E5 Murine Monoclonal Antiendotoxin Antibody in Gram-Negative Sepsis:  A Randomized Controlled Trial FREE

While knowledge and understanding of gram-negative sepsis have grown over the last 20 years, the ability to treat it successfully has not changed substantially.¹ Despite the introduction of more potent antibiotics and more sophisticated life-support technology, an estimated 200,000 Americans develop gram-negative sepsis each year with reported mortality rates of 30% to 65%.^2- 5 The prime initiator of gram-negative sepsis is endotoxin, the lipopolysaccharide component of the bacterial outer membrane.^6- 8 Endotoxin triggers the production of proinflammatory monokines (eg, tumor necrosis factor) which in turn stimulate a variety of proinflammatory and anti-inflammatory mediator cascades that result in the systemic signs and organ dysfunction that characterize clinical sepsis.⁹ Accordingly, it has been hypothesized that agents that bind endotoxin may mitigate the subsequent cascade, resulting in a decrease in the clinical manifestations of sepsis and improvement in outcome.^1,10- 11

A murine monoclonal antibody of the IgM class, E5 (XOMA Corp, Berkeley, Calif) was developed by immunizing mice to endotoxin from the J5 mutant of Escherichia coli.^12- 13 Although endotoxins vary across gram-negative species, E5 has been demonstrated in vitro to react with the core elements of endotoxin, implying specific binding to a broad range of gram-negative endotoxins.^12,14 In several animal models of sepsis, including neutropenic rats inoculated with Pseudomonas,¹⁵ rats who underwent cecal perforation,¹⁶ and mice who were exposed to lipopolysaccharide,¹⁷ E5 therapy administered after the onset of clinical signs significantly reduced mortality when compared with controls.

Prior human studies include 2 small studies (N = 9 and N = 39)^18- 19 in which safety and dosage were assessed in septic patients and 2 large efficacy trials (N = 486 and N = 847). Both efficacy trials were randomized, double-blind multicenter trials of patients with known or suspected gram-negative sepsis in which the primary end point was 30-day mortality. The first trial failed to detect a reduction in mortality overall but post-hoc analysis of patients with gram-negative infection, who were not in refractory shock at enrollment (n = 137), showed improved resolution of organ dysfunction and survival.¹⁰ The subsequent trial excluded patients in refractory shock and demonstrated improved resolution of organ failure but failed to demonstrate a significant reduction in mortality.²⁰ In both trials, E5 appeared to be well tolerated with no significant increase in adverse events.

Based on these observations, we undertook a third efficacy trial of E5 to determine whether the administration of E5 enhances survival in patients with severe sepsis due to documented or probable gram-negative infection. This trial was designed to have greater power than the 2 previous efficacy trials, to exclude patients with refractory shock, and to analyze the subgroup without nonrefractory shock. However, the study was discontinued after the second interim analysis because of insufficient effect per a prespecified stopping rule.

This prospective, double-blind, randomized, placebo-controlled trial was conducted at 136 medical centers in the United States from April 1993 to April 1997. To be eligible for the study, patients had to have documented or probable gram-negative infection, meet criteria for severe sepsis, be 18 years or older, require care in an intensive care unit, and not be improving despite standard supportive therapy and appropriate antibiotic coverage. Patients were entered into the study only after review and approval of entry criteria by a screening authorization committee on call 24 hours per day.

Gram-negative infection was considered documented by a positive culture or convincing gram stain no more than 2 calendar days prior to enrollment. A patient could be enrolled without a documented gram-negative infection in the setting of postsurgical intra-abdominal sepsis in which the intestinal tract had either been perforated or required partial resection for ischemia (provided that >1 day but <7 days had elapsed since surgery). Where frank spillage of stool into the abdomen resulted from these events, the patient could qualify without a gram stain or culture and without the 24-hour waiting period.

Sepsis, severe sepsis, and septic shock were defined as per the recommendations of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference.²¹ Specifically, sepsis was defined as the presence of any 2 of the following: temperature higher than 38°C or lower than 36°C; heart rate greater than 90/min, respiratory rate greater than 20/min, minute ventilation greater than 10 L/min, or PaCO₂ of less than 32 mm Hg; and white blood cell count greater than 12 × 10⁹/L or less than 4 × 10⁹/L, or greater than 10% immature forms (bands).

Severe sepsis was defined as sepsis and either hypotension or evidence of hypoperfusion and organ dysfunction. Hypotension was defined either as: systolic blood pressure of less than 90 mm Hg or a reduction of greater than 40 mm Hg from baseline despite administration of greater than 500-mL crystalloid solution or greater than 250-mL colloid suspension, or vasopressor therapy to maintain a systolic blood pressure of greater than 90 mm Hg despite an appropriate fluid challenge.

Hypoperfusion or organ system dysfunction was defined as one of the following: blood lactate level greater than 2 mmol/L; renal dysfunction (>44.2 µmol/L [>0.5 mg/dL] increase in serum creatinine or urine output <0.5 mL/kg for >2 hours despite hydration in patients with normal baseline renal function or >76.26 µmol/L [>1 mg/dL] increase in serum creatinine in patients with chronic renal impairment); disseminated intravascular coagulation (abnormally low or acutely decreased [>25%] platelet count with elevated prothrombin time, elevated partial thromboplastin time, or clinical bleeding, and absence of confounding factors such as liver failure or anticoagulant therapy); hepatobiliary dysfunction (serum bilirubin of 34.2 µmol/L [>2.0 mg/dL] with elevation of alkaline phosphatase, γ-glutamyl transferase, or serum transaminases beyond twice the upper limit of normal and no evidence of preexisting hepatobiliary disease); acute lung injury (PaO₂<70 mm Hg on room air or ratio of PaO₂ to fraction of inspired oxygen [FIO₂] <280 mm Hg in the absence of congestive heart failure or primary pulmonary pathology such as lower respiratory tract infection, pulmonary embolism, or interstitial lung disease). In patients presenting with pneumonia, pulmonary dysfunction could not be used as the sole criterion for organ dysfunction. Septic shock was defined as sepsis with hypotension plus hypoperfusion and organ dysfunction.

Patients were excluded from participation if any of the following conditions were present: granulocyte count of less than 1 × 10⁹/L prior to the onset of sepsis; infections associated with burns, pregnancy, or lactation; prior therapy with, or known allergy to, murine antibodies; human immunodeficiency virus infection; documented or suspected recent acute myocardial infarction; refractory shock (systolic blood pressure <90 mm Hg despite maximum vasopressor therapy); treatment with another investigational therapy; or lack of commitment to full life-support measures by the primary physician.

Patients were required to meet all entry criteria (except gram stain and culture documentation) within the 12 hours prior to commencing study medication. The study protocol was approved by each study center's institutional review board. Informed consent was obtained from the patient, next of kin, or legally authorized representative prior to enrollment.

The formulation of E5 that was used in the study contained a clear solution of 2-mg murine monoclonal antibody per milliliter of 5 mmol/L of sodium phosphate per 0.15 mmol/L of sodium chloride buffer, containing 0.01% polysorbate 80. Placebo consisted of a 0.1 mg/mL human serum albumin solution that was identical in appearance to E5. The dose of study medication was 2 mg/kg administered by intravenous infusion over 1 hour as 1 mL/kg of E5 solution. The full dose was administered twice, first on study day 1, and again 24 hours later on study day 2. This dosage regimen was based on prior pharmacokinetic studies in septic patients and was the same as that used in the 2 prior efficacy trials.^10,20

Clinical data were collected on standardized case report forms and included specific entry criteria, date and time of study drug infusion, and relevant data concerning adverse clinical events and efficacy end points. Each case report form was verified against the medical record by a study monitor and reviewed by a medical monitor, both of whom were blinded to treatment allocation. Adverse events were independently reviewed by an independent data safety and monitoring board.

Mortality at day 14 was the primary study end point. However, survival was determined through day 28, and adverse events were monitored until day 32. Patients discharged before day 28 were contacted by telephone. In the event that a patient was inaccessible at day 28, at least 2 additional telephone calls were made, and a telegram was sent within the next 2 weeks to ascertain survival status.

Study medication was discontinued and the patient was withdrawn from the study if a serious adverse event occurred; the patient was determined to be ineligible for the study following assignment of study medication; or if the investigator, patient, or patient's representative decided to withdraw consent.

For sample size calculation, the 14-day, all-cause mortality for documented, severe gram-negative sepsis was assumed to be 28% for placebo patients. A sample size of 1700 was calculated as adequate to show a 25% or greater relative risk reduction in 14-day all-cause mortality with at least 90% power at the 4.5% significance level. Because prior studies suggested patients in severe sepsis without shock were more likely to benefit from E5, we also decided prospectively to analyze this subgroup. In a previous study, 43% of the patients with documented gram-negative sepsis were not in shock at time of treatment. Assuming a similar proportion, we estimated 730 patients in the nonshock subset, providing 80% power to detect a reduction in mortality from 25% to 14.5% with α = .01.

The planned interim analyses were based on the approach of Lan and DeMets.²² The stopping rules were derived using the method of Pampallona, Tsiatis, and Kim, an extension of the Lan and DeMets approach to control for statistical power, which preserves both type I error at .0225 (under the null hypothesis of no treatment difference) and type II error at .0225 (under the alternative hypothesis of a differential mortality rate of 0.0909 [placebo − E5]). The interim analyses were reviewed by the data safety and monitoring board.

Data for patients were considered evaluable and included in the efficacy and safety analyses if any amount of study medication was administered. Baseline characteristics and mortality rates were compared between treatment groups using the 2 sample t tests and Pearson χ² tests as appropriate. Patients who received study medication, who had incomplete dose administration, or who did not receive study medication were summarized by reason. Patient mortality was also reviewed by individual sites for each treatment group, and Kaplan-Meier survival probabilities were determined and compared by the log-rank test. In addition to the primary analyses of all patients who received treatment and the subgroup who presented without shock, we also conducted post-hoc analyses using intention-to-treat and in subgroups classified by the presence or absence of major comorbidities, organ failure, site of infection, and infectious origin. The incidence of serious adverse events among randomized and treated patients was summarized by treatment group using the World Health Organization dictionary.²³

Significance for the primary end point was based on the stopping rule above. Significance for distribution of baseline characteristics, adverse events, and post-hoc analyses was assumed at P<.05.

A total of 1102 patients were randomly assigned to receive either E5 or placebo (Figure 1). Of those, 1090 (98.9%) received some amount of study medication and were therefore considered to have been treated. For the 12 patients who did not receive treatment (8 in the placebo arm and 4 in the E5 arm), reasons cited included the development of a serious adverse event between enrollment and initiation (6 in the placebo arm and 3 in the E5 arm), error in intravenous administration (1 in the placebo arm), and exclusion criteria not met (1 in the placebo arm). No reason was given for failure to administer therapy for 1 patient in the E5 arm.

The mean number of patients enrolled per site was 8 (range, 1-50). Baseline characteristics of the cohort are described in Table 1. Patients ranged in age from 18 to 95 years with a mean age of 60.7 years. Hypotension or need for vasopressors was present in three quarters of the patients at presentation, shock and renal dysfunction were each present in half, pulmonary dysfunction and lactic acidosis were each present in a third, and disseminated intravascular coagulation was present in a quarter. Hepatobiliary dysfunction was only noted in 5% of the patients at presentation. Most patients (84%) had acute dysfunction of at least 1 organ system at enrollment.

Most patients presented with primary bacteremia, intra-abdominal infection, pneumonia, or urinary tract infection. Gram-negative infection was confirmed by culture in 915 patients (83.9%). In 736 cases (67.5%), a single gram-negative organism was isolated. E coli was isolated most commonly (39.0% of cases), followed by Klebsiella (18.6%), Pseudomonas (13.7%), Enterobacter (11.7%), Bacteroides (5.2%), Proteus (3.8%), Citrobacter (3.4%), Serratia (3.4%), Acinetobacter (2.7%), Haemophilus (2.3%), and Xanthomonas (2.1%) species. The microbiologic origin was generally similar across sites of infection though Pseudomonas species were the most common isolate in patients presenting with respiratory tract infection (32.4%).

There were no significant differences in baseline characteristics between the E5 group and the placebo group with the exception of lactic acidosis, which was more common in the E5 group (P = .04). A total of 546 patients received E5 and 544 patients received placebo. Approximately 90% of patients in each treatment group completed both doses of study medication. For 61 patients in the E5 group (11.1%) and 46 patients in the placebo group (8.3%), dose administration was incomplete. Four patients in the E5 group and 8 patients in the placebo group had no infusion of study medication. The most common reason for incomplete infusion was death, which occurred in 5.5% of randomized patients in the E5 group and 5.4% of patients in the placebo group. Among patients who received no infusion, the most common reason was the occurrence of a serious adverse event for 0.5% of patients in the E5 group and 1.1% of patients in the placebo group.

Mortality rates are shown in Table 2. No patients were lost to follow-up. There were no observed differences between E5- and placebo-treated patients at 14 or 28 days. Mortality at 14 days was 29.7% (162 deaths) and 31.1% (169 deaths) in the E5 and placebo groups, respectively (P = .67). This net difference (1.4%) fell within the formal stopping boundaries and hence led to termination of the study by the data safety and monitoring board. Mortality at 28 days was 38.5% (210 deaths) and 40.3% (219 deaths) in the E5 and placebo groups, respectively (P = .56). Kaplan-Meier analysis confirmed the lack of a statistically significant difference in survival during the first 28 days (Figure 2).

Not surprisingly, patients without shock (n = 469) had a lower mortality rate than those with shock (n = 621); (30.9% vs 45.7% on day 28, respectively; P<.001). In the prospectively defined post-hoc analysis of those without shock, mortality was lower in those treated with E5 but the difference was not significant (mortality at 28 days was 28.9% in the E5 group and 32.6% in the placebo group, P = .32; Table 2 and Figure 3). Other post-hoc analyses, including intention-to-treat analysis and subgroup analyses based on comorbidity, organ failure, site of infection, and organism, failed to demonstrate any significant difference between treatment groups (Table 2).

Both serious (54.1%) and nonserious (56.7%) adverse events were commonly reported in the study. However, there were no statistically significant differences between treatment arms and most events appeared to be complications of sepsis. In particular, allergic reactions to E5 were not apparent in this cohort. Serious adverse events occurring with an incidence greater than 1% are presented in Table 3.

In this third phase 3 study of the E5 monoclonal antiendotoxin antibody, we failed to demonstrate that E5 reduces mortality in patients with gram-negative sepsis. Furthermore, though mortality in those without shock was lower when treated with E5, as had been hypothesized previously, neither this difference nor any other post-hoc analysis was statistically significant. These results are disappointing and raise important issues regarding the role of antiendotoxin therapies, our understanding of sepsis, and the evaluation of potential antisepsis therapies.

There have been several large clinical efficacy trials of antisepsis strategies that have failed to demonstrate a beneficial effect on mortality.^24- 25 The many reasons cited for the failure of prior trials can be categorized under 3 domains: choice of therapy, study design,²⁶ and the prevailing conceptual model of sepsis.²⁷

Problems with choice of therapy include insufficient knowledge regarding the biologic effects and the appropriate dosage and duration of therapy. With the exception of E5, HA-1A antiendotoxin monoclonal antibody,¹¹ and taurolidine,²⁸ most antisepsis strategies tested in the last decade have targeted endogenous inflammatory mediators.²⁴ However, these mediators have multiple effects and the degree of suppression or enhancement required to optimize survival is complex and poorly understood.^1,9 In contrast, endotoxin is an exogenous, toxic stimulus and its neutralization in the acute setting is likely to be beneficial to the host. Unlike some antiendotoxin agents, E5 was shown in preclinical studies to not only bind endotoxin^{12- 14,29- 31} but to neutralize it with subsequent attenuated inflammatory response and improved outcome.^{15- 17,30- 34} The considerable prior experience with E5 has also led to a good understanding of its underlying pharmacokinetic and pharmacodynamic properties, and 2 prior large clinical trials have used E5 at the same dosage with encouraging results in similar populations.^10,20 We do not, however, know the optimal duration of therapy. One might hypothesize that E5 should have been administered over a longer period since sepsis and organ dysfunction can often last for many days, during which recurrent endotoxemia may occur.

Attempting to learn from potential design flaws in previous trials, we undertook several steps in the design and conduct of this trial to minimize the chance of failure due to study design. First, we shortened the time window during which patients had to present with severe sepsis from 24 to 12 hours. Second, only patients failing to improve during the 12-hour period could be enrolled. Third, we targeted only patients with strong evidence of gram-negative infection and sepsis (ie, those most likely to benefit from an antiendotoxin strategy). Fourth, patients who appeared too sick to benefit (ie, were experiencing refractory shock or deemed unlikely to survive) were excluded.

Each of the above steps was monitored and tracked prospectively through the use of a screening authorization center staffed 24 hours per day by experts familiar with sepsis. The screening center authorized enrollment only after discussion with the clinical site investigators to determine suitability. The entire enrollment process was then reviewed separately by a patient evaluation and review committee to ensure compliance with study goals. In addition, the 2 prior phase 3 studies provided valuable information regarding potentially important subgroups in which benefit might be maximal. We used this information to construct entry criteria that would maximize enrollment of particular subgroups, to determine sample size, and to specify prospective subgroups for analysis.

A potential limitation of this study was our ability to detect a treatment effect in the subgroup of patients with gram-negative sepsis without shock, who were identified in previous studies as the group most likely to benefit. Even if we had completed enrollment, we would only have been able to detect a significant effect in this subgroup if mortality were reduced by almost half. Because not all patients had confirmed gram-negative infection and the trial was stopped after the second interim analysis, we had little statistical power to determine whether the observed decrease in mortality with E5 was significant. Another potential limitation of our study was the large number of sites and long enrollment period. Both of these factors may have increased the variability in management and outcome of sepsis and organ dysfunction. However, analyses of time trends and interinstitutional comparisons failed to demonstrate significant differences in outcome (data not shown). Thus, we believe our study design was generally sound with regard to most prior criticisms of sepsis trial design. Therefore, we conclude E5 either has no effect or has an effect too small to be detected using our standard approach to sepsis trial design and sample size.

If there is an important effect of E5, or of other unsuccessful antisepsis strategies, we may have to consider a more radical change in sepsis study design. For example, we may wish to study more homogeneous patient groups. As with other sepsis studies, our cohort varied widely with respect to site of infection, microorganism, and number and type of organ failures. It is possible that E5, or other antisepsis strategies, may work in particular subgroups in which mortality is most prominently driven by the septic process. To isolate this effect would likely require more focused enrollment, higher sample size, or both. Successful cardiology trials, for example, have been targeted at a relatively focused group of patients with acute myocardial infarction and have been powered to detect much smaller changes in mortality than most sepsis trials. If we had powered our study to find the same absolute risk reductions in 28-day mortality as the Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries (GUSTO I) study of thrombolytic therapy in acute myocardial infarction,³⁵ we would have required 76,000 patients. Other design modifications might include the study of combination therapies, or therapies titrated to particular elements of the inflammatory response that characterizes sepsis and are documented in all enrolled patients. Finally, choosing end points other than all-cause, short-term mortality may allow easier determination of therapies that have an important effect on septic morbidity.

In conclusion, despite prior encouraging results regarding the potential effects of both E5 and other antiendotoxin antibodies, and despite our efforts to conduct a study of a large cohort of carefully selected patients, we have failed to prove that E5 is beneficial in the treatment of sepsis. This trial concludes, at least for now, more than 20 years of antiendotoxin research without providing any definitive answers about mortality reduction despite a nagging tendency to show beneficial effects in post-hoc analyses. If we are to avoid such an indeterminate outcome from future endeavors, it behooves us not only to look to better and more realistic discovery programs and laboratory research but also to focus on improved clinical study design.