Backbone and side-chain resonance assignments of the NISTmAb-scFv and antigen-binding study

Abstract

Monoclonal antibodies (mAbs) therapeutics are the largest and fastest growing class of biologic drugs, amongst which, the vast majority are immunoglobulin G1 (IgG1). Their antigen binding abilities are used for the treatment of immunologic diseases, cancer therapy, reversal of drug effects, and targeting viruses and bacteria. The high importance of therapeutic mAbs and their derivatives has called for the generation of well-characterized standards for method development and calibration. One such standard, the NISTmAb RM 8621 based on the antibody motavizumab, has been developed by the National Institute of Standards and Technologies (NIST) in the US. Here, we present the resonance assignment of the single chain variable fragment, NISTmAb-scFv, that was engineered by linking the variable domains of the heavy and light chains of the NISTmAb. Also, addition of a peptide, corresponding to the target antigen of motavizumab, to samples of NISTmAb-scFv has induced chemical shift perturbations on residues lining the antigen binding interface thereby indicating proper folding of the NISTmAb-scFv.

Keywords: Monoclonal antibody; Motavizumab; NISTmAb; NMR spectroscopy; Respiratory syncytial virus; Single-chain variable fragment.

Fig. 1

Two-dimensional 15N-HSQC spectrum of 13C-15N-NISTmAb-scFv at 700 MHz recorded at 35 °C. Peaks are assigned according to the residue number of our construct (see text)

Fig. 2

a Phe82 (represented in CPK) in the NISTmAb-Fab domain adopts a rotamer that is locked due the proximity of the constant light (CL) domain (green surface).b In the NISTmAb-scFv, the absence of the CL domain allows Phe226 (stick) to adopt a rotamer in NISTmAb-scFv that induces a shielding effect on Asp224 amide resonance (blue sphere)

Fig. 3

a Overlay of 2D-15N-HSQC spectra of 13C-15N-NISTmAb-scFv free (blue) and peptide-bound (red) at 700 MHz recorded at 35 °C. Residues showing chemical shift perturbation upon peptide binding are indicated with a color code (red, green, blue, brown, yellow, grey-white and purple. b Ribbon diagram of the variable domains (VH and CL) of the NISTmAb-Fab X-ray structure (PDBID: 3IXT). Residues experiencing CSP depicted in panel A are mapped on the structure using the same colors