Project Description

Project Overview

Our project addresses the problematic time and resources required in developing specific antibodies for use in assays like rapid antigen tests (RAT). During the pandemic, development and manufacture was a bottleneck restricting the effective implementation of RATs. These limitations stemmed from problems upscaling the production of monoclonal antibodies (mAbs) in tests, which requires animal experimentation followed by tedious purification and processing.

Our Solution

The objective of our project is to fundamentally improve the methods used to generate and select for novel nanobodies, which have near limitless potential for implementation in the fields of research and therapeutics. We have successfully designed a procedure (Figure 1(a)) that can produce nanobodies with specific binding to a targeted antigen in a fraction of the time and resources required to produce the same results with conventional (Figure 1(b)) in vivo methods (3-4 months) associated with the isolation of nanobodies from camelied B cell lines (Pardon et al. 2014). This greatly lowers the barrier between this technology and its potential user-bases by reducing the cost of the development.

Our solution utilises DNA shuffling to produce novel nanobodies, which are smaller, more stable, and more easily manufactured compared to mAbs. DNA shuffling involves in vitro random recombination of genes to rapidly generate a large mutant gene library. As a proof of concept, we will shuffle anti-sfGFP nanobodies to produce new nanobodies specific to fuGFP (a highly mutated variant of sfGFP that is not patented). Shuffled nanobodies will be cloned into surface display plasmids expressed in E. coli. Displayed novel nanobodies can bind to fuGFP fused to cellulose binding domains (CBDs) immobilised on a cellulose matrix, allowing them to be isolated for characterisation or further shuffling.

workflow of in-vivo antibody generation from conventional methods
Workflow of our in-vitro generation of confirmation nanobodies. More detailed information can be found in the experiments page of this wiki.
Figure 1. (a) Workflow (top) for the generation of conformational Nanobodies in vivo by Pardon et al. (2014). (b) Our workflow (bottom) for the generation of conformational nanobodies in vitro.
The success of our project is a gateway to cost-effective and rapid approaches for generating novel antibodies against emerging diseases and disease variants.

The goal is to create E. coli which presents anti-fuGFP surface nanobodies. We first constructed a plasmid which contains regions coding for anti-sfGFP nanobody expression, along with a complex allowing for surface display of that nanobody. This complex is based on a paper by Salema & Fernández (2017), who successfully made nanobody-presenting E. coli. The binding affinity between anti-sfGFP E. coli and sfGFP can be quantified through fluorescence spectroscopy which is used as a control. The binding of fuGFP and anti-sfGFP E. coli was then tested to compare the affinity of the nanobodies between GFPs.

Nanobody Surface Display & Shuffling

The image compares genetic distances generated from random mutagenesis and DNA shuffled libraries. It shows that the variation from the parental sequence seen from the libraries is greater for DNA shuffling, due to the increased space between mutagen genotypes.
Figure 2. Comparison of the genotypic distance generated from random mutagenesis library (left) and DNA-shuffled library (right) illustrated in a 2 dimensional plane by Meyer et al. (2015). The black dot represents the starting parental sequence which will be anti-sfGFP in our study. The gray dots are the mutated nanobodies synthesised from either site-directed mutagenesis, which only involves point-mutations (left), or DNA shuffling (right). The star will be the new anti-fuGFP sequence which is our aim. Notably, DNA shuffling uses multiple parental sequences and recombines them to result in a wider sequence space which covers anti-fuGFP unlike random mutagenesis, making it more time and resource efficient.

Anti-GFP plasmid recombinants that are more selective for fuGFP than sfGFP can be created using DNA shuffling techniques, described by Stemmer (1994), to be compiled into a library of shuffled variants. The protocol is divided into 4 main steps: DNAse I fragmentation, PCR with and without primers, product cloning and analysis, and whole plasmid reassembly. In practice, the steps can be performed over multiple cycles to create a library of DNA sequences that qualitatively reports on the recombinants’ efficacy, allowing the selection of the best one for the next cycle (Figure 2).

The output of DNA shuffling is full length sequences of shuffled nanobodies which can be purified and inserted into plasmids. The plasmids can be transformed into E. coli for surface display and isolation with CBD bioprocessing.

Free-use GFP & Cellulose Binding Domains

CBDs were used in conjunction with E. coli display to isolate functionally active anti-fuGFP nanobodies produced by DNA shuffling of anti-sfGFP nanobodies. This novel approach involves immobilising fuGFP-CBD fusion proteins on a cellulose matrix and binding an E. coli library with surface displayed shuffled sfGFP nanobodies. E. coli that remain bound were eluted for characterisation of the shuffled nanobody gene. They could also be shuffled again to potentially increase its binding.

References

Meyer, A.J., Ellefson, J.W. and Ellington, A.D., 2014. Library generation by gene shuffling. Current protocols in molecular biology, 105(1), pp.15-12.

Pardon, E., Laeremans, T., Triest, S., Rasmussen, S.G.F., Wohlkönig, A., Ruf, A., Muyldermans, S., Hol, W.G.J., Kobilka, B.K., and Steyaert, J., 2014. A general protocol for the generation of Nanobodies for structural biology. Nature Protocols, 9(3), 674–693.

Stemmer, W.P., 1994. Rapid evolution of a protein in vitro by DNA shuffling. Nature, 370(6488), pp.389-391.