(E-171) A novel DNA display technology that facilitates the engineering of nanobodies

Introduction:: Enzyme-based therapeutics have the potential to address a wide variety of clinical needs, but their development has been challenging because nonhuman enzymes elicit unwanted immune responses. Nanobodies, which can be evolved for catalytic activity, exhibit high specificity and provide a significantly lower risk of immunogenicity. As such, engineering nanobodies is an avenue towards the development of a promising novel class of therapeutics. Current methods used in protein evolution include phage and mRNA display, but both these methods have constraints. Phage must be grown in bacteria, meaning that a plasmid containing the genetic sequence for phage must be transformed into bacteria. Transformation efficiency decreases as the size of the plasmid to be transformed increases, thus constraining the library size for phage display to 109 variants. Furthermore, in vivo technologies put selection pressure against poorly expressed and rapidly degraded proteins. In vitro technologies like mRNA display have been developed to avoid these issues, but preparing mRNA for mRNA display is labor intensive and the technique is further complicated by the inherent instability of mRNA. As such, we propose the development of a novel platform to rapidly engineer catalytic nanobodies with therapeutic enzyme activities. Specifically, we propose the development of a multiplexable, in vitro DNA display technology that leverages transcriptional and translational coupling to be used for the engineering of this potentially novel class of therapeutics.


Materials and Methods:: A fundamental part of this design is confirming that nanobodies can be expressed in vitro. Since disulfide bonds are critical to nanobody structure and function, nanobody expressions are being tested in various redox environments. The redox environment of each condition is adjusted by adding varying amounts of dithiothreitol, glutathione, and glutathione disulfide. DsbC is a known chaperone that facilitates proper disulfide formation, so each redox environment is also being tested with and without the addition of the chaperone. The protein capture complex will consist of catalytically inactive Cas9 conjugated to some structure that will catch the peptide tags attached to the protein of interest. Several factors have been considered in designing the protein capture complex, most important of which are the binding affinity between the tag-tag catcher and ease of assembly. Transcriptional and translational coupling will be restored following strategies previously demonstrated in literature, including but not limited to lowering incubation temperatures and using mutant polymerases and ribosomes.


Results, Conclusions, and Discussions:: Protein gels evaluating the first set of in vitro expressions of the GFP-minimizer nanobody were inconclusive. To get a conclusive result, a sample of the same reaction was tested through a Western blot with anti-His antibodies. The presence of bands at 14kDa is indicative of nanobody expression. This result, in conjunction with the inability to visualize a clear band through protein gels, leaves room for optimization in the in vitro expression. Expression could be potentially improved through the addition of other redox chaperones like Erv1p, the addition of secondary energy sources and amino acids, and an increase in incubation time/temperature. Further studies that define required process parameters to restore transcriptional/translational coupling in vitro are needed to understand potential constraints that must be considered when optimizing nanobody expression in vitro. Hypergeometric distribution analysis detailing the likelihood of conjugating a streptavidin/biotin complex in an ideal ratio reports a maximum success rate of ~40%, suggesting the construction of complexes reliant on the conjugation of multiple components to be highly inefficient. Future iterations may explore the idea of creating a dCas9 fusion to the tag-catcher. Expression of a fusion protein can be done on a single plasmid and avoids issues related to low conjugation efficiency. Moreover, the use of dCas9 fusions is well established in epigenome editing applications, and there exists literature describing the optimization of flexible linkers to be used in dCas9 fusion proteins.


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