Characterization of immunotoxins active against ovarian cancer cell lines.

Pirker, Robert; FitzGerald, David J.; Hamilton, Thomas C.; Ozols, Robert F.; Laird, W; Frankel, Arthur E.; Willingham, Mark C.; Pastan, Ira

doi:10.1172/jci112082

Cited by 73 publications

(29 citation statements)

References 16 publications

(14 reference statements)

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“…Cell lines OVCAR-2, -3, -4, and -5 were obtained from T. C. Hamilton and R. F. Ozols, National Cancer Institute, and grown as described (7). The human fibroblast line WI-38 was from American Type Culture Collection.…”

Section: Methodsmentioning

confidence: 99%

Pseudomonas exotoxin coupled to a monoclonal antibody against ovarian cancer inhibits the growth of human ovarian cancer cells in a mouse model.

Willingham¹,

FitzGerald²,

Pastan³

1987

Proc. Natl. Acad. Sci. U.S.A.

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In an effort to devise an alternative treatment for human ovarian cancer, we have isolated a monoclonal antibody (OVB-3) that reacts with all ovarian cancers tested (10/10) but few normal tissues. An immunotoxin produced by coupling OVB-3 to Pseudomonas exotoxin kills ovarian cancer cells in tissue culture and prolongs the life of animals bearing human ovarian cancers. These data suggest that this immunotoxin should be evaluated as a treatment for ovarian cancer in women.Ovarian cancer is a common gynecologic malignancy that accounts for the death of 11,000 women per year in the United States (1). Unfortunately, less than 50% of women with this disease can be cured by surgery and chemotherapy. We have suggested that this disease is a good candidate for treatment with immunotoxins administered directly into the peritoneal cavity because the disease usually remains confined to the peritoneal cavity and death is often due to obstruction of vital organs lying within the cavity (1). Hamilton et al. (2) have developed a mouse model that closely resembles human ovarian cancer. In this model, 108 human ovarian cancer cells (OVCAR-3) are injected into the peritoneal cavity of an immunodeficient (nude) mouse; 40-60 days later the mouse dies with massive ascites, large intraperitoneal tumors, and tumor invasion of nearby tissues (2, 3). We have used this model to evaluate the activity of immunotoxins composed of antibodies to the human transferrin receptor coupled to Pseudomonas exotoxin (PE) or ricin A chain. Both immunotoxins were able to retard or prevent the growth of ovarian cancers in mice and to extend the life ofthese animals (3, 4). The PE immunotoxin was active at 0.3 ,ug per injection, whereas it required 10 ,ug per injection of the ricin A-chain immunotoxin to achieve an equivalent effect. Immunotoxins using antibodies to the human transferrin receptor are very effective cell-killing agents, but they may not be clinically useful or have limited clinical utility because they react with many normal human tissues (refs. 5 and 6 and see Table 1). Therefore, it was necessary to obtain a more tumor-specific antibody. Here we report on the isolation of such an antibody, and its antitumor activity when coupled to PE. MATERIALS AND METHODSCell lines OVCAR-2, -3, -4, and -5 were obtained from T. C. Hamilton and R. F. Ozols, National Cancer Institute, and grown as described (7). The human fibroblast line WI-38 was from American Type Culture Collection. To determine the nature of the antigen recognized by OVB-3, OVCAR-3 cells were fixed in 3.7% (vol/vol) formaldehyde in PBS (140 mM NaCl; 15 mM sodium phosphate, pH 7.0) for 10 min at 23°C, washed, and treated with neuraminidase (Sigma type X) at 200 milliunits/ml in 50 mM sodium acetate, pH 5.0, at 370C for 45 min followed by mixed glycosidases (Miles Biochemical, Naperville, IL) from Turbo cornutus at 10 mg/ml in the same buffer at 370C for 45 min. Unfixed cells were treated with 0.2% trypsin at pH 7.4 for 30 min at 370C, Pronase (100 ,ug/ml) at pH 7.2 for 30 min a...

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Section: Methodsmentioning

confidence: 99%

Pseudomonas exotoxin coupled to a monoclonal antibody against ovarian cancer inhibits the growth of human ovarian cancer cells in a mouse model.

Willingham¹,

FitzGerald²,

Pastan³

1987

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…Our laboratory has used Pseudomonas exotoxin (PE) to construct specific cell-killing reagents (2), taking advantage of the fact that treating intact PE with iminothiolane decreases the binding of PE to cells by the PE receptor. This chemical modification also introduces -SH groups into PE that are used to conjugate the toxin to a growth factor (3) or an antibody (4)(5)(6). If such a conjugate is made with epidermal growth factor (EGF), the resulting EGF-PE that is produced kills cells bearing EGF receptors (3).…”

mentioning

confidence: 99%

Activity of a recombinant fusion protein between transforming growth factor type alpha and Pseudomonas toxin.

Chaudhary¹,

FitzGerald²,

Pastan³

1987

Proc. Natl. Acad. Sci. U.S.A.

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156

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Pseudomonas exotoxin has been crystallized, and x-ray diffraction analysis of the crystal structure of PE shows that the toxin is composed of three distinct domains (7). Using the three-dimensional structure as a guide, deletion mutants of the PE structural gene were constructed and the proteins were encoded by the different domains of PE expressed in Escherichia coli (8). These studies have shown that domain I of PE is responsible for cell recognition, domain II is responsible for translocation of the toxin across membranes, and domain III is responsible for ADP-ribosylation of elongation factor 2, the step actually responsible for cell death (Fig. 1) (3.7 kb) was ligated to the 155-bp fragment. Restriction analysis using BamHI and Sph I was used to identify the plasmid with TGF-a gene in the proper orientation (pVC33).

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“…An assessment of internalization of TF12 by fluorescent microscopy was performed in MDA-MB-468 cells following the procedure in the study by Pirker et al (26). Briefly, 5 · 10 5 MDA-MB-468 cells were incubated with 5 mg of TF12 or control TF8 anti-CD22 per milliliter for 1 h at 4°C.…”

Section: Microscopic Evaluation Of Internalizationmentioning

confidence: 99%

A New Tri-Fab Bispecific Antibody for Pretargeting Trop-2–Expressing Epithelial Cancers

et al. 2012

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RS7 is an internalizing anti-Trop-2 pancarcinoma antibody capable of targeting most epithelial cancers. Because pretargeting strategies could improve the tumor localization of radionuclides, a new anti-Trop-2 · antihapten bispecific antibody for pretargeting, based on humanized RS7, was prepared and evaluated with a radiolabeled hapten-peptide in vitro and in vivo to determine whether its internalization properties would interfere with pretargeting. Methods: The anti-Trop-2 · antihapten bispecific antibody, TF12, was prepared using the modular dock-and-lock method. TF12 and humanized RS7 binding was assessed by cell binding assays and fluorescence-activated cell sorting analysis in a variety of human carcinoma cell lines. The internalization of TF12 was evaluated in vitro using a fluorescent TF12 conjugate or hapten-peptide and 111 In-labeled TF12 and RS7. The biodistribution of TF12 and its use as a pretargeting agent with an 111 In-labeled hapten-peptide were assessed in several human epithelial cancer xenografts. Dose optimization was examined in 2 tumor models. Results: TF12 internalizes, but a substantial fraction remained accessible on the tumor surface. Fluorescence-activated cell sorting analysis showed only a minor change in fluorescent signal when the tumor was probed with a fluorescent hapten-peptide over 4 h, and microscopy showed substantial membrane staining when reassessed at 24 h after TF12 exposure. Only 40.1% of 111 In-TF12 was internalized after 24 h. In vivo, excellent tumor localization of the 111 In-labeled peptide was observed in several tumor models. Conclusion: TF12 was retained sufficiently on the cell surface in several epithelial cancers, thereby making it suitable for pretargeted imaging and therapy of various Trop-2-expressing carcinomas.

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Characterization of immunotoxins active against ovarian cancer cell lines.

Cited by 73 publications

References 16 publications

Pseudomonas exotoxin coupled to a monoclonal antibody against ovarian cancer inhibits the growth of human ovarian cancer cells in a mouse model.

Pseudomonas exotoxin coupled to a monoclonal antibody against ovarian cancer inhibits the growth of human ovarian cancer cells in a mouse model.

Activity of a recombinant fusion protein between transforming growth factor type alpha and Pseudomonas toxin.

A New Tri-Fab Bispecific Antibody for Pretargeting Trop-2–Expressing Epithelial Cancers

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