Fragments antigen-binding (Fabs) are antibody derivatives that are sought after reagents for both therapeutic and basic science applications. The traditional methodologies for Fab production utilize proteolytic enzymes such as papain to cleave the flexible elbow region, located between the Fab and FC portions of the antibody. Due to the low sequence specificity of papain and other widely used proteases this process is rarely efficient and requires optimization for each individual monoclonal antibody (MAb).
To overcome this difficulty we incorporated a genetically engineered human rhinovirus 3C (HRV3C) protease site into the elbow region of recombinant MAb constructs. We demonstrated efficient cleavage of MAbs containing the introduced HRV3C site, with no off-target proteolytic digestion of the FC or Fab products in the presence of a recombinant HRV3C protease. Due to the high sequence specificity of the HRV3C protease we hypothesised that this system would allow for the digestion of antibodies in the presence of bound antigen without compromising the integrity of the antigen-Fab complex. In this format the antibody-antigen complex is purified using protein A/G affinity chromatography and proteolytic release of the Fab bound antigen is achieved on-resin with the addition of the HRV3C protease.
To demonstrate this approach we utilized two well established viral antigens, DIII from dengue virus (DENV) and F protein from respiratory syncytial virus (RSV). We introduced the HRV3C site into the recombinant MAbs constructs 4E11 and Motavizumab, specific for DENV DIII and RSV F respectively. Recombinant DENV DIII was obtained from a crude periplasmic E. coli fraction and RSV F obtained from supernatant of a stable CHO cell line expressing the F ectodomain. After antigen capture on recombinant MAbs immobilized on protein A resin, the antigen-Fab complexes were released with the addition of the HRV3C protease, while the FC portion remained immobilized. We also demonstrated the removal of the recombinant HRV3C protease through use of the incorporated GST HRV3C fusion tag. We then validated antigen-Fab capture through SDS-PAGE and Western blot analysis. Antigen-Fab complexes were then further confirmed using negative stain transmission electron microscopy (TEM). We anticipate that this one-step procedure will greatly facilitate antigen-Fab complex generation and accelerate the structural determination of antibody-antigen interactions.