Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells

MARTÍN ROSSOTTI; SOFÍA TABARES; LUCÍA ALFAYA; CARMEN LEIZAGOYEN; GABRIEL MORON; GUALBERTO GONZÁLEZ-SAPIENZA

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2015 vol. 1850 p. 1397 - 1404

0304-4165

Background: Owing to theirminimal size, high production yield, versatility and robustness, the recombinantvariable domains (nanobodies) of camelid single chain antibodies are valued affinity reagentsfor research, diagnostic, and therapeutic applications. While their preparationagainst purified antigens is straightforward, the generationof nanobodies to difficult targets such as multi-pass orcomplex membrane cell receptors remains challenging. Here we devised a platformfor high throughput identification ofnanobodies to cell receptor based on the use of a biotin handle. Methods: Using abiotin-acceptor peptide tag, the in vivo biotinylation ofnanobodies in 96well culture blocks was optimized allowing their parallelanalysis by flow cytometry and ELISA, and theirdirect use for pull-down/MS target identification. Results: The potential ofthis strategy was demonstrated by the selection and characterization of panelsof nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte commonantigen (CD45) receptors, from a VHH library obtained from a llama immunizedwith mouse bone marrow derived dendritic cells. By on and off switching of theaddition of biotin, the method also allowed the epitope binning of the selectedNbs directly on cells. Conclusions: This strategystreamlines the selection of potent nanobodies to complex antigens, and theselected nanobodies constitute ready-to-use biotinylated reagents. Generalsignificance: This method willaccelerate the discovery of nanobodies to cell membrane receptors which comprisethe largest group of drug and analytical targets.