Interleukin 2 receptor-targeted cytotoxicity. Interleukin 2 receptor-mediated action of a diphtheria toxin-related interleukin 2 fusion protein.

The IL-2 toxin-mediated inhibition of protein synthesis in high affinity IL-2-R-positive murine and human T cell lines has been examined. Both excess free IL-2 and mAb to the Tac epitope of the p55 subunit of IL-2-R are shown to block the action of IL-2 toxin; whereas, agents that interact with other receptors or antigens on the T cell surface have no effect. We show that IL-2 toxin, like diphtheria toxin, must pass through an acidic vesicle in order to intoxicate target T cells. Finally, we demonstrate that the IL-2 toxin-mediated inhibition of protein synthesis in both human and murine T cells that bear the high affinity IL-2-R is due to the classic diphtheria toxin fragment A-catalyzed ADP ribosylation of elongation factor 2.

Prevention of smooth muscle cell outgrowth from human atherosclerotic plaque by a recombinant cytotoxin specific for the epidermal growth factor receptor.

Smooth muscle cell proliferation in the intima of arteries is a principal event associated with vascular narrowing after balloon angioplasty and bypass surgery. Techniques for limiting smooth muscle cell proliferation, however, have not as yet yielded any therapeutic benefit for these conditions. This may reflect the present lack of sufficiently potent and specific inhibitors of smooth muscle cell proliferation. DAB389 EGF is a genetically engineered fusion protein in which the receptor-binding domain of diphtheria toxin has been replaced by human epidermal growth factor. We evaluated the effect of this fusion toxin on human vascular smooth muscle cells in culture. Incubation of proliferating cells with DAB389 EGF yielded a dose-dependent inhibition of protein synthesis, as assessed by uptake of [3H]leucine, with an IC50 of 40 pM. The cytotoxic effect was inhibited in the presence of excess EGF or with monoclonal antibody to the EGF receptor. We further studied the effect of the fusion toxin on smooth muscle cell outgrowth from human atherosclerotic plaque. Outgrowth was markedly inhibited after as little as 1 h of exposure to the fusion protein. Furthermore, complete inhibition of proliferation of cells within the plaque could be attained. These results demonstrate that DAB389 EGF is highly cytotoxic to human smooth muscle cells proliferating in culture and can prevent smooth muscle cell outgrowth from "growth-stimulated" human atherosclerotic plaque. DAB389 EGF may therefore be of therapeutic value in vascular diseases characterized by smooth muscle cell accumulation.

Pivotal phase III trial of two dose levels of denileukin diftitox for the treatment of cutaneous T-cell lymphoma.

PURPOSE: The objective of this phase III study was to determine the efficacy, safety, and pharmacokinetics of denileukin diftitox (DAB389IL-2, Ontak [Ligand Pharmaceuticals Inc, San Diego, CA]) in patients with stage Ib to IVa cutaneous T-cell lymphoma (CTCL) who have previously received other therapeutic interventions. PATIENTS AND METHODS: Patients with biopsy-proven CTCL that expressed CD25 on > or = 20% of lymphocytes were assigned to one of two dose levels (9 or 18 microg/kg/d) of denileukin diftitox administered 5 consecutive days every 3 weeks for up to 8 cycles. Patients were monitored for toxicity and clinical efficacy, the latter assessed by changes in disease burden and quality of life measurements. Antibody levels of antidenileukin diftitox and anti-interleukin-2 and serum concentrations of denileukin diftitox were also measured. RESULTS: Overall, 30% of the 71 patients with CTCL treated with denileukin diftitox had an objective response (20% partial response; 10% complete response). The response rate and duration of response based on the time of the first dose of study drug for all responders (median of 6.9 months with a range of 2.7 to more than 46.1 months) were not statistically different between the two doses. Adverse events consisted of flu-like symptoms (fever/chills, nausea/vomiting, and myalgias/arthralgias), acute infusion-related events (hypotension, dyspnea, chest pain, and back pain), and a vascular leak syndrome (hypotension, hypoalbuminemia, edema). In addition, 61% of the patients experienced transient elevations of hepatic transaminase levels with 17% grade 3 or 4. Hypoalbuminemia occurred in 79%, including 15% with grade 3 or 4 changes. Tolerability at 9 and 18 microg/kg/d was similar, and there was no evidence of cumulative toxicity. CONCLUSION: Denileukin diftitox has been shown to be a useful and important agent in the treatment of patients whose CTCL is persistent or recurrent despite other therapeutic interventions.

Organ-specific binding of a thyrotropin-releasing hormone-diphtheria toxin complex after intravenous administration to rats.

We have previously demonstrated that a hybrid protein consisting of TRH linked to CRM45, a fragment of diphtheria toxin which lacks its native cell-binding moiety, specifically binds to TRH receptors in vitro. In this study we have examined its in vivo binding and shown that after iv injection, this complex labeled with 125I is selectively concentrated in the normal anterior pituitary and that concomitant administration of cold TRH reduces its uptake. Displaceable uptake was also demonstrated in the hypothalamus and testis, whereas nondisplaceable binding exceeding that of CRM45 alone was shown in the ovary and the breast parenchyma of lactating rats. Similar experiments with tritiated TRH were performed. We found that although there was uptake of the material by many tissues, almost all of the radioactivity was in the form of TRH degradation products. Therefore, we conclude that TRH linked to a large carrier like CRM45 may be a more revealing indicator of in vivo binding affinities than native TRH.

Genetic assembly and selective toxicity of diphtheria-toxin-related polypeptide hormone fusion proteins.

The reports of Miyanohara et al. (1986) and Murphy et al. (1986) were the first to describe the genetic construction, expression, and receptor-specific selective toxicity of a chimaeric toxin. In the present report, we have extended these earlier observations and have shown that the fusion of a modified gene encoding IL-2 to a truncated diphtheria toxin gene also results in the expression of a biologically active chimaeric IL-2 toxin. In both instances we have used receptor-binding-domain substitution and have genetically coupled those portions of the diphtheria toxin structural gene that encode the ADP-ribosyl transferase activity of fragment A and lipid-associating domains of fragment B to modified genes which encode either the polypeptide hormone alpha-MSH or the T-cell growth factor IL-2. The chimaeric toxins expressed from these gene fusions have been shown to be selectively targeted to those eukaryotic cells that carry specific surface receptors for the ligand compounds of the hybrid. For example, in the case of the IL-2 toxin, it is clear that the selective action of this hybrid protein is based upon both its diphtheria-toxin and IL-2-related components. Following binding to the IL-2R on activated and/or malignant T-cell, IL-2 toxin is internalized by receptor-mediated endocytosis. Upon acidification of the endosome, diphtheria toxin fragment B portions of the chimaeric toxin facilitate the delivery of fragment A to the cytosol where it catalyses the ADP ribosylation of EF-2. The assembly of chimaeric toxins at the level of the gene offers several advantages over chemical linkage. Since chemical linkage of the toxophore and ligand components of the conjugate toxins requires activation of the epsilon-amino moiety of lysine residues with reagents that will allow for subsequent disulphide linkage, the precise site of coupling is generally not known. In addition, there has been considerable concern over the lability of the disulphide bond between the toxophore and ligand components in vivo due to the action of disulphide reductases. The assembly of chimaeric toxins at the level of the gene allows for precise linkage of the toxophore and ligand components. Since the linkage between the toxophore and ligand is a peptide bond, the chimaeric toxin should be stable in vivo. In addition, the genetic construction of chimaeric toxins also allows for further protein engineering through site-directed mutagenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Prolongation of cardiac allograft survival in murine recipients treated with a diphtheria toxin-related interleukin-2 fusion protein.

A diphtheria toxin-related IL-2 fusion gene has been constructed that encodes a 68KD recombinant toxin in which the diphtheria toxin receptor-binding domain has been replaced with amino acids 2-133 of IL-2. This chimeric IL-2 toxin is cytotoxic for cells expressing the high-affinity IL-2 receptor but not for cells lacking this receptor. The ability of this IL-2 toxin to prolong allograft survival was examined in a murine vascularized, heterotopic heart transplant model in the strain combination B10.BR into C57B1/10. When given at a dose of 1.0 micrograms/day for 10 days, the IL-2 toxin significantly prolonged allograft survival in all recipients. CRM-45, a fragment of diphtheria toxin missing the binding domain, was ineffective, confirming the specificity of the therapy. The results demonstrate that this IL-2 toxin, which targets activated T cells expressing the IL-2 receptor, will prolong allograft survival, offering a new option for immunosuppressive therapy.

Immunotherapy of experimental autoimmune myasthenia gravis: selective effects of CTLA4Ig and synergistic combination with an IL2-diphtheria toxin fusion protein.

We examined the effects of CTLA4Ig treatment in an experimental model of myasthenia gravis (EAMG) induced by immunizing Lewis rats with purified Torpedo acetylcholine receptor (AChR). During a primary response, CTLA4Ig treatment inhibited AChR antibody production profoundly, and induced a shift of AChR antibody isotypes from the normally predominant IgG2 isotype pattern toward an IgG1 response. Challenge of rats previously treated with CTLA4Ig produced markedly lower AChR antibody responses compared to untreated controls, persistent inhibition of the IgG2b isotype, and no development of EAMG. Treatment of a secondary AChR response with CTLA4Ig or with DAB389IL2 (which kills lymphocytes expressing IL2 receptors) inhibited AChR antibody responses, and clinical EAMG moderately. In contrast, combined treatment with CTLA4Ig plus DAB389IL2 strikingly inhibited AChR antibody levels, and completely prevented EAMG. Our results suggest that the therapeutic benefit of CTLA4Ig may be due to overall inhibition of AChR antibody production as well as a shift in the antibody isotype repertoire.

Biological correlates of acute hypersensitivity events with DAB(389)IL-2 (denileukin diftitox, ONTAK) in cutaneous T-cell lymphoma: decreased frequency and severity with steroid premedication.

DAB(389)IL-2 (denileukin diftitox, ONTAK) is a cytokine-targeted fusion protein that delivers the catalytic domain of diphtheria toxin to lymphoma cells expressing the interleukin-2 receptor (IL-2R). In phase I and phase III studies of DAB(389)IL-2 in patients with cutaneous T-cell lymphoma (CTCL), non-Hodgkin's lymphoma, and Hodgkin's disease in which premedications were limited to diphenhydramine and acetaminophen, acute infusion-related hypersensitivity reactions occurred in 70% of patients and vascular leak syndrome (VLS) in 27%, resulting in discontinuation of therapy in 29% of patients. There was no correlation between the dose or half-life of DAB(389)IL-2 and the occurrence of hypersensitivity events or VLS. To explore whether steroid premedication would improve the tolerability of DAB(389)IL-2, we treated 15 patients with CTCL with either dexamethasone or prednisone prior to each dose of DAB(389)IL-2. The incidence of acute infusion events was significantly decreased, with only three patients experiencing acute infusion events (one grade 4) and only two patients developing clinically apparent VLS. Grade 3 skin rash occurred in two patients and moderately severe asthenia in nine patients. A significantly improved response rate of 60% was noted with the use of steroid premedication compared to prior studies in which steroids were prohibited. We conclude that steroid premedication significantly improves the tolerability of DAB(389)IL-2 without compromising the clinical response.

Pharmacokinetics of the recombinant fusion protein DAB486IL-2 in animal models.

The kinetics of the in vitro cytotoxicity of DAB486IL-2, a genetically engineered fusion protein containing a portion of diphtheria toxin and human interleukin-2, were examined in the C91/PL cell line, which constitutively expresses IL-2 receptors. Maximal inhibition of protein synthesis was observed by 4-6 h after DAB486IL-2 addition at a concentration of 300 ng/ml. The tissue distribution, urinary excretion, and plasma pharmacokinetics of DAB486IL-2 in the rat and its plasma pharmacokinetics in the monkey were also examined. In rats the primary site of distribution of [35S]-DAB486IL-2 outside the vasculature appears to be the liver, followed by the kidney, spleen, and lung. Persistence of radioactive material in the liver and urinary excretion of metabolic degradation products suggest that labeled protein is metabolized by hepatic tissue. Following i.v. bolus administration of DAB486IL-2, the initial serum half-life for both the rat and the monkey was approximately 5 min. The overall clearance rate of drug for the two species differed, with DAB486IL-2 being cleared from circulation 2-3 times more rapidly in the monkey. Presence of high levels of neutralizing antibodies to diphtheria toxin in the rat significantly influenced the clearance of bioactive DAB486IL-2. However, the question as to whether the presence of in vitro biological activity for the molecule is masked by the presence of antibodies cannot be clearly answered.

Fungal infections in neutropenic patients. A 8-year prospective study.

In this paper we report a eight-year prospective study designed to further characterize incidence, epidemiology, specific syndromes, treatment and prognosis associated with fungal infections in neutropenic patients. During the study period 30 fungal infections were diagnosed in 30 patients among 313 episodes of fever and neutropenia (10%). There were 15 cases of candidiasis, 5 pulmonary aspergillosis, 3 sinusitis by Aspergillus fumigatus, 5 infections by Fusarium sp., one infection by Trichosporon sp., and one infection due to Rhodotorula rubra. Blood cultures were positive in 18 cases (60%). The predisposing factors for fungal infection in multivariate analysis were the presence of central venous catheter (p < 0.001), longer duration of profound (< 100/mm3) neutropenia (p < 0.001), the use of corticosteroids (p < 0.001), gram-positive bacteremia (p = 0.002) and younger age (p = 0.03). In multivariate analysis only recovery of the neutropenia (p < 0.001) was associated with good prognosis whereas the diagnosis of infection by Fusarium sp. (p = 0.006) was strongly associated with a poor outcome. The death rate was 43%. There was no statistically significant difference in the death rate between patients who did receive (52%) or did not receive (50%) antifungal treatment. Identifying patients at risk, specific syndromes and prognostic factors may help to reduce the high mortality associated with disseminated fungal infections in neutropenic patients.

[Sézary-Baccaredda syndrome]. / Síndrome de Sézary-Baccaredda.

One case of Sezary Syndrome is presented and studied from the clinical, histopathological and hematological point of view. Clinically the patient presented besides keratodermia on the palms and a severe pruritus, an erythrodermia of very ostensive redness, like the so-called Hallopeau's red man. On the skin, by histological examination, it was observed an infiltration with some round cells, whose nucleus were hyperchromatic and irregular, as it is to be seen in the Sezary's cells. A lymph node taken from the cervical region did not show any specific infiltrate but only a dermopathic adenopathy. Lymphoid cells of folded nucleus were revealed by the hemogram, quite suggestive of Sezary's lymphoma. In the marrow, the normal cells kept on their regular proportion, but some abnormal and invasive ones stand out with their convoluted nucleus, quite suggestive of Sezary's cells. These typical Sezary's cells have been found in the skin lesions, in the blood and in the marrow. The possible origin--in the skin or in the lymph nodes, but not in the marrow--of such cells is put to a summarized discussion. Finally, the Sezary's Syndrome may be an erythrodermic or leukemic variant of "morbus fungoide". Treatment on the basis of MOPP and CHOP methods with Bleomycin association has been attempted but, so far, the results have not been satisfactory in spite of an 18 month treatment.

Systemic toxicity of diphtheria toxin-related fragments (CRM26, CRM45), a hormone-toxin hybrid protein (TRH-CRM45), and ricin A.

As a preliminary step in evaluating the use of thyrotropin releasing hormone (TRH) linked to toxin fragments for systemic treatment of pituitary disease, we have examined the metabolic degradation, tissue distribution, renal excretion, and toxicity in rats of TRH-CRM45 which consists of TRH coupled to CRM45, a diphtheria toxin-related polypeptide. For comparison, we have similarly studied CRM45, CRM26 (a smaller diphtheria toxin-related fragment), and ricin A. All four proteins were found to concentrate in the kidney and liver relative to blood (in comparison to bovine serum albumin), tissue:plasma ratios for the kidney being much higher than those observed for the liver. Radioiodinated CRM45 and ricin A were rapidly cleared from the circulation with similar patterns. Systemic toxicity studies showed that at doses greater than 2 micrograms/100 g body wt (bw), CRM45 caused a decrease in growth rate and caused renal damage. CRM45 modified with 2-pyridyldithio(propionate) groups as well as TRH-CRM45 was significantly less toxic than CRM45, as was TRH-CRM45. CRM26 had no discernible effect on the growth rate of the animals. Ricin A, at a dose of 50 micrograms/100 g bw slowed the growth rate of rats, but specific liver or kidney damage could not be detected. These findings define an upper range of doses for possible therapeutic use.

Isolation and characterization of extragenic suppressor strains of Corynebacterium diphtheriae.

The isolation and characterization of two different nonsense suppressor strains of Corynebacterium diphtheriae C7 sup+(-)tox- are described. Appropriate lysogens of these strains with corynephage beta, carrying known class II tox premature polypeptide chain termination mutations [C7sup-1(betatox-30) and C7sup-2(betatox-45)], each produce a 62,000-dalton polypeptide with nicotinamide adenine dinucleotide: elongation factor-2 adenosine diphosphate ribosyltransferase activity in addition to a chain-terminated polypeptide of 30,000 or 45,000 daltons, respectively. In addition, purified protein of 62,000 daltons, resulting from the suppression of the nonsense mutations tox-30 and tox-45, will react with antisera purified against the terminal 17,000 daltons of the toxin molecule and are immunologically identical to toxin by radial immunodiffusion. The suppression pattern of lysogenic derivatives of C7sup-1(-)tox- and C7sup-2(-)tox- with other class II and III mutants of corynephage beta was determined.

A gal region mutant that requires cAMP for growth on galactose in an adenyl cyclase negative (cya delta) background.

Strains of Escherichia coli K12 that contain a deletion of the adenyl cyclase gen (cya delta), required for the synthesis of cyclic adenosine-3';5' monophosphate (cAMP), grow on galactose-containing minimal medium. A mutant was isolated that grows on this medium only if cAMP is added. The mutation (designated galP20) is linked to the gal operon region as determined by both generalized transduction with bacteriophage P1 and specialized transduction with bacteriophage lambda. Studies with galP20 cya delta strains as well as gal delta (deletions of the gal operon) cya delta strains indicate that synthesis of the physiologically important transport mechanism for galactose (galactose permease) requires either cAMP or a function mission from both the galdelta strains and the galP20 strain.

Determination of Corynebacterium diphtheriae toxigenicity by a colorimetric tissue culture assay.

Chinese hamster ovary (CHO) cell cultures in microtiter wells are sensitive to growth inhibition and killing by picogram quantities of diphtheria toxin. In the absence of biologically active toxin, the CHO cell culture produces sufficient acidic metabolites to change the phenol red pH indicator from pink to yellow within 56 h. In the presence of 10 pg of toxin per well, growth inhibition can be observed microscopically within 24 h. Diphtheria toxin can be qualitatively assayed from culture supernatants of Corynebacterium diphtheriae or from beta-phage agar plaque plugs. The colorimetric CHO cell assay method for determining toxigenicity allows for the large-scale screening of either diphtheria toxigenicity or antitoxin titration of sera.

Toxicity of diphtheria toxin-related proteins produced by suppression of nonsense mutations.

The Mr = 62,000 diphtheria toxin-related proteins produced from the suppression of nonsense mutations within the tox gene of corynephage beta were purified by affinity chromatography. Except for the toxin 111-sup2-62, the Mr = 62,000 polypeptides were found to have the same specific toxicity as does wild type toxin. 111-sup2-62 was found to have a prolonged lag period prior to the onset of inhibition of protein synthesis and ADP-ribosylation of elongation factor 2. 111-sup2-62 differs from wild type toxin by an amino acid substitution at a site approximatley 47,000 daltons from the NH2 terminus. The data presented provide genetic support for the Boquet-Pappenheimer model (Boquet, P., and Pappenheimer, A. M. Jr (1978) J. Biol. Chem. 251, 5770-5778) of fragment A translocation into the eukaryotic cell cytosol.

Thyrotropin-releasing hormone-diphtheria toxin-related polypeptide conjugates. Potential role of the hydrophobic domain in toxin entry.

We have separately coupled a modified thyrotropin-releasing hormone (TRH) molecule to two diphtheria toxin-related polypeptides, CRM26 and CRM45. Both polypeptides are enzymatically active but only CRM45 contains the hydrophobic domain of the toxin molecule. The TRH-CRM45 conjugate caused a 50% inhibition of protein synthesis in GH3 rat pituitary cells at 3 X 10(-9) M. CRM45 alone was 200 to 500 times less toxic than the conjugate. Intoxication by TRH-CRM45 was prevented by excess TRH, preincubation with diphtheria antitoxin, or reduction of the disulfide cross-link. In addition, TRH-CRM45 was no more toxic than CRM45 itself to 3T3 cells which lack TRH receptors. In contrast, TRH-CRM26, although it retained enzymatic activity, was nontoxic for GH3 cells even at 10(-7) M. Binding experiments showed that both conjugates compete for TRH receptors with comparable affinities. The potential role of the hydrophobic domain in toxin entry is discussed.

Suppression of immunopathology in schistosomiasis by interleukin-2-targeted fusion toxin, DAB389IL-2. I. Studies of in vitro and in vivo efficacy.

Schistosomiasis causes pathology in an estimated 200 million individuals. Clinical disease is caused by a complex immunopathologic response to the parasite ova, which are deposited in the host tissues. This immunopathologic response is caused by T lymphocytes which express the high-affinity IL-2 receptor (IL-2R). DAB389IL-2 is a diphtheria toxin-IL-2 fusion toxin protein which functionally inactivates or kills cells which bear the high-affinity IL-2R. DAB389IL-2 has been used in man to suppress IL-2R-dependent immune reactivity. Therefore, we reasoned that DAB389IL-2 might suppress immunopathology in schistosomiasis. In these studies we assessed the in vitro and in vivo effects of DAB389IL-2 on the development of immunopathology in murine schistosomiasis. DAB389IL-2 suppressed IL-2, lectin mitogen (Con A), and soluble Schistosoma mansoni egg antigen-induced lymphocyte proliferation and in vitro granuloma formation. In addition, DA-B389IL-2 suppressed in vitro IL-2R expression. DA-B389IL-2 also suppressed the development of granulomas and collagen deposition in vivo in the livers of infected animals. Therefore, DAB389IL-2 may have potential for the targeted reduction of immunopathology due to schistosomiasis in man.

Anti-arthritic effects demonstrated by an interleukin-2 receptor-targeted cytotoxin (DAB486IL-2) in rat adjuvant arthritis.

DAB486IL-2 is an interleukin-2 receptor-specific cytotoxin which selectively targets and kills cells which bear the high-affinity form of the IL-2 receptor. Since elimination of activated T lymphocytes may be useful in the treatment of rheumatoid arthritis, the effect of DAB486IL-2 treatment in an animal model of arthritis was investigated. We demonstrated that rats treated with DAB486IL-2 during the induction phase of disease have delayed onset of symptoms and significantly reduced severity of inflammation as well as a depressed proliferative response to mycobacterial stimulation in vitro. In addition, the presence of preexisting antibodies to the molecule had no impact on the anti-arthritic effects observed in this model. These data suggest that DAB486IL-2 may have therapeutic potential in the treatment of rheumatoid arthritis.

Epidermal growth factor receptor-targeted cytotoxin inhibits neointimal hyperplasia in vivo. Results of local versus systemic administration.

Smooth muscle cell accumulation is a key feature of restenosis that may be inhibited by the delivery of receptor-targeted cytotoxins. DAB389EGF is a recombinant fusion protein in which the receptor-binding domain of diphtheria toxin has been replaced by human epidermal growth factor (EGF). We investigated the effectiveness of DAB389EGF to inhibit neointimal hyperplasia in the balloon-injured rat carotid artery. Incubation of rat carotid arteries with 125I-labeled EGF revealed extensive EGF binding sites in the neointima of balloon-injured arteries. Sixty rats subsequently received either saline or DAB389EGF (total dose, 0.15 mg) delivered immediately following balloon injury either systemically, via 14-day continuous osmotic pump infusion, or locally, via 30-minute intraluminal incubation. The effect of both treatment strategies was measured 2 weeks after injury by cross-sectional morphometric analysis of intimal area, the ratio of intimal/medial area (I/M), and the percent luminal narrowing (%LN). In addition, proliferative activity was assessed by immunostaining for the presence of the proliferating cell nuclear antigen (PCNA). Compared with controls, systemic delivery of fusion toxin significantly reduced intimal area, I/M, and %LN by 40%, 40%, and 29%, respectively. However, these rats exhibited 2% weight loss, indicating mild systemic toxicity. Local, intraluminal administration of DAB389EGF yielded a more pronounced reduction in intimal area, I/M, and %LN by 74%, 79%, and 72%, respectively. This inhibitory effect was preserved at 3 weeks postinjury, and PCNA immunostaining of locally treated arteries revealed a virtual absence of proliferative activity in the neointima and media at this timepoint. In contrast to systemically treated rats, rats receiving fusion toxin locally gained weight at a rate similar to controls, indicating avoidance of systemic toxicity. We conclude that DAB389EGF is a potent inhibitor of neointimal hyperplasia in vivo and that whereas an inhibitory effect may be achieved by systemic delivery, local delivery appears to be more potent, avoids systemic toxicity, and thus represents a feasible strategy to preempt restenosis.