Diphtheria toxin fused to variant interleukin-3 provides enhanced binding to the interleukin-3 receptor and more potent leukemia cell cytotoxicity.
Chemoresistance is a common cause of treatment failure in patients with acute myeloid leukemia (AML). We generated a diphtheria toxin (DT) fusion protein composed of the catalytic and translocation domains of DT (DT388) fused to interleukin-3 (IL-3). IL-3 receptors (IL-3R) are overexpressed on blasts from many AML patients. DT388IL-3 showed cytotoxicity to leukemic blasts in vitro and in vivo and minimal damage to normal tissues in nonhuman primate models. However, only a fraction of patient leukemic samples were sensitive to the agent. To enhance the potency and specificity of the DT388IL-3 molecule, we constructed variants with altered residues in the IL-3 moiety. Two of these variants, DT388IL-3[K116W] and DT388IL-3[Delta125-133], were produced and partially purified from Escherichia coli with excellent yields. They showed enhanced binding to the human IL-3R and greater cytotoxicity to human leukemia cell lines relative to wild-type DT388IL-3. Interestingly, the results support a previously hypothesized model for interaction of the C-terminal residues of IL-3 with a hydrophobic patch on the alpha-subunit of IL-3R. Rational modification of the targeting domain based on structural analysis can produce a fusion toxin with increased ability to kill tumor cells. One or both of these variant fusion proteins merit further development for therapy of chemotherapy refractory AML.
Department of Medicine and Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA.
Tie Fu
TF
Liu
Jeffrey O
JO
Urieto
Joseph E
JE
Moore
Mark S
MS
Miller
A Corinne
AC
Lowe
Andrew
A
Thorburn
Arthur E
AE
Frankel
D000208:Acute Disease; D019943:Amino Acid Substitution; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor / Q000187:drug effects; D004167:Diphtheria Toxin / Q000737:chemistry / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D006801:Humans; D007377:Interleukin-3 / Q000737:chemistry / Q000494:pharmacology*; D007951:Leukemia, Myeloid / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D016185:Receptors, Interleukin-3 / Q000187:drug effects* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D000970:Antineoplastic Agents; D004167:Diphtheria Toxin; D007377:Interleukin-3; D016185:Receptors, Interleukin-3; D011993:Recombinant Fusion Proteins; C407154:diphtheria toxin-interleukin-3 fusion protein, recombinant