Receptor-binding Domain Of Diphtheria Toxin Research Articles

#1603 A potent protein inhibitor of HB-EGF protects kidneys from glomerular lesions in a pig model of crescentic glomerulonephritis

Abstract Background and Aims Crescentic glomerulonephritis (CGN) is a severe disease that rapidly destroys the glomerular architecture. CGN is due to immuno-inflammatory aggressions triggered by anti-neutrophil cytoplasmic autoantibody-associated vasculitides, anti-glomerular basement membrane (GBM) autoantibodies, or immune complexes deposition as occurs in systemic lupus erythematosus, IgA nephropathy or infections. Treatments are based on aggressive immunosuppression. Although required, immunosuppression doesn't directly target the deleterious glomerular response to inflammation and leads to significant morbidity. Moreover, most patients still develop chronic or end-stage kidney failure, indicating that complementary strategies are needed. We showed previously that local over-expression of the growth factor HB-EGF (Heparin-binding EGF-like growth factor) and subsequent activation of EGFR lead to dedifferentiation and proliferation of parietal epithelial cells and podocytes, leading to crescentic lesions that irreversibly obliterate the glomeruli. We aimed to evaluate the therapeutic potential of a protein inhibitor of HB-EGF in a pig model of CGN. Method We engineered a protein inhibitor of HB-EGF, DTR8, from the receptor-binding domain of diphtheria toxin, a highly selective natural ligand of HB-EGF. Twelve mutations improved solubility, led to pM affinity, and reduced immunogenicity and antigenicity. Pigs received decomplemented nephrotoxic serum (NTS) from sheep immunized against pig GBM to mimic anti-GBM antibodies disease responsible for one form of CGN. Ten pigs were treated with 0.3 mg/kg of DTR8 twice a day by intramuscular route for 20 days, and 12 pigs received vehicle only starting 6 hours after NTS. Animals were sacrificed on day 21, and blood, urine and kidneys were analyzed. Five animals that had received pre-immune sheep serum served as controls. Results Pigs developed a nephritic syndrome with high proteinuria, hypoalbuminemia, kidney failure with increased BUN and blood creatinine levels, anasarca and crescentic lesions typical of CGN along with de novo HB-EGF glomerular overexpression. The vehicle-treated group had only 24.8% of histologically normal glomeruli, while the DTR8-treated group had 57.3% of healthy glomeruli. DTR8 treatment significantly reduced the percentage of crescentic lesions (P = .0365) and preserved podocyte differentiation as assessed by the expression of nephrin that was markedly reduced in vehicle-treated animals but maintained close to normal in DTR8-treated animals (P = .0028). DTR8 administration reduced EGFR phosphorylation in glomerular cells (P = .0161), suggesting local inhibition of the HB-EGF/EGFR cascade. Lymphocyte infiltration was reduced (P = .2543). Notably, the treatment group also displayed a global reduction in interstitial fibrosis (P = .0313). Conclusion The therapeutic protein candidate DTR8 protects kidney glomeruli in a CGN model in pigs.

Receptor-Mediated Enhanced Cellular Delivery of Nanoparticles Using Recombinant Receptor-Binding Domain of Diphtheria Toxin

Antibodies and peptides are often used to home nanoparticles (NPs) to specific cells. Here in this work, we have used recombinant receptor-binding domain of diphtheria toxin (RDT) as a homing molecule for NPs. Diphtheria toxin binds to heparin binding EGF-like growth factor (HB-EGF) through its receptor-binding domain. HB-EGF is often overexpressed as cell surface molecule in various types of cancer. We have prepared monodispersed, spherical PLGA NPs and coated these NPs with RDT. These NPs are characterized by FESEM and FT-IR spectroscopy. Using flow cytometry and fluorescence spectroscopy, we show that coating with RDT increases cellular uptake of PLGA NPs. We further show that RDT-coated nanoparticles are internalized through clathrin-dependent receptor-mediated endocytosis that can be reduced by specific inhibitor. These RDT-coated nanoparticles (RDT-NP) were further used for preferential delivery of Irinotecan, a chemotherapeutic agent, to cells overexpressing HB-EGF. We show that receptor-mediated enhanced uptake of RDT-NPs increases the potency of irinotecan in these cells.

Recombinant Receptor-Binding Domain of Diphtheria Toxin Increases the Potency of Curcumin by Enhancing Cellular Uptake

Diphtheria toxin (DT) binds to a specific cell surface receptor, gets internalized, and causes cytotoxicity through its catalytic domain. The toxicity of DT is used in several therapeutic molecules. Here, we have exploited the receptor-binding ability of DT to increase cellular uptake of curcumin, a hydrophobic molecule with low bioavailability and cellular uptake. We have expressed only the receptor-binding domain of DT (RDT) in Escherichia coli. Purified RDT binds to the receptor with an affinity equivalent to that of full-length DT. It also binds to curcumin forming a curcumin-RDT complex, and this increases the fluorescence intensity and fluorescence lifetime of curcumin. The curcumin-RDT complex binds to the receptor and associates with human glioblastoma cells (U-87 MG) expressing the receptor. The cellular uptake of curcumin is higher for the curcumin-RDT complex than curcumin alone. This increase in uptake enhances the antiproliferative effect of curcumin and induces apoptosis of these cells even at a lower dose.

Identification of a Human Monoclonal Antibody To Replace Equine Diphtheria Antitoxin for Treatment of Diphtheria Intoxication

Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 μg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.

22. Engineering of an HB-EGF Inhibitor from the Diphtheria Toxin Receptor–Binding Domain for the Treatment of HB-EGF-Related Diseases

22. Engineering of an HB-EGF Inhibitor from the Diphtheria Toxin Receptor–Binding Domain for the Treatment of HB-EGF-Related Diseases

Changing the Receptor Specificity of Anthrax Toxin

ABSTRACTThe actions of many bacterial toxins depend on their ability to bind to one or more cell-surface receptors. Anthrax toxin acts by a sequence of events that begins when the protective-antigen (PA) moiety of the toxin binds to either one of two cell-surface proteins, ANTXR1 and ANTXR2, and is proteolytically activated. The activated PA self-associates to form oligomeric pore precursors, which, in turn, bind the enzymatic moieties of the toxin and transport them to the cytosol. We introduced a double mutation into domain 4 of PA to ablate its native receptor-binding function and fused epidermal growth factor (EGF) to the C terminus of the mutated protein. The resulting fusion protein transported enzymatic effector proteins into a cell line that expressed the EGF receptor (A431 cells), but not into a line lacking this receptor (CHO-K1 cells). Addition of excess free EGF blocked transport of effector proteins into A431 cells via the fusion protein, but not via native PA. We also showed that fusing the diphtheria toxin receptor-binding domain to the C terminus of the mutated PA channeled effector-protein transport through the diphtheria toxin receptor. PA fusion proteins with altered receptor specificity may be useful in biological research and could have practical applications, including ablation or perturbation of selected populations of cells in vivo.

Structural determinants for membrane insertion, pore formation and translocation ofClostridium difficiletoxin B

Clostridium difficile toxins A and B bind to eukaryotic target cells, are endocytosed and then deliver their N-terminal glucosyltransferase domain after processing into the cytosol. Whereas glucosyltransferase, autoprocessing and cell-binding domains are well defined, structural features involved in toxin delivery are unknown. Here, we studied structural determinants that define membrane insertion, pore formation and translocation of toxin B. Deletion analyses revealed that a large region, covering amino acids 1501-1753 of toxin B, is dispensable for cytotoxicity in Vero cells. Accordingly, a chimeric toxin, consisting of amino acids 1-1550 and the receptor-binding domain of diphtheria toxin, caused cytotoxic effects. A large N-terminal part of toxin B (amino acids 1-829) was not essential for pore formation (measured by (86) Rb(+) release in mammalian cells). Studies using C-terminal truncation fragments of toxin B showed that amino acid residues 1-990 were still capable of inducing fluorescence dye release from large lipid vesicles and led to increased electrical conductance in black lipid membranes. Thereby, we define the minimal pore-forming region of toxin B within amino acid residues 830 and 990. Moreover, we identify within this region a crucial role of the amino acid pair glutamate-970 and glutamate-976 in pore formation of toxin B.

DAB389IL-2 (Denileukin Diftitox, ONTAK): A New Fusion Protein Technology

Genetic engineering has led to the development of fusion protein toxins, which are targeted effector molecules that combine a targeting ligand such as a growth factor with a cytocidal moiety such as a plant or bacterial toxin. The first genetically constructed family of fusion proteins used diphtheria toxin as the toxophore for receptor-binding domain substitution. Diphtheria toxin consists of three domains: an enzymatically active adenosine diphosphate (ADP) ribosyltransferase domain, a hydrophobic transmembrane domain, and a C-terminal receptor-binding domain. Introduction of the enzymatically active domain into the cytosol via receptor-mediated endocytosis results in inhibition of protein synthesis by ADP ribosylation of elongation-factor 2. DAB486IL-2, in which the native receptor-binding domain of diphtheria toxin was replaced with the full-length interleukin-2 (IL-2) gene, was capable of intoxicating high-affinity IL-2 receptor-bearing cells in vitro with an IC50 of 10−10 M; whereas, cells lacking the full component of the high-affinity IL-2 receptor (p55, p75, p64) were relatively resistant (IC50 of 10−8 M). Because of a short in vivo half-life of DAB486IL-2, efforts to reengineer the molecule were undertaken, leading to the DAB389IL-2 construct, which had a twofold to threefold higher Kd than DAB486IL-2 and a longer half-life, resulting in a tenfold increase in potency. Thus far, the clinical activity of both IL-2 chimeric fusion toxins has been similar, with the DAB389IL-2 molecule displaying more favorable pharmacokinetics.

Receptor-mediated uptake of an extracellular Bcl-x(L) fusion protein inhibits apoptosis.

Bcl-x(L), a member of the Bcl-2 family, inhibits many pathways of apoptosis when overexpressed in the cell cytosol. We examined the capacity of Bcl-x(L) fusion proteins to bind cells from the outside and block apoptosis. Full-length Bcl-x(L) protein at micromolar concentrations did not affect apoptosis when added to cell media. To increase uptake by cells, Bcl-x(L) was fused to the receptor-binding domain of diphtheria toxin (DTR). The Bcl-x(L)-DTR fusion protein blocked apoptosis induced by staurosporine, gamma-irradiation, and poliovirus in a variety of cell types when added to media. The potency of inhibition of poliovirus-induced apoptosis by Bcl-x(L)-DTR was greater than that of strong caspase inhibitors. Brefeldin A, an inhibitor of vesicular traffic between the endoplasmic reticulum and Golgi apparatus, prevented the Bcl-x(L)-DTR blockade of apoptosis induced by staurosporine, suggesting that Bcl-x(L)-DTR must be endocytosed and reach intracellular compartments for activity. Many diseases are caused by overexpression or underexpression of Bcl-x(L) homologues. Extracellular delivery of Bcl-2 family member proteins may have a wide range of uses in promoting or preventing cell death.

Production of recombinant DTctGMCSF fusion toxin in a baculovirus expression vector system for biotherapy of GMCSF-receptor positive hematologic malignancies.

The fusion toxin DTctGMCSF has been constructed by genetically replacing the native receptor-binding domain of diphtheria toxin (DT) with human granulocyte-macrophage colony stimulating factor (GMCSF). This recombinant fusion toxin preserves the catalytic (c) and membrane translocation (t) domains of DT and includes a sterically neutral peptide linker separating the toxin and growth factor domains. Previous work using DTctGMCSF produced in Escherichia coli has shown that this chimeric toxin is selectively cytotoxic to GMCSF receptor (R)-positive acute myeloid leukemia (AML) cells both in vitro and in vivo. Its clinical development has been hampered due to very low expression levels, requirements for solubilization with guanidine hydrochloride and subsequent refolding, and concerns about bacterial endotoxin contamination. These difficulties prompted us to investigate the utility of a baculovirus/insect cell expression system for the production of DTctGMCSF. Here, we report that a soluble form of DTctGMCSF can be produced in the baculovirus expression vector system (BEVS) and purified to homogeneity by column chromatography. The BEVS-derived DTctGMCSF fusion toxin caused apoptotic death in GMCSF-R-positive human AML cells at nanomolar concentrations. In contrast to the 100 microg/L yields of purified DTctGMCSF obtained from E. coli, the BEVS allows us to routinely generate 8-10 mg/L of purified DTctGMCSF. This increased capacity provided by the BEVS for the production of DTctGMCSF makes it now possible to obtain sufficient quantities to carry out preclinical and clinical trials. To our knowledge, this is the first report of the successful utilization of the BEVS for producing a therapeutic fusion toxin.

Cytotoxicity and specificity of directed toxins composed of diphtheria toxin and the EGF-like domain of heregulin beta1.

As a step in the design of directed toxins, aimed at cells that overexpress HER receptors, particularly breast carcinoma cells, we studied the properties of a chimera of diphtheria toxin (DT) and heregulin beta1. The EGF-like growth hormone heregulin is a ligand for the HER3 and HER4 receptors and their heterodimers with HER2. The 60-residue EGF-like domain (hrg) of heregulin elicits a biological response and binds to these receptors primarily through its N terminus. We tested a fusion protein in which hrg replaces the C-terminal receptor-binding domain of DT (DT(389)hrg) and an alternative design in which this domain is fused to the N terminus of DT(389). Of those two constructs, the N-terminal fusion was not active as a directed toxin but elicited a growth response. The C-terminal fusion of hrg to DT(389) yielded a functional toxin and showed cell line specific cytotoxicity that is consistent with heregulin specificity. The binding of hrg to its cognate receptor is not impaired as shown by receptor activation, direct binding, and competition with free hrg. Cytotoxicity is dependent on high-affinity binding of DT(389)hrg to HER3 and HER4 receptors and is not mediated by HER2 overexpression alone. For those cell lines exhibiting high-affinity binding sites, the level of cytotoxicity correlates with the rate of internalization. Thus DT(389)hrg chimeras offer a possible avenue toward directed toxins against cells that overexpress HER receptors.

Towards a recombinant vaccine against diphtheria toxin.

Two recombinant fragments of diphtheria toxin (DT) were fused to an engineered tandem repeat of the immunoglobulin (Ig) binding domain of protein A, called ZZ. These fragments are (i) the receptor binding domain (DTR), which comprises amino acids 382 to 535 of DT, and (ii) a linear peptide (DT(168-220)) which comprises residues 168 to 220 of the loop between fragment A and fragment B of DT. The fusion proteins were produced in Escherichia coli and purified by affinity chromatography. In vitro experiments showed that the DTR domain is responsible for the capacity of ZZ-DTR to bind to Vero cells and is capable of inhibiting the cytotoxicity of DT for these cells. These findings suggest that DTR binds to the cell surface receptors of DT and hence adopts a conformation that is similar to that of the receptor binding domain of DT. We compared the capacities of ZZ-DTR, ZZ-DT(168-220), and a chemically detoxified form of DT currently used for vaccination to elicit antibodies in rabbits. The toxoid was more immunogenic than ZZ-DT(168-220), which in turn was more immunogenic than ZZ-DTR. However, ZZ-DT(168-220) antiserum was poorly efficient at neutralizing DT cytotoxicity on Vero cells, whereas ZZ-DTR antiserum was only 15-fold less potent than anti-DT antisera.

Crystal Structure of the Complex of Diphtheria Toxin with an Extracellular Fragment of Its Receptor

We describe the crystal structure at 2.65 Å resolution of diphtheria toxin (DT) complexed 1:1 with a fragment of its cell-surface receptor, the precursor of heparin-binding epidermal-growth-factor-like growth factor (HBEGF). HBEGF in the complex has the typical EGF-like fold and packs its principal β hairpin against the face of a β sheet in the receptor-binding domain of DT. The interface has a predominantly hydrophobic core, and polar interactions are formed at the periphery. The structure of the complex suggests that part of the membrane anchor of the receptor can interact with a hinge region of DT. The toxin molecule is thereby induced to form an open conformation conducive to membrane insertion. The structure provides a basis for altering the binding specificity of the toxin, and may also serve as a model for other EGF–receptor interactions.

Structure-Function Analysis of the Diphtheria Toxin Receptor Toxin Binding Site by Site-directed Mutagenesis

Diphtheria toxin (DT) binds to the epidermal growth factor (EGF)-like domain of human membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF), the human DT receptor (DTR). DT does not bind to mouse proHB-EGF because of amino acid substitutions within the EGF-like domain. We made 10 independent mutants, replacing a single amino acid within the EGF-like domain of human DTR/proHB-EGF with the corresponding amino acid residue in mouse proHB-EGF. The mutant proteins were transiently expressed in mouse L cells either expressing or not expressing DRAP27/CD9, and DT binding was measured. DT binding activity of GST fusion proteins containing the mutated EGF-like domain was also determined by a cell-free binding assay. The largest effect was seen with E141H, and second largest effects were seen with F115Y and L127F in all of the assay systems. We conclude that Phe115, Leu127, and Glu141 are critical amino acid residues for DT binding. A computer model of the tertiary structure of the EGF-like domain of human DTR/proHB-EGF was made. The model predicts that three amino acid residues critical for DT binding activity, Phe115, Leu127, and Glu141, are all located on the same face of the EGF-like domain, suggesting that this face of DTR/proHB-EGF interacts with the receptor-binding domain of DT.

Characterization and receptor specific toxicity of two diphtheria toxin-related interleukin-3 fusion proteins DAB 389–mIL-3 and DAB 389–(Gly 4Ser) 2-mIL-3

We have constructed two fusion proteins, DAB 389–mIL-3 and DAB 389-(Gly 4Ser) 2–mIL-3, in which the receptor-binding domain of diphtheria toxin is replaced by mouse interleukin-3 (IL-3). Cytotoxic activity of the fusion toxins was observed on three out of six cell lines assayed. This toxicity was mediated through binding to the IL-3 receptor as it was inhibited in a dose-dependent manner with murine IL-3 or anti-IL-3 neutralizing antibodies. DAB 389–(Gly 4Ser) 2-mIL-3 was up to 5 times more toxic than DAB 389–mIL-3, depending on the cell line (0.8×10 −10 M<IC 50<3×10 −10 M). These proteins can be used for the detection of IL-3 receptors on mouse cells and should allow for the selective elimination of IL-3 receptor-positive pluripotent hematopoietic stem cells prior to bone marrow transplantation.

Synthesis, cytotoxic properties and effects on early and late gene induction of a chimeric diphtheria toxin-leukemia-inhibitory factor protein.

Leukemia-inhibitory factor (LIF) is a neuropoietin able to regulate the differentiation and the survival of many cell types, which include some neuronal populations. The present study describes the genetic construction, expression, purification and properties of a diphtheria-toxin-related LIF gene fusion in which the native receptor-binding domain of diphtheria toxin was replaced with a gene encoding human LIF. The fusion protein expressed from the chimeric tox gene was designated DT-(1-389)-LIF-(2-184)-peptide. This fusion protein has a deduced molecular mass of 65980 Da and is formed by fusion of the first 389 amino acids of diphtheria toxin to amino acids 2-184 of mature human LIF, using a linker of 34 amino acids that includes six consecutive histidine residues. The latter span allows for single-step purification of the fusion protein by Ni(2+)-resin affinity chromatography. This linker provides a high degree of flexibility between the diphtheria toxin and LIF domains, thereby permitting aggregation-free refolding of the chimeric protein while bound to the affinity column. Both LIF and DT-(1-389)-LIF-(2-184)-peptide induced the phosphorylation of CLIP1 and CLIP2 in LIF-responsive neuroblastoma SH-N-BE cells. DT-(1-389)-LIF-(2-184)-peptide was selectively cytotoxic for cultured neuroblastoma cells bearing the LIF receptor, and for sympathetic neurons. The cytotoxic action of DT-(1-389)-LIF-(2-184)-peptide, like that of native diphtheria toxin, required receptor-mediated endocytosis, passage through an acidic compartment, and delivery of an ADP-ribosyltransferase to the cytosol of target cells. The latter point was confirmed by the fact that, while both LIF and DT-(1-389)-LIF-(2-184)-peptide increased c-fos mRNA expression in SH-N-BE cells, only LIF induced proenkephalin and c-fos promoter activities in cells transiently transfected with c-fos-chloramphenicol acetyltransferase and proenkephalin-chloramphenicol acetyltransferase fusion genes. Mutational analysis suggested that the C-terminal helix (helix D) of human LIF may, in part, constitute or contribute to the active site for LIF receptor binding and cell activation. The cytotoxic properties of DT-(1-389)-LIF-(2-184)-peptide may be useful in selectively depleting neuronal and immune cell populations that express the LIF beta receptor.

Epidermal Growth Factor Receptor–Targeted Cytotoxin Inhibits Neointimal Hyperplasia In Vivo

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.

Epidermal Growth Factor Receptor–Targeted Cytotoxin Inhibits Neointimal Hyperplasia In Vivo

Abstract Smooth muscle cell accumulation is a key feature of restenosis that may be inhibited by the delivery of receptor-targeted cytotoxins. DAB 389 EGF 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 DAB 389 EGF to inhibit neointimal hyperplasia in the balloon-injured rat carotid artery. Incubation of rat carotid arteries with 125 I-labeled EGF revealed extensive EGF binding sites in the neointima of balloon-injured arteries. Sixty rats subsequently received either saline or DAB 389 EGF (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 DAB 389 EGF 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 DAB 389 EGF 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.

Interleukin-4 receptor expression by human B cells: Functional analysis with a human interleukin-4 toxin, DAB 389IL-4

Background: Studies of human IgE-secreting B cells have proven difficult because of the small size of this population. We have used an interleukin-4 (IL-4) fusion toxin to detect functionally IL-4 receptor (IL-4R) expression on B cells involved in IgE synthesis. Methods: In diphtheria toxin IL-4 (DAB 389IL-4) the receptor-binding domain of diphtheria toxin has been replaced with human IL-4. DAB 389IL-4 cytotoxicity depends on IL-4R binding and internalization. Results: Addition of DAB 389IL-4 inhibited IgE synthesis induced by IL-4 + anti-CD40 monoclonal antibody or hydrocortisone. IgE inhibition resulted from DAB 389IL-4 B-cell cytotoxicity because DAB 389IL-4 inhibited IL-4–independent B-cell proliferation. Thus induction of human IgE synthesis involves IL-4R + cells. In contrast, terminally differentiated, IgE-producing B cells no longer express functional IL-4R because DAB 389IL-4 only modestly inhibited ongoing IgE synthesis by B cells from patients with hyper-IgE states and only minimally affected IL-4–induced IgE synthesis in normal B cells when the toxin was added at day 7. Pokeweed mitogen–induced IgM synthesis was sensitive to early but not to late addition of DAB 389IL-4. Thus the loss of functional IL-4R immunoglobulin-secreting B cells is independent of isotype switching. Conclusions: IgE-secreting B cells no longer express functional IL-4R. Therapies for allergic disease that target the IL-4R would not affect IgE-secreting B cells but may block the recruitment of B cells into the IgE-secreting pool. For optimal benefits this approach may be combined with therapies that target IL-4R -, IgE-secreting B cells. (J A LLERGY C LIN I MMUNOL 1995;95:893-900.)

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.