ABSTRACT
Staphylococcus aureus causes serious bacterial infections with high morbidity and mortality, necessitating the discovery of new antibiotics. DSTA4637S is a novel antibody-antibiotic conjugate designed to target intracellular S. aureus that is not adequately eliminated by current standard-of-care antibiotics. DSTA4637S is composed of an anti-S. aureus Thiomab human immunoglobulin G1 (IgG1) monoclonal antibody linked to a novel rifamycin-class antibiotic (4-dimethylaminopiperidino-hydroxybenzoxazino rifamycin [dmDNA31]) via a protease-cleavable linker. Phagocytic cells ingest DSTA4637S-bound S. aureus, and intracellular cathepsins cleave the linker, releasing dmDNA31and killing intracellular S. aureus. This first-in-human, randomized, double-blind, placebo-controlled, single-ascending-dose phase 1 trial analyzed the safety, pharmacokinetics, and immunogenicity of DSTA4637S in healthy volunteers. Thirty healthy male and female volunteers, 18–65 years old, were randomized into five cohorts receiving single intravenous (i.v.) doses of 5, 15, 50, 100, and 150 mg/kg of DSTA4637S or placebo (4 active:2 placebo). Subjects were followed for 85 days after dosing. No subject withdrew from the study, and no serious or severe adverse events occurred. One moderate infusion-related reaction (150 mg/kg DSTA4637S) occurred. No clinically meaningful or dose-related changes in laboratory parameters or vital signs occurred. Pharmacokinetics of plasma DSTA4637S conjugate and serum DSTA4637S total antibody were dose proportional. Systemic exposure of unconjugated dmDNA31 was low. No DSTA4637S-induced anti-drug antibody responses were observed. DSTA4637S was generally safe and well tolerated as a single i.v. dose in healthy volunteers. DSTA4637S has a favorable safety and pharmacokinetic profile that supports future development as a novel therapeutic for S. aureus infections. (This study has been registered at ClinicalTrials.gov under identifier NCT02596399.)
INTRODUCTION
Staphylococcus aureus causes significant morbidity and mortality and is a leading cause of serious bacterial infections worldwide. Complications such as septicemia, infective endocarditis, and osteomyelitis occur following dissemination of the bacteria into the bloodstream (1). Invasive S. aureus infections present with bloodstream involvement from which deep-seated foci of infection develop, requiring source control and prolonged antimicrobial therapy to achieve a cure. In the industrialized world, the population incidence of S. aureus bacteremia ranges from 10 to 30 per 100,000 person-years (2) with a mortality rate of up to 40% that is attributed to metastatic, recurrent, and/or relapsing infections despite appropriate antibiotic treatment (2–4).
Infection with S. aureus remains difficult to treat because of the emergence of methicillin-resistant S. aureus (MRSA), biofilm formation in infections involving prosthetic devices, and toxicities that limit dosing of standard-of-care (SOC) antibiotics, such as nafcillin and vancomycin (5–10). In addition, multiple lines of evidence suggest that survival of antibiotic-susceptible subpopulations of S. aureus contributes to high rates of mortality, morbidity, and treatment failure (11, 12). This survival is likely due to multiple causes, including bacterium-specific factors, such as the reduction or cessation of growth, as observed in infective endocarditis, and survival inside host cells (13–15).
Intracellular survival of S. aureus is associated with chronic and difficult-to-treat infections, such as osteomyelitis and infective endocarditis (14, 16). Intracellular persistence inside phagocytic cells may allow S. aureus to disseminate via the bloodstream to tissue reservoirs of infection and subvert antibacterial immune mechanisms, enabling persistent or relapsing infections even in the presence of SOC antibiotics (14, 16). Once the bacteria are delivered to these tissues, other nonprofessional phagocytic cell types, such as epithelial cells, fibroblasts, and osteoblasts, may internalize S. aureus (14, 17). Killing intracellular S. aureus requires antibiotic MICs at least 25-fold higher than those needed to inhibit the growth of planktonic bacteria; however, these levels are not reached in serum with SOC antibiotics (18, 19). There is a significant need for new antibiotic therapies with novel mechanisms of action that deliver antibiotics directly to sites of infection and have the potential to eliminate S. aureus tissue reservoirs associated with SOC antibiotic failure.
DSTA4637S is a Thiomab antibody-antibiotic conjugate consisting of an engineered human immunoglobulin G1 (IgG1) anti-S. aureus monoclonal antibody and a novel antibiotic, 4-dimethylaminopiperidino-hydroxybenzoxazino rifamycin (dmDNA31), linked through a protease-cleavable valine-citrulline (VC) linker (19–21). DSTA4637S has an average drug-to-antibody ratio (DAR) of two VC-dmDNA31 molecules per antibody. dmDNA31 is a rifamycin-class antibiotic that inhibits bacterial DNA-dependent RNA polymerase with potent bactericidal activity against S. aureus (19). DSTA4637S specifically binds to the β-N-acetylglucosamine (β-GlcNAc) sugar modification of wall teichoic acid (β-WTA), a major cell wall component of S. aureus. After DSTA4637S binds β-WTA, the DSTA4637S-bound bacteria are phagocytosed, and intracellular cathepsins cleave the VC linker, releasing the active antibiotic dmDNA31 to kill the intracellular bacteria (Fig. 1) (19, 20).
DSTA4637S mechanism for killing intracellular S. aureus. Step 1, DSTA4637S binds S. aureus. Step 2, host cells internalize DSTA4637S-bound S. aureus. Step 3, fusion occurs with the phagolysosome where lysosomal cathepsins cleave the VC linker, releasing dmDNA31. Step 4, unconjugated dmDNA31 kills the intracellular bacteria.
DSTA4637S is anticipated to be safe because the target antigen is not expressed by human tissues. In addition, nonclinical repeat dose good laboratory practice (GLP) testing in rats and cynomolgus monkeys demonstrated that DSTA4637A (the liquid formulation used for nonclinical studies) was well tolerated at high exposures of up to 500 mg/kg in rats and 250 mg/kg in monkeys, with good safety margins for the doses tested in this phase 1 study (Genentech, Inc., unpublished data). In a wild-type mouse S. aureus bacteremia infection model, a single dose of DSTA4637A at 25 mg/kg and 50 mg/kg given 24 h after the infection was established substantially reduced bacterial load in target organs (kidneys, heart, and bones) at 4 days after infection (19, 21). In addition, in this model, a single dose of DSTA4637A at 50 mg/kg was superior to 3 days of treatment with vancomycin (19).
DSTA4637S is being developed as a potential therapeutic for patients with serious methicillin-sensitive S. aureus (MSSA) or MRSA infections. The primary objectives of this first-in-human phase 1 study were to test the safety, tolerability, pharmacokinetics (PK), and immunogenicity of DSTA4637S when administered as a single intravenous (i.v.) dose to healthy volunteers.
RESULTS
Subject demographics.The study took place between October 2015 and May 2016. A total of 104 healthy male and female volunteers were screened, and all 30 subjects who were enrolled completed the study. The mean age of the subjects was 41.9 years (range, 20 to 65 years), and the proportion of males (19 [63.3%]) was approximately twice that of females (11 [36.7%]) (Table 1). Subjects were predominantly white (19 [63.3%]) or black/African American (10 [33.3%]). Subject demographic characteristics, including weight, height, and body mass index (BMI), were similar between DSTA4637S-treated and placebo-treated subjects.
Subject demographics
Safety.There were no deaths, pregnancies, severe adverse events (AEs), or serious adverse events (SAEs) during the study. A total of 43 treatment-emergent adverse events (TEAEs) were reported by 21 (70.0%) subjects (Table 2; Table S1), with 37 TEAEs in 17 (85%) DSTA4637S-treated subjects and 6 TEAEs in 4 (40.0%) placebo-treated subjects (Table 2). The total number of subjects with TEAEs was higher for the DSTA4637S-treated subjects. The number of subjects with TEAEs was similar across dose cohorts, but the number of TEAEs was higher in the 100-mg/kg (16 TEAEs) and 150-mg/kg (8 TEAEs) dose groups compared with those in the lower-dose groups. All TEAEs were reversible and clinically manageable, and no withdrawals occurred due to TEAEs.
Overview of TEAEs
The most frequently reported TEAEs (reported by ≥4 subjects; Table 2) were nasal discharge discoloration (6 subjects), headache (5 subjects), and nausea (4 subjects). Two subjects reported discoloration at the i.v. administration site (Table S1). Twenty of the 43 TEAEs (47% of total TEAEs) were considered related to study treatment by the principal investigator, who was blind to study drug assignment. Discoloration of body fluids had been seen in preclinical studies and is known to be caused by the dmDNA31 component of DSTA4637S. Discoloration AEs were reported in the three highest-dose groups and were tolerable and fully reversible (Table S1).
The majority of TEAEs were mild (93%) (Table 2). The following three moderate TEAEs occurred (10%): 1 tooth infection (placebo group; not treatment related), 1 alanine aminotransferase (ALT) increase (100-mg/kg group; not treatment related), and 1 infusion-related reaction (150-mg/kg group; treatment related) that occurred approximately 26 h after the start of the infusion. It was graded as moderate due to hypoxia (oxygen saturation was 89% on room air, which increased to 95% on 2 liters/min of supplemental oxygen provided via nasal cannula), but all other signs and symptoms (anorexia, arthralgia, chills, headache, neuromuscular weakness, fever, and hypotension) were mild. The site investigator treated the subject with paracetamol, diphenhydramine, methylprednisolone sodium succinate infusion, prednisone, saline infusion, and oxygen, with resolution of symptoms after 20 h (46 h after end of infusion).
Clinical laboratory tests and vital signs.In comparison to the placebo, we observed no clinically meaningful changes in mean serum chemistry, hematology, or urinalysis parameters that were dose related. One subject in the 100-mg/kg dose group experienced a moderate ALT increase (3.8 times the upper limit of normal [ULN]) and a mild aspartate aminotransferase (AST) increase (2 times the ULN) on day 14 of the study associated with diarrhea (likely due to viral gastroenteritis) that was not accompanied by elevated bilirubin or alkaline phosphatase or by clinical jaundice (Table S1). Both ALT and AST increases were transient and recovered spontaneously without sequelae. Neither TEAE was considered treatment related.
No clinically meaningful changes occurred in vital signs or electrocardiograms (ECGs). Although not considered clinically significant, at 4 h after the end of the i.v. infusion of DSTA4637S, we observed transient numerical increases from baseline in the mean heart rate of each dosed cohort, ranging from 5.8 to 27.8 beats per minute (bpm), that were not seen in the placebo cohort. Mean heart rate values on day 2 were similar to baseline values in all dosed cohorts. The largest mean increases were seen in the 50-mg/kg and 100-mg/kg treatment cohorts but did not occur in all individuals in each cohort. Three subjects who received the study drug (2 in the 50-mg/kg cohort and 1 in the 100-mg/kg cohort) had heart rates above 100 bpm (maximum heart rate, 108 bpm) at the 4-h time point. There was no clear relationship between DSTA4637S dose and increased heart rate.
Pharmacokinetics.The following three analytes were measured to characterize the pharmacokinetics of DSTA4637S: DSTA4637S conjugate (measured as the total concentration of dmDNA31 conjugated to the antibody), DSTA4637S total antibody (all DARs of DSTA4637S, including fully conjugated, partially deconjugated, and fully deconjugated anti-S. aureus antibodies), and unconjugated dmDNA31 (dmDNA31 not conjugated to the antibody) (Fig. 2; Tables S2 to S4). Although some deconjugation may occur in circulation, DSTA4637S is designed to release free dmDNA31 inside phagolysosomes; therefore, very low levels of unconjugated dmDNA31 are expected in circulation.
Mean plasma or serum concentration-time profiles of DSTA4637S analytes. (A) DSTA4637S conjugate, LLOQ = 9.46 ng/ml. (B) DSTA4637S total antibody, LLOQ = 50 ng/ml. (C) Unconjugated dmDNA31, LLOQ = 0.185 ng/ml. No mean values for the 5-mg/kg cohort were calculated and plotted in panel C because more than one-third of the values were below the limit of quantification at any time point. Error bars indicate standard deviation (SD). Dashed lines indicate lower limit of quantification (LLOQ).
(i) DSTA4637S conjugate in plasma.DSTA4637S conjugate showed approximately dose-proportional plasma pharmacokinetics (Fig. 2A), with a mean half-life ranging from 4.3 to 6.1 days, which was similar across dose levels. The median time to attain maximum concentration (Tmax) in plasma was 2.56 to 4.81 h (Table S2). For the 5-mg/kg to 150-mg/kg dose groups, the mean apparent clearance (CL) and the mean volume of distribution at steady state (Vss) ranged from 0.683 to 0.801 liters/day and 3.82 to 5.31 liters, respectively.
(ii) DSTA4637S total antibody in serum.The serum concentration-time profiles of DSTA4637S total antibody emulated a biexponential disposition pattern similar to the PK profiles seen with typical monoclonal antibody-based therapeutics. DSTA4637S total antibody showed approximately dose-proportional serum pharmacokinetics, with a mean half-life ranging from 16.5 to 21.5 days, which was similar across all dose levels and was three to four times longer than that of the DSTA4637S conjugate (Fig. 2B; Table S3). The mean apparent clearance (CL) ranged from 0.174 to 0.220 liters/day, four times slower than that of the DSTA4637S conjugate. The mean volume of distribution at steady state (Vss) was similar to that of the DSTA4637S conjugate, ranging from 4.51 to 6.06 liters. The median time to attain maximum concentration (Tmax) occurred at 2.52 to 3.12 h. Mean peak serum concentrations (Cmax) and systemic exposures (all areas under the curve [AUC]) increased as doses increased.
(iii) Unconjugated dmDNA31 in plasma.Across all doses in the study, unconjugated dmDNA31 exposure was consistently lower than DSTA4637S conjugate exposure (Fig. 2C; Table S4). The observed unconjugated dmDNA31 mean maximum plasma concentration was 3.86 ng/ml at 150 mg/kg of DSTA4637S, of which approximately 95% is expected to be plasma protein bound (Genentech, Inc., unpublished data). The mean Cmax was approximately 10,000-fold lower than that of DSTA4637S conjugate. The mean half-life ranged from 3.9 to 4.3 days for unconjugated dmDNA31, which is comparable to that of conjugated dmDNA31. Mean peak plasma concentrations (Cmax) and systemic exposures (AUC) generally increased with an increase in dose from 5 mg/kg to 150 mg/kg for Cmax and from 15 mg/kg to 150 mg/kg for AUC.
Immunogenicity.Antidrug antibodies (ADAs) at baseline occurred in one subject in the placebo group (ADA prevalence, 3%). Postbaseline, all subjects treated with DSTA4637S were negative for ADAs (ADA incidence, 0%).
DISCUSSION
In the past several decades, there has been no significant improvement in morbidity or mortality in patients with complicated S. aureus infections (8, 22, 23). It is recognized that designing and executing clinical trials for S. aureus bacteremia is challenging (24). Daptomycin, approved in 2006, was the last antibiotic approved for the treatment of S. aureus bacteremia (8). More recent antistaphylococcal antibiotics such as ceftaroline (approved for acute bacterial skin and skin structure infection [ABSSSI] and community-acquired bacterial pneumonia [CABP] [25]) and telavancin (approved for ABSSSI and hospital-acquired bacterial pneumonia [HABP] [26]) are used as salvage therapy or in combination therapy to treat complicated S. aureus infections. Combination therapy with beta-lactams for MRSA infections results in more rapid clearance of S. aureus from the blood (27, 28), but this has not translated into improved clinical outcomes (29). The recent ARREST trial in patients with S. aureus bacteremia, which assessed the superiority of adjunctive rifampin in combination with SOC antibiotics to SOC antibiotics alone, showed a positive trend toward reducing infection recurrence in patients treated with adjunctive rifampin (23). These results support the ability of rifampin to sterilize deep foci of infection and clear tissue reservoirs. Overall, these trials have demonstrated that the focus of infection within tissues is one of strongest drivers of mortality, clinical failure, and microbiological failure in S. aureus bacteremia. Killing intracellular S. aureus could potentially eliminate both dissemination and seeding of S. aureus tissue reservoirs associated with SOC antibiotic failure (12), improving clearance of S. aureus infections.
There is a high need for agents with efficacy in difficult-to-treat S. aureus infections, such as bacteremia and endocarditis. DSTA4637S is a pathogen-specific, novel antibody-antibiotic conjugate that addresses this challenge by targeting intracellular S. aureus bacteria that are not adequately eliminated by current SOC antibiotics (14, 19). This phase 1 trial assessed the safety of 5 to 150 mg/kg DSTA4637S across a range of drug exposures in healthy volunteers and provided PK data for further clinical development. The drug was well tolerated, and all TEAEs were reversible and manageable. All TEAEs were mild, except for one moderate TEAE, an infusion-related reaction that occurred in the highest-dose cohort (150 mg/kg). There were no deaths or SAEs at any dose tested, and there were no clinically meaningful treatment-related findings in laboratory assessments, vital signs, or ECGs. A single dose of DSTA4637S did not induce ADA responses in any subject.
The selection of the phase 1a starting dose (5 mg/kg) was based on data from nonclinical toxicology studies that demonstrated no observed adverse effect levels (NOAELs) for DSTA4637A following 8 weekly i.v. doses of 500 mg/kg in rats and 250 mg/kg in cynomolgus monkeys, the highest doses tested (Genentech, Inc., unpublished data). Safety margins for the starting dose of 5 mg/kg were estimated as ≥15-fold for DSTA4637S by body surface area, dose, and exposure-based calculations (AUC and Cmax for DSTA4637S total antibody and DSTA4637S conjugate).
Based on an in vivo mouse model of S. aureus infection, the PK of DSTA4637S in healthy subjects in this report, and the predicted DSTA4637S PK in patients, we predicted the efficacious dose range of DSTA4637S when given in combination with SOC antibiotics in patients with S. aureus bacteremia. In a systemic in vivo mouse model of MRSA infection, 15 to 100 mg/kg of a single dose of DSTA4637A given in combination with 110 mg/kg vancomycin dosed twice daily for 3 days resulted in a ≥75% probability of a reduction in CFU below the lower limit of detection (250 CFU) in kidney at day 4 after infection (Genentech, Inc., unpublished data). Higher doses of DSTA4637A (up to 100 mg/kg) in combination with vancomycin (110 mg/kg) showed a higher probability of achieving bacterial clearance in target organs, with less variability in response (Genentech, Inc., unpublished data). Clearance of bacteria in target organs was observed within 3 days of dosing DSTA4637A, suggesting that initial systemic concentrations of DSTA4637A drive efficacy in the S. aureus bacteremia mouse model (19). In the in vivo mouse model, the PK of DSTA4637A is similar in infected and noninfected mice (i.e., in the presence and absence of the target antigen, respectively), indicating that S. aureus infection should have a minimal effect on the PK of DSTA4637A (21).
In modeling the predicted efficacious dose range of DSTA4637S, we assumed that the exposure-response relationship in the mouse infection model is similar to the exposure-response relationship in patients with S. aureus bacteremia and that the PK of DSTA4637S should be similar between healthy volunteers and patients with S. aureus bacteremia. The initial exposure (Cmax and minimum concentration at day 7 [Ctrough, day 7]) and overall exposure (AUC from 0 h to infinity [AUC0–∞]) observed in the mouse models were chosen as the most conservative target exposures to estimate the efficacious dose in humans. Using the human PK parameters from healthy volunteers reported in this phase 1 study in conjunction with the target exposure range (Cmax, Ctrough, day 7, and AUC0–∞) established in mouse infection models, we predict that weekly i.v. dosing, ranging from approximately 7 to 90-mg/kg DSTA4637S, will achieve target efficacious exposures in humans.
While in vivo studies in the acute S. aureus infection mouse model show the efficacy of DSTA4637A, there is considerable uncertainty around the translatability of the efficacy and exposure-response relationships observed in acute mouse infection models to patients with S. aureus infections. This uncertainty is due to critical differences in various parameters, such as duration of infection, bacterial load, timing of DSTA4637S therapy relative to disease onset, presence of metastatic foci of infection at the time of treatment, sites of infection, and differences in measured outcomes (clearance of abscesses versus mortality, microbiological failure, and clinical failure). Similarly, volume of distribution, clearance, organ dysfunction, and host immune response may affect the exposure of DSTA4637S in patients with S. aureus bacteremia differently than in healthy volunteers. Based on the inherent limitations with predicting the exposure-efficacy relationship in patients, we selected doses of DSTA4637S up to 150 mg/kg in this study to maintain high concentrations throughout the treatment period and to cover the anticipated exposure from multiple doses up to 100 mg/kg. The expected DSTA4637S exposure in this study for a single 150-mg/kg dose of DSTA4637S was covered by the nonclinical toxicology studies, where no significant safety events were seen.
Consistent with the PK of DSTA4637A in the in vivo mouse model (21), the concentration-time profiles of DSTA4637S conjugate (Fig. 2A) and DSTA4637S total antibody (Fig. 2B) in humans were biexponential, characterized by a short distribution phase and a longer elimination phase, as expected for a monoclonal antibody-based therapeutic. In addition, the half-life, mean apparent clearance (CL), and mean volume of distribution at steady state (Vss) for DSTA4637S conjugate in plasma and DSTA4637S total antibody in serum were similar across all doses, demonstrating dose-proportional PK. The PK of DSTA4637S total antibody in humans were also comparable to the PK of an unconjugated monoclonal antibody (30), indicating that dmDNA31 conjugation had a minimal impact DSTA4637S PK. The Vss values of DSTA4637S conjugate and DSTA4637S total antibody are similar, signifying that the distribution of DSTA4637S conjugate was primarily governed by the distribution of the antibody. Therefore, consistent with the current understanding of antibody-drug conjugate (ADC) PK (30), DSTA4637S total antibody and DSTA4637S conjugate each behave more like an antibody than like a small molecule.
Unconjugated dmDNA31 demonstrated a similar mean half-life to that of the DSTA4637S conjugate across all dose levels. The similar half-lives between these two analytes suggests that dmDNA31 follows formation rate-limited kinetics, where the deconjugation rate is much slower than the elimination rate of dmDNA31. It is expected that systemic levels of unconjugated dmDNA31 levels would be low because cleavage of DSTA4637S should occur within phagolysosomes due to the stable linker. As our results show, unconjugated dmDNA31 exposure (mean Cmax) was approximately 10,000-fold lower than DSTA4637S conjugate exposure. Even at 150 mg/kg DSTA4637S, the estimated mean Cmax of unconjugated dmDNA31 was 3.86 ng/ml, of which approximately 95% was expected to be bound to plasma protein (Genentech, Inc., unpublished data). This exposure is at least 1,000-fold lower than systemic exposures expected from oral rifampin at standard doses of 600 mg to 900 mg daily (31).
As with other rifamycin-class antibiotics, a potential concern for treating S. aureus infections with DSTA4637S is the development of resistance to the antibiotic, dmDNA31. DSTA4637S is designed as an adjunctive therapy and, similarly to other rifamycin-class molecules, should not be given as monotherapy in S. aureus infections. The in vitro frequency of spontaneous resistance to dmDNA31 is approximately 3.9 × 10−7 (unpublished data), which is similar to that for rifampin (7.7 × 10−7, unpublished data). While theoretically possible, the chance of dmDNA31 resistance emerging intracellularly during DSTA4637S treatment is likely very low due to the expected concentration of S. aureus inside phagolysosomes. Any dmDNA31-resistant S. aureus bacteria that did develop would remain susceptible to conventional SOC antibiotics once released from host cells.
In conclusion, DSTA4637S was safe and well tolerated in healthy volunteers in this phase 1 single-ascending-dose study. DSTA4637S has a favorable safety and PK profile that supports future development as a novel therapeutic for S. aureus infections.
MATERIALS AND METHODS
Participants.Subjects were healthy male and/or female volunteers, as determined by medical history, 12-lead ECG, and vital signs, and were 18 to 65 years old, with BMIs of 18 to 32 kg/m2. Subjects were excluded if they had any diseases, metabolic dysfunction, physical examination finding, or clinical laboratory finding indicating a disease or condition that could affect the interpretation of the results or render the subject at high risk from treatment complications; used tobacco or nicotine products; received any vaccine within 14 days prior to screening; were pregnant, lactating, or intending to become pregnant within 3 months after screening; received oral antibiotics within 4 weeks before initiation of dosing on day 1 or i.v. antibiotics within 8 weeks before initiation of dosing; had a history of anaphylactic or hypersensitivity drug reaction; had any exposure to any biological therapy or investigational biological agent within 90 days prior to the screening visit or had received any other investigational treatment 30 days prior to the screening visit (or within 5 half-lives of the investigational product, whichever was greater); and/or had any history of hypersensitivity or allergy to rifampin or other rifamycin analogs.
Study design.This phase 1, randomized, double-blind, placebo-controlled, single-ascending dose study (ClinicalTrials.gov identifier NCT02596399) in healthy male and female volunteers planned to enroll approximately 30 subjects into a single site in a clinic environment (Lenexa, KS). The planned treatment allocation was approximately 4 DSTA4637S:2 placebo, with at least 20 subjects receiving DSTA4637S. Subjects were screened for eligibility within 28 days before receiving their first dose. Eligible subjects checked into the clinic on day −1, and site personnel administered a single dose of study drug (DSTA4637S or placebo) to each subject on day 1. Subjects were confined at the clinical site from day −1 through day 3 (at least 48 h after dosing for close safety monitoring and collection of initial PK samples, for a total of 4 days and 3 nights). Subjects returned to the clinic on day 4 and at regular intervals up to day 85 (±4 days) for follow-up visits.
For the 5 ascending-dose cohorts (A to E) of 6 subjects each, the intent was to dose at least 4 subjects in each cohort with active drug (A, 5 mg/kg; B, 15 mg/kg; C, 50 mg/kg; D, 100 mg/kg; and E, 150 mg/kg). The first 2 subjects from each cohort received a sentinel dose (1 DSTA4637S:1 placebo) and were observed for 24 h to ensure safety during and immediately after infusion. The remaining subjects in the cohort were dosed at a ratio of 3 DSTA4637S:1 placebo. Escalation to higher-dose cohorts occurred after a safety monitoring committee (SMC) reviewed at least 14 days of safety data for all previously dosed subjects from the preceding cohort.
The clinical study protocol and informed consent forms were reviewed and approved by an institutional review board. The study was conducted in accordance with the principles of the Declaration of Helsinki in place at the time of study conduct and with the International Council for Harmonisation E6 Guideline for Good Clinical Practice. All subjects provided written, informed consent.
Interventions.DSTA4637S (Genentech, Inc., South San Francisco, CA), supplied as a sterile, blue, lyophilized powder (500 mg/vial) was reconstituted with 9.6 ml sterile water to yield a concentration of 50 mg/ml in 20 mM histidine hydrochloride (pH 5.8), 240 mM sucrose, and 0.02% (wt/vol) polysorbate 20. The placebo had the same excipient composition but was not blue. DSTA4637S was administered as a single i.v. infusion with a maximum rate of 2 ml/(kg · h) over approximately 2 to 4 h, depending on the dose.
Randomization and blinding.A statistician external to the study team who was not blind to study conditions randomly assigned each subject to receive DSTA4637S or matching placebo in a double-blind fashion using a simple randomization list generated with SAS version 9.4 or higher (SAS Institute, Inc., Cary, NC). Subjects, investigators, and other site personnel were blind to study drug assignment until the study was completed. The SMC members were blind to treatment assignment and remained blind for dose-escalation decisions, but were unblinded when necessary to ensure ongoing safety and tolerability. Personnel responsible for performing PK assays were also not blind to subject treatment assignments in order to identify appropriate PK samples to be analyzed. PK samples were collected from placebo-treated subjects to maintain blind treatment assignment.
Given the blue color of DSTA4637S and lack of a color-matched placebo, designated personnel directly involved in the preparation and dispensing of the study drug were not blind to the treatment. To minimize the risk of unblinding due to the color of the active drug, i.v. bags and tubing used for all study drugs were masked. Staff aware of treatment assignment recorded any potentially unblinding AEs, and subjects were instructed to report any AEs involving discoloration only to unblinded staff. Unblinded staff members did not discuss the unblinding AEs with any remaining staff who were blind to treatment assignment, including the blinded site investigator who was responsible for determining whether AEs were attributable to study drug.
Sample collection for PK and immunogenicity characterization.Lithium heparin plasma and serum PK samples were collected on days 1 (30 min prior to infusion and 30 min and 4 h after the end of infusion), 2, 3, 4 (±1), 8 (±1), 11 (±2), 15 (±2), 22 (±2), 29 (±2), 57 (±2), and 85 (±2). Serum samples were collected for ADA analysis at baseline and posttreatment on days 29, 57, and 85.
Bioanalytical and immunogenicity assays.Following the same approach as for antibody-drug conjugates (32, 33), three analytes were measured to characterize the PK of DSTA4637S using validated ligand-binding, liquid chromatography with tandem mass spectrometry (LC-MS/MS), and hybrid ligand-binding LC-MS/MS methods.
DSTA4637S conjugate (the total concentration of dmDNA31 conjugated to the antibody) was measured in lithium heparin plasma samples using protein A resin to affinity capture DSTA4637S from plasma, followed by enzyme-mediated release of dmDNA31 from DSTA4637S. The released dmDNA31 was analyzed by an LC-MS/MS method with electrospray ionization. This method had a lower limit of quantification (LLOQ) of 9.46 ng/ml.
DSTA4637S total antibody (all DARs in DSTA4637S, including fully conjugated, partially deconjugated, and fully deconjugated anti-S. aureus antibodies) was determined in serum using an enzyme-linked immunosorbent assay (ELISA). This assay used capture and detection mouse monoclonal antibodies against the complementarity-determining region in DSTA4637S. The LLOQ was 50 ng/ml in neat serum.
Unconjugated dmDNA31 (measured as dmDNA31 not conjugated to antibody) in lithium heparin plasma was measured using an LC-MS/MS method with electrospray ionization and an LLOQ of 0.185 ng/ml.
A bridging ELISA was validated to analyze ADAs in serum samples. Sample testing followed a tiered approach commonly used for other antibody-drug conjugates, which consists of screening, confirmatory, and characterization assays (34). The specificity of positive responses in the screening assay was confirmed using the same assay format after a competition step with DSTA4637S. The titer and domain specificity of ADAs were determined in confirmed-positive samples.
Statistical methods.The sample size was based on the dose-escalation rules described in the study design and was not based on any statistical criteria. We screened a sufficient number of subjects to ensure that approximately 6 subjects were enrolled in each cohort. We determined that 4 subjects dosed with active drug in each of the cohorts were sufficient to characterize the single-dose safety and tolerability of DSTA4637S.
For continuous data, summary statistics included the arithmetic mean, arithmetic standard deviation (SD), median, minimum, maximum, number of subjects, and number of observations. For categorical data, frequency count and percentages are presented. All subjects were grouped according to treatment actually received. All subjects who received any amount of study drug (DSTA4637S or placebo) were included in the analyses, even those who withdrew prematurely from the study. Data analysis was performed using SAS version 9.4 (SAS Institute, Inc., Cary, NC).
The safety population consisted of all subjects who received at least 1 dose of DSTA4637S or placebo. Safety parameters were listed by subject and treatment using descriptive statistics (incidence rates, means, and percentiles). The frequency of TEAEs was summarized using the Medical Dictionary for Regulatory Activities (MedDRA; version 18.1), and TEAEs were categorized by system organ class and preferred term. The onset time postdose was calculated relative to the infusion start time on day 1.
The PK population consisted of all subjects with sufficient data to enable estimation of at least 1 PK parameter. PK parameters were summarized using descriptive statistics (geometric mean and geometric coefficient of variation [CV%]). Noncompartmental analysis was performed using Phoenix WinNonlin version 6.3 (Certara, L.P., Princeton, NJ) using linear-up log-down calculations. Nominal dosing and actual sampling time were used for analysis. Below the limit of quantification (BLQ) values were set to BLQ/2 for the calculation of means. If more than one-third of the values were BLQ at a time point, then no mean value was calculated for that time point.
The immunogenicity population consisted of all subjects with at least 1 baseline and 1 postbaseline ADA assessment. The numbers and proportions of ADA-positive subjects and ADA-negative subjects during both the treatment and follow-up periods were summarized by treatment group. ADA prevalence was determined from all subjects with ADA-positive responses at baseline. Postbaseline ADA incidence was determined from the subjects with treatment-induced and treatment-enhanced ADA responses.
Outcomes.The primary outcome was to assess the safety of DSTA4637S by measuring the nature, frequency, severity, and timing of AEs, including SAEs, adverse events of special interest (AESIs), and dose-limiting adverse events (DLAEs). Changes in vital signs, physical findings, ECG, and clinical laboratory results were monitored during and after DSTA4637S administration. Clinical laboratory parameters (serum chemistry, hematology, urinalysis) were measured at screening, day −1, day 3, day 8, and day 15. The following events were considered suspected DLAEs if they occurred during the study and were assessed as causally related to study drug: (i) severe allergic or hypersensitivity reactions requiring treatment and occurring during or within 24 h of completion of the i.v. infusion, regardless of the site investigator’s assessment of causality, and (ii) any clinically significant moderate or severe (grade 2 or above) AE. Subjects who experienced suspected DLAEs were unblinded; if the subject had received DSTA4637S and the DLAE satisfied causality criteria, the DLAE was considered confirmed.
Secondary outcomes were the assessment of DSTA4637S PK and immunogenicity. To characterize the PK of DSTA4637S the following three key analytes were measured: DSTA4637S conjugate, DSTA4637S total antibody, and unconjugated dmDNA31. The following plasma and serum PK parameters were estimated using standard, noncompartmental methods, as data allowed: Cmax, Tmax, AUC0–last (area under the curve from time 0 to time of last measurable concentration), AUC0–∞ (AUC from time 0 to infinity), half-life (t1/2), Vss, and total CL.
Immunogenicity to DSTA4637S was evaluated by the incidence of ADA formation after treatment relative to the baseline prevalence. Subjects were considered ADA negative if they were ADA negative at baseline and for all samples postbaseline or if they were ADA positive at baseline and their ADA titer remained unchanged or increased by less than 0.6 titer units after treatment. Subjects were considered ADA positive if they were ADA negative at baseline but at least one sample tested positive after treatment (treatment induced) or if they were ADA positive at baseline and their titer increased by 0.6 titer units or more after treatment (treatment enhanced). At day 85, the level of DSTA4637S total antibody in all dosing groups was within the ADA assay tolerance.
ACKNOWLEDGMENTS
We thank all the subjects who participated in this study. We also thank Wendy Halpern, Nicola Stagg, Daniel Sheinson, Wouter Hazenbos, and Min Xu (Genentech, Inc.) for their critical review of the manuscript.
All authors, except for D. Dickerson and M. Leonardelli, were employees of Genentech, Inc., a member of the Roche group, at the time this work was performed and own Roche stock and/or options. Aide Castro is currently an employee of Calico Life Sciences, LLC, and Lisa Teufel is currently an employee of IDEAYA Biosciences, Inc. D. Dickerson and M. Leonardelli are employees of PRA Health Sciences.
Editing and writing assistance for the manuscript was provided by Deborah Solymar (Genentech, Inc.) and was funded by Genentech, Inc.
This study was supported by Genentech, Inc. Genentech, Inc., was involved in the study design, data interpretation, and the decision to submit for publication, in conjunction with the authors.
FOOTNOTES
- Received 19 December 2018.
- Returned for modification 15 January 2019.
- Accepted 17 March 2019.
- Accepted manuscript posted online 25 March 2019.
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02588-18.
- Copyright © 2019 American Society for Microbiology.
