ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2006, p. 3289–3296
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 50, No. 10
Potent Antiviral Synergy between Monoclonal Antibody and
Small-Molecule CCR5 Inhibitors of Human
Immunodeficiency Virus Type 1
Jose D. Murga, Michael Franti, Daniel C. Pevear,† Paul J. Maddon, and William C. Olson*
Progenics Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591
Received 6 June 2006/Returned for modification 13 July 2006/Accepted 28 July 2006
The chemokine receptor CCR5 provides a portal of entry for human immunodeficiency virus type 1
(HIV-1) into susceptible CD4?cells. Both monoclonal antibody (MAb) and small-molecule CCR5 inhib-
itors have entered human clinical testing, but little is known regarding their potential interactions. We
evaluated the interactions between CCR5 MAbs, small-molecule CCR5 antagonists, and inhibitors of
HIV-1 gp120, gp41, and reverse transcriptase in vitro. Inhibition data were analyzed for cooperative effects
using the combination index (CI) method and stringent statistical criteria. Potent, statistically significant
antiviral synergy was observed between the CCR5 MAb PRO 140 and the small-molecule CCR5 antago-
nists maraviroc (UK-427,857), vicriviroc (SCH-D), and TAK-779. High-level synergy was observed con-
sistently across various assay systems, HIV-1 envelopes, CCR5 target cells, and inhibition levels. CI values
ranged from 0.18 to 0.64 and translated into in vitro dose reductions of up to 14-fold. Competition binding
studies revealed nonreciprocal patterns of CCR5 binding by MAb and small-molecule CCR5 inhibitors,
suggesting that synergy occurs at the level of receptor binding. In addition, both PRO 140 and maraviroc
synergized with the chemokine RANTES, a natural ligand for CCR5; however, additive effects were
observed for both small-molecule CCR5 antagonists and PRO 140 in combination with other classes of
HIV-1 inhibitors. The findings provide a rationale for clinical exploration of MAb and small-molecule
CCR5 inhibitors in novel dual-CCR5 regimens for HIV-1 therapy.
The armamentarium for human immunodeficiency virus
type 1 (HIV-1) infection currently includes 22 antiretroviral
agents drawn from four mechanistic treatment classes: nucle-
oside reverse transcriptase inhibitors (NRTI), nonnucleoside
reverse transcriptase inhibitors (NNRTI), protease inhibitors,
and fusion inhibitors. The standard of care for HIV-1 infection
involves combination use of three or more antiretroviral
agents. Where available, such therapies have markedly re-
duced HIV-1 morbidity and mortality (34). However, current
therapies are limited by the emergence of multidrug-resistant
virus, by treatment-related toxicities, by unfavorable drug-drug
interactions, and by often-complex dosing regimens that can
reduce adherence to therapy. Consequently, many patients
eventually exhaust their treatment options, and there is an
urgent need for new agents that can be deployed in novel
In 1996, we and others demonstrated that the chemokine
receptor CCR5 serves as an entry coreceptor for HIV-1 (1, 10,
12). HIV-1 entry proceeds through a cascade of events medi-
ated by the HIV-1 envelope glycoproteins gp120 and gp41:
gp120 sequentially binds CD4 and then CCR5 or another co-
receptor molecule, thereby triggering gp41-mediated fusion of
the viral and cellular membranes. CCR5 has emerged as an
important target for novel HIV-1 therapies (reviewed in ref-
erence 35). Both small-molecule and monoclonal antibody
(MAb) inhibitors of CCR5 have entered human testing, and
the first of these has demonstrated potent antiviral effects in
HIV-infected individuals (14, 21).
PRO 140 is a humanized CCR5 MAb that has entered phase
1b testing for HIV-1 therapy. PRO 140 and the parent mouse
MAb (PA14) broadly and potently block CCR5-mediated
HIV-1 entry in vitro (32, 33, 45). Although PRO 140 and
small-molecule CCR5 antagonists target the same protein,
their properties are complementary in a number of important
respects. Whereas the available small-molecule CCR5 inhibi-
tors potently block the natural activity of CCR5 (11, 39, 40, 48),
antiviral concentrations of PRO 140 do not block CCR5 func-
tion in vitro (33). In addition, preliminary studies indicate that
PRO 140 is highly active against viruses that are resistant to
small-molecule CCR5 antagonists (20, 27). These functional
differences are likely related to the distinct differences in
CCR5 binding. Small-molecule CCR5 antagonists bind a hy-
drophobic pocket formed by the transmembrane helices of
CCR5 and inhibit HIV-1 via allosteric mechanisms (13, 30, 47,
48), while PRO 140 binds an extracellular epitope on CCR5
and appears to act as a competitive inhibitor (33).
Given the mechanistic differences between PRO 140 and
small-molecule CCR5 antagonists in clinical development and
the need for novel combination regimens, we examined the
interactions between these agents in vitro. PRO 140, structur-
ally diverse small-molecule CCR5 antagonists, and other
classes of HIV-1 inhibitors were tested alone and in combina-
tion for the ability to inhibit HIV-1 membrane fusion and viral
entry. Surprisingly, we observed potent antiviral synergy for
PRO 140 in combination with each of several small-molecule
CCR5 antagonists but not for PRO 140 in combination with
* Corresponding author. Mailing address: Progenics Pharmaceuti-
cals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591. Phone:
(914) 789-2800. Fax: (914) 789-2807. E-mail: firstname.lastname@example.org.
† Present address: Novartis Institute for Biomedical Research, 500
Technology Square, Cambridge, MA 02139.
agents that target different stages of HIV-1 entry. Both PRO
140 and small-molecule CCR5 antagonists synergized with
RANTES (CCL5), a natural ligand for CCR5, but purely ad-
ditive effects were observed when different small-molecule
CCR5 antagonists were combined. Competition binding ex-
periments were conducted and offer a mechanism for the
cooperative effects observed. Coupled with the available
viral resistance data, these findings indicate that PRO 140
and small-molecule CCR5 drugs may represent distinct sub-
classes of CCR5 inhibitors.
MATERIALS AND METHODS
Inhibitors. PRO 140 was expressed in mammalian cells and purified by protein
A, ion exchange, and hydroxyapatite chromatographies. Maraviroc (UK-427,857;
Pfizer) (11), vicriviroc (SCH-D; Schering-Plough Corporation) (39), TAK-779
(Takeda Pharmaceuticals) (3), enfuvirtide (T-20; Trimeris/Roche) (49), BMS-
378806 (Bristol-Myers Squibb) (23), and PRO 542 (CD4-IgG2; Progenics) (2)
were prepared according to published methods. Zidovudine (azidothymidine),
RANTES, the CCR5 MAb 2D7, and the CD4 MAb Leu-3A were purchased
from Sigma Chemicals (St. Louis, MO), R&D Systems (Minneapolis, MN),
Pharmingen (San Diego, CA), and Becton Dickinson (Franklin Lakes, NJ),
respectively. Maraviroc and vicriviroc were radiolabeled with tritium by GE
Healthcare (Piscataway, NJ), and PRO 140 was conjugated to phycoerythrin
(PE) by Southern Biotech, Inc. (Birmingham, AL).
HIV-1 membrane fusion assay. HIV-1 envelope-mediated membrane fusion
was examined using a fluorescence resonance energy transfer (RET) assay (24)
with modifications. Briefly, HeLa cells that stably express HIV-1JR-FLgp120/
gp41 (24) and CEM.NKR-CCR5 cells (NIH AIDS Research and Reference
Reagent Program) (38, 46) were labeled separately overnight with fluorescein
octadecyl ester (F18; Molecular Probes, Eugene, OR) and rhodamine octadecyl
ester (R18; Molecular Probes), respectively. Cells were washed in phosphate-
buffered saline containing 15% fetal bovine serum and coseeded at 15,000
cells/well into a 384-well plate. Inhibitors were added, and the plates were
incubated in phosphate-buffered saline containing 15% fetal bovine serum
plus 0.5% dimethyl sulfoxide for 4 h at 37°C prior to measurement of RET
using a Victor2plate reader (PerkinElmer, Boston, MA) as previously de-
scribed (24). The CD4 MAb Leu3a was used as a control inhibitor, and
percent inhibition was calculated as follows: (RET in the absence of inhibitor ?
RET in the presence of inhibitor)/(RET in the absence of inhibitor ? RET in the
presence of Leu3a) ? 100.
HIV-1 pseudovirus assay. A self-inactivating vector was derived from the
pNL4-3?Env-luciferase vector (12) by deleting 507 base pairs in the U3 region of
the 3? long terminal repeat so as to remove the TATA box and transcription
factor binding sites. The human cytomegalovirus promoter was inserted up-
stream of the luciferase gene to enable expression of luciferase following inte-
Reporter viruses pseudotyped with HIV-1JR-FLor HIV-1SF162envelopes were
generated by cotransfection of 293T cells with the self-inactivating vector and the
appropriate pcDNA Env-expressing vector as previously described (12). U87-
CD4-CCR5 cells (8,000/well; NIH AIDS Research and Reference Reagent Pro-
gram) were infected with 125 to 375 pg of HIV-1 pseudoviruses in 384-well plates
in the presence or absence of inhibitor(s). Cultures were incubated for 72 h at
37°C in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum,
1 mg/ml puromycin, 0.3 mg/ml Geneticin, antibiotics, and 0.5% dimethyl sulfox-
ide. Luciferase activity (relative light units [RLU]) was measured using Bright-
Glo reagent (Promega, Madison, WI) according to the manufacturer’s instruc-
tions. Percent inhibition was calculated as follows: (1 ? RLU in the presence of
inhibitor/RLU in the absence of inhibitor) ? 100. Fifty percent inhibitory con-
centration (IC50) and IC90were used to denote the respective concentrations
required for 50% and 90% inhibition of HIV-1.
Synergy determinations. Experimental design and data analysis were based on
the combination index (CI) method (6, 7). Compounds were tested individually
and in combination at a fixed molar ratio over a range of serial dilutions. Entry
inhibitors were combined in equimolar amounts, whereas a 1:10 molar ratio was
used for PRO 140 in combination with azidothymidine and nevirapine. Dose-
response curves were fit using a four-parameter sigmoidal equation with upper
and lower inhibition values constrained to 100% and 0%, respectively, in order
to calculate concentrations required for 50% (IC50) and 90% (IC90) inhibition
(GraphPad Prism; GraphPad Software, San Diego, CA). CI values for 50%
(CI50) and 90% (CI90) inhibition were calculated as previously described (6, 7).
For example, CI50was calculated as
where IC50(1,alone)and IC50(1,comb)denote the concentrations of compound 1
required for 50% inhibition when the compound is used alone and in combina-
tion, respectively, and IC50(2,alone)and IC50(2,comb)have the corresponding
meanings for compound 2. The mutually exclusive CI formula (? ? 0) was used
for combinations of CCR5 inhibitors, while the mutually nonexclusive formula
(? ? 1) was utilized for combinations of inhibitors to distinct targets (6). Again,
using 50% inhibition and compound 1 as an example, the dose reduction was
calculated as IC50(1,comb)/IC50(1,alone). Each test was conducted 4 to 12 times.
Synergy, additivity, and antagonism are indicated by CI values of ?1, 1, and ?1,
Competition binding assays. To examine inhibition of PRO 140 binding,
CEM.NKR-CCR5 cells were suspended in phosphate-buffered saline with 0.1%
sodium azide (PBSA) and incubated with various concentrations of unlabeled
CCR5 antagonists at ambient temperature for 30 min. Azide was added to block
CCR5 internalization during the assay. Cells were washed in PBSA and incu-
bated with 5 nM PRO 140-PE for an additional 30 min prior to washing and
analysis by flow cytometry using a FACSCalibur instrument (Becton Dickinson).
The extent of PRO 140-PE binding was measured in terms of both the mean
fluorescence intensity (MFI) and the percentage of cells gated for positive
To examine inhibition of maraviroc binding, CEM.NKR-CCR5 cells were
preincubated with unlabeled CCR5 inhibitors as described above prior to the
addition of 2 nM3H-maraviroc for an additional 30 min. The cells were washed
in PBSA and lysed with 0.5 N HCl prior to scintillation counting using a
Wallac1410 instrument. An additional study reversed the order of addition in
order to examine the stability of maraviroc binding over the course of the assay.
Cells were preincubated with 2 nM3H-maraviroc for 30 min prior to washing, the
addition of unlabeled inhibitors, and processing as described above. Fifty percent
effective concentration (EC50) and EC90were used to denote the concentrations
of unlabeled compound required to inhibit binding of labeled compound by 50%
and 90%, respectively.
Statistical analyses. Two-tailed t tests were used to test mean CI50and CI90
values for the null hypothesis H0(CI ? 1 [additivity]) using GraphPad Prism
software. P values were corrected for multiple comparisons from an initial sig-
nificance level (?) of 0.05 according to the Bonferroni method (9), excluding the
PRO 140/PRO 140 mock combination that was included as an assay control. In
the Bonferroni correction, P ? ?/n, where n is the number of comparisons.
Twenty-two synergy comparisons (11 compounds ? 2 CI values) were made
based on data generated in the membrane fusion assay, resulting in a corrected
P value of 0.0023. In the pseudovirus assay, 32 synergy comparisons (8 com-
pounds ? 2 viruses ? 2 CI values) resulted in a corrected P value of 0.0016.
Inhibition of HIV-1 membrane fusion. PRO 140 and mara-
viroc were used individually and together to inhibit HIV-1JR-FL
envelope-mediated membrane fusion in the RET cell-cell fu-
sion assay, and representative dose-response curves for the
individual agents and combination are illustrated in Fig. 1A.
Although both PRO 140 and maraviroc individually blocked
HIV-1 fusion at low nanomolar potency, the combination was
markedly more potent. In this assay, 50% inhibition was ob-
tained using 2.9 nM PRO 140 alone, 5.0 nM maraviroc alone,
or 2.1 nM of the combination (1.05 nM PRO 140 plus 1.05 nM
maraviroc). This supra-additive effect is indicative of antiviral
synergy between the two agents.
In contrast, the combination of vicriviroc and maraviroc was
no more potent than individual agents (Fig. 1B). In this exam-
ple, the dose-response curves for the individual inhibitors and
the combination were overlapping, with 50% inhibition requir-
ing 9.7 nM maraviroc, 5.5 nM vicriviroc, and 6.1 nM of the
combination. The data suggest purely additive effects for these
3290 MURGA ET AL.ANTIMICROB. AGENTS CHEMOTHER.
These studies were extended to additional CCR5 (TAK-779,
RANTES, and 2D7), gp120 (BMS-378806 and PRO 542), and
gp41 (enfuvirtide) inhibitors and were repeated four or more
times for each condition. CI50and CI90values were calculated
for each condition and averaged across the independent assays.
Cooperativity was assessed using t tests to determine whether
the CI50and CI90values were significantly different from 1. As
a test of our methods, a PRO 140/PRO 140 mock combination
was examined by adding PRO 140 to the assay wells in two
separate additions. CI50and CI90values for the PRO 140/PRO
140 combination were 0.97 and 0.96, respectively (Table 1);
therefore, purely additive effects were observed for this mock
combination as expected.
Potent synergy was observed for PRO 140 in combination
with each of three small-molecule CCR5 antagonists (maravi-
roc, vicriviroc, and TAK-779), and the findings were statisti-
cally significant even when the data were corrected for multiple
comparisons via the Bonferroni method (Table 1). CI values
ranged from 0.36 to 0.61, and these synergies translated into
dose reductions ranging from three- to eightfold across the
different conditions. Synergies were greater at 90% inhibition
than at 50% inhibition. Synergy between PRO 140 and small-
molecule CCR5 antagonists was robust in that it was observed
at both the 50% and 90% inhibition levels in every instance.
The exception was TAK-779, which did not mediate 90% in-
hibition when used individually, and therefore, a CI90was not
determined. Similarly, potent synergy was observed when
RANTES was used in combination with either PRO 140 or
maraviroc (Table 1).
Additional tests examined combinations of two small-mole-
cule CCR5 antagonists (vicriviroc/maraviroc and vicriviroc/
TAK-779) or two CCR5 MAbs (PRO 140/2D7). No significant
synergy was observed for these combinations, although the
vicriviroc/maraviroc CI90values trended towards significance.
The findings are consistent with prior observations of overlap-
ping binding sites for PRO 140 and 2D7 (33) and for vicriviroc
and TAK-779 (37).
PRO 140 was also tested in combination with the gp41
fusion inhibitor enfuvirtide and with the gp120 attachment
inhibitors PRO 542 and BMS-378806 (Table 1). CI values
ranged from 0.84 to 1.28, and none of these combinations
demonstrated synergy that met our criteria for statistical sig-
nificance. For the PRO 140/BMS-378806 combination, modest
antagonism was observed at 50% but not 90% inhibition. The
biological significance of this result is unclear.
Inhibition of HIV-1 pseudoviruses. Next, we used single-
cycle HIV-1 reporter viruses to examine whether the synergis-
tic effects were limited to cell-cell fusion or whether they ex-
tended to other modes of HIV-1 entry. Signals in this assay
require both viral entry and reverse transcription, enabling us
to include NRTI and NNRTI in the analyses. Each combina-
tion was tested against reporter viruses pseudotyped with en-
velopes from HIV-1JR-FLand HIV-1SF162in at least four
independent assays per virus. A PRO 140/PRO 140 mock
combination was again included as an assay control and
demonstrated additive effects against both HIV-1JR-FLand
HIV-1SF162pseudoviruses as expected (Table 2).
PRO 140 potently synergized with both maraviroc and vicri-
viroc in blocking virus-cell fusion, and the results met our
criteria for statistical significance. Comparable levels of syn-
ergy were observed against both HIV-1JR-FLand HIV-1SF162
pseudoviruses at 50% and 90% inhibition (Table 2), with CI
values ranging from 0.18 to 0.64. These synergies translated
into dose reductions of up to 14-fold. These results are in good
agreement with those obtained with the cell-cell fusion assay
(Table 1). Neither TAK-779 nor RANTES mediated consis-
tent, high-level inhibition of HIV-1 pseudovirus entry, and
therefore, these compounds were not included in this analysis
(data not shown).
Additive effects were observed for both the maraviroc/vicri-
viroc and PRO 140/2D7 combinations (Table 2). Similarly,
FIG. 1. Dose-response curves for inhibition of HIV-1JR-FLenve-
lope-mediated membrane fusion by combinations of CCR5 inhibitors.
Dilutions were analyzed in triplicate wells, and the data points depict
the means and standard deviations of replicates. (A) PRO 140 and
maraviroc (UK-427,857) were tested individually and in a 1:1 fixed
molar ratio over the indicated range of concentrations. In the experi-
ment whose results are depicted, IC50and IC90values were 2.9 nM and
11 nM for PRO 140, 5.0 nM and 21 nM for maraviroc, and 2.1 nM and
4.6 nM for the combination, respectively. CI50and CI90values were
0.58 and 0.32, respectively. (B) Vicriviroc (SCH-D) and maraviroc
were tested individually and in a 1:1 fixed molar ratio over the indi-
cated range of concentrations. In the experiment whose results are
depicted, IC50and IC90values were 5.5 nM and 34 nM for vicriviroc,
9.7 nM and 59 nM for maraviroc, and 6.1 nM and 31 nM for the
combination, respectively. CI50and CI90values were 0.87 and 0.73,
VOL. 50, 2006 SYNERGY BETWEEN MAb AND ORAL CCR5 DRUGS FOR HIV 3291
additivity was observed for PRO 140 used in combination with
the gp120 inhibitors PRO 542 and BMS-378806. No antago-
nism was observed for the PRO 140/BMS-378806 combination
against either virus. Overall, these findings are consistent with
those seen for cell-cell fusion. Lastly, additive effects were
observed for PRO 140 in combination with either zidovudine
(NRTI) or nevirapine (NNRTI).
Competition binding studies. As described above, additive
antiviral effects were observed for inhibitors known (PRO 140
and 2D7) or inferred (maraviroc and vicriviroc) to compete for
CCR5 binding; however, little is known regarding the compet-
itive binding of synergistic compounds (e.g., PRO 140/maravi-
roc and PRO 140/vicriviroc). Since noncompetitive binding
provides a possible mechanism for synergy between CCR5
inhibitors, we explored this issue by using labeled forms of
maraviroc and PRO 140.
Flow cytometry was used to examine inhibition of PRO
140-PE binding to CEM.NRK-CCR5 cells by unlabeled PRO
140, maraviroc, and vicriviroc. PRO 140-PE binding was effi-
ciently inhibited by unlabeled PRO 140, as expected. Com-
plete inhibition was observed in terms of both MFI values
(Fig. 2A) and the percentages of cells gated for positive
binding (Fig. 2B). The EC50based on MFI data was 2.5 nM
(Fig. 2A), and this value compares favorably with the anti-
viral IC50of PRO 140 (Tables 1 and 2). Since the percentage
of cells gated is a readout for essentially complete inhibition
of binding, the EC90value was calculated to be 17 nM, and
this value is similar to the antiviral IC90values observed for
PRO 140 (Tables 1 and 2). 2D7 also completely inhibited
binding of PRO 140-PE to CEM.NKR-CCR5 cells (data not
shown). The CCR5 specificity of PRO 140-PE was also dem-
onstrated by its inability to bind parental CEM.NKR-CCR5
cells (data not shown).
In sharp contrast, modest levels of inhibition were observed
for maraviroc and vicriviroc (Fig. 2). Micromolar concentra-
tions of maraviroc and vicriviroc reduced PRO 140-PE MFI
values by 50% or less (Fig. 2A). More dramatically, maraviroc
and vicriviroc had little impact on the percentage of cells gated
for positive binding of PRO 140-PE (Fig. 2B). The findings
suggest that maraviroc and vicriviroc partially reduce the num-
TABLE 1. CI values for inhibition of HIV-1JR-FLenvelope-mediated membrane fusiona
0.97 ? 0.07
0.61 ? 0.05
0.51 ? 0.05
0.38 ? 0.08
0.59 ? 0.08
0.48 ? 0.03
0.86 ? 0.03
1.3 ? 0.18
1.0 ? 0.14
0.84 ? 0.16
0.96 ? 0.17
1.3 ? 0.19
0.96 ? 0.14
0.40 ? 0.06
0.36 ? 0.06
0.43 ? 0.05
0.18 ? 0.01
0.75 ? 0.02
1.9 ? 0.61
0.89 ? 0.20
0.94 ? 0.19
1.1 ? 0.22
aStatistically significant results (P ? 0.0023 after application of the Bonferroni correction for multiple comparisons) are indicated in italicized bold text. IC50and
IC90denote values for the first inhibitor. NA, not applicable. TAK-779 did not consistently achieve 90% inhibition in the assay. CI values represent the means and
standard deviations of 4 to 12 independent assays.
TABLE 2. CI values for inhibition of HIV-1 reporter viruses pseudotyped with envelopes from HIV-1JR-FLand HIV-1SF162
First inhibitorTarget HIV-1 envelope IC50(nM)
P value CI90
PRO 140 CCR5JR-FL
1.2 ? 0.32
1.0 ? 0.27
0.47 ? 0.15
0.60 ? 0.17
0.44 ? 0.06
0.64 ? 0.07
0.71 ? 0.11
0.87 ? 0.06
1.5 ? 0.25
1.1 ? 0.47
1.2 ? 0.32
0.98 ? 0.28
1.2 ? 0.38
1.1 ? 0.28
1.2 ? 0.38
1.2 ? 0.34
1.1 ? 0.38
0.99 ? 0.27
0.90 ? 0.15
0.86 ? 0.33
0.18 ? 0.04
0.28 ? 0.11
0.24 ? 0.11
0.31 ? 0.11
1.2 ? 0.15
0.86 ? 0.28
1.0 ? 0.16
1.0 ? 0.18
0.64 ? 0.26
0.74 ? 0.23
0.82 ? 0.21
0.73 ? 0.28
0.63 ? 0.19
0.85 ? 0.26
1.0 ? 0.38
PRO 542gp120 2.9
aStatistically significant results (P ? 0.0016 after application of the Bonferroni correction for multiple comparisons) are indicated in italicized bold text. IC50and
IC90refer to values for the first inhibitor. RT, reverse transcriptase; NA, not applicable. 2D7 did not consistently achieve 90% inhibition in the assay. CI values represent
the means and standard deviations of four or more independent assays.
3292 MURGA ET AL.ANTIMICROB. AGENTS CHEMOTHER.
ber of PRO 140-PE molecules bound per cell; however, these
compounds do not reduce the number of cells that bind mea-
surable amounts of PRO 140-PE. Therefore, maraviroc and
vicriviroc represent partial antagonists of PRO 140 binding,
and this finding provides a mechanism for the antiviral synergy
observed between PRO 140 and these small-molecule CCR5
Next, we examined inhibition of3H-maraviroc binding by
unlabeled maraviroc, vicriviroc, and PRO 140. Binding of3H-
maraviroc to CEM.NKR-CCR5 cells was efficiently inhibited
by unlabeled maraviroc (Fig. 3A). The EC50for binding was
4.3 nM and is similar to the antiviral IC50values observed for
maraviroc (Tables 1 and 2).
Vicriviroc also blocked3H-maraviroc binding to background
levels (Fig. 3A). However, there was no correlation between
the compounds’ antiviral potency and their potency in blocking
3H-maraviroc binding. For example, whereas vicriviroc dem-
onstrated equal or slightly greater antiviral potency than ma-
raviroc (Tables 1 and 2), vicriviroc was less potent in blocking
3H-maraviroc binding (EC50? 17 nM) (Fig. 3A). This result is
consistent with minor differences in the CCR5 binding sites of
Surprisingly, PRO 140 also blocked3H-maraviroc binding to
background levels (Fig. 3A), and this result contrasts with the
modest inhibition of PRO 140-PE binding by maraviroc (Fig.
2). PRO 140 inhibited3H-maraviroc binding with an EC50of
14 nM, which is 5- to 10-fold higher than the antiviral IC50of
PRO 140 (Tables 1 and 2).
A final experiment examined the stability of maraviroc bind-
ing to CEM.NKR-CCR5 cells under the conditions of the
competition assay. For this, we preincubated cells with
maraviroc and then examined its dissociation in the presence
of unlabeled maraviroc, vicriviroc, and PRO 140. As indicated
in Fig. 3B, there was minimal dissociation of
over 30 min at ambient temperature, and maraviroc wasn’t
displaced by either PRO 140 or vicriviroc. Therefore, the in-
ability of maraviroc to efficiently compete for PRO 140 binding
to CCR5 (Fig. 2) is not due to rapid dissociation of maraviroc
from CCR5 during the course of the assay. Collectively, the
data indicate that PRO 140 can bind CCR5 in the presence of
FIG. 2. Inhibition of PRO 140-PE binding to CEM.NKR-CCR5
cells by unlabeled PRO 140, maraviroc, and vicriviroc. CEM.NKR-
CCR5 cells were incubated with various concentrations of unlabeled
PRO 140, maraviroc, or vicriviroc for 30 min at room temperature in
PBSA buffer prior to the addition of 5 nM PRO 140-PE for an addi-
tional 30 min. Cells were washed and then analyzed by flow cytometry
for both the MFI of binding and the percentage of cells gated for
positive binding of PRO 140-PE. Inhibition was assessed on the basis
of both MFI (A) and the percentage of cells gated (B).
FIG. 3. Inhibition of3H-maraviroc binding by unlabeled maraviroc,
vicriviroc, and PRO 140. (A) CEM.NKR-CCR5 cells were preincu-
bated with various concentrations of unlabeled maraviroc, vicriviroc,
or PRO 140 for 30 min in PBSA buffer at ambient temperature prior
to the addition of 2 nM3H-maraviroc for an additional 30 min. Cells
were washed and then analyzed for radioactivity by scintillation count-
ing. (B) The stability of maraviroc binding under the assay conditions
was examined by preincubating CEM.NKR-CCR5 cells with 2 nM
3H-maraviroc prior to washing, the addition of unlabeled compounds
for 30 min, and processing as described above.
VOL. 50, 2006 SYNERGY BETWEEN MAb AND ORAL CCR5 DRUGS FOR HIV3293
This study is the first to examine combinations of CCR5
drugs that are currently in development for HIV-1 therapy.
Surprisingly, we observed potent antiviral synergy between the
CCR5 MAb PRO 140 and each of three structurally distinct
small-molecule CCR5 antagonists. Consistent, high-level syn-
ergy was observed across various assay systems, viral isolates,
target cells, and inhibition levels. PRO 140 and small-molecule
CCR5 antagonists were more potently synergistic when used
together rather than in combination with inhibitors that block
other stages of HIV-1 entry. In contrast, additive effects were
observed for combinations of two small-molecule CCR5 an-
tagonists. Competition binding studies revealed complex and
nonreciprocal patterns of CCR5 binding by MAb and small-
molecule CCR5 inhibitors and suggest that the synergistic in-
teractions occur at the level of receptor binding. Our findings
have implications for the potential use of novel dual-CCR5
regimens for HIV-1 therapy.
Robust synergy between MAb and small-molecule CCR5
inhibitors was observed in this study, and this finding is con-
sistent with that of a recent report (36). Potent synergy was
observed for both cell-cell and virus-cell fusion, and there was
a good concordance of findings in these two well-established
assay systems. Comparable levels of synergy were observed for
PRO 140 in combination with each of three small-molecule
CCR5 antagonists from unrelated chemical series. In addition,
consistent synergy was observed for each of two CCR5 target
cells and two well-characterized HIV-1 envelopes. HIV-1JR-FL
was isolated from the brain of an AIDS patient at autopsy (31).
Like other late-stage neurovirulent viruses (17), HIV-1JR-FL
encodes an envelope with high binding affinity for CCR5 (36).
Lastly, similar levels of synergy were observed when PRO 140
and maraviroc were tested against HIV-1BaLpseudoviruses
(15) in preliminary studies (data not shown).
Synergy increased with increasing levels of viral inhibition
and translated into in vitro dose reductions of up to 14-fold.
Viewed alternatively, this degree of synergy provides a corre-
sponding increase in antiviral pressure at a given concentration
of drugs, thereby improving viral suppression and potentially
delaying the emergence of drug-resistant virus.
We also observed potent synergy for RANTES used in com-
bination with either maraviroc or PRO 140. Endogenous levels
of RANTES may afford some protection against HIV-1 disease
progression during natural infection (16, 25), and therefore,
our finding of synergy has important and positive implications
for CCR5-targeted therapies of HIV-1. Antiviral synergy be-
tween RANTES and PRO 140 is not surprising based on our
prior observation that RANTES signaling is not blocked by
antiviral concentrations of murine PRO 140 (PA14) (33). Syn-
ergy between RANTES and maraviroc is less easily explained
given that maraviroc is a potent CCR5 antagonist. However,
our findings are consistent with prior observations of synergy
between the small-molecule CCR5 antagonist SCH-C and ami-
nooxypentane-RANTES (44), a RANTES derivative that has
been evaluated as a potential topical microbicide (19).
In contrast to the robust synergy observed between MAb
and small-molecule CCR5 antagonists, additive effects were
observed for combinations of small-molecule CCR5 antago-
nists. A lack of cooperativity is consistent with the view that
these molecules compete for binding to a common pocket on
CCR5 (13, 30, 47, 48). However, synergy is not required to
derive clinical benefit from combination therapy. No interfer-
ence was observed between small-molecule CCR5 antagonists,
and this finding has relevance when considering combining
such agents in the clinic.
Similarly, we did not observe potent synergy between PRO
140 and inhibitors of HIV-1 attachment (PRO 542 and BMS-
378806), fusion (enfuvirtide), or reverse transcriptase (zido-
vudine and nevirapine), and these findings underscore the
significance of the synergy observed for PRO 140 and
small-molecule CCR5 antagonists. A number of prior studies
have examined interactions between various small-molecule
CCR5 antagonists (maraviroc, SCH-C, TAK-220, TAK-652,
and E913) and drugs from each of the existing HIV-1 treat-
ment classes. Most (42–44) but not all (11, 26) studies have
reported broad synergy between CCR5 inhibitors and the
other HIV-1 treatment classes, and the divergent results may
reflect differences in the compounds and methods used for
antiviral testing as well as differences in the methods used for
data analysis. When maraviroc was tested against 20 licensed
antiretroviral agents, additive effects were observed in all but
three cases, in which modest synergy was reported (11). This
result is consistent with our findings for combinations of PRO
140 and HIV-1 inhibitors that do not target CCR5.
Synergy between anti-HIV-1 drugs may stem from a variety
of mechanisms. In mixed virus cultures, one compound may
inhibit virus resistant to a second compound (18), and NRTI/
NNRTI combinations may overcome specific reverse tran-
scriptase-mediated resistance mechanisms (4, 5). Metabolic
interactions between inhibitors may increase their effective
intracellular drug concentrations (28), and synergistic entry
inhibitors may disrupt interdependent steps in the entry cas-
cade (29, 41). The present study examined clonal viral enve-
lopes rather than mixed populations, and the extracellular na-
ture of the target argues against metabolic interactions.
Multiple domains of gp120 contribute to CCR5 binding (8),
but it is unclear at present whether these interactions represent
separate or discrete events during infection.
Our findings indicate that antiviral synergy between MAb
and small-molecule CCR5 inhibitors may occur at the level of
the receptor, although the mechanism remains poorly defined.
As discussed above, MAbs and small molecules bind distinct
loci on CCR5 (13, 30, 33, 47, 48). When preincubated with
CCR5 cells in the present study, PRO 140 completely blocked
subsequent binding of maraviroc to the receptor, although the
PRO 140 concentrations were higher than those needed to
block HIV-1 entry into the same cells. In contrast, preincuba-
tion of CCR5 cells with supersaturating concentrations of ma-
raviroc or vicriviroc reduced PRO 140 binding by 50% or less.
As one possible explanation, PRO 140 could recognize CCR5
conformers that are not bound by maraviroc or vicriviroc.
Although cell surface CCR5 exists in multiple conformations
(22), it seems unlikely that the small-molecule antagonists
could demonstrate potent antiviral activity while failing to bind
a significant fraction of cell surface CCR5. In this regard, it
is important to note that a common cellular background
(CEM.NKR-CCR5 cells) was used for competition binding
and antiviral studies, and therefore, the findings are not related
to cell-specific differences in CCR5 expression.
3294MURGA ET AL.ANTIMICROB. AGENTS CHEMOTHER.
In our view, a more plausible explanation for our findings is
that PRO 140 is capable of forming a ternary complex with
maraviroc-bound CCR5, and this ternary complex provides an
increased barrier to HIV-1 entry. Within the context of this
model, PRO 140 may bind maraviroc-bound CCR5 somewhat
less efficiently than free CCR5, as evidenced by the modest
reduction in PRO 140 binding in the presence of maraviroc.
However, we note that our studies do not demonstrate the
existence of a ternary complex, and additional studies will be
required to test this model and to further define the mecha-
nisms of synergy between MAb and small-molecule CCR5
inhibitors. To this end, time-of-addition studies have indicated
that murine PRO 140 (PA14) acts at a later stage of the fusion
cascade than does the small-molecule CCR5 antagonist
SCH-C (36). This report proposed that SCH-C blocks gp120
binding to CCR5, while PA14 prevents ill-defined postbinding
The combination index method is widely used to assess drug-
drug interactions. In this method, cooperativity is often defined
on the basis of empirical CI values (e.g., ?0.9 for synergy and
?1.1 for antagonism) irrespective of interassay variability. Sta-
tistical analyses are performed infrequently, and even more
rarely are adjustments made for multiple comparisons. In the
absence of such analyses, there is increased potential to over-
estimate the number of synergistic combinations.
We adopted a rigorous approach for identifying synergistic
effects. CI values were tested for statistical significance against
the null hypothesis of additivity (CI ? 1). In addition, our
studies determined 20 to 30 different CI values per experiment
(Tables 1 and 2), as is common in synergy studies. In order to
reduce the potential for spurious positive results, we reduced
the significance level using the Bonferroni correction. We also
evaluated a mock combination as a control. We conclude that
numerous apparent synergies (CI ? 0.9) could not be distin-
guished from interassay variation based on the available data.
Nevertheless, PRO 140 and small-molecule inhibitors demon-
strated significant synergy under every test condition, lending
credence to this finding. Combinations with CI values that
trended towards significance in the present survey, such as the
PRO 140/enfuvirtide combination, could be explored in future
A growing body of data indicates that MAb and small-mol-
ecule CCR5 antagonists represent distinct subclasses of CCR5
inhibitors, and a number of important parallels can be drawn
between NRTI and NNRTI on the one hand and between
MAb and small-molecule CCR5 antagonists on the other. In
each instance, there are distinct binding loci for the inhibitors
on the target protein (reverse transcriptase or CCR5). One set
of inhibitors (NNRTI or small-molecule CCR5 antagonists)
acts via allosteric mechanisms, while the other set (NRTI or
CCR5 MAbs) acts as a competitive inhibitor. Like NRTI and
NNRTI, MAb and small-molecule CCR5 inhibitors are syner-
gistic and possess complementary patterns of viral resistance in
vitro in preliminary testing (20, 27). NRTI and NNRTI repre-
sent important and distinct treatment classes even though they
target the same protein, and MAb and small-molecule CCR5
inhibitors similarly may offer distinct HIV-1 treatment modal-
This work was supported by Public Health Service grants AI046871
and AI066329 from the National Institute of Allergy and Infectious
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