Characterization of RFB4-Pseudomonas exotoxin A immunotoxins targeted to CD22 on B-cell malignancies.
To develop an immunotoxin for the treatment of B-cell malignancies, we constructed several candidate conjugates with RFB4, a B-cell specific anti-CD22 IgG1, and truncated forms of Pseudomonas exotoxin (PE). The four versions of PE included PE35 and PE35KDEL, which were linked to RFB4 via a disulfide bond, and PE38 and PE38KDEL, which were linked via a thioether bond. The PE35 truncated forms, which are fully active in ADP ribosylation and lack receptor binding sequences, do not require intracellular proteolytic cleavage in order to be active. PE35KDEL has the consensus endoplasmic reticulum retention signal, KDEL, replacing the wild type PE C-terminal sequence, REDLK. The PE38 forms retain all of domain II and therefore require cleavage to be active within cells. Cytotoxicity experiments on CD22-positive cell lines revealed that the PE35 conjugates were more active than the PE38 versions and the presence of the KDEL sequence generally enhanced toxicity by 5-10-fold compared to that of REDLK. The RFB4-PE35KDEL immunotoxin was most active in cytotoxicity assays against Burkitt's lymphoma cell lines such as Daudi and CA46 (IC50 = 0.2 ng/mL) and displayed little cytotoxicity toward human vascular endothelial cells (IC50 > 20 micrograms/mL). Results of experiments conducted in nude mice showed that both RFB4-PE35KDEL and RFB4-PE35 could inhibit the development of subcutaneous CA46 tumors.
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
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Mansfield
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Pastan
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FitzGerald
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000276:immunology*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D015815:Cell Adhesion Molecules; D002470:Cell Survival / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects; D005098:Exotoxins / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry*; D037102:Lectins; D016393:Lymphoma, B-Cell / Q000276:immunology*; D051379:Mice; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011550:Pseudomonas aeruginosa; D051918:Sialic Acid Binding Ig-like Lectin 2; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; D001427:Bacterial Toxins; D015815:Cell Adhesion Molecules; D005098:Exotoxins; D000922:Immunotoxins; D037102:Lectins; D011065:Poly(ADP-ribose) Polymerases; D051918:Sialic Acid Binding Ig-like Lectin 2; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C497867:CD22 protein, human; C497868:Cd22 protein, mouse