Nanobodies as potential tools for microbiological testing of live biotherapeutic products
Robert J. Dorosky,
Jeremy E. Schreier,
Stephanie L. Lola,
Rosa L. Sava,
Michael P. Coryell,
Adovi Akue,
Mark KuKuruga,
Paul E. Carlson,
Sheila M. Dreher-Lesnick,
Scott Stibitz
Affiliations
Robert J. Dorosky
Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration
Jeremy E. Schreier
Department of Marine Sciences, University of Georgia
Stephanie L. Lola
Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration
Rosa L. Sava
Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration
Michael P. Coryell
Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration
Adovi Akue
Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration
Mark KuKuruga
Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration
Paul E. Carlson
Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration
Sheila M. Dreher-Lesnick
Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration
Scott Stibitz
Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration
Abstract Nanobodies are highly specific binding domains derived from naturally occurring single chain camelid antibodies. Live biotherapeutic products (LBPs) are biological products containing preparations of live organisms, such as Lactobacillus, that are intended for use as drugs, i.e. to address a specific disease or condition. Demonstrating potency of multi-strain LBPs can be challenging. The approach investigated here is to use strain-specific nanobody reagents in LBP potency assays. Llamas were immunized with radiation-killed Lactobacillus jensenii or L. crispatus whole cell preparations. A nanobody phage-display library was constructed and panned against bacterial preparations to identify nanobodies specific for each species. Nanobody-encoding DNA sequences were subcloned and the nanobodies were expressed, purified, and characterized. Colony immunoblots and flow cytometry showed that binding by Lj75 and Lj94 nanobodies were limited to a subset of L. jensenii strains while binding by Lc38 and Lc58 nanobodies were limited to L. crispatus strains. Mass spectrometry was used to demonstrate that Lj75 specifically bound a peptidase of L. jensenii, and that Lc58 bound an S-layer protein of L. crispatus. The utility of fluorescent nanobodies in evaluating multi-strain LBP potency assays was assessed by evaluating a L. crispatus and L. jensenii mixture by fluorescence microscopy, flow cytometry, and colony immunoblots. Our results showed that the fluorescent nanobody labelling enabled differentiation and quantitation of the strains in mixture by these methods. Development of these nanobody reagents represents a potential advance in LBP testing, informing the advancement of future LBP potency assays and, thereby, facilitation of clinical investigation of LBPs.
