Engineered CH2 domains (nanoantibodies)

Abstract

Currently, almost all FDA approved therapeutic antibodies (except ReoPro, Lucentis and Cimzia which are Fabs), and the vast majority of those in clinical trials are full-size antibodies mostly in IgG1 format of about 150 kDa size. A fundamental problem for such large molecules is their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules (e.g., on the HIV envelope glycoprotein) which are fully accessible only by molecules of smaller size. Therefore, much work especially during the last decade has been aimed at developing novel scaffolds of much smaller size and high stability. Here I briefly describe a proposition to use the immunoglobulin (Ig) constant CH2 domain (CH3 for IgE and IgM) as a scaffold. CH2 is critical for the Ig effector functions. Isolated CH2 is stable monomer in contrast to all other constant domains and most of the variable domains. CH2 and engineered CH2 domains with improved stability can be used as scaffolds for construction of libraries containing diverse binders to various antigens. Such binders based on a CH2 scaffold could also confer some effector functions. Because the CH2 domains are the smallest independently folded antibody domains that can be engineered to contain simultaneously antigen-binding sites and binding sites mediating effector and stability functions, and to distinguish them from domain antibodies which are used to denote engineered VH or VL domains or nanobodies which are used to denote camelid VHH, I termed them nanoantibodies (nAbs).

Keywords: CH2; Fabs; antibodies; nanoantibodies; scaffold.

Figure 1

Crystal structure of Fc (PDB code 1IIS)14 (the two heavy chains are in light and dark blue and the N-linked oligosaccharides attached to Asn297 is in red) indicating weak carbohydrate-mediated inter-chain CH2 interactions in contrast to CH3.

Figure 2

Structure of the C_H2 antibody domain and structural comparison with the corresponding region in the Fc and IgG structures. (A) Ribbon diagram of the isolated, unglycosylated C_H2 domain from IgG g1 is shown. (B) The isolated C_H2 domain (green) was superimposed by a least-squares algorithm using the Cα traces of the C_H2 domains of Fc structure (blue; PDB code 2DTQ). (C) Superposition of the isolated C_H2 structure (green) with that of C_H2 portions of an intact IgG (PDB code 1HZH) using the Cα trace alignment. Heavy and light chains of IgG are shown in red and blue respectively. Carbohydrate moieties in between the C_H2 domains of Fc and IgG structures in (B) and (C) are omitted for clarity.