The specific Î± subunit of the interleukin-3 receptor (IL-3RÎ±, CD123) is strongly expressed in AML, B-cell lineage acute lymphocytic leukemia (B-ALL), and hairy cell leukemia. The expression of CD123 is elevated in the primitive leukemia stem cell population (CD34+/CD38â\#8364;“), but not detectable in normal hematopoietic stem cells . Thus CD123 appears to be an excellent target for the therapy of leukemias. To develop immunotoxins targeting CD123, three anti-CD123 hybridomas, 26292, 32701 and 32716 were selected on the basis of their affinity for CD123. Total RNAs were extracted from three anti-CD123 hybridomas and used to clone the Fvs. The Fvs were assembled into single chain Fvs and fused to a 38 kDa 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 to 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 ng/ml to about 40 ng/ml by mutating the REDLK sequence at the C terminus to KDEL. 26292(Fv)-PE38-KDEL was found to be 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 AML and other CD123 expressing malignancies.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA