The expression and purification of the fab fragment of ibalizumab: a monoclonal anti-CD4 antibody with potent HIV inhibitory effects

Abstract

Infection with Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immunodeficiency Syndrome (AIDS), remains one of the most significant healthcare challenges facing South Africa. The development of alternative therapeutic strategies targeting different stages of the viral life cycle remains an important ongoing biomedical research objective. Recently, a monoclonal antibody which binds CD4, the primary cellular host receptor of HIV, has shown outstanding antiviral efficacy in Phase II clinical trials. However, the high costs associated with conventional therapeutic monoclonal antibody production, which involves expression, assembly and secretion of full-length antibodies using large-volume mammalian cell cultures; limit the clinical use of such compounds in the South African public healthcare setting. Accordingly, this study investigated the feasibility of generating a functional Ibalizumab Fab (IBLZFab) fragment using an E. coli-based expression system. Heavy and Light Variable (VH/VL) Ibalizumab chains were expressed in BL21 E. coli and functional Fab fragments were reconstituted by oxidative refolding in the bacterial periplasm. The success of reconstitution and efficacy of the Fab fragment was assessed in vitro by analytical SDS-PAGE, Surface Plasmon Resonance (SPR) and cell culture models of HIV infection. The calculated CD4-binding affinity (KD) of IBLZFab was 11.3 nM, which compares favorably with the (KD) values of most antibody-antigen interactions, which are typically in the low nanomolar (nM) range. Viral inhibition studies were also performed to evaluate whether the Fab fragment had any detectable neutralization activity. Even though neutralization activity was obtained at a high concentration the bacterially expressed IBLZFab fragment was able to inhibit CAP210 viral entry, illustrating the feasibility of expressing and purifying functional Ibalizimab Fab fragments using a bacterial expression system. Ongoing efforts are focused on improving yields of purified IBLZFab through optimization of the expression and purification methods employed.