A fold-back single-chain diabody format enhances the bioactivity of an anti-monkey CD3 recombinant diphtheria toxin-based immunotoxin.
T-cell depleting anti-CD3 immunotoxins have utility in non-human primate models of transplantation tolerance and autoimmune disease therapy. We recently reported that an affinity matured single-chain (scFv) anti-monkey CD3 antibody, C207, had increased binding to T-cells and increased bioactivity in a diphtheria toxin (DT)-based biscFv immunotoxin compared with the parental antibody, FN18. However, FN18 scFvs and their mutant derivatives such as C207 did not exhibit robust bivalent character in the biscFv format. We now report that C207 in a diabody format exhibits a 7-fold increase in binding to T-cells over scFv (C207) indicating considerable divalent character for the diabody. This construct was formed by reducing the V(L)/V(H) linker to five residues and was secreted from Pichia pastoris as the non-covalent dimer. An immunotoxin based on this diabody format was secreted as a non-covalent dimer but was devoid of bioactivity and failed to bind T-cells, suggesting steric hindrance from the two large closely positioned truncated DT moieties. We constructed a single-chain diabody immunotoxin by fusing to the truncated DT C-terminus L1-VL-L1-VH-L2-VL-L1-VH where L1 is a five-residue linker and L2 is the longer (G4S)3 linker permitting interactions between the distal and proximal VL/VH domains. This 'fold-back' immunotoxin was secreted predominantly as the monomer and exhibited a 5- to 7-fold increase in bioactivity over DT390biscFv(C207) and depleted monkey T-cells in vivo.
Kim
Geun-Bae
GB
Section on Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, Bldg 10, Room 3D46, 10 Center Drive, Bethesda, MD 20892-1216, USA.
Wang
Zhirui
Z
Liu
Yuan Yi
YY
Stavrou
Scott
S
Mathias
Askale
A
Goodwin
K Jeanine
KJ
Thomas
Judith M
JM
Neville
David M
DM
D000818:Animals; D017252:CD3 Complex / Q000737:chemistry*; D019281:Dimerization; D004167:Diphtheria Toxin / Q000737:chemistry*; D000882:Haplorhini; D007128:Immunoglobulin Fragments / Q000737:chemistry; D000922:Immunotoxins / Q000737:chemistry*; D010843:Pichia / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013601:T-Lymphocytes / Q000378:metabolism; D013997:Time Factors
D017252:CD3 Complex; D004167:Diphtheria Toxin; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins