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A First-in-Human Study To Assess the Safety and Pharmacokinetics of Monoclonal Antibodies against Human Cytomegalovirus in Healthy Volunteers Kiran Dole,a Florencia Pereyra Segal,a Adam Feire,a,b Baldur Magnusson,c Juan C. Rondon,d Janardhana Vemula,e Jing Yu,a Yinuo Pang,a Peter Pertela Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, USAa; Novartis Institutes for BioMedical Research, Emeryville, California, USAb; Novartis Pharmaceuticals AG, Basel, Switzerlandc; Elite Research Institute, Miami, Florida, USAd; Novartis Pharmaceuticals, Hyderabad, Indiae
Human cytomegalovirus (HCMV) can cause significant disease in immunocompromised patients and treatment options are limited by toxicities. CSJ148 is a combination of two anti-HCMV human monoclonal antibodies (LJP538 and LJP539) that bind to and inhibit the function of viral HCMV glycoprotein B (gB) and the pentameric complex, consisting of glycoproteins gH, gL, UL128, UL130, and UL131. Here, we evaluated the safety, tolerability, and pharmacokinetics of a single intravenous dose of LJP538 or LJP539 or their combination in healthy volunteers. Adverse events and laboratory abnormalities occurred sporadically with similar incidence between antibody and placebo groups and without any apparent relationship to dose. No subject who received antibody developed a hypersensitivity, infusion-related reaction or anti-drug antibodies. After intravenous administration, both LJP538 and LJP539 demonstrated typical human IgG1 pharmacokinetic properties, with slow clearances, limited volumes of distribution, and long terminal half-lives. The pharmacokinetic parameters were linear and dose proportional for both antibodies across the 50-fold range of doses evaluated in the study. There was no apparent impact on pharmacokinetics when the antibodies were administered alone or in combination. CSJ148 and the individual monoclonal antibodies were safe and well tolerated, with pharmacokinetics as expected for human immunoglobulin.
H
uman cytomegalovirus (HCMV) can cause significant disease in immunocompromised individuals, including transplant patients and neonates infected in utero (1). Therapies available to treat HCMV disease in transplant recipients, including ganciclovir, cidofovir, and foscarnet, are effective but associated with serious toxicities; there are no approved treatments for congenital HCMV (2). Passive immunization with HCMV hyperimmunoglobulin, which is polyclonal IgG from human plasma pools, is also used to prevent HCMV disease in some transplant recipients. It is safe and well tolerated but is less effective than other antiviral therapies. Preliminary data from a combination of anti-HCMV monoclonal antibodies, showed a modest, therapeutic benefit in renal transplant recipients (3–6). CSJ148 is a combination of two fully human anti-HCMV IgG1 monoclonal antibodies, LJP538 and LJP539 (7), and offers the potential to be a safe and well-tolerated alternative to available therapies for the prevention and treatment of HCMV disease. Each antibody binds to and inhibits the function of viral glycoproteins essential for HCMV infectivity. In vitro studies demonstrated that LJP538 and LJP539 were up to 100 to 1000-fold more potent, respectively, than HCMV-hyperimmunoglobulin at inhibiting infection of various cell lines. Furthermore, LJP538 and LJP539 were active against a panel of clinical isolates in vitro and demonstrated minor to moderate synergy in combination (A. Patel et al., unpublished data). Importantly, viruses with reduced susceptibility to either LJP538 or LJP539 remained susceptible to the other antibody, and no cross-resistance with ganciclovir was observed in vitro. In addition, no resistance to either antibody was seen when HCMV was cultured in the presence of LJP538 and LJP539 in combination for ⬎400 days (Patel et al., unpublished). In human tissue cross-reactivity studies, LJP538 and LJP539 exhibited no off-target binding. Scattered binding was noted in human tissues,
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but occurred only in cells confirmed to be positive for HCMV DNA or RNA by in situ hybridization (A. Hey et al., unpublished data). Using the combination of antibodies in CSJ148 has several advantages. Each antibody targets a separate glycoprotein epitope; LJP538 binds the viral gB protein, while LJP539 binds the gH/gL/ UL128/UL130/UL131a pentameric complex. Although antibodies directed against gB correlate with neutralizing activity (8), the major neutralizing antibody response is directed against the pentameric complex, both in natural infections and in HCMV hyperimmunoglobulin (9, 10). In vitro, LJP538 inhibited HCMV infection of all cell types tested, while LJP539 blocked infection of cell types likely required for systemic spread of the virus (Patel et al., unpublished). Furthermore, in vitro data suggest that the combination of LJP538 and LJP539 will significantly decrease the development of resistance (Patel et al., unpublished). The objectives of this first-in-human study were to evaluate the safety, tolerability and pharmacokinetics of a single intravenous (i.v.) dose of CSJ148 in healthy volunteers.
Received 6 November 2015 Returned for modification 30 December 2015 Accepted 20 February 2016 Accepted manuscript posted online 29 February 2016 Citation Dole K, Segal FP, Feire A, Magnusson B, Rondon JC, Vemula J, Yu J, Pang Y, Pertel P. 2016. A first-in-human study to assess the safety and pharmacokinetics of monoclonal antibodies against human cytomegalovirus in healthy volunteers. Antimicrob Agents Chemother 60:2881–2887. doi:10.1128/AAC.02698-15. Address correspondence to Florencia Pereyra Segal, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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MATERIALS AND METHODS Study design. This was a randomized, double-blind, placebo-controlled, single ascending intravenous dose study conducted at a single center. It was designed to determine the safety, tolerability, and pharmacokinetics of LJP538, LJP539, and their combination (CSJ148) in healthy subjects. The study consisted of a 20-day screening period, one baseline visit, a 1-day treatment period, with approximately eight follow-up visits, and an end-of-study visit on day 105. The planned enrollment was 30 male and female volunteers randomized to sequential cohorts of five subjects, with four receiving antibody, and one receiving placebo. Subjects in cohort 1 received LJP538 (1 mg/kg) or matching placebo, and those in cohort 2 received LJP539 (0.1 mg/kg) or a placebo. Subjects in cohorts 3 to 6 received single ascending doses of CSJ148 (1 to 50 mg/kg LJP538 and 0.1 to 5 mg/kg LJP539) or the placebo. LJP538 and/or LJP539 and/or placebo were administered as single i.v. doses on the morning of day 1. Study drug was administered in two separate i.v. lines over a period of 2 h. In cohorts 1 and 2 only, the first two subjects were dosed with study drug at least 24 h before the remaining three subjects were dosed. The sponsor and the principal investigator reviewed safety data through day 14 to ensure that escalation to the next higher dose was appropriate and acceptable. The study protocol was approved by the Institutional Review Board (IRB) for the center. All subjects provided written informed consent. Subjects. Healthy male and female (of nonchildbearing potential) subjects aged 19 to 55 years were eligible. Good health was determined by past medical history, physical examination, vital signs, electrocardiogram, and laboratory tests at screening. Subjects had to weigh at least 50 kg and have a body mass index (BMI) between 18 and 32 kg/m2. Exclusion criteria included history of immunodeficiency disease, hyperviscosity (including cryoglobulinemia, monoclonal gammopathies, or marked hypertriglyceridemia at screening and baseline), and any surgical or medical condition that might significantly alter the disposition of i.v. monoclonal antibodies. Subjects were also excluded if they had a history of some malignancies, impaired renal function (creatinine clearance ⬍ 90 ml/min), liver disease, cardiovascular disease, or history of allergy to monoclonal antibodies. HCMV seropositive and seronegative individuals were eligible to participate; HCMV DNA quantification was performed in all subjects. Safety assessments. Safety assessments consisted of collecting all adverse events, with their severity and relationship to study drug, serious adverse events, and pregnancies. There was also regular monitoring of hematology and blood and urine chemistry, as well as assessments of vital signs, physical condition, electrocardiograms, and body weight. Pharmacokinetic and immunogenicity assessments. Blood samples for pharmacokinetic and immunogenicity evaluations (3 to 5 ml per time point) were collected from a forearm vein contralateral to the infusion site. Samples for determination of drug concentrations were collected predose, as well as 2, 2.5, 3, 4, 6, and 10 h postdose on day 1, and on days 2, 4, 7, 14, 21, 28, 42, 56, 77, and 105. Free LJP538 and LJP539 in the serum were quantified using a Meso Scale Discovery (MSD; Rockville, MD)based assay, which uses anti-idiotypic antibodies specific for either LJP538 or LJP539 as capture reagents. The lower limits of quantification (LLOQ) were 10 ng/ml for both assays. Samples for assessment of immunogenicity (anti-drug antibodies) were collected predose and on days 14, 28, 56, and 105. The presence of anti-LJP538 and anti-LJP539 antibodies in serum was assessed using an internal homogenous bridging MSD-based electrochemiluminescence assay with three tiers (initial screening, confirmation, and titration). Statistical methods. Summary statistics are provided for pharmacokinetic parameters by treatment and sampling time point. Concentrations below LLOQ were treated as zero in summary statistics. A geometric mean was not reported if the data set included zero. Noncompartmental pharmacokinetic analysis was performed on unbound LJP538 and LJP539 serum concentration-time profiles using the actual infusion durations and elapsed sampling times for each subject. The following pharmacokinetic parameters were generated using WinNonlin Phoenix (version 6.2; Pharsight, Mountain View, CA): area under the serum concentration-time
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curve from time zero to the last time point with quantifiable concentration (AUClast), area under the serum concentration-time curve from time zero to infinity (AUCinf), maximum observed serum concentration (Cmax), systemic clearance (CL), apparent volume of distribution at steady state (Vss), and terminal-phase half-life (t1/2). The Cmax and AUC were assessed by fitting a dose proportionality model to the data based on the power model: pharmacokinetic parameter ⫽ ␣ ⫻ dose. The estimate of the exponent  and its 90% CI values were determined, using this linear model of log-transformed pharmacokinetic parameters and log dose. Dose proportionality was concluded if the 90% CI interval for  (slope estimate) was completely contained within the prespecified critical region (0.943 to 1.057) (11).
RESULTS
Subject demographics. Overall, 32 subjects were enrolled and 28 completed the study. Two subjects were lost to follow-up: one on day 14 who received LJP539 (0.1 mg/kg) and one on day 19, who received the placebo. Both of these subjects were replaced. Two subjects withdrew consent: one who received LJP538 (50 mg/kg) and LJP539 (5 mg/kg) on day 60 and one who received placebo on day 34. Neither of these subjects was replaced. The demographic data for all enrolled subjects are shown in Table 1. The subjects were predominantly male (65.6%) and Caucasian (93.8%); all subjects identified their ethnicity as Hispanic/Latino. The study population had a mean BMI of 27 kg/m2, and the mean age was 43 years. A total of 28 of the 32 subjects (87.5%) had serological evidence of prior HCMV infection. Safety and tolerability. There were no deaths during the study and no subject discontinued due to an adverse event. There was 1 serious adverse event, a grade 1 transient ischemic attack on day 58, which developed in a subject who received 1 mg/kg LJP538 57 days earlier. The subject presented to the emergency department complaining of right sided paresthesias. He was admitted to the hospital for additional work-up, during which a computed tomography of the head did not show any evidence of thrombosis or bleeding, and his symptoms resolved without treatment. The event was considered by the investigator to be unrelated to study drug, which is supported by the lack of temporal association between administration of study drug and adverse event onset. The subject reported no further symptoms or concomitant medications during follow-up and completed the study as planned. Seventeen treatment-emergent adverse events occurred during the study period: 14 among 7 subjects who received antibody and 3 among 2 subjects who received placebo. Adverse events by dosing group and preferred term are shown in Table 2. The percentages of subjects who developed adverse events were similar among those administered antibody (7/25; 28.0%) or placebo (2/7; 28.6%). Adverse events occurred sporadically and without any apparent relationship to treatment group or dose. All adverse events were assessed as grade 1 in severity and all resolved, with only three events (transient ischemic attack, dizziness, and an influenza-like illness) in two subjects requiring action. All but one adverse event were assessed as not related to study drug. The one event suspected to be related was palpitations, which developed in a subject who received 20 mg/kg LJP538 and 2 mg/kg LJP539 the day before. This subject also developed myalgia and headache that same day (day 2). All three events resolved after 2 h without any action taken. The myalgia and headache were not assessed as related to study drug. The most common adverse events were dizziness (n ⫽ 3) and palpitations (n ⫽ 2). Dizziness developed in two subjects who
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TABLE 1 Demographic summary by treatment group Treatment Parameter
LJP538
LJP539
LJP538/LJP539
LJP538/LJP539
LJP538/LJP539
LJP538/LJP539
Pooled placebo
Dose(s), mg/kg n
1 4
0.1 5
1/0.1 4
5/0.5 4
20/2 4
50/5 4
7
Age (yr) Mean (SD) Range
39 (17.0) 19–55
41 (11.2) 24–53
32 (8.8) 22–43
54 (1.4) 52–55
39 (12.0) 28–51
50 (3.6) 45–53
44 (8.5) 27–52
Gender, no. (%) Male Female
4 (100) 0
4 (80.0) 1 (20.0)
2 (50.0) 2 (50.0)
1 (25.0) 3 (75.0)
3 (75.0) 1 (25.0)
1 (25.0) 3 (75.0)
6 (85.7) 1 (14.3)
Race, no. (%) Caucasian Black Asian
4 (100) 0 0
5 (100) 0 0
3 (75.0) 0 1 (25.0)
3 (75.0) 1 (25.0) 0
4 (100) 0 0
4 (100) 0 0
7 (100) 0 0
Wt (kg) Mean (SD) Range
81 (5.7) 76–87
71 (7.0) 64–81
76 (6.9) 69–82
76 (11.0) 66–90
79 (21.1) 54–104
70 (4.7) 64–74
82 (11.3) 68–100
Ht (cm) Mean (SD) Range
169 (6.1) 160–174
168 (6.0) 160–176
168 (10.5) 154–179
164 (9.3) 154–176
169 (13.8) 150–180
159 (7.6) 153–170
170 (8.1) 160–178
BMI (kg/m2) Mean (SD) Range
28 (1.9) 26–30
25 (1.6) 22–26
27 (3.1) 23–30
28 (1.5) 27–30
27 (3.4) 24–32
27 (2.0) 25–29
28 (2.4) 25–32
received antibody and one subject who received placebo. The first subject received 0.1 mg/kg LJP539, and he developed dizziness on day 53; no action was taken. This subject had also developed palpitations on day 28. The second subject received 1 mg/kg LJP538 and 0.1 mg/kg LJP539, and she developed dizziness and syncope
on day 88; the dizziness was treated with promethazine. This subject also developed an influenza-like illness on day 87 and menorrhagia on day 40. The third subject received the placebo, and he developed grade 1 dizziness on day 1 shortly after completion of infusion; no action was taken. Palpitations developed in two sub-
TABLE 2 Incidence of adverse events by treatment group Treatment Parameter or adverse event (preferred term)
LJP538
LJP539
LJP538/LJP539
LJP538/LJP539
LJP538/LJP539
LJP538/LJP539
Pooled placebo
Parameter Dose(s), mg/kg n
1 4
0.1 5
1/0.1 4
5/0.5 4
20/2 4
50/5 4
7
No. (%) of adverse events (preferred term)a Any event Dizziness Palpitations Diarrhea Headache Hypoglycemia Influenza-like illness Menorrhagia Myalgia Pruritus Rash erythematous Syncope Transient ischemic attack Viral upper respiratory tract infection Vomiting
1 (25.0) 0 0 0 0 0 0 0 0 0 0 0 1 (25.0) 0 0
2 (40.0) 1 (20.0) 1 (20.0) 0 0 0 0 0 0 0 0 0 0 1 (20.0) 0
1 (25.0) 1 (25.0) 0 0 0 0 1 (25.0) 1 (25.0) 0 0 0 1 (25.0) 0 0 0
2 (50.0) 0 0 1 (25.0) 0 1 (25.0) 0 0 0 0 0 0 0 0 1 (25.0)
1 (25.0) 0 1 (25.0) 0 1 (25.0) 0 0 0 1 (25.0) 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 (28.6) 1 (14.3) 0 0 0 0 0 0 0 1 (14.3) 1 (14.3) 0 0 0 0
a
Adverse events are arranged in order of decreasing total frequency and then alphabetically.
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FIG 1 Serum logarithmic views of LJP538 (A) and LJP539 (B) concentration-versus-time profiles. Cohort 1, LJP538 (1 mg/kg)/LJP539 (0 mg/kg); cohort 2, LJP538 (0 mg/kg)/LJP539 (0.1 mg/kg); cohort 3, LJP538 (1 mg/kg)/LJP539 (0.1 mg/kg); cohort 4, LJP538 (5 mg/kg)/LJP539 (0.5 mg/kg); cohort 5, LJP538 (20 mg/kg)/LJP539 (2 mg/kg); cohort 6, LJP538 (50 mg/kg)/LJP539 (5 mg/kg).
jects who received antibody; both subjects are discussed above. No treatment site or infusion-related reactions were reported, although one subject who received the placebo developed pruritus and erythematous rash on the hands within 5 h of dosing on day 1; the pruritus and rash resolved without treatment. Only one subject, who received 5 mg/kg LJP538 and 0.5 mg/kg LJP539, had a laboratory abnormality assessed as an adverse event. This subject had a grade 1 low blood glucose level on day 105. No other laboratory abnormality was assessed as clinically significant,
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and no clinically significant vital sign or electrocardiogram abnormalities were noted during the study. Pharmacokinetics. Pharmacokinetic properties were consistent across individuals within each cohort, with limited interindividual variability noted in the serum concentration versus time profiles (Fig. 1) and in the noncompartmental pharmacokinetic parameter estimates (Table 3). After the 2-h intravenous infusions of LJP538 and LJP539, serum concentrations of both antibodies increased rapidly and reached the maximum concentration
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TABLE 3 Noncompartmental pharmacokinetic parameters for each antibodya Mean (SD) Dose(s) of LJP538/LJP539 (mg/kg)
AUClast (day·g/ml)
AUCinf (day·g/ml)
Cmax (g/ml)
C28D (g/ml)
CL (ml/day/kg)
Vss (ml/kg)
t1/2 (day)
LJP538 1/0 1/0.1 5/0.5 20/2 50/5
337 (84.4) 318 (81.1) 1,610 (351) 6,620 (921) 16,100 (2,170)
345 (89.4) 331 (92.7) 1,650 (373) 6,750 (1,020) 16,700 (1,790)
34.3 (4.50) 27.5 (3.13) 164 (9.42) 614 (81.1) 1,540 (31.1)
3.97 (1.43) 3.75 (1.36) 18.6 (5.64) 78.4 (17.6) 185 (26.9)
3.08 (0.946) 3.20 (0.873) 3.16 (0.730) 3.02 (0.496) 3.03 (0.329)
62.6 (4.63) 77.1 (6.31) 67.6 (8.39) 66.0 (7.35) 66.4 (6.47)
20.1 (4.87) 22.9 (6.44) 18.9 (3.75) 18.7 (3.89) 18.6 (0.659)
LJP539 0/0.1 1/0.1 5/0.5 20/2 50/5
45.7 (7.91) 48.5 (11.1) 273 (53.4) 1,030 (147) 2,130 (347)
47.3 (8.49) 51.7 (13.8) 284 (60.1) 1,080 (173) 2,270 (294)
3.38 (0.290) 3.14 (0.360) 17.7 (1.11) 77.7 (7.47) 164 (11.5)
0.539 (0.111) 0.584 (0.153) 3.59 (0.840) 11.6 (1.99) 27.1 (0.929)
2.18 (0.460) 2.05 (0.570) 1.83 (0.423) 1.90 (0.351) 2.23 (0.330)
58.8 (9.69) 61.6 (4.42) 49.0 (4.42) 55.8 (6.19) 64.6 (4.28)
21.6 (2.61) 25.9 (6.31) 22.1 (4.28) 23.4 (4.02) 23.7 (2.31)
a
n ⫽ 4 for all summary parameters except LJP539 Cmax after the 0/0.1 dose of LJP538/LJP539.
around the end of infusion (Fig. 1). After infusion, serum concentrations decreased rapidly during the initial few days, followed by a much slower terminal elimination phase. Overall, pharmacokinetic characteristics of both LJP538 and LJP539 were typical of human IgG1 antibodies, with slow clearances, limited apparent volumes of distribution, and long terminal half-lives (Table 3). Administration of the two antibodies in combination did not appear to impact the pharmacokinetics of either individual antibody, since the pharmacokinetic profiles and parameters were generally comparable between cohorts 1 and 3 for LJP538 at 1 mg/kg and cohorts 2 and 3 for LJP539 at 0.1 mg/kg. In addition, prior infection with HCMV based on serology had no appreciable impact on the pharmacokinetics of LJP538 or LJP539. Across the 50-fold dose ranges (1 to 50 mg/kg for LJP538 and 0.1 to 5 mg/kg for LJP539), key pharmacokinetic parameter estimates for serum exposure (AUC and Cmax), clearance (CL), distribution (Vss), and terminal half-life (t1/2) were similar, indicating linear pharmacokinetics across the range of doses evaluated (Table 3). Based on the statistical analyses, dose proportionality was demonstrated for AUClast, AUCinf, and Cmax across the whole range of doses for LJP538 and LJP539 (Table 4). Immunogenicity. Among the 25 subjects administered LJP538 and/or LJP539, no anti-drug antibodies were detected either at baseline or any of the postdose time points (days 14, 28, 56, and
105). LJP538 concentrations in 19 samples from all subjects in cohorts 5 and 6 who received antibody exceeded the drug tolerance level (48.7 g/ml) for the anti-drug antibody assay at the earlier time points; above the drug tolerance level there is the potential for interference with the assay, and the presence of antidrug antibodies can therefore not be fully excluded. However, analysis of samples from the same subjects at later time points, especially on day 105, indicated that no subjects treated with either LJP538 and/or LJP539 had anti-drug antibodies by the end of the study. No anti-drug antibodies were detected in any of the samples from subjects who received placebo. DISCUSSION
The combination of LJP538 and LJP539 is designed to block the early steps in HCMV infection, as well as to decrease the risk of developing resistance. HCMV infects a broad range of tissues, and the glycoproteins required for entry vary based on cell type. The infection of all cell types requires gB. In addition, a three-member complex (gH, gL, and gO) is essential for entry into fibroblasts, whereas a pentameric complex (gH, gL, UL128, UL130, and UL131a) allows infection of epithelial, endothelial, and hematopoietic cells (12), (13). Targeting the viral gB protein by LJP538, along with blocking the pentameric complex by LJP539, is therefore expected to limit infection of multiple cell types involved in
TABLE 4 Dose proportionality of pharmacokinetic parameters Estimated increase across the dose rangea
Slope estimate Parameter
Estimate
90% CI
Increase
90% CI
Dose proportionality across the whole range based on slope
LJP538 AUClast (day·g/ml) AUCinf (day·g/ml) Cmax (g/ml)
1.00 1.00 1.00
0.95–1.05 0.95–1.05 0.97–1.03
50.50 50.72 50.03
41.79–61.03 41.65–61.77 44.52–56.21
Yes Yes Yes
LJP539 AUClast(day·g/ml) AUCinf (day·g/ml) Cmax (g/ml)
0.99 1.00 1.02
0.94–1.04 0.95–1.05 0.99–1.04
48.48 49.21 53.84
40.02–58.74 40.37–60.00 48.70–59.52
Yes Yes Yes
a
The dose ranges were 1 to 50 mg/kg for LJP538 and 0.1 to 5 mg/kg for LJP539. Estimates and 90% CI values of the exponent were determined from a simple linear regression analysis of the log-transformed values of the pharmacokinetic parameter and dose.
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HCMV pathogenesis and systemic spread in vivo. Antibodies against both gB (8) and the pentameric complex (9, 10) have been associated with neutralizing activity during natural infection. In addition, LJP538 and LJP539 demonstrated minor to moderate synergy in vitro, with no antagonism noted (Patel et al., unpublished). Importantly, LJP538 and LJP539 did not show cross-resistance with each other in vitro and no resistant virus was detected when HCMV-infected cells were passaged in the presence of the combined antibodies for more than 400 days (Patel et al., unpublished). In healthy subjects, LJP538 and LJP539 were safe and well tolerated after single i.v. doses up to 50 and 5 mg/kg, respectively. Both antibodies showed dose proportional linear pharmacokinetics, slow clearances, limited volumes of distribution, and long terminal half-lives. This was expected because LJP538 and LJP539 have intact Fc domains, and low levels of the targets for both antibodies are to be expected in healthy volunteers who have no evidence of actively replicating virus (HCMV DNA was not quantifiable in all subjects). As expected, administering the antibodies in combination did not appear to impact the pharmacokinetic parameters of either individual antibody; doses were well below the level at which saturation of the neonatal Fc receptor may influence clearance (14). The ranges of doses tested included the predicted efficacious doses for each antibody. It is likely that maintaining antibody concentrations greater than those needed to inhibit viral replication throughout the dosing interval will be required to achieve maximal efficacy in patients. Mechanistic-based pharmacokinetic-pharmacodynamic modeling, which was initially performed to define the 50% effective concentrations (EC50s) for candidate selection (15), predicts that a minimum trough serum concentration of each antibody needs to be maintained throughout the dosing interval to prevent virus rebound. The model describes antibody and viral load kinetics in plasma and peripheral tissue, as well as binding dynamics. For both antibodies, the predicted minimal trough concentrations were similar to the EC99 values obtained from in vitro HCMV neutralization experiments, with predicted i.v. doses of 5.0 mg/kg and 0.05 mg/kg for LJP538 and LJP539, respectively, given every 4 weeks. However, HCMV breakthrough in vitro is only fully inhibited when virus is serially passaged in the presence of LJP539 at concentrations that are at least 10⫻ the EC90, suggesting a need for 0.5 mg/kg LJP539 in order to suppress viral rebound in patients. For LJP538, in vitro suppression of viral breakthrough requires lower concentrations. Based on modeling, minimal trough serum concentrations of 7.4 g/ml for LJP538 and 0.74 g/ml for LJP539 need to be maintained to suppress viral replication. In this study, the observed mean serum concentrations on day 28 for subjects receiving 5 mg/kg LJP538 and 0.5 mg/kg LJP539 (cohort 4) were 2.5- and 4.9-fold higher than the predicted efficacious trough levels of LJP538 and LJP539, respectively. The mean serum concentrations on day 28 for subjects dosed with 50 mg/kg LJP538 and 5 mg/kg LJP539 (cohort 6) were 25- and 37-fold higher than the predicted efficacious trough levels. Given the possibility that the predicted efficacy by pharmacokinetic/pharmacodynamic modeling, which uses in vitro viral neutralization data, might not translate quantitatively to clinical efficacy, the model parameters and the ideal CSJ148 efficacious dose and interval will be refined based on in vivo clinical data from this study and subsequent efficacy studies. Shorter half-lives of monoclonal antibody and pooled immu-
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noglobulin preparations have been reported in stem cell and solid organ transplant recipients (16–21). Based on preclinical data (Patel et al., unpublished) and the clinical data presented here, as well as the safety profiles of other anti-infective immunoglobulins, there is a low risk of toxicity associated with CSJ148 administration. This supports the use of higher than model-predicted efficacious doses of both antibodies to maintain adequate trough concentrations in clinical situations where faster clearance may be an issue. In conclusion, both LJP538 and LJP539 alone and in combination were safe and well tolerated after intravenous administration in healthy subjects. LJP538 and LJP539 demonstrated linear pharmacokinetics that were typical for human IgG1 antibodies, and serum concentrations on day 28 were above the predicted efficacious trough levels in humans. These data support the further evaluation of CSJ148 to prevent or treat clinically significant HCMV infections. ACKNOWLEDGMENTS Financial support was provided by Novartis for the conduct of this study and the preparation of the manuscript. K.D., F.P.S., A.F., B.M., J.V., J.Y., Y.P., and P.P. are employees of Novartis and/or shareholders of Novartis stock. We thank Igor Vostiar and Kyu Suk Chung for providing bioanalytical support and Catherine Jones for editorial assistance.
FUNDING INFORMATION This work, including the efforts of Kiran Dole, Florencia Pereyra Segal, Adam Feire, Baldur Magnusson, Juan C. Rondon, Janardhana Vemula, Jing Yu, Yinuo Pang, and Peter Pertel, was funded by Novartis.
REFERENCES 1. Ho M. 2008. The history of cytomegalovirus and its diseases. Med Microbiol Immunol 197:65–73. http://dx.doi.org/10.1007/s00430-007-0066-x. 2. Biron KK. 2006. Antiviral drugs for cytomegalovirus diseases. Antiviral Res 71:154 –163. http://dx.doi.org/10.1016/j.antiviral.2006.05.002. 3. Torres-Madriz G, Boucher HW. 2008. Immunocompromised hosts: perspectives in the treatment and prophylaxis of cytomegalovirus disease in solid-organ transplant recipients. Clin Infect Dis 47:702–711. http://dx .doi.org/10.1086/590934. 4. Snydman DR. 1990. Cytomegalovirus immunoglobulins in the prevention and treatment of cytomegalovirus disease. Rev Infect Dis 12(Suppl 7):S839 –S848. 5. Snydman DR, Werner BG, Heinze-Lacey B, Berardi VP, Tilney NL, Kirkman RL, Milford EL, Cho SI, Bush HL, Jr, Levey AS. 1987. Use of cytomegalovirus immune globulin to prevent cytomegalovirus disease in renal-transplant recipients. N Engl J Med 317:1049 –1054. http://dx.doi .org/10.1056/NEJM198710223171703. 6. Ishida JH, Patel A, McBride J, Burgess T, Derby MA, Emu B, Feierbach B, Liao XC, Maia M, Deng R, Rosenberger CM, Gennaro LA, Striano NS, Tavel JA. 2015. Phase 2 randomized, double-blind, placebocontrolled trial of RG7667 for prevention of cytomegalovirus infection in high-risk kidney transplant recipients. Abstr 55th Intersci Conf Antimicrob Agents Chemother, San Diego, CA. 7. Macagno A, Bernasconi NL, Vanzetta F, Dander E, Sarasini A, Revello MG, Gerna G, Sallusto F, Lanzavecchia A. 2010. Isolation of human monoclonal antibodies that potently neutralize human cytomegalovirus infection by targeting different epitopes on the gH/gL/UL128-131A complex. J Virol 84:1005–1013. http://dx.doi.org/10.1128/JVI.01809-09. 8. Marshall GS, Rabalais GP, Stout GG, Waldeyer SL. 1992. Antibodies to recombinant-derived glycoprotein B after natural human cytomegalovirus infection correlate with neutralizing activity. J Infect Dis 165:381–384. http://dx.doi.org/10.1093/infdis/165.2.381. 9. Wang D, Li F, Freed DC, Finnefrock AC, Tang A, Grimes SN, Casimiro DR, Fu TM. 2011. Quantitative analysis of neutralizing antibody response to human cytomegalovirus in natural infection. Vaccine 29:9075–9080. http://dx.doi.org/10.1016/j.vaccine.2011.09.056.
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10. Fouts AE, Chan P, Stephan JP, Vandlen R, Feierbach B. 2012. Antibodies against the gH/gL/UL128/UL130/UL131 complex comprise the majority of the anti-cytomegalovirus (anti-CMV) neutralizing antibody response in CMV hyperimmune globulin. J Virol 86:7444 –7447. http://dx .doi.org/10.1128/JVI.00467-12. 11. Smith BP, Vandenhende FR, DeSante KA, Farid NA, Welch PA, Callaghan JT, Forgue ST. 2000. Confidence interval criteria for assessment of dose proportionality. Pharm Res 17:1278 –1283. http://dx.doi.org/10 .1023/A:1026451721686. 12. Hahn G, Revello MG, Patrone M, Percivalle E, Campanini G, Sarasini A, Wagner M, Gallina A, Milanesi G, Koszinowski U, Baldanti F, Gerna G. 2004. Human cytomegalovirus UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes. J Virol 78:10023–10033. http://dx.doi.org/10.1128/JVI.78.18.10023-10033.2004. 13. Wang D, Shenk T. 2005. Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism. Proc Natl Acad Sci U S A 102:18153–18158. http://dx.doi.org/10.1073/pnas.0509201102. 14. Jin F, Balthasar JP. 2005. Mechanisms of intravenous immunoglobulin action in immune thrombocytopenic purpura. Hum Immunol 66:403– 410. http://dx.doi.org/10.1016/j.humimm.2005.01.029. 15. Yu J, Karcher H, Feire AL, Lowe PJ. 2011. From target selection to the minimum acceptable biological effect level for human study: use of mechanism-based PK/PD modeling to design safe and efficacious biologics. AAPS J 13:169 –178. http://dx.doi.org/10.1208/s12248-011-9256-y. 16. Rand KH, Houck H, Ganju A, Babington RG, Elfenbein GJ. 1989. Pharmacokinetics of cytomegalovirus specific IgG antibody following intravenous immunoglobulin in bone marrow transplant patients. Bone Marrow Transpl 4:679 – 683.
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17. Rand KH, Gibbs K, Derendorf H, Graham-Pole J. 1991. Pharmacokinetics of intravenous immunoglobulin (Gammagard) in bone marrow transplant patients. J Clin Pharmacol 31:1151–1154. http://dx.doi.org/10 .1002/j.1552-4604.1991.tb03688.x. 18. DeRienzo SY, Chiang KY, O’Neal WM, Godder K, Abhyankar S, Christiansen NP, Bridges KD, Henslee-Downey PJ. 2000. Evaluation of the half-life of intravenous human cytomegalovirus immune globulin in patients receiving partially mismatched related donor bone marrow transplantation. Pharmacotherapy 20:1175–1178. http://dx.doi.org/10.1592 /phco.20.15.1175.34592. 19. Boeckh M, Bowden RA, Storer B, Chao NJ, Spielberger R, Tierney DK, Gallez-Hawkins G, Cunningham T, Blume KG, Levitt D, Zaia JA. 2001. Randomized, placebo-controlled, double-blind study of a cytomegalovirus-specific monoclonal antibody (MSL-109) for prevention of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transpl 7:343–351. http://dx.doi.org/10.1016/S1083 -8791(01)80005-7. 20. Andresen I, Kovarik JM, Spycher M, Bolli R. 2000. Product equivalence study comparing the tolerability, pharmacokinetics, and pharmacodynamics of various human immunoglobulin-G formulations. J Clin Pharmacol 40:722–730. http://dx.doi.org/10.1177/00912700 022009477. 21. Snydman DR, McIver J, Leszczynski J, Cho SI, Werner BG, Berardi VP, LoGerfo F, Heinze-Lacey B, Grady GF. 1984. A pilot trial of a novel cytomegalovirus immune globulin in renal transplant recipients. Transplantation 38:553–557. http://dx.doi.org/10.1097/00007890-198411000 -00026.
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A First-in-Human Study To Assess the Safety and Pharmacokinetics of Monoclonal Antibodies against Human Cytomegalovirus in Healthy Volunteers Kiran Dole,a Florencia Pereyra Segal,a Adam Feire,a,b Baldur Magnusson,c Juan C. Rondon,d Janardhana Vemula,e Jing Yu,a Yinuo Pang,a Peter Pertela Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, USAa; Novartis Institutes for BioMedical Research, Emeryville, California, USAb; Novartis Pharmaceuticals AG, Basel, Switzerlandc; Elite Research Institute, Miami, Florida, USAd; Novartis Pharmaceuticals, Hyderabad, Indiae
Human cytomegalovirus (HCMV) can cause significant disease in immunocompromised patients and treatment options are limited by toxicities. CSJ148 is a combination of two anti-HCMV human monoclonal antibodies (LJP538 and LJP539) that bind to and inhibit the function of viral HCMV glycoprotein B (gB) and the pentameric complex, consisting of glycoproteins gH, gL, UL128, UL130, and UL131. Here, we evaluated the safety, tolerability, and pharmacokinetics of a single intravenous dose of LJP538 or LJP539 or their combination in healthy volunteers. Adverse events and laboratory abnormalities occurred sporadically with similar incidence between antibody and placebo groups and without any apparent relationship to dose. No subject who received antibody developed a hypersensitivity, infusion-related reaction or anti-drug antibodies. After intravenous administration, both LJP538 and LJP539 demonstrated typical human IgG1 pharmacokinetic properties, with slow clearances, limited volumes of distribution, and long terminal half-lives. The pharmacokinetic parameters were linear and dose proportional for both antibodies across the 50-fold range of doses evaluated in the study. There was no apparent impact on pharmacokinetics when the antibodies were administered alone or in combination. CSJ148 and the individual monoclonal antibodies were safe and well tolerated, with pharmacokinetics as expected for human immunoglobulin.
H
uman cytomegalovirus (HCMV) can cause significant disease in immunocompromised individuals, including transplant patients and neonates infected in utero (1). Therapies available to treat HCMV disease in transplant recipients, including ganciclovir, cidofovir, and foscarnet, are effective but associated with serious toxicities; there are no approved treatments for congenital HCMV (2). Passive immunization with HCMV hyperimmunoglobulin, which is polyclonal IgG from human plasma pools, is also used to prevent HCMV disease in some transplant recipients. It is safe and well tolerated but is less effective than other antiviral therapies. Preliminary data from a combination of anti-HCMV monoclonal antibodies, showed a modest, therapeutic benefit in renal transplant recipients (3–6). CSJ148 is a combination of two fully human anti-HCMV IgG1 monoclonal antibodies, LJP538 and LJP539 (7), and offers the potential to be a safe and well-tolerated alternative to available therapies for the prevention and treatment of HCMV disease. Each antibody binds to and inhibits the function of viral glycoproteins essential for HCMV infectivity. In vitro studies demonstrated that LJP538 and LJP539 were up to 100 to 1000-fold more potent, respectively, than HCMV-hyperimmunoglobulin at inhibiting infection of various cell lines. Furthermore, LJP538 and LJP539 were active against a panel of clinical isolates in vitro and demonstrated minor to moderate synergy in combination (A. Patel et al., unpublished data). Importantly, viruses with reduced susceptibility to either LJP538 or LJP539 remained susceptible to the other antibody, and no cross-resistance with ganciclovir was observed in vitro. In addition, no resistance to either antibody was seen when HCMV was cultured in the presence of LJP538 and LJP539 in combination for ⬎400 days (Patel et al., unpublished). In human tissue cross-reactivity studies, LJP538 and LJP539 exhibited no off-target binding. Scattered binding was noted in human tissues,
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but occurred only in cells confirmed to be positive for HCMV DNA or RNA by in situ hybridization (A. Hey et al., unpublished data). Using the combination of antibodies in CSJ148 has several advantages. Each antibody targets a separate glycoprotein epitope; LJP538 binds the viral gB protein, while LJP539 binds the gH/gL/ UL128/UL130/UL131a pentameric complex. Although antibodies directed against gB correlate with neutralizing activity (8), the major neutralizing antibody response is directed against the pentameric complex, both in natural infections and in HCMV hyperimmunoglobulin (9, 10). In vitro, LJP538 inhibited HCMV infection of all cell types tested, while LJP539 blocked infection of cell types likely required for systemic spread of the virus (Patel et al., unpublished). Furthermore, in vitro data suggest that the combination of LJP538 and LJP539 will significantly decrease the development of resistance (Patel et al., unpublished). The objectives of this first-in-human study were to evaluate the safety, tolerability and pharmacokinetics of a single intravenous (i.v.) dose of CSJ148 in healthy volunteers.
Received 6 November 2015 Returned for modification 30 December 2015 Accepted 20 February 2016 Accepted manuscript posted online 29 February 2016 Citation Dole K, Segal FP, Feire A, Magnusson B, Rondon JC, Vemula J, Yu J, Pang Y, Pertel P. 2016. A first-in-human study to assess the safety and pharmacokinetics of monoclonal antibodies against human cytomegalovirus in healthy volunteers. Antimicrob Agents Chemother 60:2881–2887. doi:10.1128/AAC.02698-15. Address correspondence to Florencia Pereyra Segal, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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MATERIALS AND METHODS Study design. This was a randomized, double-blind, placebo-controlled, single ascending intravenous dose study conducted at a single center. It was designed to determine the safety, tolerability, and pharmacokinetics of LJP538, LJP539, and their combination (CSJ148) in healthy subjects. The study consisted of a 20-day screening period, one baseline visit, a 1-day treatment period, with approximately eight follow-up visits, and an end-of-study visit on day 105. The planned enrollment was 30 male and female volunteers randomized to sequential cohorts of five subjects, with four receiving antibody, and one receiving placebo. Subjects in cohort 1 received LJP538 (1 mg/kg) or matching placebo, and those in cohort 2 received LJP539 (0.1 mg/kg) or a placebo. Subjects in cohorts 3 to 6 received single ascending doses of CSJ148 (1 to 50 mg/kg LJP538 and 0.1 to 5 mg/kg LJP539) or the placebo. LJP538 and/or LJP539 and/or placebo were administered as single i.v. doses on the morning of day 1. Study drug was administered in two separate i.v. lines over a period of 2 h. In cohorts 1 and 2 only, the first two subjects were dosed with study drug at least 24 h before the remaining three subjects were dosed. The sponsor and the principal investigator reviewed safety data through day 14 to ensure that escalation to the next higher dose was appropriate and acceptable. The study protocol was approved by the Institutional Review Board (IRB) for the center. All subjects provided written informed consent. Subjects. Healthy male and female (of nonchildbearing potential) subjects aged 19 to 55 years were eligible. Good health was determined by past medical history, physical examination, vital signs, electrocardiogram, and laboratory tests at screening. Subjects had to weigh at least 50 kg and have a body mass index (BMI) between 18 and 32 kg/m2. Exclusion criteria included history of immunodeficiency disease, hyperviscosity (including cryoglobulinemia, monoclonal gammopathies, or marked hypertriglyceridemia at screening and baseline), and any surgical or medical condition that might significantly alter the disposition of i.v. monoclonal antibodies. Subjects were also excluded if they had a history of some malignancies, impaired renal function (creatinine clearance ⬍ 90 ml/min), liver disease, cardiovascular disease, or history of allergy to monoclonal antibodies. HCMV seropositive and seronegative individuals were eligible to participate; HCMV DNA quantification was performed in all subjects. Safety assessments. Safety assessments consisted of collecting all adverse events, with their severity and relationship to study drug, serious adverse events, and pregnancies. There was also regular monitoring of hematology and blood and urine chemistry, as well as assessments of vital signs, physical condition, electrocardiograms, and body weight. Pharmacokinetic and immunogenicity assessments. Blood samples for pharmacokinetic and immunogenicity evaluations (3 to 5 ml per time point) were collected from a forearm vein contralateral to the infusion site. Samples for determination of drug concentrations were collected predose, as well as 2, 2.5, 3, 4, 6, and 10 h postdose on day 1, and on days 2, 4, 7, 14, 21, 28, 42, 56, 77, and 105. Free LJP538 and LJP539 in the serum were quantified using a Meso Scale Discovery (MSD; Rockville, MD)based assay, which uses anti-idiotypic antibodies specific for either LJP538 or LJP539 as capture reagents. The lower limits of quantification (LLOQ) were 10 ng/ml for both assays. Samples for assessment of immunogenicity (anti-drug antibodies) were collected predose and on days 14, 28, 56, and 105. The presence of anti-LJP538 and anti-LJP539 antibodies in serum was assessed using an internal homogenous bridging MSD-based electrochemiluminescence assay with three tiers (initial screening, confirmation, and titration). Statistical methods. Summary statistics are provided for pharmacokinetic parameters by treatment and sampling time point. Concentrations below LLOQ were treated as zero in summary statistics. A geometric mean was not reported if the data set included zero. Noncompartmental pharmacokinetic analysis was performed on unbound LJP538 and LJP539 serum concentration-time profiles using the actual infusion durations and elapsed sampling times for each subject. The following pharmacokinetic parameters were generated using WinNonlin Phoenix (version 6.2; Pharsight, Mountain View, CA): area under the serum concentration-time
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curve from time zero to the last time point with quantifiable concentration (AUClast), area under the serum concentration-time curve from time zero to infinity (AUCinf), maximum observed serum concentration (Cmax), systemic clearance (CL), apparent volume of distribution at steady state (Vss), and terminal-phase half-life (t1/2). The Cmax and AUC were assessed by fitting a dose proportionality model to the data based on the power model: pharmacokinetic parameter ⫽ ␣ ⫻ dose. The estimate of the exponent  and its 90% CI values were determined, using this linear model of log-transformed pharmacokinetic parameters and log dose. Dose proportionality was concluded if the 90% CI interval for  (slope estimate) was completely contained within the prespecified critical region (0.943 to 1.057) (11).
RESULTS
Subject demographics. Overall, 32 subjects were enrolled and 28 completed the study. Two subjects were lost to follow-up: one on day 14 who received LJP539 (0.1 mg/kg) and one on day 19, who received the placebo. Both of these subjects were replaced. Two subjects withdrew consent: one who received LJP538 (50 mg/kg) and LJP539 (5 mg/kg) on day 60 and one who received placebo on day 34. Neither of these subjects was replaced. The demographic data for all enrolled subjects are shown in Table 1. The subjects were predominantly male (65.6%) and Caucasian (93.8%); all subjects identified their ethnicity as Hispanic/Latino. The study population had a mean BMI of 27 kg/m2, and the mean age was 43 years. A total of 28 of the 32 subjects (87.5%) had serological evidence of prior HCMV infection. Safety and tolerability. There were no deaths during the study and no subject discontinued due to an adverse event. There was 1 serious adverse event, a grade 1 transient ischemic attack on day 58, which developed in a subject who received 1 mg/kg LJP538 57 days earlier. The subject presented to the emergency department complaining of right sided paresthesias. He was admitted to the hospital for additional work-up, during which a computed tomography of the head did not show any evidence of thrombosis or bleeding, and his symptoms resolved without treatment. The event was considered by the investigator to be unrelated to study drug, which is supported by the lack of temporal association between administration of study drug and adverse event onset. The subject reported no further symptoms or concomitant medications during follow-up and completed the study as planned. Seventeen treatment-emergent adverse events occurred during the study period: 14 among 7 subjects who received antibody and 3 among 2 subjects who received placebo. Adverse events by dosing group and preferred term are shown in Table 2. The percentages of subjects who developed adverse events were similar among those administered antibody (7/25; 28.0%) or placebo (2/7; 28.6%). Adverse events occurred sporadically and without any apparent relationship to treatment group or dose. All adverse events were assessed as grade 1 in severity and all resolved, with only three events (transient ischemic attack, dizziness, and an influenza-like illness) in two subjects requiring action. All but one adverse event were assessed as not related to study drug. The one event suspected to be related was palpitations, which developed in a subject who received 20 mg/kg LJP538 and 2 mg/kg LJP539 the day before. This subject also developed myalgia and headache that same day (day 2). All three events resolved after 2 h without any action taken. The myalgia and headache were not assessed as related to study drug. The most common adverse events were dizziness (n ⫽ 3) and palpitations (n ⫽ 2). Dizziness developed in two subjects who
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TABLE 1 Demographic summary by treatment group Treatment Parameter
LJP538
LJP539
LJP538/LJP539
LJP538/LJP539
LJP538/LJP539
LJP538/LJP539
Pooled placebo
Dose(s), mg/kg n
1 4
0.1 5
1/0.1 4
5/0.5 4
20/2 4
50/5 4
7
Age (yr) Mean (SD) Range
39 (17.0) 19–55
41 (11.2) 24–53
32 (8.8) 22–43
54 (1.4) 52–55
39 (12.0) 28–51
50 (3.6) 45–53
44 (8.5) 27–52
Gender, no. (%) Male Female
4 (100) 0
4 (80.0) 1 (20.0)
2 (50.0) 2 (50.0)
1 (25.0) 3 (75.0)
3 (75.0) 1 (25.0)
1 (25.0) 3 (75.0)
6 (85.7) 1 (14.3)
Race, no. (%) Caucasian Black Asian
4 (100) 0 0
5 (100) 0 0
3 (75.0) 0 1 (25.0)
3 (75.0) 1 (25.0) 0
4 (100) 0 0
4 (100) 0 0
7 (100) 0 0
Wt (kg) Mean (SD) Range
81 (5.7) 76–87
71 (7.0) 64–81
76 (6.9) 69–82
76 (11.0) 66–90
79 (21.1) 54–104
70 (4.7) 64–74
82 (11.3) 68–100
Ht (cm) Mean (SD) Range
169 (6.1) 160–174
168 (6.0) 160–176
168 (10.5) 154–179
164 (9.3) 154–176
169 (13.8) 150–180
159 (7.6) 153–170
170 (8.1) 160–178
BMI (kg/m2) Mean (SD) Range
28 (1.9) 26–30
25 (1.6) 22–26
27 (3.1) 23–30
28 (1.5) 27–30
27 (3.4) 24–32
27 (2.0) 25–29
28 (2.4) 25–32
received antibody and one subject who received placebo. The first subject received 0.1 mg/kg LJP539, and he developed dizziness on day 53; no action was taken. This subject had also developed palpitations on day 28. The second subject received 1 mg/kg LJP538 and 0.1 mg/kg LJP539, and she developed dizziness and syncope
on day 88; the dizziness was treated with promethazine. This subject also developed an influenza-like illness on day 87 and menorrhagia on day 40. The third subject received the placebo, and he developed grade 1 dizziness on day 1 shortly after completion of infusion; no action was taken. Palpitations developed in two sub-
TABLE 2 Incidence of adverse events by treatment group Treatment Parameter or adverse event (preferred term)
LJP538
LJP539
LJP538/LJP539
LJP538/LJP539
LJP538/LJP539
LJP538/LJP539
Pooled placebo
Parameter Dose(s), mg/kg n
1 4
0.1 5
1/0.1 4
5/0.5 4
20/2 4
50/5 4
7
No. (%) of adverse events (preferred term)a Any event Dizziness Palpitations Diarrhea Headache Hypoglycemia Influenza-like illness Menorrhagia Myalgia Pruritus Rash erythematous Syncope Transient ischemic attack Viral upper respiratory tract infection Vomiting
1 (25.0) 0 0 0 0 0 0 0 0 0 0 0 1 (25.0) 0 0
2 (40.0) 1 (20.0) 1 (20.0) 0 0 0 0 0 0 0 0 0 0 1 (20.0) 0
1 (25.0) 1 (25.0) 0 0 0 0 1 (25.0) 1 (25.0) 0 0 0 1 (25.0) 0 0 0
2 (50.0) 0 0 1 (25.0) 0 1 (25.0) 0 0 0 0 0 0 0 0 1 (25.0)
1 (25.0) 0 1 (25.0) 0 1 (25.0) 0 0 0 1 (25.0) 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 (28.6) 1 (14.3) 0 0 0 0 0 0 0 1 (14.3) 1 (14.3) 0 0 0 0
a
Adverse events are arranged in order of decreasing total frequency and then alphabetically.
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FIG 1 Serum logarithmic views of LJP538 (A) and LJP539 (B) concentration-versus-time profiles. Cohort 1, LJP538 (1 mg/kg)/LJP539 (0 mg/kg); cohort 2, LJP538 (0 mg/kg)/LJP539 (0.1 mg/kg); cohort 3, LJP538 (1 mg/kg)/LJP539 (0.1 mg/kg); cohort 4, LJP538 (5 mg/kg)/LJP539 (0.5 mg/kg); cohort 5, LJP538 (20 mg/kg)/LJP539 (2 mg/kg); cohort 6, LJP538 (50 mg/kg)/LJP539 (5 mg/kg).
jects who received antibody; both subjects are discussed above. No treatment site or infusion-related reactions were reported, although one subject who received the placebo developed pruritus and erythematous rash on the hands within 5 h of dosing on day 1; the pruritus and rash resolved without treatment. Only one subject, who received 5 mg/kg LJP538 and 0.5 mg/kg LJP539, had a laboratory abnormality assessed as an adverse event. This subject had a grade 1 low blood glucose level on day 105. No other laboratory abnormality was assessed as clinically significant,
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and no clinically significant vital sign or electrocardiogram abnormalities were noted during the study. Pharmacokinetics. Pharmacokinetic properties were consistent across individuals within each cohort, with limited interindividual variability noted in the serum concentration versus time profiles (Fig. 1) and in the noncompartmental pharmacokinetic parameter estimates (Table 3). After the 2-h intravenous infusions of LJP538 and LJP539, serum concentrations of both antibodies increased rapidly and reached the maximum concentration
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TABLE 3 Noncompartmental pharmacokinetic parameters for each antibodya Mean (SD) Dose(s) of LJP538/LJP539 (mg/kg)
AUClast (day·g/ml)
AUCinf (day·g/ml)
Cmax (g/ml)
C28D (g/ml)
CL (ml/day/kg)
Vss (ml/kg)
t1/2 (day)
LJP538 1/0 1/0.1 5/0.5 20/2 50/5
337 (84.4) 318 (81.1) 1,610 (351) 6,620 (921) 16,100 (2,170)
345 (89.4) 331 (92.7) 1,650 (373) 6,750 (1,020) 16,700 (1,790)
34.3 (4.50) 27.5 (3.13) 164 (9.42) 614 (81.1) 1,540 (31.1)
3.97 (1.43) 3.75 (1.36) 18.6 (5.64) 78.4 (17.6) 185 (26.9)
3.08 (0.946) 3.20 (0.873) 3.16 (0.730) 3.02 (0.496) 3.03 (0.329)
62.6 (4.63) 77.1 (6.31) 67.6 (8.39) 66.0 (7.35) 66.4 (6.47)
20.1 (4.87) 22.9 (6.44) 18.9 (3.75) 18.7 (3.89) 18.6 (0.659)
LJP539 0/0.1 1/0.1 5/0.5 20/2 50/5
45.7 (7.91) 48.5 (11.1) 273 (53.4) 1,030 (147) 2,130 (347)
47.3 (8.49) 51.7 (13.8) 284 (60.1) 1,080 (173) 2,270 (294)
3.38 (0.290) 3.14 (0.360) 17.7 (1.11) 77.7 (7.47) 164 (11.5)
0.539 (0.111) 0.584 (0.153) 3.59 (0.840) 11.6 (1.99) 27.1 (0.929)
2.18 (0.460) 2.05 (0.570) 1.83 (0.423) 1.90 (0.351) 2.23 (0.330)
58.8 (9.69) 61.6 (4.42) 49.0 (4.42) 55.8 (6.19) 64.6 (4.28)
21.6 (2.61) 25.9 (6.31) 22.1 (4.28) 23.4 (4.02) 23.7 (2.31)
a
n ⫽ 4 for all summary parameters except LJP539 Cmax after the 0/0.1 dose of LJP538/LJP539.
around the end of infusion (Fig. 1). After infusion, serum concentrations decreased rapidly during the initial few days, followed by a much slower terminal elimination phase. Overall, pharmacokinetic characteristics of both LJP538 and LJP539 were typical of human IgG1 antibodies, with slow clearances, limited apparent volumes of distribution, and long terminal half-lives (Table 3). Administration of the two antibodies in combination did not appear to impact the pharmacokinetics of either individual antibody, since the pharmacokinetic profiles and parameters were generally comparable between cohorts 1 and 3 for LJP538 at 1 mg/kg and cohorts 2 and 3 for LJP539 at 0.1 mg/kg. In addition, prior infection with HCMV based on serology had no appreciable impact on the pharmacokinetics of LJP538 or LJP539. Across the 50-fold dose ranges (1 to 50 mg/kg for LJP538 and 0.1 to 5 mg/kg for LJP539), key pharmacokinetic parameter estimates for serum exposure (AUC and Cmax), clearance (CL), distribution (Vss), and terminal half-life (t1/2) were similar, indicating linear pharmacokinetics across the range of doses evaluated (Table 3). Based on the statistical analyses, dose proportionality was demonstrated for AUClast, AUCinf, and Cmax across the whole range of doses for LJP538 and LJP539 (Table 4). Immunogenicity. Among the 25 subjects administered LJP538 and/or LJP539, no anti-drug antibodies were detected either at baseline or any of the postdose time points (days 14, 28, 56, and
105). LJP538 concentrations in 19 samples from all subjects in cohorts 5 and 6 who received antibody exceeded the drug tolerance level (48.7 g/ml) for the anti-drug antibody assay at the earlier time points; above the drug tolerance level there is the potential for interference with the assay, and the presence of antidrug antibodies can therefore not be fully excluded. However, analysis of samples from the same subjects at later time points, especially on day 105, indicated that no subjects treated with either LJP538 and/or LJP539 had anti-drug antibodies by the end of the study. No anti-drug antibodies were detected in any of the samples from subjects who received placebo. DISCUSSION
The combination of LJP538 and LJP539 is designed to block the early steps in HCMV infection, as well as to decrease the risk of developing resistance. HCMV infects a broad range of tissues, and the glycoproteins required for entry vary based on cell type. The infection of all cell types requires gB. In addition, a three-member complex (gH, gL, and gO) is essential for entry into fibroblasts, whereas a pentameric complex (gH, gL, UL128, UL130, and UL131a) allows infection of epithelial, endothelial, and hematopoietic cells (12), (13). Targeting the viral gB protein by LJP538, along with blocking the pentameric complex by LJP539, is therefore expected to limit infection of multiple cell types involved in
TABLE 4 Dose proportionality of pharmacokinetic parameters Estimated increase across the dose rangea
Slope estimate Parameter
Estimate
90% CI
Increase
90% CI
Dose proportionality across the whole range based on slope
LJP538 AUClast (day·g/ml) AUCinf (day·g/ml) Cmax (g/ml)
1.00 1.00 1.00
0.95–1.05 0.95–1.05 0.97–1.03
50.50 50.72 50.03
41.79–61.03 41.65–61.77 44.52–56.21
Yes Yes Yes
LJP539 AUClast(day·g/ml) AUCinf (day·g/ml) Cmax (g/ml)
0.99 1.00 1.02
0.94–1.04 0.95–1.05 0.99–1.04
48.48 49.21 53.84
40.02–58.74 40.37–60.00 48.70–59.52
Yes Yes Yes
a
The dose ranges were 1 to 50 mg/kg for LJP538 and 0.1 to 5 mg/kg for LJP539. Estimates and 90% CI values of the exponent were determined from a simple linear regression analysis of the log-transformed values of the pharmacokinetic parameter and dose.
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HCMV pathogenesis and systemic spread in vivo. Antibodies against both gB (8) and the pentameric complex (9, 10) have been associated with neutralizing activity during natural infection. In addition, LJP538 and LJP539 demonstrated minor to moderate synergy in vitro, with no antagonism noted (Patel et al., unpublished). Importantly, LJP538 and LJP539 did not show cross-resistance with each other in vitro and no resistant virus was detected when HCMV-infected cells were passaged in the presence of the combined antibodies for more than 400 days (Patel et al., unpublished). In healthy subjects, LJP538 and LJP539 were safe and well tolerated after single i.v. doses up to 50 and 5 mg/kg, respectively. Both antibodies showed dose proportional linear pharmacokinetics, slow clearances, limited volumes of distribution, and long terminal half-lives. This was expected because LJP538 and LJP539 have intact Fc domains, and low levels of the targets for both antibodies are to be expected in healthy volunteers who have no evidence of actively replicating virus (HCMV DNA was not quantifiable in all subjects). As expected, administering the antibodies in combination did not appear to impact the pharmacokinetic parameters of either individual antibody; doses were well below the level at which saturation of the neonatal Fc receptor may influence clearance (14). The ranges of doses tested included the predicted efficacious doses for each antibody. It is likely that maintaining antibody concentrations greater than those needed to inhibit viral replication throughout the dosing interval will be required to achieve maximal efficacy in patients. Mechanistic-based pharmacokinetic-pharmacodynamic modeling, which was initially performed to define the 50% effective concentrations (EC50s) for candidate selection (15), predicts that a minimum trough serum concentration of each antibody needs to be maintained throughout the dosing interval to prevent virus rebound. The model describes antibody and viral load kinetics in plasma and peripheral tissue, as well as binding dynamics. For both antibodies, the predicted minimal trough concentrations were similar to the EC99 values obtained from in vitro HCMV neutralization experiments, with predicted i.v. doses of 5.0 mg/kg and 0.05 mg/kg for LJP538 and LJP539, respectively, given every 4 weeks. However, HCMV breakthrough in vitro is only fully inhibited when virus is serially passaged in the presence of LJP539 at concentrations that are at least 10⫻ the EC90, suggesting a need for 0.5 mg/kg LJP539 in order to suppress viral rebound in patients. For LJP538, in vitro suppression of viral breakthrough requires lower concentrations. Based on modeling, minimal trough serum concentrations of 7.4 g/ml for LJP538 and 0.74 g/ml for LJP539 need to be maintained to suppress viral replication. In this study, the observed mean serum concentrations on day 28 for subjects receiving 5 mg/kg LJP538 and 0.5 mg/kg LJP539 (cohort 4) were 2.5- and 4.9-fold higher than the predicted efficacious trough levels of LJP538 and LJP539, respectively. The mean serum concentrations on day 28 for subjects dosed with 50 mg/kg LJP538 and 5 mg/kg LJP539 (cohort 6) were 25- and 37-fold higher than the predicted efficacious trough levels. Given the possibility that the predicted efficacy by pharmacokinetic/pharmacodynamic modeling, which uses in vitro viral neutralization data, might not translate quantitatively to clinical efficacy, the model parameters and the ideal CSJ148 efficacious dose and interval will be refined based on in vivo clinical data from this study and subsequent efficacy studies. Shorter half-lives of monoclonal antibody and pooled immu-
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noglobulin preparations have been reported in stem cell and solid organ transplant recipients (16–21). Based on preclinical data (Patel et al., unpublished) and the clinical data presented here, as well as the safety profiles of other anti-infective immunoglobulins, there is a low risk of toxicity associated with CSJ148 administration. This supports the use of higher than model-predicted efficacious doses of both antibodies to maintain adequate trough concentrations in clinical situations where faster clearance may be an issue. In conclusion, both LJP538 and LJP539 alone and in combination were safe and well tolerated after intravenous administration in healthy subjects. LJP538 and LJP539 demonstrated linear pharmacokinetics that were typical for human IgG1 antibodies, and serum concentrations on day 28 were above the predicted efficacious trough levels in humans. These data support the further evaluation of CSJ148 to prevent or treat clinically significant HCMV infections. ACKNOWLEDGMENTS Financial support was provided by Novartis for the conduct of this study and the preparation of the manuscript. K.D., F.P.S., A.F., B.M., J.V., J.Y., Y.P., and P.P. are employees of Novartis and/or shareholders of Novartis stock. We thank Igor Vostiar and Kyu Suk Chung for providing bioanalytical support and Catherine Jones for editorial assistance.
FUNDING INFORMATION This work, including the efforts of Kiran Dole, Florencia Pereyra Segal, Adam Feire, Baldur Magnusson, Juan C. Rondon, Janardhana Vemula, Jing Yu, Yinuo Pang, and Peter Pertel, was funded by Novartis.
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10. Fouts AE, Chan P, Stephan JP, Vandlen R, Feierbach B. 2012. Antibodies against the gH/gL/UL128/UL130/UL131 complex comprise the majority of the anti-cytomegalovirus (anti-CMV) neutralizing antibody response in CMV hyperimmune globulin. J Virol 86:7444 –7447. http://dx .doi.org/10.1128/JVI.00467-12. 11. Smith BP, Vandenhende FR, DeSante KA, Farid NA, Welch PA, Callaghan JT, Forgue ST. 2000. Confidence interval criteria for assessment of dose proportionality. Pharm Res 17:1278 –1283. http://dx.doi.org/10 .1023/A:1026451721686. 12. Hahn G, Revello MG, Patrone M, Percivalle E, Campanini G, Sarasini A, Wagner M, Gallina A, Milanesi G, Koszinowski U, Baldanti F, Gerna G. 2004. Human cytomegalovirus UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes. J Virol 78:10023–10033. http://dx.doi.org/10.1128/JVI.78.18.10023-10033.2004. 13. Wang D, Shenk T. 2005. Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism. Proc Natl Acad Sci U S A 102:18153–18158. http://dx.doi.org/10.1073/pnas.0509201102. 14. Jin F, Balthasar JP. 2005. Mechanisms of intravenous immunoglobulin action in immune thrombocytopenic purpura. Hum Immunol 66:403– 410. http://dx.doi.org/10.1016/j.humimm.2005.01.029. 15. Yu J, Karcher H, Feire AL, Lowe PJ. 2011. From target selection to the minimum acceptable biological effect level for human study: use of mechanism-based PK/PD modeling to design safe and efficacious biologics. AAPS J 13:169 –178. http://dx.doi.org/10.1208/s12248-011-9256-y. 16. Rand KH, Houck H, Ganju A, Babington RG, Elfenbein GJ. 1989. Pharmacokinetics of cytomegalovirus specific IgG antibody following intravenous immunoglobulin in bone marrow transplant patients. Bone Marrow Transpl 4:679 – 683.
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17. Rand KH, Gibbs K, Derendorf H, Graham-Pole J. 1991. Pharmacokinetics of intravenous immunoglobulin (Gammagard) in bone marrow transplant patients. J Clin Pharmacol 31:1151–1154. http://dx.doi.org/10 .1002/j.1552-4604.1991.tb03688.x. 18. DeRienzo SY, Chiang KY, O’Neal WM, Godder K, Abhyankar S, Christiansen NP, Bridges KD, Henslee-Downey PJ. 2000. Evaluation of the half-life of intravenous human cytomegalovirus immune globulin in patients receiving partially mismatched related donor bone marrow transplantation. Pharmacotherapy 20:1175–1178. http://dx.doi.org/10.1592 /phco.20.15.1175.34592. 19. Boeckh M, Bowden RA, Storer B, Chao NJ, Spielberger R, Tierney DK, Gallez-Hawkins G, Cunningham T, Blume KG, Levitt D, Zaia JA. 2001. Randomized, placebo-controlled, double-blind study of a cytomegalovirus-specific monoclonal antibody (MSL-109) for prevention of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transpl 7:343–351. http://dx.doi.org/10.1016/S1083 -8791(01)80005-7. 20. Andresen I, Kovarik JM, Spycher M, Bolli R. 2000. Product equivalence study comparing the tolerability, pharmacokinetics, and pharmacodynamics of various human immunoglobulin-G formulations. J Clin Pharmacol 40:722–730. http://dx.doi.org/10.1177/00912700 022009477. 21. Snydman DR, McIver J, Leszczynski J, Cho SI, Werner BG, Berardi VP, LoGerfo F, Heinze-Lacey B, Grady GF. 1984. A pilot trial of a novel cytomegalovirus immune globulin in renal transplant recipients. Transplantation 38:553–557. http://dx.doi.org/10.1097/00007890-198411000 -00026.
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