title: Recombinant toxins containing the variable domains of the anti-Tac monoclonal antibody to the interleukin-2 receptor kill malignant cells from patients with chronic lymphocytic leukemia.
abstract: We have previously shown that the variable domains of the monoclonal antibody anti-Tac [anti-Tac(Fv)] can be fused to derivatives of Pseudomonas exotoxin (PE) or diphtheria toxin (DT) to produce recombinant immunotoxins that kill interleukin-2 (IL-2) receptor-bearing cells. We now report that two of these single-chain recombinant immunotoxins, anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), are cytotoxic toward peripheral blood mononuclear cells (PBMCs) from patients with chronic lymphocytic leukemia (CLL). In anti-Tac(Fv)-PE40KDEL, anti-Tac(Fv) is genetically fused to the amino terminus of PE40KDEL, a recombinant form of PE which contains amino acids 253-608 of PE and the -KDEL mutation at the carboxyl terminus. In DT388-anti-Tac(Fv), anti-Tac(Fv) is fused to the carboxyl terminus of the first 388 amino acids of DT. PBMCs from 14 patients were incubated with the recombinant toxins for 60 hours, and [3H]-leucine incorporation was measured. Anti-Tac(Fv)-PE40KDEL was cytotoxic to 7 of the 14 patient samples, with half-maximal inhibition of protein synthesis (IC50) achieved at 1.2 to 9 ng/mL (1.8 to 13 x 10(-11) mol/L). DT388-anti-Tac(Fv) was cytotoxic to 11 of the 14 samples, with IC50s ranging from less than 1 to 250 ng/mL. DT388-IL-2, in which the first 388 amino acids of DT are attached to IL-2, was marginally cytotoxic toward only 4 of 13 CLL samples tested with IC50s ranging from 100 to 550 ng/mL. Trypan blue staining of cells from several patients indicated that inhibition of protein synthesis correlated with cell death. Binding assays using [3H]-anti-Tac indicated that the CLL cells from nine of the patients contained between 400 and 2,500 sites per cell. Cells from another patient, which were resistant to both anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), had less than 100 sites per cell. We conclude that anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv) can kill CLL cells which have low numbers of IL-2 receptors, and should be investigated further for therapy of this disease.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
authors.forename: R J
authors.initials: RJ
authors.lastname: Kreitman
authors.forename: V K
authors.initials: VK
authors.lastname: Chaudhary
authors.forename: R W
authors.initials: RW
authors.lastname: Kozak
authors.forename: D J
authors.initials: DJ
authors.lastname: FitzGerald
authors.forename: T A
authors.initials: TA
authors.lastname: Waldman
authors.forename: I
authors.initials: I
authors.lastname: Pastan
mesh_terms: D036002:ADP Ribose Transferases; D000368:Aged; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001427:Bacterial Toxins; D002470:Cell Survival / Q000187:drug effects; D005098:Exotoxins / Q000235:genetics / Q000494:pharmacology*; D005260:Female; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood*; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000187:drug effects* / Q000473:pathology; D011550:Pseudomonas aeruginosa; D015375:Receptors, Interleukin-2 / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A