Molecular modification of a recombinant, bivalent anti-human CD3 immunotoxin (Bic3) results in reduced in vivo toxicity in mice.
A novel bivalent single chain fusion protein, Bic3, was assembled consisting of the catalytic and translocation domains of diphtheria toxin (DT(390)) fused to two repeating sFv molecules recognizing human CD3 epsilon of the human T-cell receptor. Historically, problems with these constructs include low yield, toxicity, and reduced efficacy. Instead of using conventional Gly(4)Ser linkers to connect heavy/light chains, aggregation reducing linkers (ARL) were used which when combined with a new SLS-based refolding method reduced aggregation and enhanced the yield of final product. Toxicity was reduced at least 25-fold by repeating the two sFv molecules and adding a portion of the hinge-CH2-CH3 human constant regions. The resulting Bic3 was just as cytotoxic to HPB-MLT.UM T leukemia cells in vitro (IC(50)=4 pmol) as a monovalent construct made with the same DT and sFv. In vivo, Bic3 was effective in a new and aggressive therapy model in which it significantly prolonged survival of scid mice with established human T-cell leukemia (p<0.0001 compared to controls). Importantly, no toxicity measured by weight loss, enzyme function, or histology was observed at the highest dose of Bic3 tested (2000 ug/kg). Bic3 warrants investigation as a new drug for treating T-cell malignancy and other T-cell related disorders.
Vallera
Daniel A
DA
Department of Therapeutic Radiology-Radiation Oncology, University of Minnesota Cancer Center, Section on Molecular Cancer Therapeutics, MMC: 367, Minneapolis, MN 55455, USA. valle001@umn.edu
Todhunter
Deborah
D
Kuroki
David W
DW
Shu
Yanqun
Y
Sicheneder
Andy
A
Panoskaltsis-Mortari
Angela
A
Vallera
Vincent D
VD
Chen
Hua
H
D000818:Animals; D000911:Antibodies, Monoclonal; D017252:CD3 Complex / Q000276:immunology; D004195:Disease Models, Animal; D006801:Humans; D007074:Immunoglobulin G; D000922:Immunotoxins / Q000009:adverse effects* / Q000737:chemistry*; D007668:Kidney / Q000473:pathology; D015458:Leukemia, T-Cell / Q000188:drug therapy*; D051379:Mice; D016513:Mice, SCID; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins / Q000009:adverse effects* / Q000138:chemical synthesis* / Q000737:chemistry; D013601:T-Lymphocytes / Q000187:drug effects
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D007074:Immunoglobulin G; D000922:Immunotoxins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins