26 
Dr. Clones asked Dr. Murphy why he used a morphological test for toxicity 
testing rather than a more specific quantitative protein inhibition assay. 
Dr. Murphy said he has recently employed protein inhibition tests to show 
that protein synthesis in human malignant melanoma cells is inhibited 50 
percent by approximately 2 nanomoles of the a-MSH-diphtheria toxin fusion 
protein. Diphtheria toxin sensitive Chinese Hamster Ovary cells are not 
affected by the hybrid toxin protein. Dr. Murphy said the fusion protein 
appears to be directed only against cells having a-MSH receptors on their 
surface. 
Dr. Cohen asked hew the feet most of the U.S. population is irmtunized 
against diphtheria would affect the therapeutic effectiveness of the hybrid 
protein. 
Dr. Murphy said two years ago his group published a monoclonal antibody 
study of the interaction of 43 different monoclonal antibodies with diph- 
theria toxin. The study demonstrated that only those monoclonal antibodies 
that prevented diphtheria toxin from binding to its receptor were neutra- 
lizing antibodies. Antibodies binding to other portions of the toxin 
molecule did not neutralize cytotoxic activity. Preliminary experiments 
with polyclonal antisera show that concentrations more than 100- fold greater 
than would be expected to neutralize diphtheria toxin activity are needed 
to reduce the potency of the a-MSH-diphtheria toxin fusion protein on 
human malignant melanoma cells. 
Dr. Davis asked whether the diphtheria toxin fragment used to construct 
the fusion protein has any toxicity. 
Dr. Murphy said the fragment codes for all of diphtheria toxin except for 
67 amino acids which have been deleted from the carboxyl-terminal end. 
It is not currently knewn whether the diphtheria toxin receptor region, is 
in that 67 amino acid section or whether that region is necessary to main- 
tain the appropriate configuration for receptor binding. However, the 
fusion protein in which the carboxyl-terminal 67 amino acids are replaced 
with a 13 amino acid sequence of a-MSH is not cytotoxic for cells exquis- 
itely sensitive to diphtheria toxin. He inferred from this observation 
that the carboxyl-terminal region carries either the binding domain or 
segments necessary for the appropriate molecular configuration. 
Dr. Martin asked Dr. Murphy whether he had tested the diphtheria toxin 
fragment without the a-MSH ligand for receptor activity and toxicity. 
Dr. Murphy said he had only studied the fusion protein. Dr. Martin said 
the ligand might affect toxin binding; the toxin fragment might be toxic 
if the ligand is removed. 
[408] 
