A synthetic library for rapid isolation of humanized single-domain antibodies

Easily Established and Multifunctional Synthetic Nanobody Libraries as Research Tools

“…Humanization of this framework was also conducted to minimize immunogenicity to humans [19]. Some relatively early published synthetic nanobody libraries such as the Yan, Wang, Wei, and Ju libraries preferred using this framework or the humanized version as research on the universal nanobody scaffold was very limited [10][11][12][13]. To choose the scaffold for the NaLi-H1 library, Moutel et al screened several hundred clones from immune or naïve llama VHH libraries to find a nanobody with excellent solubility and stability [20,21].…”

“…Unlike other libraries with a huge gap between the designed and achieved varieties, the Ju library tried to constrain the designed variety to a manageable size using phage display. The designed variety was around 10 11 by using short-length CDR3 containing seven amino acids and limiting amino acid diversity within three different amino acids at moderately variable positions and within nine at five highly variable positions [10] (Figure 3). Libraries with a manageable diversity are designed to allow complete control of the library contents, while libraries with a higher designed diversity can introduce more opportunities to find high-binding affinity nanobodies.…”

Immunogenicity and humanization of single‐domain antibodies

“…A synthetic phage display library of humanized V H Hs, NaLi-H1, with a complexity of 3×10 9 was described by Moutel et al [131]. To construct the library, a consensus V H H optimized for intracellular expression and stability (hs2dAb) was partially humanized at A similar CDR randomization approach was employed by Ju et al [136] to generate a synthetic humanized V H H phage display library based on the "universal" humanized scaffold, hNbBcll10 FGLA , of Vincke et al [102]. Several humanized V H Hs against each of three model antigens were identified by phage ELISA and DNA sequencing.…”

Selection of Functional Intracellular Nanobodies

“…Phage display, the most extensively used screening method, can generally manage libraries of 10 10 to 10 12 clones, which is much less than the >10 30 different VHH sequences that would be produced from full CDR randomization. 47…”

“…To cope with the limitations involved in synthetic VHH library production Man-Seok Ju et al 47 analyzed naturally occurring CDR sequences and limited the variation within the CDR loops to a select group of amino acids that most often occur in nature. Stop codons and cysteines were excluded to prevent premature termination or addition of unwanted disulfide bonds.…”