JOURNAL OF VIROLOGY, July 2010, p. 6935–6942
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 14
Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal
Antibody with Anti-HIV-1 Activity in Infected Patients?
Ruijiang Song,† David Franco,† Chia-Ying Kao, Faye Yu, Yaoxing Huang, and David D. Ho*
Aaron Diamond AIDS Research Center, The Rockefeller University, 455 First Avenue, 7th Floor, New York, New York 10016
Received 1 March 2010/Accepted 29 April 2010
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) epi-
demic continues to spread at the alarming rate of approxi-
mately 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 ex-
traordinary diversity of the virus, its capacity to evade adaptive
immune responses, and the inability to induce broadly neutral-
izing antibodies against HIV-1 represent unprecedented chal-
lenges for vaccine development (3). Alternatively, the strategy
of preexposure prophylaxis (PrEP) with antiretroviral drugs or
even virus-specific immunoglobulins (Igs) (11) is gaining trac-
tion. Protection of rhesus macaques from challenge with sim-
ian 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 activ-
ities against HIV-1 without unwanted side effects.
The CD4 molecule, a cell surface glycoprotein found pri-
marily on T lymphocytes, is the primary receptor for the HIV-1
envelope gp160 glycoprotein (7, 18). A member of the immu-
noglobulin superfamily (19), CD4 consists of an extracellular
segment composed of four tandem immunoglobulin-like do-
mains (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-deter-
mining 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 cy-
totoxicity 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 iso-
lates, including CCR5-tropic and CXCR4-tropic strains from
multiple subtypes, with 50% inhibitory concentrations (IC50s)
of 0.0004 to 0.152 ?g/ml (4, 5). In vivo, treatment with ibali-
zumab prominently reduced plasma viremia in rhesus ma-
caques 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 sub-
stantial 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 bind-
ing 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 anti-
body 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
* Corresponding author. Mailing address: Aaron Diamond AIDS
Research Center, 455 First Avenue, New York, NY. Phone: (212)
448-5100. Fax: (212) 725-1126. E-mail: email@example.com.
† R.S. and D.F. contributed equally to this work.
?Published ahead of print on 12 May 2010.
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