Most importantly, the plant‐produced VHH 7 and VHH 7‐ F c antibodies were functional in detecting PSA and could thus be used for diagnostic applications.