Epitope mapping of ibalizumab, a humanized anti-CD4 monoclonal antibody with anti-HIV-1 activity in infected patients

Abstract

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.

Figures

FIG. 1.

The epitope of ibalizumab is likely to be conformational. (A) Binding of ibalizumab to soluble hCD4, CD4-IgG2, PR14 (aa 121 to 134), or TG26 (aa 115 to 140) as measured by ELISA. (B) Competition for ibalizumab binding to sCD4 in solution by CD4-IgG2 or PR14 as measured by ELISA. (C) Binding titers of mouse anti-PR14 antiserum to hCD4 or PR14. (D) FACS analysis of cell surface staining by ibalizumab in control 293T cells or cells transfected with hD2-mCD4 or hCD4 plasmid DNA.

FIG. 2.

Map of the ibalizumab epitope obtained by constructing and testing mouse-human chimeric CD4 molecules in binding studies using FACS. Sequence identity between mouse and human CD4 is denoted by an asterisk above the amino acid alignment. The β-strands defined by Ryu et al. (24) are shown below the sequence alignment.

FIG. 3.

Results of fine-mapping the ibalizumab epitope by point mutations in the chimeric mCD4-hGACFG (A) or hCD4 (B). Amino acids in regions of interest for ibalizumab binding were mutated to alanine, glycine, or the corresponding mouse amino acid as shown by single-letter amino acid symbols above the CD4 sequence alignment. Mutations with no impact on CD4 expression or ibalizumab binding are unmarked. Residues critical to ibalizumab binding are enclosed by boxes. A gray dot denotes the amino acid that partially affected ibalizumab binding. The asterisks indicate mutations that resulted in a substantial reduction (to

FIG. 4.

Localization of ibalizumab and M-T441 epitopes on three-dimensional structures of hCD4. The epitopes of ibalizumab (pink) and M-T441 (yellow) are highlighted on CD4 (blue) complexed to the core of gp120 (brown), with two views of the structure rotated 90° around a central vertical axis (A), or space-filling model of three CD4s (blue) bound to the trimer of gp120 core (brown), before and after a conformational rearrangement (B), as derived from the cryo-electron microscopy study of Liu et al. (17). The red dot in D1 denotes F43, a critical component that binds HIV-1 Env. The parts in green highlight the V5 loop in gp120.

FIG. 5.

Comparison of ibalizumab with M-T441 in virus neutralization and competitive binding to hCD4. (A) Neutralizing activity of ibalizumab versus M-T441 as tested against four HIV-1 isolates (JRCSF, WT-1a, MDR-1a, and MDR-5a). (B) Two-way binding competitions between ibalizumab and M-T441 based on FACS analysis.

FIG. 6.

Results of fine-mapping of the M-T441 epitope by point mutations in the backbone of hD2-mCD4 (A) or hCD4 (B). Unmarked mutations have no impact on CD4 expression or M-T441 binding. Mutations enclosed by boxes substantially decreased M-T441 binding.

FIG. 7.

Binding affinity of ibalizumab and M-T441 to hCD4 as assessed in a Biacore assay.