Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary 
receptor for human immunodeficiency virus type 1 (HIV-1). With its unique 
specificity for domain 2 of CD4, this antibody potently and broadly blocks HIV-1 
infection in vitro by inhibiting a postbinding step required for viral entry but 
without interfering with major histocompatibility complex class II 
(MHC-II)-mediated immune function. In clinical trials, ibalizumab has 
demonstrated anti-HIV-1 activity in patients without causing immunosuppression. 
Thus, a characterization of the ibalizumab epitope was conducted in an attempt 
to gain insight into the underlying mechanism of its antiviral activity as well 
as its safety profile. By studying mouse/human chimeric CD4 molecules and 
site-directed point mutants of CD4, amino acids L96, P121, P122, and Q163 in 
domain 2 were found to be important for ibalizumab binding, with E77 and S79 in 
domain 1 also contributing. All these residues appear to cluster on the 
interface between domains 1 and 2 of human CD4 on a surface opposite the site 
where gp120 and the MHC-II molecule bind on domain 1. Separately, the epitope of 
M-T441, a weakly neutralizing mouse monoclonal antibody that competes with 
ibalizumab, was localized entirely within domain 2 on residues 123 to 125 and 
138 to 140. The results reported herein not only provide an appreciation for why 
ibalizumab has not had significant adverse immunological consequences in 
infected patients to date but also raise possible steric hindrance mechanisms by 
which this antibody blocks HIV-1 entry into a CD4-positive cell.