Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

About authors

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

Correspondence should be addressed: Stepan P. Chumakov
Miklukho-Maklaya, 16/10, Moscow, 117997; moc.liamg@lukhtah

About paper

Funding: the work was funded by MESR, project code RFMEFI60716X0156.

Author contribution: Kravchenko YuE — Alpaca blood collection and processing, RNA extraction, ELISA; Ivanov SV — construction of libraries of VHH-fragments of antibodies, selection by phage display; Kravchenko DS — protein purification, experiments with eucaryotic cell cultures; Frolova EI — research planning, animal immunization, manuscript editing; Chumakov SP — research planning, selection by ribosome display, analysis of the results, writing a manuscript.

Received: 2018-12-08 Accepted: 2018-12-22 Published online: 2019-02-25

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

Keywords: nanobodies, VHH antibodies, ribosome display, phage display, biopanning, PDLIM4