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Originally published online as doi:10.1189/jlb.0306139 on August 21, 2006

Published online before print August 21, 2006
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(Journal of Leukocyte Biology. 2006;80:1175-1182.)
© 2006 by Society for Leukocyte Biology

Anti-HIV-1 immunotoxin 3B3(Fv)-PE38: enhanced potency against clinical isolates in human PBMCs and macrophages, and negligible hepatotoxicity in macaques

Paul E. Kennedy*, Tapan K. Bera{dagger}, Qing-Cheng Wang{dagger}, Maria Gallo{dagger}, Wendeline Wagner{ddagger}, Mark G. Lewis{ddagger}, Edward A. Berger*,1 and Ira Pastan{dagger}

* Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases and
{dagger} Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA; and
{ddagger} Southern Research Institute, Frederick, Maryland, USA

1 Correspondence: Laboratory of Viral Diseases, NIAID National Institutes of Health Building 4, Room 237 Bethesda, MD 20892, USA. E-mail: edward_berger{at}nih.gov

ABSTRACT

Highly active antiretroviral therapy (HAART) against human immunodeficiency virus type 1 (HIV-1) infection dramatically suppresses viral load, leading to marked reductions in HIV-1 associated morbidity and mortality. However, infected cell reservoirs and low-level replication persist in the face of suppressive HAART, leading invariably to viral rebound upon cessation of treatment. Toxins engineered to target the Env glycoprotein on the surface of productively infected cells represent a complementary strategy to deplete these reservoirs. We described previously highly selective killing of Env-expressing cell lines by CD4(178)-PE40 and 3B3(Fv)-PE38, recombinant derivatives of Pseudomonas aeruginosa exotoxin A containing distinct targeting moieties against gp120. In the present report, we compare the in vitro potency and breadth of these chimeric toxins against multiple clinical HIV-1 isolates, replicating in biologically relevant primary human target cell types. In PBMCs, 3B3(Fv)-PE38 blocked spreading infection by all isolates examined, with greater potency than CD4(178)-PE40. 3B3(Fv)-PE38 also potently inhibited spreading HIV-1 infection in primary macrophages. Control experiments demonstrated that in both target cell types, most of the 3B3(Fv)-PE38 activity was due to selective killing of infected cells, and not merely to neutralization by the antibody moiety of the chimeric toxin. High-dose treatment of rhesus macaques with 3B3(Fv)-PE38 did not induce liver toxicity, whereas equivalent dosage of CD4(178)-PE40 induced mild hepatotoxicity. These findings highlight the potential use of 3B3(Fv)-PE38 for depleting HIV-infected cell reservoirs persisting in the face of HAART.

Key Words: HAART • reservoirs persistence • targeted killing

INTRODUCTION

The current paradigm of highly active anti-retroviral therapy (HAART) for treatment of human immunodeficiency type 1 (HIV-1) infection involves combinations of inhibitors of specific viral encoded enzymes, in particular the reverse transcriptase and protease [1 ]. HAART promotes dramatic reductions in viral load in blood and lymphoid tissues; the antiviral effect is accompanied by significant recovery of CD4+ T lymphocyte counts and immune system function, leading to major reductions in morbidity and mortality. However, enthusiasm for the current therapeutic regimens has been tempered by the realization that despite years of suppressive HAART, replication-competent HIV-1 still persists in the body. This finding is evidenced by the presence of reservoirs of infected cells and ongoing low-level virus replication [2 ]. As a result, cessation of therapy is invariably followed by rapid return of viral load, typically to pretreatment levels. Long-term (perhaps lifelong) treatment will therefore be required with the current drug regimens, a daunting prospect in view of the emerging toxicities of these drugs, the development of drug resistance, the difficulties in adhering to complex multidrug regimens, and the high financial burden.

This awareness highlights the compelling need for therapeutic interventions that eliminate, or at least control, the persistent infected cell reservoirs so that antiviral therapy can be terminated without re-emergence of high viral loads [3 ]. One approach is to intensify the therapeutic regimen with new drugs that block additional steps in the HIV replication cycle such as virion entry, capsid uncoating, genome integration, and particle assembly [4 ], or with agents that enhance the efficacy of current antiretrovirals [5 ]. Another proposed strategy is to deliberately activate latently infected cells with agents such as cytokines (e.g., interleukin-2, interleukin 7), anti-CD3 monoclonal antibody, non-tumorigenic phorbol esters (e.g., prostratin), or histone deacetylase inhibitors (e.g., valproic acid); the idea is that the infected cell reservoirs would be purged as a result of viral cytopathic effects and HIV-specific effector mechanisms [6 7 8 9 ]. Although enhanced antiviral activities have been demonstrated in some clinical studies [10 11 12 13 ], impact on viral rebound after cessation of therapy was negligible. As a complement to these pharmacological approaches, therapeutic vaccination efforts are being developed to boost specific anti-HIV immune responses in HAART-treated individuals [14 15 16 ]. While some favorable immunological responses have been observed in HIV-infected people [17 ], viral rebound after treatment cessation was unaffected.

Another approach is based on an actively studied strategy in the cancer field, namely the design of bifunctional toxins targeted to selectively bind to and kill cells expressing specific surface molecules [18 19 20 ]. We and others have proposed the use of such hybrid toxins targeting the HIV-1 Env glycoprotein at the surface of productively infected cells as a means to selectively deplete the infected cell reservoirs persisting after HAART [21 , 22 ]; the rationale is that an agent that kills already-infected cells would be an especially useful complement to current regimens that block viral dissemination by inhibiting specific steps in the HIV replication cycle. We have developed recombinant derivatives of Pseudomonas aeruginosa exotoxin A (PE) containing an Env-targeting moiety linked to the PE translocation and cytotoxic domains. Our first agent, designated CD4(178)-PE40, contains the first two extracellular domains of CD4 for binding to the gp120 subunit of HIV Env [23 ]. Despite the highly promising activities of this agent in vitro, Phase 1 clinical trials were discontinued due to dose-limiting hepatotoxicity that prevented accumulation of efficacious levels in plasma [24 , 25 ]. Subsequently we developed 3B3(Fv)-PE38, a recombinant immunotoxin containing a single-chain Fv fragment (SCFv) of the 3B3 antibody, which also is directed against the highly conserved CD4 binding site of HIV-1 gp120 [26 ]. The 3B3 SCFv was originally derived using phage-display affinity evolution of Fab b12, and exhibits enhanced binding affinity and improved potency and breadth of neutralization [27 ]. Using direct cell killing assays [26 ], we demonstrated previously that the 3B3(Fv)-PE38 immunotoxin specifically kills cell lines constitutively expressing the Env glycoproteins of laboratory-adapted HIV-1 isolates. Importantly, the specific cytotoxic potency of 3B3(Fv)-PE38 was >10-fold higher than that of CD4(178)-PE40. In the present report, we expand previous findings by comparing the in vitro potency and breadth of these recombinant Env-targeted toxins against phenotypically distinct clinical HIV-1 isolates, replicating in biologically relevant primary human target cell types. We also examine the in vivo hepatotoxicity of these agents in rhesus macaques. Our results indicate that 3B3(Fv)-PE38 is a highly improved agent for potential clinical use in the depletion of infected cell reservoirs that persist after HAART.

MATERIALS AND METHODS

Proteins
Recombinant immunotoxins were expressed in inclusion bodies in E. coli and purified by denaturation and renaturation as described previously for CD4(178)-PE40 [23 ], 3B3(Fv)-PE38 [26 ], and the control immunotoxin SS(Fv)-PE38 directed against mesothelin [28 ]. The CD4(178)-PE40 and soluble CD4 were generous gift from S. J. Johnson, Pharmacia and Upjohn, Inc. (Kalamazoo, MI). The recombinant 3B3 SCFv [27 ] was donated by C. F. Barbas, Scripps Research Institute (La Jolla, CA).

Cells and viruses
Primary human cell types were prepared from healthy HIV seronegative donors and plated for individual experiments in flat-bottom 96-well microtiter plates. PBMCs were stimulated with phytohemagglutinin (M form) for 3–4 days in RPMI 1640 medium (Quality Biologicals, Gaithersburg, MD) supplemented with 10% fetal bovine serum, then infected with the indicated viral isolates. Primary macrophages were prepared by countercurrent centrifugation elutriation of PBMCs and differentiation of the monocyte fraction in the absence of exogenous cytokines [29 ]. Phenotypic analyses (not shown) indicated that the differentiated cell population contained <1% contamination with T and B cells (flow cytometry); at least 95% of the cells stained positive for nonspecific esterase in the presence of 0.4 M sodium fluoride (indicating mature macrophages) and essentially all the cells stained positive in the absence of inhibitor (indicating that the remaining cells represented monocytes); this cell preparation supported efficient replication of R5 but not X4 HIV-1 isolates. All HIV-1 isolates were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH; the primary isolates were originally from the UNAIDS Network for HIV Isolation and Characterization, and the Division of AIDS, NIAID.

Replication assays
All cell cultures were maintained at 37° in 5% CO2. Phytohemagglutinin-stimulated PBMCs were suspended at a density of 107 cells/ml in medium [RPMI 1640 containing 10 mM HEPES, pH 7.0 and 10% (v/v) fetal bovine serum, plus antibiotics]. Cells were infected with the designated primary isolates at a m.o.i. of 0.5 TCID50/1000 cells. Following 3 h adsorption, cells were diluted with 9 vol of IL-2 medium (medium supplemented with 20 U/ml interleukin-2). Duplicate 0.1 ml samples were transferred to individual wells of 96-well tissue culture plates containing 0.1 ml IL-2 medium with the designated drug at the indicated final concentrations. The initial cell density was 5 x 105 cells/ml (100,000 cells/0.2 ml). At 3 to 4 day intervals, 0.05 ml aliquots of culture supernatant were removed for subsequent analysis of virus production. The remaining cells were uniformly suspended, and 0.1 ml samples were transferred to individual wells of fresh 96-well plates containing 0.1 ml of IL-2 media plus drug at the same concentrations. Cultures were maintained in this fashion for 15 days, by which time virus production was essentially complete. Virus production was monitored by ELISA assay of p24 in culture supernatants (Beckman Coulter HIV-1 p24 Antigen EIA, catalog # 626391). Nonspecific effects of PE-based toxins in PBMC experiments were assessed by comparing the activities of the specific Env-targeted toxins with the control immunotoxin SS(Fv)-PE38.

For infection experiments with monocyte-derived macrophages, 7 x 104 cells were seeded in 96-well flat-bottom plates in medium [DMEM containing 10% (v/v) human AB serum, plus antibiotics]. Cells were infected with the Ba-L isolate at a m.o.i. of 0.5 TCID50/1000 cells. Following 3 h adsorption, cells were diluted with 4 vol of medium, with or without drug. Every 3 to 4 days, 0.1 ml of culture supernatant was removed for subsequent analysis of virus production and replaced with fresh media with the designated drug at the same final concentrations. Cultures were maintained in this fashion for up to 50 days. Virus production was monitored by assay of p24 in culture supernatants. Nonspecific effects of Env-targeted toxins on viability of uninfected macrophages were assessed by measuring cleavage of the yellow tetrazolium salt [3-(4,5-dimethylthiozol-2yl)-2,5-diphenyltetrazolium bromide] (MTT, Cell Proliferation Kit I, Roche, Basel, Switzerland) into purple formazan crystals by metabolic active cells. Culture media was removed and 110 µl of MTT labeling reagent plus media was added and incubated for 4 h at 37°C; 100 µl of solubilization solution was added, and after overnight incubation the samples were mixed and 200 µl aliquots were transferred to fresh 96-well flat-bottom plates for measurement of absorbance at 570 nm (Thermomax, Molecular Devices, Sunnyvale, CA).

All virus replication and cell viability data are presented as the mean of duplicate wells, with error bars indicating the individual values. In cases where error bars are not apparent, it is because the individual data points were so close as to be obscured by the symbols in the graphs.

Hepatotoxicity studies
Hepatoxicity was analyzed in rhesus macaques (two animals per group). The immunotoxins (prepared at 0.5 mg/ml in PBS) were administered intravenously on days 0, 2, and 4; sham controls (no drug) were treated with the equivalent volumes of PBS. The animals were bled at the indicated times, and serum levels of alanine transferase were determined. All animals (sham and drug-treated) were sedated with ketamine (intramuscular, 5 mg/kg) prior to each dosing and bleeding procedure.

RESULTS

Effects of Env-targeted toxins on spreading infection by primary HIV-1 isolates in PBMC
We previously used direct cell killing assays to demonstrate the selective and potent activity of 3B3(Fv)-PE38 against continuous cell lines constitutively expressing Envs from T cell line-adapted HIV-1 strains [26 ]. We now wished to test the effects of the immunotoxin against spreading infection by primary HIV-1 isolates in PBMC. We focused on Clade B isolates, which predominate in geographic regions where HAART has been most successfully used to date. The isolates examined (Table 1 ) covered the range of coreceptor usage phenotypes, i.e., CCR5-specific (R5), CXCR4-specific (X4), and dual-tropic (R5X4) strains. Figure 1 demonstrates that 100 nM 3B3(Fv)-PE38 potently inhibited replication of all isolates examined, including strains from the United States, France, Brazil, Thailand, and Haiti. The effects observed for the primary isolates were comparable with those for the laboratory-adapted isolates LAI and Ba-L strains. CD4(178)-PE40 at 100 nM also inhibited spreading infection of the isolates tested, consistent with the previously reported breadth of activity of this agent against diverse isolates and different target cell types [30 31 32 33 ]. At this concentration, 3B3(Fv)-PE38 was as effective or more so than CD4(178)-PE40.


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  		Table 1. HIV-1 Isolatesa

 

Figure 1
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  		Figure 1. Effects of Env-targeted toxins on replication of primary HIV-1 strains in PBMCs. Activated PBMCs were infected with the indicated HIV-1 strains, in the absence (filled squares) or presence of 100 nM 3B3(FV)-PE38 (open circles) or CD4(178)-PE40 (open diamonds). Virus replication was monitored by measurement of p24 levels in culture supernatants. Each data point represents the mean of duplicate culture wells, with error bars indicating the individual values.

 
We next performed dose-response analyses of replication inhibition by measuring p24 production by the 92TH014 primary isolate in the presence of varying concentrations of either 3B3(Fv)-PE38 or CD4(178)-PE40. Figure 2A shows that both agents inhibited the spreading infection in dose-dependent fashion, with 3B3(Fv)-PE38 showing greater potency over the entire time course. For example at day 11 (Fig. 2B) , the responses to the two agents were found to be significantly different (P=0.001), with the IC50 values calculated as 0.08 nM for 3B3(Fv)-PE38 and 8.3 nM for CD4(178)-PE40. These results are consistent with the relative potencies previously reported in direct killing assays with cells expressing Envs from laboratory-adapted strains, i.e., 15 to 40-fold greater potency for 3B3(Fv)-PE38) [26 ].


Figure 2
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  		Figure 2. Comparative potencies of Env-targeted toxins for inhibiting replication of a primary HIV-1 strain in PBMCs. Activated PBMCs were infected with the 92TH014 primary HIV-1 isolate in the presence of various concentrations of the indicated Env-targeted toxins. Virus replication was monitored by measuring p24 levels in culture supernatants. Each data point represents the mean of duplicate culture wells, with error bars indicating the individual values. (A) Time courses of p24 production in the presence of 3B3(FV)-PE38 (upper panel) or CD4(178)-PE40 (lower panel) at the following concentrations: 0 (no drug, filled squares); 0.1 nM (open circles); 1.0 nM (open triangles); 10 nM (open diamonds); 100 nM (open squares). (B) Dose response of p24 production at day 11 in the presence of the indicated concentrations of 3B3(FV)-PE38 (open squares) or CD4(178)-PE40 (filled diamonds). The responses to the two agents were found to be different as determined by two-way ANOVA with drug treatment and concentration as factors (P=0.001); IC50 values were calculated by fitting the data to a generalized linear model of the binomial family with logit link function.

 
The 3B3 SCFv moiety of the immunotoxin is directed against conserved determinants at the CD4 binding site of gp120, and the free 3B3 Fab shows broad cross-reactive neutralization of diverse primary isolates [27 ]. It was, therefore, essential to compare the activities of 3B3(Fv)-PE38 with the free 3B3 Fab in order to assess the contribution of targeted cell killing to the observed inhibitory activity of the immunotoxin. Table 2 shows the effects of these agents (at 50 nM) against spreading infection in PBMC with two primary HIV-1 isolates, 92US660 and 92TH014. For both strains, the immunotoxin caused dramatic inhibition (>95%); the free Fab also neutralized, as expected, but the inhibition was not as effective (50–74%). Parallel phenomena were observed in comparisons of CD4-based agents (Table 2) , consistent with previously reported results [30 31 32 33 ]; the CD4(178)-PE40 caused inhibition [although less effectively than 3B3(Fv)-PE38, as noted above]; whereas sCD4 had no effect (consistent with the well-known insensitivity of primary HIV-1 strains to the soluble receptor [34 ]). As an additional control, the SS(Fv)-PE38 control immunotoxin directed against an irrelevant antigen (mesothelin) expressed on the surface of human mesothelial cells produced minimal effects. From this series of experiments, we conclude that the enhanced activity of the 3B3(Fv)-PE38 immunotoxin compared with the free 3B3 Fab in PBMCs is a reflection of the selective killing of HIV-infected cells by the former agent.


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  		Table 2. Comparison of Env-Targeted Toxins With Control Proteinsa

 
Effects of 3B3(Fv)-PE38 on HIV-1 replication in primary macrophages
Cells of the monocyte/macrophage lineage have long been recognized as targets and reservoirs for HIV-1 infection, and recent findings have highlighted the potential contribution of these cell types to HIV-1 persistence despite suppressive HAART (reviewed in [35 , 36 ]). The results shown in Fig. 3 demonstrate the activity and potency of 3B3(Fv)-PE38 against spreading HIV-1 infection in primary monocyte-derived macrophages. Inhibition occurred throughout the time course (Fig. 3A) and in dose-dependent fashion (Fig. 3B) , with a potent IC50 of {approx}0.1 – 1 nM. The free 3B3 Fab also produced some inhibition, consistent with its known neutralizing activity; however, the potency was much weaker (IC50{approx}0 nM). 3B3(Fv)-PE38 displayed minimal effects on viability of uninfected macrophages over the time and dose ranges used in these experiments (Fig. 3C) , ruling out nonspecific cytotoxicity as an explanation for the observed antiviral activity. Thus, as interpreted above for PBMC target cells, the enhanced activity of the immunotoxin compared with the free Fab against spreading HIV-1 infection in primary macrophages is a reflection of toxin-mediated killing of infected cells. These findings parallel our previous report of effective suppression of HIV-1 replication in primary macrophages by CD4(178)-PE40, but not by sCD4 [31 ].


Figure 3
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  		Figure 3. Effects of 3B3(Fv)-PE38 on HIV-1 replication in monocyte-derived macrophages. Primary macrophages were infected with the Ba-L isolate, and virus replication was monitored by measurement of p24 levels in culture supernatants. Each data point represents the mean of duplicate culture wells, with error bars indicating the individual values. (A) Time course in the absence (squares) or presence of 5 nM 3B3(Fv)-PE38 (diamonds) or free 3B3 Fab (circles). (B) Dose response: The effects of the indicated concentrations of either 3B3(Fv)-PE38 (squares) or free 3B3 Fab (diamonds) at day 29 were determined. (C) Primary macrophages were cultured in the absence (open bars) or presence (filled bars) of 5 nM 3B3(Fv)-PE38. Cell viability was assessed by the MTT assay at the indicated time points.

 
Negligible hepatotoxicity of 3B3(Fv)-PE38 in rhesus macaques
Previous Phase I clinical trials of CD4(178)-PE40 in the pre-HAART era were terminated due to dose-limiting reversible hepatotoxicity [24 , 25 ]. At the maximum tolerated dose in the clinical trials (10 µg/kg for single-dose escalation study), the peak plasma levels were suboptimal based on in vitro efficacy data and no significant reductions in viral burden were observed. We, therefore, wished to examine the in vivo hepatotoxicity of the Env-targeted toxins in a rhesus macaque model. As shown in Fig. 4 , high-dose 3B3(Fv)-PE38 (250 µg/kgx3) did not cause elevation of plasma alanine transferase levels; whereas the same high dose of CD4(178)-PE40 (but not a lower dose of 15 µg/kgx3, not shown) caused modest elevation.


Figure 4
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  		Figure 4. Hepatotoxity of Env-targeted cytotoxins in rhesus macaques. Rhesus macaques were given the indicated intravenous treatments on days 4, 2, and 0; serum levels of alanine transferase were monitored. Each panel shows data from 2 animals. (Left panel) no drug (sham); (center panel) high-dose CD4(178)-PE40 (250 µg/kgx3); (right panel) high-dose 3B3(Fv)-PE38 (250 µg/kgx3). The minor alanine transferase elevation observed with the sham controls reflects a typical response to muscle tissue breakdown resulting from the intramuscular anesthesia injection.

 
DISCUSSION

Immunotoxins have great potential for selective cell depletion in a variety of clinical settings including cancer, graft vs. host disease, autoimmune disorders, allograft rejection, and viral infections. Such bifunctional proteins have received the greatest attention for rational design of anti-cancer therapeutics [18 19 20 ]; indeed recent Phase I clinical trials have reported major responses against several hematologic malignancies [37 38 39 40 41 ], including many complete remissions in cases of chemotherapy-resistant hairy cell leukemia. Moreover, a conceptually related agent (Mylotarg) consisting of a cytoxic antibiotic chemically coupled to a recombinant monoclonal antibody has been approved for treatment of relapsing acute myelocytic leukemia in individuals over 60 years of age who are not considered candidates for cytotoxic chemotherapy [42 ]. These developments provide encouragement for the notion that the targeted toxin strategy might prove useful in attacking the reservoirs of HIV-infected cells persisting after potent antiviral therapy. In addition to the diverse chimeric toxins directed against the viral Env glycoprotein on the surface of productively infected cells [23 , 26 , 43 44 45 46 47 48 49 50 ], anti-HIV activities have been reported for analogous agents targeted to host cell molecules such as the interleukin-2 receptor on activated T-cells [51 52 53 ], the CD45RO antigen on memory T cells [54 55 56 ], and the CCR5 coreceptor on T cells and monocytes [57 ].

The data in the present report highlight the improved anti-HIV activity and nonspecific hepatotoxicity profile of the Env-targeted immunotoxin 3B3(Fv)-PE38. Beyond the previously reported direct killing of cell lines expressing Envs from laboratory-adapted strains [26 ], we show here that this agent blocks spreading infection and is effective against all primary HIV-1 isolates tested. We observed this activity in both of the major target cell types derived from peripheral blood, namely PBMC and monocyte-derived macrophages. The latter case is particularly encouraging in view of recent evidence that HIV-1 assembly in macrophages occurs predominantly in the late endosomal compartment rather than at the cell surface; even by this route, it appears that Env is still expressed at the cell surface at sufficient levels to render infected macrophages sensitive to killing by the immunotoxin.

The potency of the 3B3(Fv)-PE38 against spreading infection greatly exceeded that of CD4(178)-PE40, consistent with the previous direct killing assay results [26 ]. Importantly, 3B3(Fv)-PE38 showed no in vivo hepatotoxicity in rhesus macaques at extremely high doses, a particularly encouraging finding in view of the dose-limiting hepatoxicity observed with CD4(178)-PE40 in clinical trials, conducted in the pre-HAART era [24 , 25 ]. Studies of PE-based immunotoxins in a murine model have indicated that marked reductions in nonspecific liver toxicity without compromising specific cell killing can be achieved by mutating the Fv moiety to reduce the isoelectric point (from 10.2 to 6–8) [58 , 59 ]. Thus, the high isoelectric point of sCD4 (8.86) might have been a major factor contributing to the observed clinical hepatotoxicity of CD4(178)-PE40; this speculation is consistent with the emerging human clinical trial data on PE-based cancer immunotoxins, which indicate major anti-tumor responses with acceptable liver toxicity profiles [38 39 40 41 ]. The likely reduced hepatotoxicity of 3B3(Fv)-PE38 compared with CD4(178)-PE40, coupled with the present findings of its superior in vitro potency against diverse primary HIV-1 isolates replicating in biologically relevant cell types, suggest a significantly improved therapeutic window in a clinical setting.

Taken together, the present efficacy and toxicity data suggest that 3B3(Fv)-PE38 is a promising agent in strategies to deplete reservoirs of HIV-infected cells persisting after HAART, in the hope that viral rebound after treatment cessation will be significantly delayed (prevented?). Indeed the particular benefit of combining a drug regimen that blocks HIV replication with a targeted toxin that selectively kills already infected cells has been experimentally verified both in vitro and in vivo. Thus, CD4(178)-PE40 displayed marked synergistic activity with RT inhibitors in cultured cells, and the combination of both types of drugs resulted in complete elimination of infectious virus [60 ]. In the thy/liv-SCID-hu mouse model of HIV-1 infection, complementation of standard anti-retroviral drugs with either CD4(178)-PE40 or 3B3(Fv)-PE38 dramatically suppressed viral rebound after cessation of therapy [61 ]. Success of this approach will depend on many variables and unknowns, including the possible requirement to deliberately activate viral expression from latently infected cells, the compartmentalization of infected cells in multiple anatomical sanctuaries that are inaccessible to the immunotoxin (e.g., brain, testes), and the question of whether every infected cell must be eliminated to achieve a sustained therapeutic benefit. The use of Env-targeted immunotoxins does not preclude additional strategies to accelerate elimination of the infected cell reservoirs, e.g., immuno-modulatory interventions such as therapeutic vaccines. These issues provide a focus for future experimental and clinical studies to evaluate Env-targeted immunotoxins as a complement to antiretroviral therapy for driving HIV infection into drug-free remission.

ACKNOWLEDGEMENTS

We thank Shirley Leow (Pharmacia and Upjohn, Inc.) and Carlos F. Barbas (Scripps Research Institute) for generous donation of reagents, and Les Klimczak (NIAID) and Bertrand Saunier (NIAID) for statistical analysis. This work was supported in part by the Intramural Research Program of the NIH (National Institute of Allergy and Infectious Diseases; National Cancer Institute, Center for Cancer Research), by the NIH Intramural AIDS Targeted Antiviral Program [Edward A. Berger (NIAID), principal investigator] and by NIH/NIAID contract N01.

Received March 4, 2006; revised July 5, 2006; accepted July 17, 2006.

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