New immunotoxins targeting CD123, a stem cell antigen on acute myeloid leukemia cells.
The specific alpha subunit of the interleukin-3 receptor (IL-3Ralpha, CD123) is strongly expressed in various leukemic blasts and leukemic stem cells and seems to be an excellent target for the therapy of leukemias. In this study, immunotoxins were developed to target CD123 only, which bypasses the dependence on other subunits to form intact IL-3R. Three anti-CD123 hybridomas (26292, 32701, and 32716) were selected on the basis of their affinity for CD123. Total RNAs were extracted from the 3 anti-CD123 hybridomas and used to clone the fragment of variable region (Fvs). The Fvs were assembled into single chain Fvs and fused to a 38-kd fragment of Pseudomonas exotoxin A to make recombinant immunotoxins. 26292(Fv)-PE38 was found to have the highest cytotoxic activity on the CD123 expressing leukemia cell line TF-1. It bound the cells with a kd of 3.5 nM. Another immunotoxin, 32716(Fv)-PE38, belonging to a different epitope group, had a similar binding ability but was less active, demonstrating the role of epitope selection in immunotoxin action. The cytotoxic activity of 26292(Fv)-PE38 was increased from 200 to about 40 ng/mL by mutating the REDLK sequence at the C terminus to KDEL. 26292(Fv)-PE38-KDEL was specifically cytotoxic to several CD123 expressing cell lines (TF-1, Molm-13, and Molm-14) with good CD123 expression but not to ML-1 or U937 with low or absent expression. In conclusion, 26292(Fv)-PE38-KDEL shows good cytotoxic activity against CD123 expressing cell lines, and merits further development for the possible treatment of acute myeloid leukemia and other CD123 expressing malignancies.
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA.
Xing
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Du
Mitchell
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Ira
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Pastan
D036002:ADP Ribose Transferases / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use*; D000208:Acute Disease; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use; D001427:Bacterial Toxins / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D005098:Exotoxins / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins / Q000276:immunology* / Q000378:metabolism / Q000627:therapeutic use*; D053650:Interleukin-3 Receptor alpha Subunit / Q000276:immunology* / Q000378:metabolism; D007951:Leukemia, Myeloid / Q000276:immunology* / Q000378:metabolism / Q000628:therapy*; D016185:Receptors, Interleukin-3 / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D053650:Interleukin-3 Receptor alpha Subunit; D016185:Receptors, Interleukin-3; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C115022:IL3RA protein, human