A Randomized, Double-Blind, Placebo-Controlled
Assessment of BMS-936558, a Fully Human Monoclonal
Antibody to Programmed Death-1 (PD-1), in Patients
with Chronic Hepatitis C Virus Infection
David Gardiner1*, Jay Lalezari2, Eric Lawitz3, Michael DiMicco4, Rheem Ghalib5, K. Rajender Reddy6,
Kyong-Mi Chang6,7, Mark Sulkowski8, Steven O’ Marro9, Jeffrey Anderson1, Bing He1, Vikram Kansra10¤,
Fiona McPhee11, Megan Wind-Rotolo10, Dennis Grasela1, Mark Selby12, Alan J. Korman12, Israel Lowy13
1Bristol-Myers Squibb, Pennington, New Jersey, United States of America, 2Quest Clinical Research, San Francisco, California, United States of America, 3Alamo Medical
Research, San Antonio, Texas, United States of America, 4Advanced Clinical Research Institute, Anaheim, California, United States of America, 5The Liver Institute at
Methodist Hospital, Dallas, Texas, United States of America, 6University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America,
7Philadelphia Veterans Affairs Medical Center, Philadelphia, Pennsylvania, United States of America, 8Johns Hopkins University School of Medicine, Baltimore, Maryland,
United States of America, 9Springfield Clinic Infectious Diseases, Springfield, Illinois, United States of America, 10Bristol-Myers Squibb, Princeton, New Jersey, United
States of America, 11Bristol-Myers Squibb, Wallingford, Connecticut, United States of America, 12Bristol-Myers Squibb, Milpitas, California, United States of America,
13Regeneron Pharmaceuticals, Tarrytown, New York, United States of America
Expression of the programmed death 1 (PD-1) receptor and its ligands are implicated in the T cell exhaustion phenotype
which contributes to the persistence of several chronic viral infections, including human hepatitis C virus (HCV). The antiviral
potential of BMS-936558 (MDX-1106) – a fully human anti-PD-1 monoclonal immunoglobulin-G4 that blocks ligand binding
– was explored in a proof-of-concept, placebo-controlled single-ascending-dose study in patients (N=54) with chronic HCV
infection. Interferon-alfa treatment-experienced patients (n=42) were randomized 5:1 to receive a single infusion of BMS-
936558 (0.03, 0.1, 0.3, 1.0, 3.0 mg/kg [n=5 each] or 10 mg/kg [n=10]) or of placebo (n=7). An additional 12 HCV treatment-
naı ¨ve patients were randomized to receive 10 mg/kg BMS-936558 (n=10) or placebo (n=2). Patients were followed for 85
days post-dose. Five patients who received BMS-936558 (0.1 [n=1] or 10 mg/kg) and one placebo patient achieved the
primary study endpoint of a reduction in HCV RNA $0.5 log10IU/mL on at least 2 consecutive visits; 3 (10 mg/kg) achieved a
.4 log10reduction. Two patients (10 mg/kg) achieved HCV RNA below the lower limit of quantitation (25 IU/mL), one of
whom (a prior null-responder) remained RNA-undetectable 1 year post-study. Transient reductions in CD4+, CD8+and
CD19+cells, including both naı ¨ve and memory CD4+and CD8+subsets, were observed at Day 2 without evidence of
immune deficit. No clinically relevant changes in immunoglobulin subsets or treatment-related trends in circulating
cytokines were noted. BMS-936558 exhibited dose-related exposure increases, with a half-life of 20–24 days. BMS-936558
was mostly well tolerated. One patient (10 mg/kg) experienced an asymptomatic grade 4 ALT elevation coincident with the
onset of a 4-log viral load reduction. Six patients exhibited immune-related adverse events of mild-to-moderate intensity,
including two cases of hyperthyroidism consistent with autoimmune thyroiditis. Further investigation of PD-1 pathway
blockade in chronic viral disease is warranted.
Trial Registration: ClinicalTrials.gov NCT00703469
Citation: Gardiner D, Lalezari J, Lawitz E, DiMicco M, Ghalib R, et al. (2013) A Randomized, Double-Blind, Placebo-Controlled Assessment of BMS-936558, a Fully
Human Monoclonal Antibody to Programmed Death-1 (PD-1), in Patients with Chronic Hepatitis C Virus Infection. PLoS ONE 8(5): e63818. doi:10.1371/
Editor: Golo Ahlenstiel, University of Sydney, Australia
Received November 8, 2012; Accepted April 4, 2013; Published May 22, 2013
Copyright: ? 2013 Gardiner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was wholly funded by Medarex, now Bristol-Myers Squibb. The funders, Medarex (now BMS), had a primary role in study design, in
collaboration with clinical investigators and health authorities. In addition, Medarex had a primary role in the data collection and analysis, decision to publish, and
publication of the manuscript.
Competing Interests: This study was funded by Bristol-Myers Squibb. D. Grasela, JA, BH, D. Gardiner, VK, MWR, FM, MS and AK are/were employees of, and may
hold stock in, Bristol-Myers Squibb (BMS). JL is employed by Quest Clinical Research, EL by Alamo Medical Research and IL by Medarex at the time of the study.
Anti-PD-1 (BMS-936558) is a patented monoclonal antibody owned by BMS and currently in Phase 3 development as treatment for patients with chronic
malignancy. There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS
ONE policies on sharing data and materials, as detailed online in the guide for authors.
* E-mail: email@example.com
¤ Current address: TESARO, Waltham, Massachusetts, United States of America
PLOS ONE | www.plosone.org1May 2013 | Volume 8 | Issue 5 | e63818
Virus-induced suppression of host immunity contributes to the
persistence of chronic infections with clinically important viruses
including hepatitis C virus (HCV), hepatitis B virus (HBV), and
human immunodeficiency virus (HIV) [1–3]. Various immuno-
modulators have been evaluated as therapeutics for these
infections, with the goal of overcoming and/or reversing virus-
induced immunosuppression. These include interferon-alfa, which
is well-established in therapy of HBV and HCV infections, as well
as interferon-lambda, toll-like receptor 7 agonists, interleukin-2,
interleukin-7, therapeutic vaccines, and other agents [1,4–8]. In
the case of HBV and HIV infections, although antiviral therapy
provides significant clinical benefits, durable control of the
infections with immune modulation remains an unmet goal for
Multiple mechanisms of viral immune evasion may contribute
to viral persistence [3,9–11]. For example, virus interactions with
host immune cells can attenuate interferon pathways and cause
dysfunction of dendritic cells, macrophages, and natural killer cells
. Also, rapid selection of immune escape variants can evade the
adaptive immune response. While T cells play a critical role in
viral clearance, chronic immune activation resulting from
prolonged antigen expression can trigger T cell exhaustion and
dysfunction, further contributing to viral persistence [1,9,10,12].
Analysis of T cells in the lymphocytic choriomeningitis virus
(LCMV) mouse model of chronic viral infection has demonstrated
that the exhausted T cell phenotype is driven, at least in part, by
the expression and function of the inhibitory receptor, pro-
grammed death 1 (PD-1) .
The PD-1 cell surface receptor and its ligands PD-L1 (B7–H1)
and PD-L2 (B7–DC) belong to the CD28–B7 family of T-cell
regulatory pathways with a critical role in maintaining the balance
between protective immunity against foreign pathogens and
destructive autoimmunity [14–16]. PD-1 is induced upon activa-
tion on various immune cell subsets, including CD4+and CD8+T
cells, natural killer cells, B cells, monocytes and some dendritic
cells. PD-L1 is expressed on multiple lymphoid and peripheral cell
types and is induced by inflammatory cytokines commonly
associated with viral infection, such as IFN-gamma. Expression
of PD-L2 is more restricted to myeloid cells, including dendritic
cells [16,17]. Engagement of PD-1 by either of its ligands globally
reduces T cell activity through the inhibition of cytokine
production, cytolytic function and T-cell proliferation . PD-
1/PD-L1 interactions also contribute to T regulatory function and
development [18,19], and data demonstrate that the PD-1
pathway is a major mechanism utilized by human tumors to
evade immune responses [20,21]. Several solid tumors have been
shown to over-express the ligands for PD-1, PD-L1 and PD-L2,
allowing these tumors to directly suppress T-cells activated by
tumor-specific antigens [22–24]. This understanding of the
function of the PD-1/PD-L1 interaction in tumor immune evasion
has led to several approaches to restore immune response to
tumors by suppression of the PD-1 pathway.
The PD-1 pathway has been implicated in T-cell exhaustion
associated with chronic viral infections in humans, including HCV
infection [12,14]. Persistent viremia has been associated with
upregulation of PD-1 expression on virus-specific CD8+ T-cells
[25–28]. In patients with chronic HIV, HBV or HCV infections,
enhanced T cell expression of PD-1 has been associated with T-
cell exhaustion, manifested by reduced virus-specific proliferative
capacity and cytokine expression [25,26,29,30]. Importantly,
CD8+cell function could be partially restored ex vivo by blockade
of the PD-1 pathway [25,26,29,31–34]. Consistent with these
findings, treatment of simian immunodeficiency virus (SIV)-
infected macaques with anti-PD-1 antibodies resulted not only in
rapid expansion of SIV-specific CD8+ T-cells with improved
functional characteristics in vivo, but also an increase in anti-SIV
humoral immunity as well as increased survival [35,36]. Similarly,
in vivo blockade of the PD-1 pathway resulted in enhanced
antiviral effector T cell responses and control of murine LCMV
infection . Thus, the PD-1 pathway is a target with potentially
broad application for therapy of multiple chronic viral infections.
In addition, other inhibitory pathways have been implicated in
suppression of immune responses against chronic viral infections,
including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4),
T cell immunoglobulin 3 (Tim-3) and lymphocyte activation gene-
3 (Lag-3) [2,12,37,38].
BMS-936558 (MDX-1106) is a fully human monoclonal
immunoglobulin G4 (IgG4 [S228P]) that targets PD-1 and inhibits
its binding to PD-L1 and PD-L2. BMS-936558 monotherapy,
when dosed continuously on a Q2W schedule, has demonstrated
significant and durable responses in pretreated patients with
advanced non-small cell lung cancer, melanoma and renal cell
carcinoma, with generally good tolerability [21,39,40]. These early
results have led to the initiation of Phase III registrational trials
across each indication (NCT01668784, NCT01642004, and
NCT01673867). The study described herein is the first clinical
evaluation of BMS-936558 in patients with chronic HCV
infection. The study was undertaken as a Proof-of-Concept to
determine whether exploiting PD-1 blockade has potential as a
therapy for chronic viral infections in which PD-1 upregulation
contributes to viral immune escape and persistence.
Materials and Methods
The protocol for this trial and supporting CONSORT checklist
are available as supporting information; see Protocol S1 and
Checklist S1, respectively.
Written informed consent was obtained from all patients. The
study was approved by five institutional review boards (IRBs)
responsible for the seven study sites: the Western IRB (Baltimore,
MD; 3 sites); Johns Hopkins Medicine IRB (Baltimore, MD); the
University of Pennsylvania Office of Regulatory Affairs (Philadel-
phia, PA); Aspire IRB (La Mesa, CA), and the Fox Commercial
IRB Ltd (Springfield, IL). The study was conducted in compliance
with the Declaration of Helsinki, Good Clinical Practice Guide-
lines, and local regulatory requirements.
Study Design and Patients
This was a randomized, modified double-blind (see below),
placebo-controlled single ascending dose study (clinicaltrials.gov
identifier NCT00703469) evaluating the safety, pharmacokinetics
and immunogenicity of BMS-936558 in HCV-infected patients
enrolled at seven US centers (Dallas and San Antonio, Texas; San
Francisco and Anaheim, California; Baltimore, Maryland; Phila-
delphia, Pennsylvania, and Springfield, Illinois) between October
2008 and July 2009.
Eligible patients were adult ($ 18 years) men and women with
chronic HCV genotype 1 infection of at least 6 months duration
and screening serum HCV RNA $100,000 IU/mL. Both HCV
treatment-naı ¨ve patients and patients who failed prior interferon-
alfa-based therapy (treatment-experienced) were included. Treat-
ment experience was defined as lack of sustained virologic
response after at least 12 weeks of interferon-alfa or pegylated
interferon-alfa combined with ribavirin. Patients were asymptom-
BMS-936558 in Chronic Hepatitis C
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atic or with only minor HCV symptoms not restricting normal
activities. Liver biopsy results consistent with chronic HCV
infection without evidence or history of bridging fibrosis or
cirrhosis, were required within 2 years prior to study entry. Eligible
patients had adequate bone marrow, liver, and renal function
(creatinine ,1.56ULN), as well as normal blood pressure,
electrocardiograms, chest x-rays, and alfa-fetoprotein levels.
Females of child-bearing potential and all males were required
to use adequate contraception for 70 days (females) or 180 days
(males) after study drug administration. Patients coinfected with
human immunodeficiency virus, hepatitis B virus, or with other
active infections (e.g. respiratory/urinary tract, herpes simplex)
were excluded; as were patients using immunosuppressive,
antiviral, or interferon-based therapies within 28 days of study
drug administration, or with a history or evidence of autoimmune
or immunodeficiency disease, cancer, bleeding disorders, alcohol
or drug misuse, or any serious medical condition other than
In the first part of the study, treatment-experienced patients
were assigned to one of six escalating dose cohorts (0.03, 0.1, 0.3,
1.0, 3.0, or 10 mg/kg BMS-936558), with 6 patients per cohort.
Within each cohort, patients were randomly assigned in a 5:1 ratio
to receive a single dose of BMS-936558 or placebo by intravenous
infusion administered over 15 minutes (0.03 mg/kg), 30 minutes
(0.1 mg/kg) or 60 minutes (all other doses). Patients were observed
for infusion reactions at their study sites for 6 hours post-infusion.
Study visits consisted of a screening visit (Day 228 to 26), a
pre-treatment visit (Day 25 to 22) and Days 1 (dosing), 2, 3, 8, 15,
22, 29, 31 (delayed-type hypersensitivity skin testing only), 43, 57
and 85. Randomization was by computer-generated tables
provided by the sponsor. The sponsor notified the site pharmacist
(who prepared the infusion) of the treatment assignment. All other
site staff remained blinded to treatment assignments. The sponsor
was not blinded. Randomized patients were assigned to the next
available dose cohort following a safety assessment and agreement
to escalate among the investigators and the sponsor’s medical
The study began with the lowest dose (0.03 mg/kg). Each
subsequent cohort was initiated when all patients in the previous
cohort reached Day 29 without dose-limiting toxicities (DLTs;
adverse events or laboratory abnormalities of at least grade 3,
considered at least potentially related to study drug and reported
within 28 days of dosing). Dose escalations continued until the last
cohort was enrolled or the maximum tolerated dose (MTD) was
identified, defined as the highest dose without a DLT. After
completion of dose escalation in treatment-experienced patients,
treatment-naı ¨ve and additional treatment-experienced patients
were enrolled at the highest dose achieved (10 mg/kg or the
MTD) and randomized as described.
All patients were followed for 85 days after dosing. Patients with
HCV RNA declines .2 log10 IU/mL were followed for an
additional 12 months after completion of the protocol in a
separate, long-term follow-up study.
Endpoints and Assessments
The primary endpoint for pharmacologic activity was the
proportion of subjects in each dosing cohort who achieved clinical
response, defined as a decline in HCV RNA from baseline of at
least 0.5 log10IU/mL in 2 or more consecutive measurements
(COBAS TaqMan HCV test, Roche Diagnostics, Pleasanton, CA;
lower limit of quantitation of 25 IU/mL and a limit of detection of
approximately 10 IU/mL). Secondary pharmacodynamics end-
points included the magnitude and duration of HCV RNA
changes, changes in levels of serum cytokines, and clinical
immunology assessments (PBMC reactivity to HCV antigens,
type IV hypersensitivity response to Candida albicans/tetanus toxoid
antigen skin test, and anti-tetanus antibody titers). Safety
assessments included treatment-emergent adverse events and
laboratory abnormalities, physical examinations, immunogenicity
(human anti-human antibody), treatment-associated cytokine
changes, and changes in immune cell subsets by flow cytometry.
Peripheral blood mononuclear cells (PBMCs) were collected
throughout the study and stored for analysis of antigen-specific
T-cell responses via ELISpot using HCV-specific peptide pools.
IL28B genotype (rs12979860 single nucleotide polymorphism) was
assessed as part of the follow-up study in 3 individuals with .4
log10IU/mL reductions in HCV RNA, and classified as
homozygous CC, heterozygous CT or homozygous TT by a
TaqMan genotyping assay (Applied Biosystems, Foster City, CA).
Rebound viremia in subjects who experienced transient declines
in HCV RNA was assessed for potential superinfection events by
examination of the HCV NS5A gene region. HCV RNA was
extracted from plasma samples obtained at baseline and at various
postinfusion time points, and NS5A amplicons generated by RT-
PCR were sequenced. Resulting population sequences were
subjected to a Basic Local Alignment Search Tool (BLAST)
search against NS5A genotype 1a samples in the BMS clinical
database and the Los Alamos National Laboratory (LANL) HCV
database. A neighbor-joining tree was created based on the
alignment using the Molecular Evolutionary Genetics Analysis
megasoftware.net)  with a bootstrap test of 500 replicates.
The tree was rooted using a genotype 1b sequence.
HCV genotyping and protocol-defined laboratory safety assess-
ments including cytokines and flow cytometric analyses were
performed by Covance Central Laboratory Services, Inc. (India-
napolis, IN, USA). Flow cytometry for determination of immune
cell cluster of differentiation (CD) antigens and HLA-DR was
performed by Covance Inc. using fluorescently labeled monoclo-
nal antibodies from Beckton Dickinson (BD Biosciences, San Jose,
CA, USA). HCV genotype was determined by Trugene HCV
genotyping assay (Siemens Healthcare Diagnostics, Tarrytown,
A quantitative enzyme-linked immunosorbent assay (ELISA)
was used for pharmacokinetic assessments of BMS-936558.
Recombinant PD-1/Fc chimera (R&D Systems, Minneapolis,
MN) was adsorbed onto a microtiter plate to capture BMS-936558
contained in serum samples. Captured BMS-936558 was detected
using a commercially obtained, purified goat anti-human antibody
labeled with alkaline phosphatase, with a p-nitrophenyl phosphate
substrate for colorimetric readout, and compared to a standard
curve for quantitation. Serum concentrations of BMS-936558
were assessed on Day 1 pre-infusion and at 1, 1.25, 1.5, 2, 3, 4, 6,
24, and 48 hours after start of infusion. Additional samples were
taken at 0.25, 0.5, and 0.75 hours after start of infusion for patients
receiving 0.03 mg/kg, and at 0.5 and 0.75 hours after start of
infusion for subjects receiving 0.1 mg/kg. Single serum samples
for pharmacokinetic parameters were taken on Days 8, 15, 22, 29,
43, 57, and 85 for all patients. Serum concentration versus time
data were analyzed by non-compartmental methods using the
program Kinetica (Thermo Scientific, Philadelphia, PA, USA).
Actual sampling times were used for PK parameter calculations.
The study employed an initial dose escalation design of 5
patients dosed with drug and 1 with placebo dosed at each level,
and a single occurrence of a set of predefined DLTs was deemed
unacceptable. Based upon binomial probability, this design had an
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PLOS ONE | www.plosone.org3May 2013 | Volume 8 | Issue 5 | e63818
approximately 77% likelihood of observing no DLTs at a dose
level if the true rate of DLT occurrence was 5%, and a 76%
probability of observing at least one DLT if the true rate of DLT
occurrence was 25%. Additional patients were enrolled following
determination of the maximum tolerated dose. Analysis of
antiviral activity, immunobiologic findings and safety were
conducted on a modified intent-to-treat population (ITT-exposed),
comprising all patients who received at least a partial dose of study
drug. Descriptive statistics were used to summarize all antiviral
activity, safety, immunobiologic and pharmacokinetic data.
Patient Disposition and Baseline Characteristics
A total of 56 patients were randomized, of whom 54 were
treated between August 8, 2008 and July 27, 2009 (45 BMS-
936558, 9 placebo). Patient disposition is shown in Figure 1. All
treated subjects in the ITT-exposed population completed the Day
29 evaluations. One patient assigned to placebo withdrew consent
at Day 31 and discontinued the study. No patient discontinued
due to an adverse event.
There were no clinically relevant differences between dose
groups in baseline disease or demographic parameters (Table 1).
Most patients were white males with 57% (31/54) HCV genotype
1a infections overall; treatment-naı ¨ve patients were somewhat
younger than other treatment groups. Ten subjects who received
10 mg/kg of BMS-936558 and 2 placebo subjects were treatment-
naive. Among treatment-experienced patients, approximately 38%
(16/42) had prior null response to peginterferon-alfa/ribavirin
therapy, 12% (5/42) had partial response, 36% (15/42) had post-
treatment relapse, and 14% (6/42) had received incomplete or
In the ITT-exposed population, clinical response (HCV RNA
decline $0.5 log10IU/mL on at least two consecutive visits) was
observed in six patients: one of five (20%) treatment-experienced
patients who received BMS-936558 0.1 mg/kg, one of 10 (10%) in
the treatment-experienced group receiving BMS-936558 10 mg/
kg, three of 10 (30%) in the treatment-naı ¨ve group receiving BMS-
936558 10 mg/kg, and one of nine (11%) placebo recipients
(Table 2). The duration of response was $8 weeks for four of the
six responders and between 4 and 8 weeks in two.
Among the six clinical responders, three patients receiving
BMS-936558 10 mg/kg – two treatment-naı ¨ve and one prior null
responder – experienced reductions in serum HCV RNA that
exceeded 4 log10IU/mL at nadir (Figure 2 and Table 2). Of these
three, one IL28B-CC homozygous, treatment-naive patient with
HCV GT-1a from a pre-study genotype assay (patient 1; Fig. 2A)
experienced a 4.55 log10 decline in HCV RNA which subse-
quently began to return to pre-treatment levels on-study. One
prior null responder with HCV genotype 1a from pre-study assay
data showed HCV RNA that dropped below the assay lower limit
of quantitation at study Day 85 (Patient 2; Fig. 2B), subsequently
became undetectable, and remained undetectable for more than 1
year after study completion. This patient was an IL28B-TT
homozygote, consistent with prior non-response to interferon-alfa
treatment [42,43]. Finally, one treatment-naı ¨ve, IL28B-CC patient
demonstrated HCV RNA below the assay lower limit of
quantitation at Study days 57 through 85 (patient 3; Fig. 2C). In
Figure 1. Patient disposition.
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post-study follow up this patient became transiently undetectable
before HCV RNA returned to pretreatment levels. This patient
was infected with HCV genotype 1 but his subtype could not be
established. In addition, in this latter patient, population genotypic
analysis of the NS5A region from baseline and relapse samples
confirmed that re-infection had not occurred (data not shown). All
three patients were male, and all received BMS-936558 in July
2009. There were no other homogeneous factors or features
No clinically relevant changes in immunoglobulin subsets (IgM,
IgA, IgG) were observed. Mean changes from baseline were
,15% in both BMS-936558 and placebo groups for all three
subsets during follow-up.
The immunoadjuvant activity of BMS-936558 on antibody
titers to tetanus toxoid was explored. Anti-tetanus antibody levels
varied between patients but median levels were generally
comparable for those who received study drug (any dose) versus
Table 1. Baseline characteristics.
0.03 0.1 0.313 1010 (Naı ¨ve)
Age, mean (SD) years55.8 (7.3)51.6 (3.4)50.8 (5.4) 48.2 (10.4) 52.0 (10.1)53.5 (5.8) 44.3 (9.9)47.1 (4.4)
Sex, n (%) male 2 (40)2 (40)2 (40)3 (60) 3 (60)6 (60) 9 (90) 7 (77.8)
Weight, mean (SD) kg88.9 (12.9)84.0 (10.7)83.2 (16.2) 78.0 (21.7)80.1 (26.7) 75.6 (11.7)85.0 (14.3)79.8 (8.8)
Race, n (%) White4 (80) 3 (60)4 (80) 5 (100) 4 (80)8 (80) 8 (80) 6 (66.7)
Black 1 (20)2 (40) 1 (20)0 1 (20) 2 (20) 1 (10)3 (33.3)
Other000000 1 (10)0
Time since diagnosis,
mean (SD) years
12.7 (6.6) 5.5 (3.1)7.3 (2.3) 8.2 (5.0)6.8 (2.9) 10.9 (6.9)7.3 (5.3) 9.6 (8.1)
HCV RNA, mean (SD)
6.63 (0.21)6.42 (0.47) 6.45 (0.38)6.48 (0.45) 6.42 (0.51)6.31 (0.56)6.41 (0.70) 6.42 (0.65)
HCV genotype, n (%) 1a3 (60) 3 (60)3 (60)3 (60)3 (60)4 (40) 5 (50)7 (77.8)
1b 2 (40) 2 (40) 2 (40)1 (20)1 (20)4 (40)2 (20) 1 (11.1)
1 (unsubtyped)000 1 (20)0 2 (20)3 (30) 1 (11.1)
1b/2b0000 1 (20)000
Treatment experienceNaive000000 102
Table 2. Antiviral activity results.
0.030.10.3131010 (Naı ¨ve) Total
n (%)01 (20.0)0001 (10.0)3 (30.0)5 (11.1)1 (11.1)
95% CINDNDNDNDND0.3, 44.56.7, 65.2NDND
Duration of response
(weeks), n (%)
$4 to ,801 (20.0)00001 (10.0)2 (4.4)0
$8000001 (10.0)2 (20.0)3 (6.7)1 (11.1)
Max. log10HCV RNA
decrease from BL, n (%)
,0.55 (100.0)4 (80.0)5 (100.0)5 (100.0)5 (100.0)9 (90.0)7 (70.0)40 (88.9)8 (88.9)
$0.5 to ,1.0 01 (20.0)00001 (10.0)2 (4.4)1 (11.1)
$2000001 (10.0)2 (20.0)3 (6.7)0
BL, baseline; 95% CI, 95% confidence interval.
aDefined as $0.5 log decline from baseline in HCV RNA on at least 2 consecutive measures.
BMS-936558 in Chronic Hepatitis C
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placebo. Some patients in the placebo group showed transient
increases in tetanus titers at Day 8, while subjects at dose levels of
0.03 through 1 mg/kg showed only minimal changes throughout
the study. In contrast, greater than 5-fold increases in anti-tetanus
antibody titer over pre-treatment baseline were seen at Day 8 in
two patients who received 3.0 mg/kg BMS-936558 (9-fold and 11-
fold over baseline) and in four treatment-experienced patients who
received 10 mg/kg (6-fold to 36-fold over baseline). These
increases subsequently declined back towards baseline (Figure 3).
In quantitative type IV hypersensitivity skin tests, there were no
treatment-related patterns in Candida-specific or tetanus-specific
erythema or induration.
Four patients had positive responses in BMS-936558 immuno-
genicity assessments (human-anti-human antibody [HAHA] anal-
ysis) conducted pretreatment, Day 29 and Day 85. One patient
was positive pretreatment but negative thereafter, two patients
were positive only on Day 85, and one patient was positive on
Days 29 and 85. The latter three patients received low doses (0.03
or 0.3 mg/kg) of BMS-936558.
There were no apparent treatment-related trends in mean
serum levels of IL-13, IP-10, neopterin, TGF-b1, IFN-c, IL-10,
IL-12, IL-1B, IL-2, IL-4, IL-5, IL-6, IL-8, or TNF-a during
follow-up (data not shown).
Mean absolute values and percentages of CD3+, CD4+, CD8+,
CD19+, CD3+/CD4+(6 CD38+, 6 HLA-DR+), CD3+/CD8+(6
Figure 2. HCV RNA and ALT changes in patients with clinical response.
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CD38+, 6 HLA-DR+), CD4+/CD25+, CD16+/CD56+, and in the
CD4+/CD8+and CD3+/CD19+ratios on peripheral blood
lymphocytes, were examined by flow cytometry. Treatment was
associated with substantial but transient declines at Day 2 in
absolute CD4+and CD8+cells (both the memory and naı ¨ve
subsets) and in CD19+cells (Table 3) that were not dose-related.
Substantial changes in cell percentages were not evident (Table 3).
Cell counts generally returned to pretreatment levels after one
week. PBMCs were obtained for ELISpot analysis of antigen-
specific T-cell responses pre- and post-treatment. Unfortunately,
cell viability was very low (generally ,30% viable cells) and not
permissive of ELISpot analysis.
The most common adverse events were fatigue, headache,
diarrhea, and pharyngeal pain (Table 4), with no pattern
suggesting dose-related differences in frequencies. All events were
mild or moderate in intensity (grade 1 or 2) with the exception of
one severe (grade 4) ALT elevation in a treatment-naı ¨ve patient
who received BMS-936558 10 mg/kg. Infection-related adverse
events suggested neither treatment-related immune deficits nor
exaggerated immune responses during the observation period.
The grade 4 ALT elevation occurred in a 51 year-old male
diagnosed with hepatitis C in 2006 and naive to prior therapy.
ALT was normal at baseline but began increasing two days after
dosing with BMS-936558 10 mg/kg (Figure 2A). ALT levels
peaked on Day 22 at 17-fold above the upper limit of normal
(43 U/L), concurrent with the maximum reduction in HCV RNA
(4.55 log10IU/mL). Other indicators of hepatic function, such as
bilirubin levels and INR, remained stable. Serologic evaluation for
concurrent liver disease was unrevealing. This event resolved
Immune-related adverse events, defined as clinically significant
events consistent with an immune-mediated mechanism and of
otherwise unknown etiology, were identified in six BMS-936558
recipients. These events included one patient who experienced
Figure 3. Mean changes from baseline in anti-tetanus antibody titer.
Table 3. Immune cell changes from baseline at Day 2.
Mean (SD) change from baseline to Day 2
Absolute cells/mL Percentage cells
All BMS-936558 (N=45)Placebo (N=9) All BMS-936558 (N=45)Placebo (N=9)
221 (105)0 (2.4)
BMS-936558 in Chronic Hepatitis C
PLOS ONE | www.plosone.org7May 2013 | Volume 8 | Issue 5 | e63818
hyperthyroidism, hypothyroidism, urticaria and pruritus; one
patient who experienced hyperthyroidism and diarrhea; two
patients with diarrhea alone, and one patient each with blister
and rash, respectively. All events were mild to moderate in
intensity and resolved without specific intervention. Hypo- and
hyperthyroidism were established by assay of TSH, total T3 and
In addition to the grade 4 ALT elevation previously described,
two other potentially immune-related events are of particular note:
one case of hyperthyroidism in a patient on concomitant
levothyroxine that required medical intervention, and one
exacerbation of adult onset diabetes.
The hyperthyroidism event concerned an interferon-alfa-expe-
rienced 52 year-old male diagnosed with HCV infection in 1995
and hypothyroidism in 1997, requiring levothyroxine administra-
tion prior to study entry. The patient’s baseline TSH (1.96 MIU/
L) and total T3 (2.2 nmol/L) were within the normal ranges of
0.34–5.6 MIU/L and 1.2–2.3 nmol/L, respectively. The patient
received 1 mg/kg of BMS-936558 and subsequently reported
insomnia on Day 5, diarrhea and increased appetite on Day 13,
and anxiety on Day 29. Thyroid function tests on Day 29 revealed
low TSH (0.14 MIU/L) and mildly elevated total T3 (2.7 nmol/
L); the subject was diagnosed with hyperthyroidism and instructed
to discontinue levothyroxine. Antithyroglobulin antibody (ATGA)
titer was slightly high at pre-treatment baseline (17.0 IU/mL
[normal range 0.0–14.4], increased to 264 IU/mL at Day 29, and
subsequently increased further to a maximum of 397 IU/mL on
Day 57. Symptoms improved; TSH measured 26.28 MIU/L on
Day 57 and the patient was restarted on levothyroxine 25mg daily.
TSH and ATGA remained elevated through the end of the study
and the subject remained on treatment for hypothyroidism.
The exacerbation of diabetes concerned a 45 year old
Caucasian male with HCV infection since 2000 and ongoing
diabetes treated with metformin, who experienced significant
worsening of blood glucose control on study day 22 requiring the
introduction of insulin which persisted until study conclusion.
Computer-assisted tomography did not reveal a structural cause
for the changes and no coincident infection or other precipitating
cause of the exacerbation of his diabetes was identified. Anti-
insulin antibodies and other markers of autoimmune diabetes were
not examined .
BMS-936558 demonstrated dose-related increases in exposure
across the range of doses studied, with near dose-proportional
increases over the dose range of 1 to 10 mg/kg (Table 5).
Specifically, for an increase in dose in the ratio of 1.0: 3.0: 10,
there was an increase in Cmaxin a ratio of 1.0: 3.0: 7.3 and an
increase in AUCINFof 1.0: 3.4: 9.5. Furthermore, BMS-936558
exhibited a long serum half-life (TK), of 20.6 days for the 1 mg/kg
cohort, increasing slightly to 23.7 days for the 10 mg/kg cohort.
The variability of derived exposure measures Cmaxand AUCINF
was consistently modest (coefficients of variation between 13% and
20%) at doses from 1 to 10 mg/kg, but higher between 0.03 and
0.3 mg/kg, most likely due to assay limitations for quantifying the
low serum levels obtained at the lower doses.
These data describe the proof-of-concept evaluation of an anti-
PD-1 monoclonal antibody in patients with chronic viral infection.
Following administration of a single dose of BMS-936558, HCV
RNA reductions $0.5 log10IU/mL were observed on $2
consecutive visits (protocol-defined clinical response) in five of 45
(11.1%) patients. At the highest administered dose (10 mg/kg),
HCV RNA reductions .4 log10IU/mL were observed in three
patients. Thus, in this high-dose group, 3/20 patients, or 15%,
experienced significant reductions of HCV RNA. Suppression of
HCV replication persisted more than eight weeks in most patients,
consistent with the hypothesized immunologic mode of action as
well as the long serum half-life of BMS-936558 . HCV RNA
was below the assay lower limit of quantitation (25 IU/mL) in two
patients at the end of the 12-week study follow-up, one of whom, a
prior null responder to interferon-alfa, progressed to undetectable
HCV RNA and remained so over one year later.
HCV RNA generally varies ,0.5 log10IU/mL in patients with
established HCV infection and spontaneous remissions are rare
[38,45], therefore it appears that the observed reductions in virus
titer are related to treatment with BMS-936558. While conclusions
are limited by the small number of patients treated in this study,
responders did not have a higher rate of immune-related adverse
events and neither BMS-936558 exposure nor immunogenicity
predicted response. The modest rate of response may have been
due, at least in part, to the administration of only a single dose to
establish proof of concept. In addition, several other processes
affecting the HCV-specific host immune response may conceiv-
ably have contributed to these observations, though data are
lacking in this small patient set. For example, response may be
influenced by levels of PD-1 ligand expression in the liver or
lymphoid organs. The effectiveness of anti-PD-1 blockade could
be limited by the emergence of HCV variants with T cell escape
mutations conferring reduced immunogenicity and recognition by
Table 4. Adverse events reported in $5% BMS-936558 (combined) or placebo recipients.
0.03 0.1 0.313 1010 (Naı ¨ve) Total
N55555 1010 459
Any AE, n (%)3 (60) 4 (80)4 (80)4 (80) 4 (80)6 (60) 10 (100)35 (78)6 (67)
Fatigue, n (%)0 2 (40)00 2 (40)2 (20) 3 (30)9 (20) 1 (11)
Headache, n (%)0 3 (60) 1 (20) 1 (20)01 (10)2 (20)8 (18)1 (11)
Diarrhea, n (%)0 2 (40)02 (40)2 (40)006 (13) 1 (11)
Pharyngeal pain, n (%)0 1 (20)0 1 (20)1 (20)02 (20)5 (11) 1 (11)
Cough, n (%)01 (20)0 2 (40)1 (20)004 (9)1 (11)
Upper abdominal pain, n (%)01 (20)00 2 (40)003 (7)0
BMS-936558 in Chronic Hepatitis C
PLOS ONE | www.plosone.org8May 2013 | Volume 8 | Issue 5 | e63818
T cells targeting the original virus. Finally, other cell surface
inhibitory receptors in addition to PD-1–such as lymphocyte
activation gene-3 (LAG-3), T cell immunoglobulin mucin-3 (TIM-
3), and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) –
are implicated in the co-regulation of the T cell exhaustion
phenotype [46,47], and their influence may also limit the
effectiveness a single-locus blockade of PD-1. It is noteworthy
that in ex vivo studies of T-cells from patients with chronic HCV
infection, the modest functional impairment of circulating CD8+
T-cells could be reversed by PD-1 blockade alone, whereas the
marked dysfunction of intrahepatic CD8+T-cells, which expressed
very high levels of PD-1, could not . However, impairment of
both circulating and intrahepatic CD8+cells in HCV infection
could be reversed by simultaneous blockade of both PD-1 and
Several immunologic findings were noteworthy. First, as might
be expected from a human monoclonal antibody, human-anti-
human (HAHA) antibody responses were not detected in most
patients through 85 days post-dose, suggesting such responses did
not influence safety or antiviral activity. Second, a transient
decrease in CD4+, CD8+and CD19+lymphocytes was observed
post treatment. These were changes were generally transient and
not associated with adverse events suggesting immune deficits.
Similar observations have been made in cancer patients following
anti-PD-1 administration  and were hypothesized to represent
redistribution of lymphocyte subsets into tumor and tissue
compartments. Finally, the immunoadjuvant activity of BMS-
936558 on anti-tetanus toxoid antibody levels was explored.
Marked increases in anti-tetanus antibody levels over pre-
treatment baseline were seen in several patients who received
3.0 mg/kg BMS-936558 and 10 mg/kg which exceeded changes
observed in patients who received lower doses or placebo. A
finding of an effect of anti-PD-1 blockade on antibody titers would
be consistent with a role for PD-1 in B cell as well as T follicular
helper cell function as has been described [48,49]. Increases in
anti-SIV titers have previously been shown in SIV-infected
macaques treated with an anti-PD-1 antibody .
A single dose of BMS-936558 was generally well tolerated; most
common adverse events were non-specific in nature. Immune-
related adverse events were anticipated with this intervention; such
events could result from a break in host tolerance to self-antigens
or from a liver-directed antiviral T-cell response following reversal
of immune suppression. Overall, immune-related events were
infrequent, mostly mild to moderate in intensity, and generally
similar to those reported in studies of BMS-936558 in patients with
solid tumors . Most immune-related events resolved sponta-
Endocrine abnormalities, including thyroid abnormalities, can
be autoimmune in nature and potentially indicate altered
immunologic self-tolerance. Two patients experienced low TSH
levels (hyperthyroidism) followed by abnormally high TSH; one
also experienced increased titers of ATGA and symptoms
consistent with hyperthyroidism. This pattern resembles some
cases of immune-mediated thyroiditis seen in clinical practice and
would be consistent with reduced self-tolerance to thyroid specific
antigens. Administration of anti-PD-1 therapy requires a vigilant
follow up for signs and symptoms consistent with autoimmune
One patient who received BMS-936558 10 mg/kg and
achieved a significant clinical response had a marked grade 4
ALT elevation. In studies of solid tumors, ALT elevations have
been observed in the absence of identifiable hepatic metastases,
indicating the potential for non-specific hepatic inflammation. The
ALT elevation in this subject occurred concomitant with a .4
log10reduction of HCV RNA, a finding consistent with activation
of HCV-specific, liver-directed T cell-mediated immunity in acute
HCV infection . Thus, it is possible that this ALT elevation
was a specific result of the predicted mechanism of action of BMS-
936558. Importantly, bilirubin and measures of hepatic function
remained stable and the event resolved without intervention,
concomitant with a return of HCV RNA towards baseline levels.
In summary, this study establishes proof-of-concept that PD-1
blockade with BMS-936558 can lead to persistent suppression of
HCV replication in some patients with chronic infection, including
those who do not respond to interferon-alfa therapy. While the
therapeutic application of this approach in HCV infection is
limited by the recent, ongoing development of highly effective new
treatments, the promising results of this study suggest that further
exploration of PD-1 pathway blockade is warranted across other
chronic viral diseases, possibly in combination with other
immunomodulatory or direct-acting antiviral agents. The poten-
tial for autoimmune adverse events, or of activity-related hepatic
inflammation in liver infections, will require further evaluation in a
larger dataset following multiple-dose administration.
Table 5. Summary of pharmacokinetic parameters.
g. mean (% CV)
g. mean (% CV)
g. mean (% CV)
3 1.41 (17) 3.25 (1.8–6.3)4.20 (151)NR NR
0.15 3.52 (129) 0.63 (0.5–3.0)113 (99) NRNR
0.35 6.86 (21)1.03 (1.0–3.0)1597 (35) NRNR
15 26.8 (13)1.27 (1.0–4.0) 7377 (17)8537 (19)20.6 (2.81)
3580.4 (14)1.50 (1.3–3.0) 26,189 (17)28,799 (19) 23.2 (4.69)
1020195 (20) 1.75 (1.0–48.0)74,378 (17)81,022 (20)23.7 (4.41)
aTwo subjects in the 0.03 mg/kg dose cohort had serum drug concentrations below the assay lower limit of quantitation for all time points and were excluded from
pharmacokinetic statistical analyses.
NR=Not reported; the AUCINFextrapolated area was .20% of the total area resulting in undue potential for error.
BMS-936558 in Chronic Hepatitis C
PLOS ONE | www.plosone.org9May 2013 | Volume 8 | Issue 5 | e63818
The following employees of BMS and Medarex played key roles in the
design, execution and analysis of the study: Diane Sherman (Clinical
Operations, BMS), Zhang-Hua Lan (Clinical Science, Medarex) and
Areceli Doronila (Clinical Operations, Medarex). Editorial assistance with
the drafting of the manuscript in association with the authors was provided
by two professional medical writers (Nicholas Fitch and Richard Boehme)
with funding from BMS.
Conceived and designed the experiments: D. Gardiner IL JA BH VK FM
MWR D. Grasela M. Selby AJK. Performed the experiments: EL RG M.
Sulkowski JL KRR KMC MD SO VK FM MWR. Analyzed the data: D.
Gardiner IL JA BH VK FM MWR M. Selby AJK. Wrote the paper: DGr.
Critically reviewed and edited the manuscript for content: D. Gardiner EL
MD RG KRR KMC M. Sulkowski SO JA BE VK FM MWR D. Grasela
M. Selby AJK IL.
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