NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies

Abstract

In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.DOI: http://dx.doi.org/10.7554/eLife.16228.001

Escherichia coli surface display for the selection of nanobodies

“…albumin) and the therapeutic target, thus resulting in increased serum half‐lives (Tijink et al ., ; Vosjan et al ., ). In addition, the stability and small size of Nbs make them ideal candidates for targeting intracellular proteins and show great potential as intrabodies (Lobato and Rabbitts, ; Blanco‐Toribio et al ., ; Bouchet et al ., ; Van Impe et al ., ; Bethuyne et al ., ; Fulcher et al ., ; Moutel et al ., ). Other applications of Nbs have been recently reviewed (Bruce et al ., ).…”

“…Libraries of VHHs are commonly generated through camel immunization, often dromedaries ( Camelus dromedarius ) or llamas ( Lama glama ; Arbabi Ghahroudi et al ., ; Conrath et al ., ; Koch‐Nolte et al ., ; Hassanzadeh‐Ghassabeh et al ., ), but these may also be obtained from naïve (non‐immune) animals or be from synthetic origin by in vitro CDR randomization (Monegal et al ., ; Olichon and de Marco, ; Moutel et al ., ). In most cases, the VHHs are cloned into a vector that allows their expression on the surface of a biological entity, usually a bacteriophage or a microbial cell (i.e.…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…When the sequence of a nanobody is available after panning, its expression should be planned considering the final application of the resulting construct. We developed and validated a large array of modular vectors based on pET vector scaffolds that share: i) a conserved cloning site for the nanobody sequence (NcoI/NotI); ii) a further cloning cassette for inserting a "functional tag" such as fluorescent proteins, Avitag, SNAP, free cysteine, SpyTag …; iii) a poly-His tag for affinity purification [13,44,47,62,[126][127][128][129][130][131]. This concept enables to exchange the modules and adapt existing constructs to design new vector versions.…”

Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos

“…As long as a researcher has purified antigen of interest, it is possible to screen and evolve binders from synthetic libraries. Such libraries are available from several companies (Appendix) or have been generated by independent labs (Fridy et al, 2014; Moutel et al, 2016). Of note, protocols for a fully in vitro synthetic nanobody discovery platform, based on yeast surface display, are now available open source through the Kruse Lab (McMahon et al, 2018).…”