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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session A

Poster I9 - DEVELOPMENT OF HIGH-AFFINITY NANOBODIES FOR POINT OF CARE DIAGNOSTICS

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  With the health risks associated with exposure to toxins or epidemic outbreaks, point-of-care diagnostics (POCD) are necessary to aid in rapid medical intervention and triaging. A highly potent toxin, referred to here as Toxin X, has been identified as a target protein of interest for POCD development. Due to its potency and the need for rapid detection and effective intervention, Toxin X represents a significant focus for developing new diagnostic tools. However, the development of POCD requires the generation of high-affinity antibodies, which is a traditionally time- and resource-intensive process. Previous studies have demonstrated the applications of yeast display technologies in generating antibody clones and their potential for affinity maturation in a more cost- and time-effective manner.

Materials and

Methods:  Using yeast display, previously our lab panned nanobody (Nb) libraries selective against Toxin X through a series of Magnetic Activated Cell Sorting (MACS) and Fluorescent Activated Cell Sorting (FACS) to identify Nbs that bind to Toxin X. Subsequently, we characterized the binding affinities of 6 unique Nb clones through surface plasmon resonance (SPR), and performed subsequent epitope binning to determine complementary binding. Then, we integrated the Nbs into autonomous hypermutation yeast surface display (AHEAD), a technology developed by Elon et. al. that mimics somatic hypermutation by employing an error-prone polymerase to induce mutations within the genome, offering a potentially more cost-effective and faster method of affinity maturing Nbs compared to traditional animal immunization approaches. Yeast cells with Nb inserts were enriched through consecutive rounds of culturing, in which cells demonstrating high-binding and expression were selected through FACS. Cells were incubated with serial dilutions of the target antigen, with flow cytometry informing binding affinity after each round of selection.

Results, Conclusions, and Discussions: 

Results:
Initial nanobody (Nb) clones obtained through down-selection of Nb libraries via yeast display were further characterized through SPR and were determined to have KD in the nanomolar ranges, with one Nb having the lowest KD of ~24 nM. Subsequent epitope binning demonstrated potential complementary binding between several distinct pairs of Nbs. After obtaining SPR results, 2 constructs were chosen and successfully integrated into AHEAD with their preliminary results depicting an increase in binding affinity after consecutive rounds of culturing and enrichment.

Conclusions and Discussions:
Here we show an experimental pipeline that shows potential in developing potent binders towards a toxic protein of interest, with broad applicability towards developing diagnostics across other toxins or deadly diseases. Future work consists of analysis of next-generation sequencing to depict enrichment across each round of FACS and identification of mutations in residues that may influence Nb interactions with the target antigen. Subsequent purification and characterization of mutant Nbs through SPR will be necessary to identify fold changes in binding affinity, and ultimately access the potential improved sensitivity and reliability of the Nbs in diagnostic applications. Overall, improving methods for developing POCD with high sensitivity and specificity is crucial for effective detecting exposure to toxins, thereby aiding in timely therapeutic interventions and ultimately reducing patient mortality rates.

Acknowledgements (Optional):