Introduction

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Pokeweed antiviral protein (PAP), a 30-kDa plant protein with broad spectrum antiviral activity against plant and human viruses (Irvin and Uckun, 1992), was recently discovered to have anti-human immunodeficiency virus (HIV) activity as well (Zarling et al., 1990; Rajemohan et al., 1999). Targeting PAP to HIV-infected cells by a monoclonal antibody resulted in inhibition of HIV replication at picomolar concentrations of the immunoconjugates (Zarling et al., 1990). PAP immunoconjugates were effective against clinical HIV-1 isolates from acquired immunodeficiency syndrome patients both in vitro (Erice et al., 1993) and in HIV-infected Hu-PBL-SCID mice in vivo (Uckun et al., 1998). The lead immunoconjugate, TXU (anti-CD7)-PAP, was very well tolerated by cynomolgus monkeys at systemic exposure levels capable of abrogating HIV replication in human PBMCs (Waurzyniak et al., 1997; Uckun et al., 1998). In cynomolgus monkeys, TXU-PAP showed favorable pharmacokinetics with an elimination half-life of 8.1 to 8.7 h. The monkeys treated with TXU-PAP at dose levels of 50 µg/kg/day × 5 days or 100 µg/kg/day × 5 days tolerated the therapy well, without any significant clinical compromise or side effects, and at necropsy, no gross or microscopic lesions were found (Waurzyniak et al., 1997). The purpose of the present study was to evaluate the toxicity and pharmacokinetics of TXU-PAP in HIV-infected chimpanzees and adult patients.

	Materials and Methods

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Animals. Six adolescent Pan troglodytes chimpanzees were treated according to a contract services agreement at the Coulston Foundation (Alamogordo, NM) or Southwest Foundation (San Antonio, TX). Chimpanzees were fed PMI Fiber-Plus Chimpanzee Diet 5049 or 5037 Jumbo. A veterinarian or designated technician observed each animal daily for signs of illness or distress. The selected daily TXU-PAP dose was administered i.v. over 1 h. Animals were sedated with ketamine or other appropriate anesthetic agents to obtain body weights, physical examination parameters, and collect blood samples. Animals were incubated and maintained on inhalant anesthesia (isoflurane) for the duration of the infusion. Blood samples from all chimpanzees were collected before each infusion and sent for determination of a compete blood cell count with differential and platelets, serum albumin, liver enzyme levels, coagulation parameters, blood urea nitrogen, serum creatine, and serum electrolytes. Branched-chain DNA (Chiron HIV-1 Quantiplex version 2.0) signal amplification method was used to detect and quantify the HIV-1 viral load, as measured by HIV-1 RNA copies/ml plasma, in peripheral blood of chimpanzees at the indicated time points. Vital signs (including heart rate, respiratory rate, systolic blood pressure, and diastolic blood pressure), as well as overall well-being, were monitored at least twice daily. Body weight was determined daily. Clinical and laboratory data were analyzed and tabulated using the National Cancer Institute toxicity grading system. The chimpanzees were not euthanized after completion of the study.

Patients. Nine HIV-1-infected patients (three African-American women, one African-American man, one Hispanic woman, and four Hispanic men; age range, 27-57 years, median age, 42 years) who were diagnosed with HIV disease and had detectable HIV burden despite antiretroviral therapy were enrolled in this clinical pharmacokinetics study of TXU-PAP between July 14, 1998, and August 22, 1998. These patients were initially diagnosed between 1991 and 1997 (i.e., 1-7 years before study entry), and for all of them, previous treatment programs using combinations of nucleoside inhibitors, protease inhibitors, and/or non-nucleoside inhibitors had failed. To be eligible for this study, patients had to 1) be older than 13 years; 2) have HIV-1 infection as documented with a licensed enzyme-linked immunosorbent assay (ELISA) kit and confirmed by polymerase chain reaction (PCR) detection of HIV-1 RNA in the plasma within 30 days before study entry; 3) have received and failed prior therapy with available licensed anti-HIV drugs; 4) have a CD4⁺ T cell count of >300/µl within 30 days before study entry; 5) have a Karnofsky performance score of 70 within 14 days before study entry; 6) have a plasma hemoglobin level of 9.0 g/dl, white blood cell count of 3000/µl, absolute neutrophil count of 1200/µl, and a platelet count of 80,000/µl; 7) have adequate renal function (serum creatine of 1.5 × normal or glomerular filtration rate of 70 ml/min/1.73 m²); adequate liver function (total bilirubin of  1.5 × normal and alanine aminotransferase (ALT) <5 × normal); adequate cardiac function with ejection fraction 50%; adequate pulmonary function (no dyspnea at rest or exercise intolerance and oxygen saturation by pulse oximetry >94% in room air); and 8) be -human chorionic gonadotrophin negative (if female) within 14 days before study entry and practice adequate birth control to prevent pregnancy while receiving the study medication and for 3 months thereafter. Patients with: 1) lymphoma, 2) malignancy (e.g., Kaposi's sarcoma) requiring systemic therapy, 3) higher than grade II bilateral peripheral neuropathy, 4) history of acute or chronic pancreatitis, 5) active uncontrolled opportunistic infections or unexplained temperature of >38.5°C, 6) severe chronic diarrhea defined as more than three liquid stools/day persisting for 15 days within the last 30 days before study entry, 7) uncontrolled diabetes mellitus or other serious medical conditions within 14 days before study entry, or 8) a history of an experimental therapy, interferon or interleukin therapy, or HIV vaccine exposure within 30 days before study entry were not eligible. Patients were treated and followed at the Hughes Institute (St. Paul, MN) and South Florida Bioavailability Clinic (Miami, FL). The clinical protocol was approved by the institutional review boards of the participating Institutions. Informed consent was obtained from all patients or their guardians according to U.S. Department of Health and Human Services Guidelines.

Standard Laboratory Tests in Patients. The CD4⁺ T cell count and CD56⁺ natural killer (NK) cell count were determined by flow cytometric immunophenotyping, as described previously (Uckun and Ledbetter, 1988; Uckun et al., 1997). All immunophenotypic analyses were done at the centralized Immunophenotyping and Flow Cytometry Laboratory of the Fairview University Medical Center (St. Paul, MN). Blood samples from all nine patients were also collected before each infusion and sent for determination of a compete blood cell count with differential and platelets, serum albumin, liver enzyme levels, coagulation parameters, blood urea nitrogen, serum creatine, and serum electrolytes. The determination of the plasma HIV-1 RNA burden by quantitative PCR was performed at the ViroMed Laboratories, Inc. (St. Paul, MN). Reverse transcription-PCR (AMPLICOR HIV-1 MONITOR, Roche Diagnostics) is a target amplification method that simultaneously reverse transcribes and amplifies viral RNA and an internal quantitation standard (QS) RNA and then detects the amplified material in a microwell plate format. The signal strength from the specimen's RNA is compared with that of the QS, and the concentration of viral RNA is determined. The quality control procedures were as follows: Each batch of specimens tested included one high positive and one low positive control as well as a negative control. The expected range for each of the positive controls was provided with each kit Data Card. If positive controls were out of the published Data Card range, the laboratory supervisor or designee was called on to determine whether the run was valid. A QS was added to each specimen, and two wells containing QS only were included as an assay control. The QS is a synthetic RNA molecule with primer sites that are identical with the HIV target and a unique probe sequence specific for the QS molecule. A known number of QS copies were added to the specimen early in the processing stage such that the QS and the specimen RNA undergo the same reverse transcription, amplification, hybridization, and detection steps. HIV-1 RNA levels were determined by comparing the absorbance of the specimen with that of the QS. The QS controls for any inhibitory influences present in the patient specimen. If either the QS or HIV-1 values were outside of expected limits, the assay was repeated. All raw data and calculated data were verified by an individual who did not perform the test or calculations; the proofed results were entered into the laboratory information management system. Results entered into the laboratory information management system were subsequently reviewed by one of the following before their release: Supervisor of Molecular Biology, Director of Clinical Virology and Molecular Biology, Scientific Director, President of ViroMed Laboratories, Inc.

Dosage and Drug Administration. The procedures used for the large-scale production and purification of TXU-PAP have been previously described in detail (Myers et al., 1997). The composition and physicochemical properties of TXU-PAP were previously reported (Myers et al., 1997). The endotoxin level of TXU-PAP was determined by the limulus amebocyte lysate assay. The endotoxin levels of three lots of TXU-PAP that have been produced were <0.24 EU/mg, <0.59 EU/mg, and <0.58 EU/mg (material used in the present study). The specification for TXU-PAP final product is 3 EU/mg. The maximum human dose (M) administered in a single 1-h period was 0.005 mg/kg. Therefore, the acceptable endotoxin limit was K/M (where K = 5.0 EU/kg), which is 5.0/0.005 EU/mg = 1000 EU/mg. Thus, the endotoxin level of the TXU-PAP patient lot was >1000-fold lower than the acceptable limit. The murine DNA contamination was <171 pg DNA/ml (<342 pg DNA/mg protein). The TXU-PAP preparation contained <13% free TXU monoclonal antibody and <1% free PAP, as determined by immunoblotting using anti-PAP and anti-mouse IgG antibodies. TXU-PAP preparation was not pyrogenic in rabbits and was found to be sterile when tested for bacterial and fungal contamination with the direct inoculation method at Microbiological Associates, Inc. (Rockville, MD). The sterility testing was performed in compliance with the U.S. Food and Drug Administration Good Laboratory Practice regulations (21 CFR 58) and Good Manufacturing Practices Regulations, Title 21 CFR 211 and 610. The purpose of the sterility test was to detect the presence of one or more species of bacterial and fungal contaminants in the test article. The direct inoculation method meets or exceeds USP 23 and/or 21 CFR610 requirements. TXU-PAP did not cause turbidity in the fluid thioglycollate, soybean-casein digest, and peptone yeast glucose broths. No growth was observed on the Sabouraud-dextrose agar slants. The bacterial positive controls did cause turbidity in all broths while growth was observed in the fungal positive control tubes. The uninoculated and the inoculated negative control broth tubes and the agar slants remained clear. The general safety test in mice and guinea pigs was satisfactory. TXU-PAP immunotoxin was formulated as a sterile solution in 150 mM sodium chloride and 40 mM sodium phosphate buffer, pH 7.5, and was stable over 48 months at 4°C as determined by Western blotting. The same TXU-PAP lot was used in chimpanzees and human subjects.

Pretreatment and Follow-Up Evaluation of Patients. Medical histories, physical examinations, and laboratory studies were performed before enrollment and throughout therapy to monitor the toxic effects of TXU-PAP. Laboratory studies included a complete blood cell count with differential, serum electrolytes, blood urea nitrogen, creatine, liver function tests (bilirubin, ALT, aspartate aminotransferase, alkaline phosphatase), urine analysis, chest radiography, pulse oximetry, electrocardiography, gated cardiac pool scan or echocardiography, and a pulmonary function test. Chest radiographs and echocardiograms were repeated on day 14 and whenever clinically indicated. Toxicities were evaluated according to the National Cancer Institute Common Toxicity Criteria. The clinical and laboratory evaluations of patients for toxicity were performed daily on days 1 through 7 and then weekly until day 91 (i.e., days 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91).

Pharmacokinetic Studies and Pharmacokinetic Modeling. In the preclinical study, blood samples were collected from the chimpanzees before infusion and at 15 min, 30 min, 1 h, 12 h, and 24 h after completion of the first infusion on day 1 and last infusion on day 10. In addition, preinfusion and 1-h postinfusion blood samples were collected on days 2 through 9. In the clinical study, blood samples were obtained from the HIV-1-infected patients before and at 1, 2, 4, 9, 12, 18, and 24 h after the 1-h TXU-PAP infusion. The concentration of the intact TXU-PAP immunoconjugate in the plasma samples from chimpanzees and patients was determined by a quantitative "double sandwich" solid-phase ELISA detection system, as described in detail (Waurzyniak et al., 1997). The intact TXU-PAP concentrations in the plasma samples were determined from standard curves that were generated by linear regression analysis using varying amounts of purified TXU-PAP standard. The lower limit of detection for this assay is 1 ng/ml.

Anti-HIV Activity of Plasma Samples from TXU-PAP-Treated Adult Patients. We examined the ability of the 1-h postinfusion samples (1:2 to 1:100 dilution) from four HIV-1-infected patients to inhibit the replication of the HIV-1 strain HTLV_IIIB in normal PBMCs, as described previously for cynomolgus monkey plasma samples (Uckun et al., 1998). The concentration of TXU-PAP needed to inhibit viral replication by 50% was based on p24 production assays (IC₅₀ [p24]). Percent viral inhibition was calculated by comparing the p24 values from the test plasma-treated infected cells with p24 values from untreated infected cells (i.e., virus controls; Uckun et al., 1998; Sudbeck et al., 1998).

	Results

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Pharmacokinetics, Toxicity, and Biological Activity of TXU-PAP in Chimpanzees. Four HIV-1-infected chimpanzees (C-291, C-294, C-881, and C-1150) were treated with 10 µg/kg/day TXU-PAP i.v. for 10 days. The therapy was very well tolerated by three of the chimpanzees without any clinical signs of toxicity. One chimpanzee (C-881, Table 1), who did not tolerate the anesthesia well, showed signs of moderate-severe dehydration during days 11 to 21 (weight loss, hypoactivity, increased serum sodium levels, increased levels of blood urea nitrogen, increased heart rate), which was attributed to the side effects of anesthesia, but then fully recovered. All four chimpanzees showed a transient grade I to III elevation of the liver enzyme ALT between days 2 and 14 without a concomitant rise in total bilirubin levels (Table 1). The serum albumin levels decreased between days 1 and 5 without any concomitant weight gain or peripheral edema. This hypoalbuminemia, which we attributed to a very mild presumed grade I vascular leak syndrome (VLS), did not resolve for 2 to 3 weeks (Table 1).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   A, plasma TXU-PAP concentration-time curves in two chimpanzees (C-291 and C-294) after the i.v. infusion of 10 µg/kg TXU-PAP over 1 h on the first day of a 10-day treatment course. B, plasma TXU-PAP concentration-time curves in C-291 and C-294 after i.v. infusion of 10 µg/kg TXU-PAP over 1 h on the last day of a 10-day treatment course.

Pharmacokinetics, Toxicity, and Biological Activity of TXU-PAP in HIV-1-Infected Adult Patients. We examined the pharmacokinetics of TXU-PAP in nine HIV-1-infected adult patients. A one-compartment PK model was used to analyze the plasma concentration-time curves. TXU-PAP showed a slow elimination with a mean ± S.E. plasma elimination half-life of 12.4 ± 1.4 h, an MRT ± S.E. of 17.9 ± 2.0 h, and a mean ± S.E. systemic clearance of 2.7 ± 0.7 ml/h/kg. The mean value for the AUC was 3059 ± 721 ng · h/ml (Table 2). The pharmacokinetic parameters obtained from "pooled data modeling" were very similar to these mean values: T_1/2 = 14.2 h; MRT = 20.4 h; clearance = 1.4 ml/h/kg; AUC = 3516 ng · h/ml. The composite plasma concentration-time curve for all patients is depicted in Fig. 2. All nine patients were evaluated for toxicity and developed no significant adverse reactions to TXU-PAP. In particular, no patient developed VLS, allergic reactions, or myalgias.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Composite plasma TXU-PAP concentration-time curve for nine HIV-infected patients treated with a single 5 µg/kg TXU-PAP administered i.v. over 1 h.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   In vitro anti-HIV activity of plasma samples from TXU-PAP-treated patients. The ability of serially diluted plasma samples from TXU-PAP treated patients to inhibit HIV replication in normal PBMCs was examined using p24 antigen ELISA, as described in Materials and Methods. VC, viral control.

	Discussion

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We evaluated the toxicity, pharmacokinetics, and clinical potential of the TXU (anti-CD7)-PAP immunoconjugate as a new biotherapeutic anti-HIV agent in HIV-1-infected chimpanzees and adult patients. TXU-PAP showed favorable pharmacokinetics in both chimpanzees and adult patients with relatively long elimination half-lives and was very well tolerated at the applied dose levels without any clinical signs of significant toxicity in chimpanzees or adult patients. At 2 months after initiation of the daily ×10 TXU-PAP infusions at a 10 µg/kg/day dose level, the HIV-1 burden was reduced to below detection levels in three of the four chimpanzees. In the remaining chimpanzee, the HIV burden became undetectable at 2 weeks but returned to the pretreatment levels by 2 months. We examined the in vitro anti-HIV activities of 1-h postinfusion plasma samples from four patients treated with a single 5 µg/kg dose of TXU-PAP. These plasma samples showed potent anti-HIV activity and inhibited HIV replication in normal PBMCs even at a 1:100 dilution. Furthermore, treatment with TXU-PAP at this very low dose level was capable of reducing the viral burden and increasing circulating NK cell numbers in six of six patients evaluated. On the basis of its anti-HIV activity in preclinical models, favorable pharmacokinetics, and relative lack of toxicity, we hypothesize that the incorporation of this immunoconjugate into clinical treatment protocols may improve the prognosis of HIV-infected patients. A phase I dose-escalation study is now under way to determine the maximum tolerated dose of TXU-PAP when administered as a single infusion per day for 10 consecutive days.

TXU-PAP immunoconjugate delivers the broad-spectrum antiviral agent PAP to CD7 antigen carrying T cells and monocytes (Zarling et al., 1990; Uckun et al., 1998). PAP is a site-specific RNA N-glycosidase that catalytically removes a single adenine base from a highly conserved loop of the large rRNA species in eukaryotic (28S rRNA) and prokaryotic (23S rRNA) ribosomes (Monzingo et al., 1993; Hudak et al., 1999). This depurination of the SR loop results in irreversible inhibition of protein synthesis at the translocation step (Ussery et al., 1977; Aron and Irvin, 1980; Kurinov et al., 1999) by impairing both the elongation factor-1-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of elongation factor-2 to the affected ribosome. Our initial studies had suggested that the anti-HIV activity of PAP can be attributed to its ability to reduce viral protein synthesis in HIV-infected cells (Zarling et al., 1990; Uckun et al., 1998). PAP has also been shown to inhibit ribosomal frameshifting and retrotransposition, a molecular mechanism used by many RNA viruses, including HIV-1, to produce Gag-Pol fusion proteins (Tumer et al., 1998). More recent studies indicate that the potent antiviral activity of PAP may at least in part be due the unique ability of PAP to extensively depurinate viral RNA, including HIV-1 RNA (Rajamohan et al., 1999). The emergence of resistance to the antiretroviral agents continues to be a major obstacle to an effective treatment of HIV-infected patients (Carpenter et al., 1998). Because the molecular mechanism of action of TXU-PAP is different from those of the currently available anti-HIV agents, we postulate that the combination of TXU-PAP with other anti-HIV drugs may enhance their anti-HIV activity and reduce the likelihood for the emergence of drug-resistant HIV strains.