Construction and characterization of human CD7-specific single-chain Fv immunotoxins

To develop novel therapeutic agents for treatment of human T cell malignancies, we constructed two single-chain Fv (sFv) immunotoxins specific for the T cell-associated Ag CD7. The sFv fragments were derived from the murine hybridomas 3A1e and 3A1f and were expressed as soluble proteins in Escherichia coli. Surface plasmon resonance analyses demonstrated that the purified 3A1e and 3A1f sFv fragments specifically bound CD7 with high affinity, 8.1 and 1.8 nM, respectively. The difference in affinity is chiefly due to a slower dissociation rate for the 3A1f sFv fragment. Despite this difference, both monovalent sFv fragments were comparably internalized by CD7+ human T leukemic cells within 30 min. These data support findings of previous studies suggesting that CD7 internalization does not require cross-linking. The sFv immunotoxins were assembled by linking ricin toxin A chain to the C termini of the sFv fragments via disulfide bonds. Both sFv immunotoxins were comparably potent in their ability to inhibit protein synthesis in vitro in CD7+ Jurkat cells (50% inhibiting concentration = 15 pM). Further preclinical studies on the use of the 3A1e and 3A1f sFv immunotoxins to treat human T cell diseases therefore appear warranted.