Monoclonal antibody hNM01 in HIV-infected patients: a phase I study

Objectives and study design: The aim of this study was to investigate the clinical responses of the individuals when treated with the humanized antibody NMO1. In this phase I study, four HIV-1 infected patients with CD4 counts between 50 and 500 cells/μl received a total of four doses of hNM01 in an intrapatient dose escalation fashion: day 1–0.2 mg/kg, day 15–1 mg/kg, day 29–5 mg/kg, and day 43–5 mg/kg. Patients were required to have virus that reacted to hNM01 by a virion capture assay and to have a viral load ≥15,000 copies/mL. Results and conclusion: The antibody was well-tolerated; no significant adverse events were observed even at the highest dose tolerated. None of the patient developed either human anti-hNM01 (anti-idiotype) or human anti-rat antibodies. The mean elimination half-life was 153 h (6.4 days). During hNMO1 therapy effects were observed on CD4 cell counts and plasma viral loads and further dose finding trials are necessary to better determine the therapeutic activity of hNM01 in HIV-infected individuals. Keywords Humanized monoclonal antibody HIV infection Clinical study 1 Introduction The administration of neutralizing antibodies may complement our current approach to the treatment of HIV-1 infection ( Armbruster et al., 2002 ). The murine monoclonal antibody mNM01 binds to both purified HIV-1 as well as forms budding at the surface of the infected cell membrane, leading to the activation of human complement and subsequent disruption of the viral envelope ( Nakamura et al., 1993 ). Experimental data show an ID 50 of <1 μg/mL for both MN and IIIB isolates, when evaluated in H9 lymphoblastic cell lines. Further, it has been shown that mNM01 also blocks the infectivity of these isolates in MT-2 cells, as measured by the inhibition of syncytium formation ( Ohno et al., 1991 ). Unfortunately, the potential for the use of murine antibodies in clinical practice may be limited by their short half-lives and their immunogenicity. These limitations may be partially circumvented by the generation of chimeric or humanized constructs ( Isaacs et al., 1992; Junghans et al., 1996; LoBuglio et al., 1989 ). The humanized version of mNM01 (hNM01) is a construct of the IgG 1 kappa class which binds to the V 3 loop of gp120 (GPGRAF), one of the principal neutralizing determinants of the virus ( Nakamura et al., 2000 ). Amino acids in the V 3 loop (particularly R and F) have been identified as essential to the process of viral entry, due to their participation in chemokine co-receptor utilization. The hNM01 antibody reacts equally well with loop peptides from the MN, IIIB, RF and CDC4 isolates (all of which have the entire GPGRAF sequence) as well as at least 20 clade B clinical isolates that have been evaluated ( Nakamura et al., 2000; Ohno et al., 1991 ). Based on supportive in vivo pre-clinical data, a phase I dose escalation study was designed to evaluate the safety and efficacy of hNM01 in HIV-1-infected individuals. The study was conducted at the Beth Israel Deaconess Medical Center. Prior to the performance of any study activities, written witnessed informed consent was obtained from all participants in accordance with human experimentation guidelines of the United States Department of Health and Human Services, and the local Human Investigations Committee. 2 Methods 2.1 Patients Patients with CD4 counts of 50–500 cells/μL and plasma viral loads > 15,000 copies/mL (branched DNA assay, Chiron Corporation, Emeryville, CA) were eligible for participation if they met the following inclusion criteria: absolute neutrophil count > 1000 cells/mm 3 , hemoglobin > 9.0 g/dL, platelets > 75,000/mm 3 , transaminase levels < 3.0 × upper limit of normal (ULN), bilirubin <2.0 mg/dl, and creatinine <1.5 × ULN. Patients were excluded if they had received cytotoxic chemotherapy, interferon treatment, or radiation therapy within the preceding 21 days, if they had used of any investigational drug within 30 days, or any investigational biologic agent within 42 days. Patients received antiretroviral therapy and were on a stable regimen for 6 weeks before study entry; this therapy was continued for the duration of the clinical trial. Patients received prophylactic therapy for Pneumocystis carinii if their CD4 counts were <200 cells/μL. Before a patient's eligibility for study participation was finalized, circulating viral strains were evaluated with respect to their ability to react with hNM01. For this purpose, used was a virion capture assay ( Cantin et al., 2001 ). The subject's plasma was incubated with magnetic beads coated with hNM01. The supernatant was separated and viral RNA was quantitated (bDNA assay, Chiron). This value was compared to that obtained from the patient's plasma sample without incubation, to generate a capture ratio (viral load after versus before incubation). Enrollment in the trial was restricted to patients with a positive capture ratio (>45%). Informed consent was obtained from patients. Human experimentation guidelines of the US Department of Health and Human Services and Beth Israel Deaconess Medical Center were followed. 2.2 Study design Patients were evaluated at screening and baseline visits and on days 1, 2, 4, 8, 15, 16, 22, 29, 30, 36, 43, 44 and 57. A complete medical history and physical examination were done at the time of the initial visit. Complete blood count, serum electrolytes, liver and renal function tests, glucose, and urinalysis were done at each visit. The hNM01 was administered by intravenous infusion over 1 h, beginning at 0.2 mg/kg on day 1, and then escalating to 1 mg/kg on day 15, and 5 mg/kg on days 29 and 43. The biweekly schedule was chosen to reflect half-lives typical of other antibodies. The doses were selected based on expected blood levels that could be achieved. A relatively rapid intra-patient dose escalation was employed due to concerns related to escape mutants ( Yoshida et al., 1997 ). Samples were obtained for pharmacokinetic measurements at the first and fourth infusions. Plasma was collected just prior to the infusion and 15 min, 4, 24, and 96 h after the infusion ended. At the second and third infusions, samples were obtained at baseline and again 15 min and 24 h after the infusion ended. Random pharmacokinetic samples levels were drawn on days 8, 22, 36 and 57. Data analysis included measurement of the elimination half-life of hNM01, and the apparent volume of distribution (dose divided by plasma concentration at zero time and by patient's body weight). Plasma viral load (bDNA assay, Chiron) and circulating CD4 cell counts were measured on days 1 (prior to infusion), 29 (prior to infusion), and 57. Serum for the measurement of human anti-rat and anti-idiotype antibodies was obtained on days 1, 8, 15, 22, 29, 36, 43, and 57. 3 Results 3.1 Safety A total of four patients were enrolled in the study (three males; three Caucasians and one African–American). The median age was 46 (37–51) years. Three of the patients had received multiple antiretroviral therapy regimens in the past and were on protease inhibitor-based therapy at the time of enrollment. One patient was naïve to antiretroviral therapy. A clinically defined maximum tolerated dose was not reached. Although stopping rules were built into the protocol specifying treatment discontinuation for grade 3–4 toxicity (National of Allergy and Infectious Diseases standardized toxicity grading system), no such events occurred, and all four patients received all four doses of study drug. One patient developed grade 1 hypotension during the infusion and one patient developed grade 1 hypertension. Both of these events were transient in nature and resolved without any medical intervention. No patient developed either anti-idiotype or human anti-rat antibodies at any of the measured time points. 3.2 Antiviral response At baseline, the median CD4 count was 240 (82–536) cells/μl, and the median plasma viral load 54,110 (16,510–124,700) copies/mL. CD4 counts and viral loads are shown In Table 1 . Interestingly, three of four patients had a decrease in their plasma viral load measurements, with the fourth individual having values consistently above 100,000 copies/mL. 3.3 Humanized monoclonal antibody NMO1 pharmacokinetics Pharmacokinetic data at a dose of 5 mg/kg are shown in Table 2 . The mean elimination half-life of 153 h (6.4 days) is robust. The mean C max of 4.3 μg/mL was below the IC 95 (12–18 μg/mL) of this antibody when tested against a panel of clinical isolates. 4 Discussion As the model of care for HIV infection is now based on life-long antiretroviral therapy ( Sande et al., 1993 ), the use of adjunctive approaches (such as immune-based interventions) is quite relevant. They may enhance the activity of the regimens we prescribe, and be part of innovative induction/maintenance strategies, which may eventually allow us to discontinue treatment while maintaining good virologic control and immune function. As such, the evaluation of a novel agent, such as hNM01 is quite pertinent. In a small study of four patients, multiple doses were extremely well tolerated, and all patients successfully completed the entire study protocol. Pharmacokinetic studies showed that, based on the half-life, it will not be necessary to administer hNM01 more than once a week in future trials. At the highest dose evaluated in this protocol, the C max may still have been in the sub-therapeutic range. This may explain why there was no consistent virologic or immunologic benefit of this intervention. Although this study was limited to four patients due to administrative constraints, its results are somewhat encouraging. The hNM01 antibody, binding to an immuno-dominant epitope at the tip of the V 3 loop on the viral envelope, is the first antibody binding to this domain to have been evaluated in clinical trials. Studies of higher doses in larger numbers of patients are planned to evaluate the toxicity and efficacy of this compound in a more definitive manner. Acknowledgements Financial support was provided by Nissin Food Products Ltd., and General Clinical Research Center; Grant number RR01032. References Armbruster et al., 2002 C. Armbruster G. Stiegler B. Vcelar W. Jager N. Michael N. Vetter H. 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