Recombinant Toxins as Novel Therapeutic Agents

Pastan, Ira; Chaudhary, Vijay; FitzGerald, David J.

doi:10.1146/annurev.bi.61.070192.001555

Cited by 306 publications

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“…Immunotoxins must be internalized upon binding to their receptor in order to kill cells (Pastan et al, 1992). As we have shown above (Figure 2), AG 1478 treatment inhibits the activation-induced downregulation of the EGFRvIII by the Cbl proteins.…”

Section: The Cytotoxicity Of An Egfrviii-specific Immunotoxin Is Antamentioning

confidence: 83%

“…The IC 50 of MR1-1(scFv)-PE38 in this study (approximately 10 ng/ml) is similar to previously reported values . To function, immunotoxins must be internalized upon binding to their receptors (Pastan et al, 1992); indeed anti-EGFRvIII monoclonal antibodies -including MR1-1(scFv)-PE38 -are rapidly internalized by EGFRvIII-expressing cells (Reist et al, 1995;Kuan et al, 2000). These internalized antibodies become localized to vesicles in the perinuclear Golgi region and are rapidly catabolized, suggesting that the internalized EGFRvIII:monoclonal antibody complex is trafficked to the lysosome.…”

Section: Discussionmentioning

confidence: 99%

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EGFRvIII undergoes activation-dependent downregulation mediated by the Cbl proteins

et al. 2006

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The overexpression or mutation of tyrosine kinases (TKs), such as the epidermal growth factor receptor (EGFR), can lead to the development of cancer. The most common mutation of the EGFR in glioblastomas is the deletion of exons 2-7 known as the EGFRvIII. This mutant receptor cannot bind EGF but, instead, is constitutively active. The Cbl family of ubiquitin ligases (Cbl, Cbl-b, and Cbl-c) targets the activated EGFR for degradation. As the EGFRvIII is transforming, we investigated whether it could be downregulated by the Cbl proteins. The over-expression of all three Cbl proteins resulted in the ubiquitination and degradation of the EGFRvIII. As with the wild-type EGFR, the TK-binding domain and the RING finger of Cbl-b are sufficient for the downregulation of the EGFRvIII. Also, we found that Cbl-b is recruited to the EGFRvIII and inhibits the transformation of NIH 3T3 cells by the EGFRvIII. Mutation of the Cbl-binding site (Y1045F) in the EGFRvIII inhibits its ubiquitination and downregulation by Cbl-b and enhances its ability to transform. Furthermore, the EGFR TK inhibitor, AG 1478, prevents the downregulation of the EGFRvIII by the Cbl proteins and antagonizes the ability of an immunotoxin directed against the EGFRvIII to kill cells expressing this receptor. In conclusion, the EGFR-vIII does not transform by escaping regulation by Cbl proteins and this activation-induced downregulation of the EGFRvIII has an important role in mediating the toxicity of anti-EGFRvIII immunotoxins.

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Section: The Cytotoxicity Of An Egfrviii-specific Immunotoxin Is Antamentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

EGFRvIII undergoes activation-dependent downregulation mediated by the Cbl proteins

et al. 2006

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“…Knowledge about how this watersoluble protein crosses lipid bilayers may provide insight into the general principles of protein transport across biological membranes. Another active area of DT research is the production of immunotoxins, in which DT is linked to antibodies or other ligands that recognize cells targeted for destruction, such as cancer cells (Pastan et al, 1992). These areas of research depend on knowing the 3-dimensional structure of DT at atomic resolution.…”

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confidence: 99%

Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

1994

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The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections ( F > l o ( F ) ) , yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp.The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.Keywords: ADP-ribosyltransferase; diphtheria; membrane insertion Diphtheria toxin (DT) is secreted from toxic strains of the bacterium Corynebacterium diphtheriae. Toxigenic conversion of C. diphtheriae occurs by lysogenization with corynephage 0 (Freeman, 1951), which carries the DT gene encoding a 535-residue protein (M, 58,342) (Uchida et al., 1971; Greenfield et al., 1983). DT kills eukaryotic cells by inactivating an essential component of the protein translation machinery, elongation factor 2 (EF-2) (Collier, 1975).DT is produced as a protomer of 2 fragments connected by a loop containing a proteolysis site and a disulfide bond. Limited proteolysis with trypsin and reduction of the disulfide separates the 2 fragments, which are denoted fragment A and fragment B (Drazin et al., 1971). Fragment A consists of an independent folding domain, the catalytic (C) domain (residues 1-190) (Choe et al., 1992). Fragment B consists of 2 folding domains, the transmembrane (T) domain (residues 191-378) and receptor-binding (R) domain (residues 379-535) (Choe et al., 1992).

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“…Immunotoxins (ITs) are anti-cancer agents consisting of a binding antibody and a toxin. 7,8 The toxin and antibody are either covalently linked via a chemical linker or generated by recombinant DNA fusion technology.That anti-CD30 -toxin chemical conjugates can be used to eliminate HRS cells has been established in several studies in vitro 9 -11 as well as in vivo. 12,13 In a clinical trial, an IT containing the anti-CD30 MAb Ber-H2 and saporin-6 demonstrated some efficacy.…”

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confidence: 99%

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Isolation of new anti-CD30 scFvs from DNA-immunized mice by phage display and biologic activity of recombinant immunotoxins produced by fusion with truncatedpseudomonas exotoxin

et al. 2001

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To target CD30 on Hodgkin's disease and anaplastic largecell lymphoma, anti-CD30 single-chain antibodies were obtained by DNA immunization of mice with the complete human CD30 cDNA. Spleens were isolated from mice with high anti-CD30 titer, and the RNA was used for the production of an scFv-displaying phage library. Specific phages were enriched by 3 rounds of panning on soluble CD30 or CD30 ؉ K562 cells. Recombinant immunotoxins (rITs) were made from 3 ELISA-positive scFv phages by fusion to a 38 kDa truncated mutant of Pseudomonas exotoxin (PE38) with or without a KDEL mutant sequence at the C terminus. In vitro cytotoxicity of purified anti-CD30 rITs was measured on CD30-transfected A431 cells. IC 50 values ranged from 3 to 7 ng/ml (50 -110 pM) for PE38 rITs and 0.1 ng/ml (2 pM) for the PE38-KDEL IT on A431-CD30 cells. The parental A431 cells were resistant, indicating that the cytotoxicity was specific and CD30-mediated. rITs were tested for anti-tumor activity in a nude mouse model. Most newly diagnosed patients with adult Hodgkin's disease are curable with modern radiation therapy and/or combination chemotherapy regimens. 1,2 Nevertheless, about 20% of Hodgkin's disease patients overall will die from their disease, 3 and about 19% of patients who are cured will develop second malignancies within 15 years after treatment. Therefore, new therapeutic agents with greater selectivity for malignant Reed-Sternberg cells (HRS) are still needed. HRS cells consistently express high numbers of CD30 antigens, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. 4 Tumors from patients with nonHodgkin's lymphoma (NHL) are often CD30 ϩ , particularly in anaplastic large-cell lymphomas (ALCLs), which constitute 5% of all NHLs. 5 In one study, mediastinal large B-cell lymphomas were CD30 ϩ in 69% of cases. 6 Because of its restricted expression in only a small subset of normal lymphocytes, the CD30 antigen is an excellent candidate for selective targeting by CD30-specific antibodies. Immunotoxins (ITs) are anti-cancer agents consisting of a binding antibody and a toxin. 7,8 The toxin and antibody are either covalently linked via a chemical linker or generated by recombinant DNA fusion technology.That anti-CD30 -toxin chemical conjugates can be used to eliminate HRS cells has been established in several studies in vitro 9 -11 as well as in vivo. 12,13 In a clinical trial, an IT containing the anti-CD30 MAb Ber-H2 and saporin-6 demonstrated some efficacy. 14 Our laboratory has utilized Pseudomonas exotoxin (PE) for the development of ITs. PE is a 3-domain protein. 15 Recombinant ITs (rITs) are constructed by removing the binding domain (domain I) of PE and replacing it with an antibody or cytokine that reacts with an antigen on a tumor cell. Domain II is the translocation domain, which enables PE to cross from the endocytic compartment into the cytosol. Domain III contains the ADP ribosylation activity that inactivates elongation factor 2. We can increase the cytotoxic activity by replacing the RED...

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Recombinant Toxins as Novel Therapeutic Agents

Cited by 306 publications

References 1 publication

EGFRvIII undergoes activation-dependent downregulation mediated by the Cbl proteins

EGFRvIII undergoes activation-dependent downregulation mediated by the Cbl proteins

Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

Isolation of new anti-CD30 scFvs from DNA-immunized mice by phage display and biologic activity of recombinant immunotoxins produced by fusion with truncatedpseudomonas exotoxin

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