Open-Label, Dose Escalation Study of the Safety and Pharmacokinetic Profile of Tefibazumab in Healthy Volunteers

Tefibazumab (Aurexis) is a humanized monoclonal antibody beingevaluated as adjunctive therapy for the treatment of Staphylococcusaureus infections. This open-label, dose escalation study evaluatedthe safety and pharmacokinetics of tefibazumab in 19 healthyvolunteers aged 18 to 69 years. Each subject received a singleadministration of tefibazumab at a dose of 2, 5, 10, or 20 mg/kgof body weight infused over 15 min. Plasma samples for pharmacokineticassessments were obtained before infusion as well as 1, 6, 12,and 24 h and 3, 4, 7, 21, 28, 42, and 56 days after dosing.Plasma concentrations of tefibazumab were detected 1 h afterthe end of the infusion, with a mean maximum concentration ofdrug in serum (C_max) of 59, 127, 252, and 492 µg/ml followingdoses of 2, 5, 10, and 20 mg/kg, respectively. The median timeto maximum concentration of drug in serum (T_max) was 1.0 h foreach dose. The mean elimination half-life (t_1/2) was approximately22 days. The volume of distribution (V) was 4.7, 6.7, 7.2, and7.2 liters after doses of 2, 5, 10, and 20 mg/kg, respectively.Clearance (CL) was 6.0, 9.2, 10.2, and 9.9 ml/hr, respectively.At the highest dose, plasma levels of tefibazumab were >100µg/ml for 21 days. On day 56, the mean plasma concentrationswere 6.3, 10.0, 16.4, and 30.5 µg/ml for the 2, 5, 10,and 20 mg/kg doses, respectively. Tefibazumab exhibited linearkinetics across doses of 5, 10, and 20 mg/kg. No anti-tefibazumabantibodies were detected after dosing in any subject. Therewere no serious adverse events, and tefibazumab was well toleratedover the entire dose range.

INTRODUCTION

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Introduction

Infections due to Staphylococcus aureus continue to be a majormedical and public health problem. Despite presently availableantibiotics and infection control measures, mortality rateshave not declined over the past 15 years (4). Factors that contributeto the growing problem of staphylococcal infections includean increased use of intravascular devices, increased incidenceof antibiotic-resistant strains of S. aureus in both hospitaland community-acquired infections, and limitations of presentantibiotic therapies. Within the spectrum of numerous infectionscaused by S. aureus, bacteremia carries a significant burdenof morbidity and mortality by causing septic shock or seedingsecondary sites and/or medical devices.

While the introduction of new antibiotics for the treatmentof methicillin-resistant S. aureus (MRSA) is important, thisstrategy has limitations. For example, shortly after the introductionof linezolid, a new antibiotic for the treatment of methicillin-resistantS. aureus and vancomycin-resistant enterococcal infections,strains of linezolid-resistant S. aureus were identified (10,15). The ever-changing epidemiology and ecology of S. aureusand the continued morbidity and mortality associated with infectionsdue to S. aureus highlight the need for novel therapies in additionto present antimicrobial approaches.

MSCRAMM (for "microbial surface components recognizing adhesivematrix molecules") proteins are a family of cell surface adhesinsthat recognize and specifically bind to distinct extracellularmatrix components within host tissues or to serum-conditionedimplanted biomaterials such as catheters, artificial joints,and vascular grafts (2, 9). Once S. aureus successfully adheresto and colonizes host tissues, the expression of specific genesis altered, resulting in a phenotype that is more resistantto antibiotics (1). Therefore, interventions that impact earlyevents in the infectious process may lead to an improved clinicaloutcome.

Clumping factor A (ClfA) is an MSCRAMM protein expressed byS. aureus that promotes binding of fibrinogen and fibrin tothe bacterial cell surface (5, 6). The biological role of ClfAas a virulence factor and the therapeutic benefit of anti-ClfAantibodies have been evaluated in experimental animal modelsof septic arthritis and infective endocarditis (3, 8, 12, 13).These data indicate that ClfA is a valid target for the developmentof novel immunotherapeutic agents.

Tefibazumab is a humanized monoclonal antibody (immunoglobulinG1 kappa) with a variable antigen binding region composed ofhuman (>98%) and murine (<2%) amino acid sequences. Itspecifically recognizes and has a high affinity for ClfA expressedby S. aureus.

We conducted a single-center, open-label, dose escalation, phaseI study to evaluate the safety and pharmacokinetic profile oftefibazumab after single intravenous doses to healthy adults.

MATERIALS AND METHODS

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Materials and Methods

Patients. Healthy men and nonpregnant women who were 18 to 70 years old,inclusive, were eligible to participate if they signed an informedconsent, had a body mass index of 19 to 30 (inclusive), hada negative urine drug screen, and had negative blood screeningresults for hepatitis B surface antigen, hepatitis C antibody,and human immunodeficiency virus antibody. Subjects were excludedif they had an active medical condition that was under evaluationor treatment, had had major surgery within 4 weeks before dosing,or had a history of hypersensitivity to immune globulin preparations.All medications except oral contraceptives, nonprescriptionanalgesics, or vitamins were discontinued at least 2 weeks beforedosing. Subjects could not receive an investigational drug ordonate blood within 30 days or consume alcohol within 72 h beforedosing. The use of additional medications, immune globulins,blood products, or immunizations was avoided, where possible,for the 56-day course of the study. The protocol and writteninformed consent form were approved by the institutional ethicscommittee before the study was initiated.

Randomization. cohorts of four subjects were sequentially assigned to one offour escalating dose cohorts (2, 5, 10, or 20 mg of tefibazumabper kg of body weight).

Study drug. Tefibazumab for injection was supplied as a sterile, nonpyrogenic,phosphate-buffered solution at a concentration of 10 mg/ml.Each subject received a single dose by intravenous infusionover 15 min through a 0.22-micron-pore-size, low-protein-binding,in-line filter. Subjects were closely monitored, and the infusionwas permanently discontinued for intravenous line extravasationor any signs of anaphylaxis. Subjects who did not receive acomplete dose were replaced.

Safety monitoring. The first cohort received tefibazumab at a concentration of2 mg/kg. Enrollment of sequential, dose-escalating cohorts proceededfollowing review of day 7 safety data from the prior cohort.Clinical adverse events (AE) were graded as mild, moderate,or severe-serious. Two additional subjects were added to a cohortif one of the four initial subjects experienced a moderate AEconsidered at least possibly related to tefibazumab. Dose escalationwas halted, defining the maximum tolerated dose, if two or moresubjects in a cohort experienced moderate AEs or if any subjectexperienced a serious adverse event that was possibly or definitelyrelated to the study drug.

Screening evaluations included medical history, physical examination,clinical laboratory assessments, electrocardiogram, chest Xray, urine drug screening, hepatitis and human immunodeficiencyvirus screening, and pregnancy test, if applicable. Physicalexaminations were repeated on days 7 and 56, and pregnancy testingwas repeated on day 56, for women of childbearing potential.Subjects were monitored during and for 24 h following the infusionof tefibazumab in a clinical research unit. Subjects returnedto the unit for follow-up evaluations on study days 3, 4, 7,14, 21, 28, 42, and 56.

Blood samples were obtained for analysis of anti-idiotypic antibodiesto tefibazumab using an enzyme-linked immunosorbent assay atscreening and on days 28 and 56. Anti-tefibazumab antibody titerswere determined using twofold dilutions covering a range of1:10 to 1:20,480. The assay included four positive and fournegative controls. The cut-point value to identify positiveanti-tefibazumab plasma samples was defined as the mean absorbancevalue of 1:10 dilutions of the negative controls and plate blanksplus three standard deviations.

Pharmacokinetic parameters. The plasma concentration of tefibazumab was determined usingan enzyme-linked immunosorbent assay that was linear (r² = 0.999)between the lower (2 ng/ml) and upper (125 ng/ml) limits ofquantitation. Three quality control samples (12.5, 37.5, and93.8 ng/ml) were included in each run. The coefficient of variationwas 3 to 16% over 3 days of analysis. If necessary, plasma sampleswere diluted with phosphate-buffered saline, 0.1% bovine serumalbumin, and 0.05% Tween so that the measured concentrationswere within the linear range.

Plasma concentration-time data were used to determine peak concentrationof drug in serum (C_max), time to peak concentration of drugin serum (T_max), minimum concentration of drug in serum (C_min),area under the plasma concentration-time curve (AUC) from immediatelyprior to dosing (time 0) to the last sample [AUC_(0-last)] andextrapolated to infinity [AUC_(0-inf)], the apparent terminalelimination half-life (t_1/2), volume of distribution (V), andsystemic clearance (CL). Actual sampling times were used forthe calculation of pharmacokinetic parameters, and nominal samplingtimes were used for the creation of mean concentration tablesand mean concentration-time figures. All calculations were basedon nonmissing plasma concentrations. Pharmacokinetic calculationswere performed using a noncompartmental model (WinNonlin softwareprogram; Pharsight Corporation, Cary, N.C.). Dose proportionalitywas investigated by plotting the point estimate of the meanAUC_(0-last) and C_max by dose. Formal statistical analysis wasnot performed, because the dose range and the number of subjectsin each cohort were too small for meaningful analysis.

RESULTS

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Results

Enrollment. The planned study enrollment was 16 subjects divided into fourcohorts. One subject in cohort 1 (dosage, 2 mg/kg of body weight)developed a headache of moderate intensity that was consideredto be possibly related to the administration of the study drug.Two additional subjects were enrolled into cohort 1. One subjectin cohort 2 experienced an extravasation of study drug and wasnot included in the pharmacokinetic assessments. An additionalsubject was enrolled as a replacement. Thus, 19 subjects participatedin the study and were included in safety analyses. Eighteensubjects received complete doses and were included in pharmacokineticanalyses.

The study population consisted of 7 women and 12 men rangingin age from 18 to 69 years (mean ± standard deviation,41.6 ± 17.9 years; median, 41 years). Sixteen subjectswere Caucasian, 2 were African-American, and 1 was Hispanic-Caucasian.The mean body mass index was 25.9 ± 2.7, with a rangeof 20.0 to 29.0 and a median of 26.0.

Safety results. With the exception of the infusion stopped due to extravasation,all infusions of study drug were completed without interruption,and all were well tolerated. Table 1 summarizes the distributionof adverse events by cohort for AEs that were at least possiblyrelated to study drug administration and by severity grade forall AEs regardless of the relationship to the study drug.

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TABLE 1. Summary of treatment-emergent adverse events by dose cohort

Treatment-emergent AEs, regardless of the relationship to thestudy drug, were reported by 14 of 19 (73.7%) subjects. Of the31 treatment-emergent AEs, 30 were mild and 1, that of a subjectwith a headache treated with acetaminophen, was moderate. Headachewas reported by four subjects, one in each dose cohort, andback pain was reported by two subjects in cohort 3. No othertreatment-emergent AE was reported by more than one subject.

A total of 6 of 31 (19.4%) treatment-emergent AEs were consideredto be possibly related to the administration of the study drug.These AEs were reported by two subjects each in cohorts 1, 2,and 4 and included headache (three subjects), neutropenia, gastroesophagealreflux, and erythema (one subject each). No clinical laboratoryfindings were considered to be clinically significant or relatedto the study drug. The subject who experienced neutropenia hadon-therapy values that were marginally lower than screeningvalues and the lower limit of the normal range (1.8 x 10⁹/liter).This subject's absolute neutrophil count decreased from 3.0x 10⁹/liter on day –15 to 1.8 x 10⁹/liter on day –7and 1.5 x 10⁹/liter on day 6 before returning to normal on day13 without intervention. Total white blood cell counts for thissubject decreased from 5 x 10⁹/liter on day –15 to 3.7and 3.0 x 10⁹/liter on day –7 and day 6, respectively,before increasing to within normal limits. Erythema was describedas scattered pinprick red points on both cheeks that started12.5 h after dosing and resolved within 3 days without intervention.Although the investigator indicated it was unlikely to be relatedto study drug, a causal relationship could not be ruled outdefinitively.

Pharmacokinetic evaluation. Plasma concentrations of tefibazumab were detected in all samplesbeginning 1 h after dosing (first postdose collection) throughthe final sample collection on day 56. For each dose, the meanplasma concentration was largest 1 h after dosing and graduallydiminished throughout the sampling interval (Fig. 1). The lastsampling times at which the mean plasma concentration of tefibazumabwas >100 g/ml were 12, 168, and 336 h for the 5, 10, and20 mg/kg doses, respectively. For the 2 mg/kg dose, the meanplasma concentration was <100 µg/ml at all samplingtimes.

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FIG. 1. Mean plasma concentration of tefibazumab following single intravenous doses of 2 (•), 5 ( ${circ}$ ), 10 ( ${blacksquare}$ ), or 20 ( ${square}$ ) mg/kg of body weight to healthy subjects.

The C_max and AUC_(0-inf) appeared to increase proportionallywith tefibazumab doses of 5, 10, and 20 mg/kg, but the clearanceand volume of distribution results were similar across all doses.The median T_max was 1.0 h for each dose, and the mean eliminationt_1/2 was approximately 22 days (Table 2). The t_1/2 was calculatedfrom the beta phase using the data points that describe theterminal log-linear decline in the regression. Only concentrationsafter C_max, with a minimum of three points, were used to describethe elimination phase. This occurred approximately between 7and 56 days postdose.

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TABLE 2. Mean pharmacokinetic parameters by dose

All plasma samples collected on day 28 and day 56 were negativefor anti-tefibazumab antibodies, indicating that the study subjectshad no detectable immune response to tefibazumab.

DISCUSSION

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Discussion

The increased incidence of antibiotic-resistant strains of S.aureus in both nosocomial and community-acquired infectionsand the significant morbidity and mortality associated withthese infections highlight the need for novel therapies in additionto present antimicrobial approaches. The reduced infectivityof isogenic mutants unable to express ClfA in animal modelsof septic arthritis and infective endocarditis and protectionagainst S. aureus septic arthritis and sepsis-induced deathafforded by passive immunization of mice with anti-ClfA antibodies(3, 8, 12, 13) indicate that ClfA is a valid target for thedevelopment of novel immunotherapeutic agents.

Tefibazumab was well tolerated following administration of singleintravenous doses of 2, 5, 10, and 20 mg/kg of body weight tohealthy adults. All but one of the treatment-emergent AEs wasclassified as mild, and 25 of 31 treatment- emergent AEs wereconsidered not to be related to the administration of the studydrug. None of the subjects experienced dose-limiting toxicity;therefore, the maximum tolerated dose of tefibazumab was notestablished in this study.

There was no evidence of dose-related adverse events in thissmall study. The numbers of both treatment-emergent AEs andthose considered to be at least possibly related to the studydrug administration were distributed across the dose cohortsand did not appear to be associated with the dose of tefibazumab.No subject experienced a serious adverse event during the study.No clinically significant abnormalities were observed in clinicallaboratory tests.

Dose-proportional increases in C_max and AUC_(0-inf) were observedfollowing the administration of doses of 5, 10, and 20 mg/kg,but the V and CL were similar across all doses. These resultsindicate that tefibazumab exhibited linear pharmacokineticsacross the dose range of 5 to 20 mg/kg. The relatively smallV value may indicate that tefibazumab enters the interstitialfluid but does not cross the cell membrane into the intracellularfluids.

Plasma concentrations of tefibazumab were detected 1 h afterdosing and remained detectable through day 56, the first andlast collection points after dosing, respectively. The prolongedt_1/2 of tefibazumab is characteristic of immunoglobulin G monoclonalantibodies (7, 11, 14). The time during which the mean plasmaconcentration was >100 µg/ml increased as the doseincreased. This may prove to be an important consideration indose selection. In the rabbit infectious endocarditis model,doses of tefibazumab that provided serum concentrations >100 µg/ml at all times after dosing afforded significantreductions in bacteremia and the bacterial colony counts inthe heart vegetation, kidney, and, in one study, the spleen(unpublished data).

The results of this phase I dose escalation study and the nonclinicaldata suggesting that tefibazumab may be effective in preventingand treating staphylococcal infections indicate that furtherinvestigation of this novel immunotherapeutic agent is warranted.

ACKNOWLEDGMENTS

We thank Brenda Ames and Elena Gorovits for development of theAurexis plasma assay and Ben Crumley, Andrea Smith, and HeatherHughes for performing the assay on all PK samples and compilingthe data for analysis. We thank the Inhibitex QA departmentfor auditing of PK data. We thank Amy Morris for her expertiseand guidance during the execution of this trial. The authorsacknowledge the assistance of George H. D'Addamio in preparingthe manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Inhibitex, Inc., 1165 Sanctuary Parkway, Suite 400, Alpharetta, GA 30004. Phone: (678) 746-1108. Fax: (678) 746-1265. E-mail: bbennett{at}inhibitex.com.

REFERENCES

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