Overcoming low yields of plant-made antibodies by a protein engineering approach
The commercial development of plant-based antibody production platforms is often limited by low and variable yields, but little is known about the factors that affect antibody accumulation during and after translation. Here, we present a strategy to identify yield-limiting regions in the transcript and protein. We exchanged variable heavy chain (VH) domain sequences between two human antibodies at structurally conserved positions, thus creating ten chimeric VH domains containing sequences from M12 (similar to 1000 mu g/g leaf fresh weight [FW]) and 4E10 (similar to 100 mu g/g FW). After transient expression in Nicotiana benthamiana leaves, we measured mRNA and protein levels by quantitative real-time PCR and surface plasmon resonance spectroscopy, respectively. Transcript levels were similar for all constructs, but antibody levels ranged from similar to 250 mu g/g to over 2000 mu g/g FW. Analysis of the expression levels showed that: (i) 4E10 yields were only marginally increased by suppression of post-transcriptional gene silencing; (ii) the complementarity determining region 3 (CDR3) of 4E10 contains a protease site; and (iii) a bipartite, yield-limiting region exists in the CDR2/CDR3. Our findings highlight the strong impact of cotranslational and posttranslational events on antibody yields and show that protein engineering is a powerful tool that can be used to overcome the remaining limitations affecting antibody production in plants.