Discovery of novel candidate therapeutics and diagnostics based on engineered human antibody domains

Abstract

The smallest independently folded antibody fragments, the domains, are emerging as promising scaffolds for candidate therapeutics and diagnostics that bind specifically targets of interest. The discovery of such binders is based on several technologies including structure-based design and generation of libraries of mutants displayed on phage or yeast, next-generation sequencing for diversity analysis, panning and screening of the libraries, affinity maturation of selected binders, and their expression, purification, and characterization for specific binding, function, and aggregation propensity. In this review, we describe these technologies as applied for the generation of engineered antibody domains (eAds), especially those derived from the human immunoglobulin heavy chain variable region (VH) and the second domain of IgG1 heavy chain constant region (CH2) as potential candidate therapeutics and diagnostics, and discuss examples of eAds against HIV-1 and cancer-related proteins.

Fig. (1).

A flow diagram indicating various stages and technologies involved in the development of eAds.

Fig. (2).

Ribbon diagrams illustrating (A) a model of the human antibody VH domain, m0, and (B) the crystal structure of the human IgG1 CH2 domain, which have been used as scaffolds for eAd library construction. The β-strands are labeled through A-G, having the same topology and similar structures except there are two additional strands, C′ and C″, in the VH domain. The CDRs or loop regions between those β-strands are at the same end of the barrel. The CDR1–3 in VH and BC, DE, and FG loops in CH2 are marked.

Fig. (3).

Analysis of sequence diversity of the VH-based library m8l by 454 sequencing. (A) Frequencies of site-directed mutations to A, D, S or Y at specific positions in the CDR1. (B) CDR2 length distribution. (C) CDR3 length distribution.

Fig. (4).

Analysis of sequence diversity of the VH-based library m9l by 454 sequencing. (A) CDR1 (LCDR3) length distribution. (B) CDR2 length distribution. (C) CDR3 length distribution.

Fig. (5).

Analysis of sequence diversity of the CH2-based library m01sl by 454 sequencing. (A) Frequencies of site-directed mutations at eight residues in the BC loop. (B) Frequencies of site-directed mutations at five residues in the DE loop. (C) FG loop (HCDR3) length distribution. AA, amino acid.

Fig. (6).

Temperature induced unfolding of the recombinant human IgG1 CH2 domain. The graph was obtained using Avacta Optim1000 instrument. The ratio of 350 nm/330 nm indicated that the two tryptophan residues in CH2 were exposed as the protein unfolded at 50–60 °C. The unfolding transition thermal mid points (Tm) was calculated to be 56.2 °C. The two tryptophan residues in the CH2 domain are buried inside the folded protein as seen in the crystal structure [26], inset of the figure.