Nanobody Engineering and Detection through Directed Molecular Evolution and Microfluidic Screening

Our goal was to develop a protein engineering platform which is able to quickly and cost-effectively develop nanobodies with optimal binding affinity for any antigen of interest. These engineered nanobodies are desirable for protein therapeutics and diagnostic purposes.

Background Results

Alex Boone, Garrett Hovander, Xue Zou, Junyi Jiang, Kydric Luyen. PI: Prof. Xiaohua Huang

Research DesignInstrumentation Design

Microfluidic Screening Device

This is an integrated system that can be utilized for fluorescent and phase-contrast imaging of single molecules in our flow cell microfluidic screening device.

MBBS

MBPF

TL

Zeiss Axio Observer Microscope w/ autofocus & motorized XY stages (not shown)

Zeiss TIRF 3

Liquid light guide

Sutter DG5 (300 W Xenon)

488 nm

405 nm

532 nm

642 nm

EMCCDcameras

Dual-cam adaptor

BPF

Single-mode fiber Four lasers +Optical fiber

Dual-cam adaptor with2 Andor IXon3 EMCCDs

Andor IXon+ EMCCD

Sutter DG5

Ludl XY stagesZeiss autofocus

Zeiss TIRF3Slider

Zeiss Axio Observer

A manufactured microfluidic device can be used to screen nanobodies against novel antigens. Our proof of concept was demonstrated through the adherence of biotin-labeled antigens (α-Synuclein) that were covalently bound to a biotin, avidin coated glass substrate, allowing for our nanobody (NbSyn2) to successfully bind.

Nanobody Mutagenesis & Gene Synthesis

The nanobody moieties show the variable amino acid regions that were altered using directed mutagenesis.

The DNA stain shows evident 1kb gene synthesis of 10 oligonucleotides.

100 bases

1 kilobases

Primers

1kb GeneResearch Design

References

Conclusions

Acknowledgments

Biopharmaceuticals, also known as biologics, are terms used to refer to engineered macromolecular products such as proteins that may be used for medicinal purposes. Many antibody biologics are currently very expensive methods used to treat certain diseases. Not only are antibodies an expensive form of treatment, but their thermostability, accessibility to cryptic epitopes, and ease of manufacturability calls forth the need for a replacement protein biopharmaceutical. Thus, much research is being conducted in the field of nanobody synthesis. Nanobodies posses the aforesaid positive characteristics which allow for the generation of cheaper biopharmaceuticals which are overall more effective and cheaper for the patient. Through targeted directed mutagenesis, oligonucleotide synthesis may be performed in order to develop a high-quality DNA library for gene synthesis. Following gene synthesis, a ribosomal-display complex allows for the attachment of both mRNA and protein. This complex is achieved through the integration of a hairpin mRNA conformation that impedes the translation of mRNA. Therefore, the ribosome stays attached to both the mRNA and protein. By screening this complex at varying conditions (temperature) against a novel antigen, the mRNA within the ribosomal-display complex may be reverse transcribed to cDNA, amplified, and sequenced to determined genetic relationships.

CDR 2Asparagine-52; Valine-57; Lysine-58

CDR 3Phenylalanine-101; Serine-102; Tyrosine-105; Cysteine-106; Serine-109; Tryptophan-110; Serine-111; Asparagine-112

All the aforesaid mutation regions were determined through parallel camelid sequence alignment.

Gene Construct

Amino Acid Mutations

Following the synthesis of our gene, statistical analysis validated that our product had achieved a 2.4:1 fluorescence ratio of the 1kb to .2kb band synthesized by the T4 system. This yield was much greater than the Q5 system which merely yielded a 1:1 fluorescence ratio. Following the transcription and translation of our gene, we amplified our antigen product in E. Coli. Our antigen was purified and covalently bound to metallic beads. With real-time imaging, we were able to quantify the signal ratio between samples with the nanobody and samples without the nanobody. In conclusion, the samples that contained the engineered nanobody yielded approximately 1.5 times the signal than the samples that did not contain the nanobody. These results indicate that the engineered NbSyn2 mutated nanobody is viable for targeted binding.

Specific nanobodies have shown to be highly stable molecular proteins in extreme conditions (pH, temperature, etc.). As demonstrated, large libraries of mutated nanobodies may be artificially created for high-throughput screening. Furthermore, the microfluidics device is highly reliable since there are few factors to consider when performing these processes. These factors include the use of the appropriate antigen, DNA library, and thermal/chemical conditions. Our screening process is relatively simple with the use of a protocol for the biomolecular techniques in combination with the microfluidics device. Seeing that our results have shown enhanced favourable binding of our NBSyn2 nanobody to the Alpha-Synuclein antigen, there is promise of the capability of developing artificial nanobodies in order to target novel antigens.

1) S. Wiecek, Andrew. (2010). Nanobodies: Going single-domain. BioTechniques.2) Stefan Zielonka, et al, Shark Attack: High affinity binding proteins derived from shark cNARdomains by stepwise in vitro affinity maturation. J. Biotechnol. (2014)3) Douthwaite, Julie A., and Ronald H. Jackson. Ribosome Display and Related Technologies: Methods and Protocols. New York: Humana, 2012. Print.3) Douthwaite, Julie A., and Ronald H. Jackson. Ribosome Display and Related Technologies: Methods and Protocols. New York: Humana, 2012. Print.

We would like to recognize Dr. Xiaohua Huang and Dr. Pedro Cabrales for providing the resources and guidance to successfully complete this senior design research project.

Experimental Group: With Nanobody

Control Group: Without Nanobody