Selective depletion of activated T cells by recombinant immunotoxin containing anti-CTLA-4 single-chain fragment of variable antibody and N-terminal fragment of perforin.
We constructed a novel immunotoxin, hS83P34, by fusing the fragment containing the N-terminal 34 amino acids of human perforin to the C-terminal of humanized anti-CTLA-4 single chain fragment of variable antibody. In vitro cytotoxicity assays demonstrated that CTLA-4-positive activated human T cells and 6T-CEM were sensitive to hS83P34, while CTLA-4-negative resting T cells and endothelial cell ECV-304 were resistant to hS83P34. The IC50s of hS83P34 for activated T cells and 6T-CEM were about 0.2 micromol/L and 1.0 micromol/L, there was no obvious cytotoxicity of ECV-304 as detected at 8 micromol/L of hS83P34. In tumor graft rejection models, after treatment with 1.2 mg/kg immunotoxin every day for 12 days, the transplanted tumor cells were rescued by immunotoxin. The tumor weights of grafts of the rejection control group, nonrejection control group, and test group were 0.006 +/- 0.014 g, 0.261 +/- 0.048 g, and 0.135 +/- 0.056 g, respectively. In the early 3 days posttransplantation, there were a lot of CD4- and CD8-positive T cells infiltrating into the tumor grafts of the rejection control group, while only a few T cells were detected in the tumor grafts of the test group. According to these results, we concluded that immunotoxin hS83P34 selectively depleted activated T cells in vitro as well as in vivo in an acute rejection model.
Zeng
L
L
Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu 610041, People's Republic of China.
Wan
L
L
Chen
L
L
Li
S
S
Lu
Y
Y
Huang
Q
Q
Wang
L
L
Li
Y
Y
Cheng
J
J
Lu
X
X
D015703:Antigens, CD / Q000276:immunology*; D000943:Antigens, Differentiation / Q000276:immunology*; D060908:CTLA-4 Antigen; D003208:Concanavalin A; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry; D006801:Humans; D008213:Lymphocyte Activation; D008212:Lymphocyte Depletion; D008562:Membrane Glycoproteins / Q000737:chemistry*; D010446:Peptide Fragments / Q000276:immunology; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D052899:Pore Forming Cytotoxic Proteins; D011993:Recombinant Fusion Proteins; D003208:Concanavalin A; D054353:Perforin