Federal Register / Vol. 50, No. 226 / Friday, November 22, 1985 / Notices 
48347 
"E. coli host vector systems 
expressing the Shiga-like toxin gene 
may be moved from BL3 + EK1 to 
BL2 + EK1 containment conditions 
provided that: (1) The amount of toxin 
produced by the modified host-vector 
systems be no greater than that 
produced by the positive control strain 
Shigella dysenteriae 60R, grown and 
measured under optimal conditions; and 
(2) the cloning vehicle is to be an EKl 
vector preferably belonging to the class 
of poorly mobilizable plasmids such as 
pBR322, pBR328, and pBR325." 
I accept these recommendations and 
Appendix F-IV-H has been modified 
accordingly. 
B. Request To Clone a Hybrid Toxin 
Gene in E. coli K-12 
Dr. John Murphy of the University 
Hospital of Boston University Medical 
Center in a letter dated July 30, 1985, 
requested permission to construct a 
hybrid molecule in which the gene 
coding for interleukin-2 (IL— 2) is joined 
to a segment of the gene encoding 
diphtheria toxin. The diphtheria gene 
toxin segment encodes the A subunit 
and portions of the B subunit of the 
toxin. The hybrid gene would be cloned 
in E. coli K-12 host-vector systems. 
The goal of the project is the 
development and testing of novel 
therapeutic agents targeted at the 
interleukin 2 (IL— 2) receptor to combat 
rejection and/or create a state of 
tolerance in organ transplantation. As a 
by-product, some of the agents may 
have therapeutic potential for patients 
with leukemia and lymphoma. 
This proposal was published in the 
August 19, 1985, Federal Register for 
public comment. No comments were • 
received on the proposal during the 
comment period. 
On August 16, 1985, the RAC Working 
Group on Toxins considered this 
proposal. 
Although not part of the action 
requested in this proposal, it should be 
noted that Dr. Murphy was previously 
granted permission (Appendix F-IV-I, 
49 FR 46279) to propagate under BL4 
containment a hybrid molecule 
composed of the gene coding for a- 
melanocyte stimulating hormone (a- 
MSH) and a segment of the diphtheria 
toxin gene. On August 16. 1985, the RAC 
Working Group on Toxins considered a 
request from Dr. Murphy to remove 
these clones from BL4 containment. Dr. 
Murphy submittted data on the effects 
on guinea pigs challenged with: (1) The 
viable and boiled E. coli K-12 host- 
vector system (the control); (2) the 
viable and boiled E. coli K-12 host- 
vector system carrying and expressing 
the hybrid gene; and (3) the conjugate 
protein. After thorough discussion, the 
working group recommended Dr. 
Murphy be permitted to proceed with 
experiments involving clones carrying 
the a-MSH-diphtheria toxin gene at BL2 
containment w'ith BL3 practices. I 
previously accepted this 
recommendation. 
The portion of the minutes of the 
August 16, 1985, meeting of the Working 
Group on Toxins pertaining to Dr. 
Murphy’s proposal to construct and 
clone a hybrid gene composed of the 
genes coding for IL— 2 and portion of 
diphtheria toxin reads as follows: 
"Dr. Gottesman said in this proposal. 
Dr. John Murphy of the University 
Hospital of the Boston University 
Medical Center requests permission to 
construct a hybrid gene composed of the 
gene coding for interleukin-2 (IL— 2) and 
the Sphl segment of the diphtheria toxin 
gene. The hybrid gene would be cloned 
in E. coli K-12 host-vector systems. 
“The long-term goal of this research is 
to develop novel 1L-2 receptor-targeted 
cytotoxic agents to combat organ 
rejection and to create a state of graft 
"tolerance” in organ transplantation. 
Some of the products may have 
therapeutic potential for treating 
leukemia and lymphoma. 
“Dr. Gottesman said the primary 
difference between the a-MSH- 
diphtheria toxin hybrid molecule 
proposal and the IL-2-diphtheria toxin 
hybrid molecule proposal is the type of 
cell postulated to be sensitive to the 
conjugate problem. 
"Dr. John Williams of Beth Israel 
Hospital said IL— 2 receptors are found 
on proliferating, antigen-activated T 
cells and to a lesser extent on activated 
B cells. These cells would be the targets 
of the IL-2-diphtheria toxin hybrid 
protein. Resting or memory lymphocytes 
do not express IL-2 receptors. There is 
no evidence of IL-2 receptors on tissues 
outside of the lymphoid compartment. 
"Dr. Williams said the goal of this 
project is to selectively remove 
activated T cells and activated B cells 
during a well controlled time period 
following organ transplantation. It is 
hoped the IL-2-diphtheria toxin 
molecule would be more selective than 
the immunosuppressive techniques 
currently in use. Experiments in mice 
using anti-IL-2 receptor-monoclonal 
antibody conjugates to suppress cells 
which cause rejection in cardiac 
transplants support this hypothesis. In 
these experiments, the life of the graft in 
mice was extended from 10-14 days to 
three months or more in 80 percent of 
the animals. 
“Dr. Gill said the ‘worse case' 
scenario he could conjecture would be 
that an animal's T cells and B cells 
would be eliminated by this hybrid 
protein. A long observation of test 
animals might be necessary before such 
an effect would be detected. Dr. Habig 
said a long observation period might 
also be necessary for animals treated 
with the a-MSH-diphtheria hybrid 
protein. 
"Dr. Levine said the IL-2-diphtheria 
toxin hybrid protein could be an 
important therapeutic agent, but it 
would be a new toxin and could be 
harmful. He suggested containment be 
initially set at the BL4 level. 
“Dr. Martin said the largest BL4 
facility at the Frederick Cancer 
Research Facility is run by NIAID. Any 
request by Dr. Murphy for use of the 
facility will need to be considered by 
NIAID in relation to other competing 
requests. 
"Dr. Levine said since little is known 
about the potential toxicity of the 
proposed hybrid protein, the working 
group approach should be conservative. 
If BI/4 containment is not available, the 
experiment should be permitted under 
BL3 containment with a recognition that 
concern exists about the nature of the 
proposed hybrid protein. 
"Dr. Gottesman suggested 
containment could be set at BL3 with a 
requirement for the use of EK2 host- 
vector systems. Dr. Levine said he 
would accept the suggestion to specify 
EK2 vectors; he would not accept the 
suggestion to specify EK2 hosts. 
"Dr. Gottesman asked whether any 
type of risk assessment experiment 
could be performed which would 
answer basic questions about the nature 
of the protein. 
"Dr. Gill said risk assessment 
experiments with this hybrid protein 
would differ from previous risk 
assessment protocols because acute 
lethality is not a likely outcome. Tests 
would have to be devised to look for 
chronic impairment of the immune 
system. This type of data and the 
protocols necessary to generate it are 
different from that generally required for 
toxins. 
“Dr. Gill offered a motion to 
recommend permission to proceed under 
BL3 conditions; EK2 vectors would be 
used. Dr. Levine said he would prefer 
the language of the motion specified the 
use of ‘poorly mobilizable plasmid 
vectors such as the EK2 certified 
plasmids.’ Dr. Gill agreed to this 
modification. Dr. Levine seconded the 
motion. 
"By a vote of five in favor, none 
opposed, and no abstentions, the 
working group agreed this 
recommendation should be forwarded to 
the RAC." 
[529] 
