11 
toxicity of the Sph l fragment. Is the Sph l fragment an active toxin if the 
a-MSH moiety is cleaved off? Is the level of toxicity of the Sph l fragment 
similar to that of the CFM 45 fragment? He noted that the CFM 45 fragment is 
smaller than Sph l but is toxic injected intracerebral ly or intraneurally. 
Hr. Gill pointed out that the CFM 45 fragment is 100 times less toxic than 
diphtheria- toxin. If the Sph l fragment is 100 times less toxic than diphtheria 
toxin, experiments involving the cloning of the fragment would be classified 
in the BL1 category. 
Dr. Cbllier asked Dr. Murphy to conpare the Sph l fragment to the CFM fragments 
in terms of size. Dr. Murphy said the Sph l fragment lacks 67 amino acids of 
the mature diphtheria toxin. It is larger than the largest kncwn CFM fragment. 
Dr. Gill asked if any data exist on the toxicity of a diphtheria toxin fragment 
of that size. Dr. NOrphy said no data acist but felt the Sph l fragment 
would be as toxic as the CFM 45 fragment. 
Dr. I/evine asked whether the challenged guinea pigs mounted an antibody response 
against the a-MSH-diphthena toxin hybrid protein. Dr. Murphy said he had 
not tested for an antibody response but assumed antibodies would be produced . 
Dr. Habig asked Aether the conjugate protein could cause an antibody response 
against a-MSH itself. Dr. Irvine said another question is vh ether the 
conjugate would be effective in humans if more than one course of therapy was 
necessary since most humans would mount an antibody response against the a-MSH- 
diphthena toxin hybrid molecule. 
Dr. Habiq said SO per cent of the immunized population have some antibodies to 
the A chain portion of the diphtheria molecule. He questioned vhether the 
a-MSH-diphthena toxin molecule would be therapeutically effective in mminized 
populations. Dr. Murphy hypothesized that it would. He said only those mono- 
clonal antibodies that block toxin binding to the receptor neutrailize the 
toxin; monoclonal antibodies that bind to the toxin but do not block receptor 
binding do not neutralize the toxin. He hoped that only antitoxin antibodies 
capable of blocking receptor binding would be able to neutralize the cc-NEH- 
diphthena toxin molecule in vivo. 
Dr. Habig said the a-MSH-diphthena toxin hybrid might be neutralized in the 
animal by the formation of polyvalent complexes. Tfetanus toxin can be neutral- 
ized by lattice formation even if the receptor binding site is not blocked by 
antibodies . 
Dr. Gottesman asked vhether fragment Sph l has an internal stop codon. Wculd 
fusion proteins result if "read-through" occurred?' Dr. Murphy said the Sphl 
frapment has no interna] stop codon. A fusion protein results vhen the Sph l 
gene fragment -is linked directly to the vector DMA. 
Dr. Gottesman asked Dr. Murphy vhether any toxicity data exist for randan Sph l 
fusion molecules. Dr. Murphy said he had no toxicity data on Sph l fusion 
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