Epitope Mapping of Ibalizumab,a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients

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.The human immunodeficiency virus type 1 (HIV-1) epidemic continues to spread at the alarming rate of approximately 2.5 million new cases per year, despite intensive efforts from the scientific community. A safe and effective HIV-1 vaccine would be a key weapon to fight this epidemic; however, vaccine development has not yet proven successful. The extraordinary diversity of the virus, its capacity to evade adaptive immune responses, and the inability to induce broadly neutralizing antibodies against HIV-1 represent unprecedented challenges for vaccine development (3). Alternatively, the strategy of preexposure prophylaxis (PrEP) with antiretroviral drugs or even virus-specific immunoglobulins (Igs) (11) is gaining traction. Protection of rhesus macaques from challenge with simian immunodeficiency virus (SIV) has been observed after passive administration of anti-gp120 or anti-gp41 monoclonal antibodies, such as b12, 2G12, 2F5, and 4E10 (2, 20). However, the application of these antibodies as PrEP has been hindered due to their lack of potency or breadth or both. To this end, PrEP strategies could also consider antibodies to CCR5 (13) or CD4 (8, 12, 14), which have potent and broad inhibitory activities against HIV-1 without unwanted side effects.The CD4 molecule, a cell surface glycoprotein found primarily on T lymphocytes, is the primary receptor for the HIV-1 envelope gp160 glycoprotein (7, 18). A member of the immunoglobulin superfamily (19), CD4 consists of an extracellular segment composed of four tandem immunoglobulin-like domains (D1, D2, D3, and D4), a single transmembrane span, and a short C-terminal cytoplasmic tail (15, 24). It is worth noting that both human major histocompatibility complex (MHC) class II (26) and HIV-1 gp120 (16, 24) bind to the same surface on the first domain (D1) of the CD4 molecule.Ibalizumab (formerly known as TNX-355) is a humanized IgG4 monoclonal antibody that blocks HIV-1 entry by binding to human CD4 (8, 12, 14, 33). It was engineered from its mouse progenitor (5A8) by grafting the mouse complementary-determining region (CDR) onto a human IgG4 construct (4, 5). The IgG4 isotype was chosen to minimize the chances for CD4⁺ T-cell depletion by antibody- and complement-dependent cytotoxicity mediated by binding to Fc receptors. Ibalizumab or 5A8 blocks CD4-dependent virus entry and inhibits a broad spectrum of both laboratory-adapted and clinical HIV-1 isolates, including CCR5-tropic and CXCR4-tropic strains from multiple subtypes, with 50% inhibitory concentrations (IC₅₀s) of 0.0004 to 0.152 μg/ml (4, 5). In vivo, treatment with ibalizumab prominently reduced plasma viremia in rhesus macaques infected with SIV, because this monoclonal antibody has equal affinity for rhesus CD4 (22, 23). In HIV-1 patients, single as well as multiple doses of ibalizumab resulted in substantial reductions (∼10-fold) in viral loads and increases in CD4⁺ T-cell counts without evidence of serious adverse effects or immunologic impairments (12, 14).Efforts were made years ago to delineate the antibody binding site of 5A8 on human CD4 (hCD4) (5). Two stretches, amino acids (aa) 121 to 124 and aa 127 to 134, in domain 2 (D2) were found to be critical for binding. Since then, however, little work has been done to fine-map this epitope, and whether other parts of hCD4 are involved in the binding of this antibody has remained unexplored. The fact that an anti-hCD4-D2 antibody can noncompetitively, yet potently, block HIV-1 entry is intriguing, as viral gp120 binds to D1 of hCD4 (16, 24). Therefore, to gain a better understanding of the mechanism by which ibalizumab inhibits HIV-1 infection while avoiding undesired side effects, we sought to fine-map the epitope of this unique monoclonal antibody.