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.

Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies

“…The procedure can be repeated by re-equilibrating the column with the denaturing buffer, then the cycle of denaturant removal via a gradient and protein elution is repeated to obtain additional amounts of folded protein. This method has been used successfully for the high-yield production of cytokines [ 30 – 32 ] and the NISTmAb-scFv fragment [ 21 ]. While three cycles of refolding were needed for cytokines, the NISTmAb-scFv required up to 10 cycles.…”

“…This concept had been tested for the soluble expression of Fab fragments [ 11 , 18 – 20 ], however, we added the capability to remove the linker by taking advantage of the papain cleaving site in the hinge region of the heavy chain and by adding a thrombin cleaving site at the amino-terminal end of the light chain. In addition, a cleavable polyhistidine tag was added to the amino-terminal end of the heavy chain to allow on-column refolding [ 21 ] among other refolding methods. This single chain strategy was tested on four Fab fragments: adalimumab-Fab (Humira®), NISTmAb-Fab (RM 8671), rituximab-Fab (Rituxan®), and trastuzumab-Fab (Herceptin®).…”