In HIV-1–infected patients, reductions in viral
load have been observed after one infusion of a
single bnAb, thus demonstrating the biological
activity of HIV-1 bnAbs ( 31–34). A modest extension of viral rebound was also observed when
individual bnAbs were infused after antiretroviral
drugs were discontinued in previously suppressed
HIV-1–infected subjects ( 32, 33). NHP and human
passive transfer studies have also suggested that
such bnAbs can enhance antiviral immunity that
may contribute to improved viral control ( 35, 36).
In addition, NHP studies demonstrate the importance of mAb potency and prolonged antibody half-life in mediating protection against
infection ( 26, 29). The generation of trispecific
Abs with improved potency and breadth may
further enhance the efficacy of either passive immunity or passive-active immunization strategies.
Although bnAbs show exceptional breadth and
potency, resistant viral strains have been de-
tected in patients who make these Abs ( 6, 37)
and among natural viral isolates ( 38–40), raising
the concern that resistance and escape mutations
may arise. Such escape mutations are produced
frequently with antiviral drug therapy ( 41), and
countermeasures to reduce the likelihood of
escape would increase the likelihood of develop-
ing a globally relevant therapy. Such breadth of
coverage might alternatively be generated by
administering multiple bnAbs, and protective
efficacy in a NHP model has recently been dem-
onstrated against a mixture of SHIV viruses using
an antibody cocktail ( 42), providing further sup-
port for the multitargeting concept. Combination
mAb therapy increases the complexity, develop-
ment pathway, cost, and regulatory burdens of
their use for treatment or prevention, in contrast
to a single biologic therapy. The potency of the
trispecific Abs described here also exceeds that
of a broad and potent recombinant form of CD4
( 43), termed eCD4-Ig (fig. S4), and this latter
molecule is also directed to a single, albeit highly
conserved, HIV-1 Env epitope. The availability of a
single protein that targets multiple independent
epitopes on virus also reduces the potential
generation of escape mutations. This advantage
could be related, in part, to the presence of three
independent binding specificities at all times, in
contrast to mixtures of antibodies where selec-
tive pressure by individual mAbs with shorter
half-lives may wane.
The trispecific Abs have not yet been evaluated
for safety and efficacy in humans. Initial characterization of their half-life in NHPs suggests
that they behave similarly to conventional antibodies, but it remains unknown whether they
could be immunogenic in vivo. The administration of a bispecific antibody to the human cytokines IL- 4 and IL- 13, which uses a related format
and linkers ( 44), may provide guidance in this
regard. This bispecific antibody has been evaluated in humans, where single subcutaneous doses
of SAR156597, ranging from 10 to 300 mg/kg,
were well tolerated in healthy subjects, with low
titers of anti-drug antibody (ADA) in only 4 of
36 subjects ( 44). This trial showed a mean half-life
of about 2 weeks ( 44), similar to natural mAbs.
Although further human trials are needed
to assess the full potential of the trispecific Ab
platform, the data from the NHP challenge study
described here, as well as the previous experience
in humans with bispecific Abs ( 44), suggest that
the approach merits further clinical investiga-
tion. Studies in HIV-infected subjects, alone or in
combination with other immune interventions,
will address the potential of trispecific Abs to
provide durable protective immunity against in-
fection or sustained viral control in HIV-infected
subjects during drug holidays or in the absence
of antiretroviral therapy. The recognition of in-
dependent target sites with multispecific anti-
bodies can also be applied to other infectious
diseases, cancer, and autoimmunity. These anti-
bodies can promote recognition and binding to
critical antigenic determinants on target cells
while simultaneously allowing engagement of
immune cells that can stimulate relevant effec-
tor function without the complications and ex-
pense of delivering multiple recombinant proteins.
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Fig. 6. Trispecific and broad neutralizing antibody sensitivity of SHIVs, plasma antibody
levels, and viremia in rhesus macaques. (A) IC50 neutralizing titers, in mg/ml, of VRC01, PGDM1400,
and VRC01/PGDM1400-10E8v4 against replication-competent SHIV BaLP4 or SHIV 325c, with colors
as in Fig. 1. (B) Plasma levels of VRC01, PGDM1400, and VRC01/PGDM1400-10E8v4 in rhesus
macaques (n = 8 on each arm, done in two separate experiments with four animals each). All animals
were administered 5 mg/kg of the indicated antibody intravenously. Each data point represents the
mean ± SEM of the values from all eight animals per group. (C) Plasma viral loads in rhesus macaques
(n = 8 per group) challenged with a mixture of SHIV BaLP4 and SHIV 325c, 5 days after intravenous
administration of VRC01, PGDM1400, or VRC01/PGDM1400-10E8v4.