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

Abstract

BACKGROUND Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domain (nanobody) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. METHODS Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct used for pull-down/MS target identification. RESULTS The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. CONCLUSIONS This strategy streamline the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. GENERAL SIGNIFICANCE This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.

Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays

“…In order to select nanobodies reacting with native epitopes of the enzyme, the library was panned against chemically biotinylated sEH immobilized on streptavidin coated magnetic beads. After two rounds of panning, the DNA sequence of the VHHs were transferred “ en masse ” to the pINQ-BtH6 vector, which allows high yield expression of the nanobodies fused to a 15-mer biotin-acceptor-peptide (BtAP) tag 27 . The reactivity of 96 clones with sEH was assayed by ELISA, figure 1.…”

“…Under these conditions, the biotin binding capacity of the wells was saturated with BtNb concentrations ≥ 0.5 µg/mL, figure 3. We recently reported the optimization of the in vivo biotinylation of nanobodies in 96 well culture plates 27 , and typically, the yield of expression of different VHHs in 96 culture plates is well above this value, however to compensate for loses during the purification step and assure full saturation, six replicas of a culture block with 96 sEH-ELISA positive clones were grown in parallel and the cell extracts were combined in the Ni-NTA spin plates. This step serves a double purpose; it concentrates the BtNb and eliminates the free biotin before their incubation in the avidin wells.…”

Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications

“…In the context of this peptide the side chain of its Lys residue is coupled with biotin by overexpression of the biotin ligase BirA of Escherichia coli during the production of the Nb ( 47 ). We recently adopted this strategy to develop a method for high-throughput screening of Nbs to cell receptors ( 48 ). The in vivo biotinylation facilitates the parallel analysis of hundreds of Nb clones by cell cytometry, because there is no need of blocking the interactions of the primary or secondary antibodies in case of cells expressing Fc receptors.…”

“…The strong biotin–streptavidin binding also allows for stringent conditions during pull-down experiments yielding neat finger printings for mass spectrometry identification. Moreover, by adding or not adding biotin during the expression of the Nbs, each clone can be produced with or without this label, which allows to perform competitive epitope binning of the selected Nbs directly on the cells ( 48 ). Once selected, the biotinylated Nbs are ready-to-use reagents for cell cytometry diagnosis or immunohistochemistry ( 49 ).…”

Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain

“…The VHHs V36, G7, and 49 specific for the CD11b chain of Mac-1, the mouse Ly-5 leukocyte common antigen (CD45), and the b chain of the class II MHC, respectively, were recently isolated and characterized in our laboratory. 30 The bispecific antibodies were prepared and their functionality tested by ELISA and flow cytometry. Figure 5 shows the ELISA titration of equal amounts of nanobodies against TetC.…”