Selective destruction of nerve growth factor receptor-bearing cells in vitro using a hybrid toxin composed of ricin A chain and a monoclonal antibody against the nerve growth factor receptor.
A hybrid toxin composed of ricin A chain and a monoclonal antibody directed against the rat nerve growth factor (NGF) receptor (192-IgG) was prepared using the heterobifunctional cross-linking agent N-succinimidyl-3-(2-pyridyldithio)-propionate and purified by affinity chromatography. Characterization studies showed that the hybrid, 192-s-s-A, displaced bound 125I-labeled 192-IgG from rat superior cervical ganglion (SCG) membranes with an IC50 3-5 times lower than that of unconjugated 192-IgG. When incubated with cultured rat SCG neurons, 192-s-s-A inhibited protein synthesis in a concentration-dependent fashion. The effect of 192-s-s-A on these neurons was reversed by coincubation with an excess of 192-IgG. The IC50 of 192-s-s-A on protein synthesis in rat SCG neurons was 4 nM. Intact ricin and ricin A chain inhibited protein synthesis in these neurons with IC50 values of 5 pM and 500 nM, respectively. The 192-s-s-A hybrid had no effect on mouse SCG neurons or a human melanoma cell line known to have NGF receptors. This is consistent with the finding that 192-IgG recognizes only the rat NGF receptor. Also, 192-s-s-A did not inhibit protein synthesis in primary cultures of rat skeletal muscle or Vero cells, which do not have cell surface receptors for NGF. 192-s-s-A was able to inhibit protein synthesis in PC12 cells but the potency was 10-100 times less in these cells compared to rat SCG neurons. Ricin and A chain were also 10-100 times less potent in PC12 cells than neurons. Rat SCG neurons exposed to 192-s-s-A lost their refractile appearance under phase-contrast optics, showed granular degeneration of neurites, and died. Thus the decreased protein synthesis caused by the hybrid toxin correlated with the morphological destruction of the neurons. 192-s-s-A represents a potentially powerful tool by which to selectively destroy NGF receptor-bearing cells in vitro. The hybrid toxin may prove useful as an in vivo toxin.
DiStefano
P S
PS
Schweitzer
J B
JB
Taniuchi
M
M
Johnson
E M
EM
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005728:Ganglia, Sympathetic; D009414:Nerve Growth Factors / Q000276:immunology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D009474:Neurons / Q000187:drug effects*; D051381:Rats; D011956:Receptors, Cell Surface / Q000276:immunology*; D017475:Receptors, Nerve Growth Factor; D012276:Ricin / Q000008:administration & dosage* / Q000633:toxicity
D000911:Antibodies, Monoclonal; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor; D012276:Ricin