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Alternatives to Monoclonal Antibody Production (Proceedings)

The Responsibilities and Needs of the IACUC in Advising Investigators About Alternatives to in vivo Monoclonal Antibody Production

Marcia E. Etheridge
Johns Hopkins University School of Medicine
 Division of Comparative Medicine
 Baltimore, MD

Good afternoon. I would like to address three questions in my presentation today. First, "Is the IACUC responsible for advising investigators about alternatives to in vivo methods of monoclonal antibody production?"; secondly, "How does our IACUC discharge its responsibility in this matter ?" And lastly, "What has been the experience of monoclonal antibody users at the Johns Hopkins University regarding in vivo vs in vitro methodologies?"

In addition to its other responsibilities (CFR 1985, PHS 1996, Guide 1996) the IACUC functions to assist investigators in reducing the number of animals used in research, to help refine experimental procedures so that pain and distress to the animals are minimized, and to recommend the replacement of animals with in vitro methods whenever possible ( Russell and Burch, 1959). In light of these responsibilities, therefore, the answer to the first question is "Yes, the IACUC is responsible for informing investigators of valid alternatives to in vivo procedures." (This does not absolve investigators of their obligation, in the conduct of responsible science, to also seek non-animal alternatives to experimental procedures). These objectives must be accomplished without compromising the validity of experimental results (IRAC, 1985). Therefore, the IACUC has the dual responsibility of safeguarding the welfare of animals used in research without unduly hindering the progress of investigations which are valuable to the health and welfare of both the public and domestic animals. So how does our IACUC discharge its responsibility in this matter? Our IACUC relies upon information from a number of sources, including published, objective data from scientifically designed research trials, as well as feedback from actual monoclonal antibody users on their experiences with both in vivo and in vitro methodologies. The IACUC for the Johns Hopkins University School of Medicine recommends, but does not mandate, the use of in vitro methods whenever possible; and investigators submitting protocols which describe in vivo monoclonal antibody techniques are requested to justify this choice over in vitro methods. The IACUC recognizes, based upon published data (Amyx, 1987; Jackson and Fox, 1995), that it is possible to produce monoclonal antibodies in vivo with minimal pain and discomfort to the animal if certain procedures are followed. The potential for pain and distress in monoclonal antibody production is associated primarily with: (1) immunization procedures and "priming" agents, (2) excessive abdominal distension due to ascites, (3) intra-abdominal growth of large tumors, and (4) abdominal paracentesis. As do many other institutions, our IACUC makes recommendations to investigators to refine these procedures in order to reduce the level of discomfort. For example, the initial step in monoclonal antibody production is the generation and collection of stimulated cells from the spleen or lymph nodes. This typically involves initial immunization with antigen plus adjuvant, followed by 1 or more boosts, blood sampling for antibody titer, and finally euthanasia for harvest of immune cells. In an effort to minimize the discomfort associated with immunization, our IACUC sets limitations on: the use of certain adjuvants (e.g. CFA may be used for the initial immunization, but not for subsequent boosts), the total volume of injected material (e.g. 2 ml for IP injections), and the volume and frequency of blood sampling for antibody titers. Deviation from these recommendations must be scientifically justified. We do not, however, tell investigators that they may only inject 0.2 ml (vs. 0.5 ml) of Freunds adjuvant IP, or that they must limit their injections of the priming agent, pristane, to 0.2 ml. However, in light of growing documentation that lesser amounts of these agents may be as effective in generating antibody titers as larger volumes, while simultaneously sparing the mouse the pain and discomfort associated with larger volumes (Jackson and Fox, 1995) the IACUC may consider asking investigators to justify their use of larger volumes, i.e., to submit objective data that smaller amounts were ineffective with their particular antigen. This may, however, have the unfortunate side effect of causing more, rather than fewer, animals to be used in the initial stages of antibody production. Other potential sources of pain and distress in monoclonal antibody production are excessive abdominal distension, growth of large solid tumors in the abdomen, and abdominal paracentesis. Clearly, abdominal distension that is excessive and prolonged will not only cause chronic distress, but may also impinge upon the thorax and compromise breathing (McGuill and Rowan, 1989). Our IACUC, therefore, recommends daily observation once ascites develops and timely taps to prevent excessive distension. Three abdominal taps will usually harvest all of the fluid, with euthanasia following the third tap. If, however, an investigator is obtaining antibody-rich ascites beyond the third tap, and there is no evidence of a large tumor on abdominal palpation, then we feel additional taps are justified. The mouse must, however, appear healthy and be in good physical condition.

In addition to using published information, our IACUC solicits feedback from monoclonal antibody users as to their experiences with the various techniques. Their responses have been very useful to us in making recommendations that will be beneficial to the animals and yet not impede productive research. I'd like to share one of these responses with you.

"This letter is to describe to you the explorations my lab has made toward getting away from the use of live mice in the production of monoclonal antibodies. ...Our lab uses large quantities of several different monoclonal antibodies for affinity purification of antigens. We therefore try to produce 100 mg or so of monoclonal antibody per production run. Currently, our protocol involves growing the hybridoma cells as ascites tumors in mice. We recover ascites fluid from the abdominal cavity and obtain roughly 10 mg per mouse, typically in a volume of about 1-2 ml of fluid. We would be interested in getting away from live animal production if this were really feasible. In fact, we invested approximately $5,000 in a system for growing the hybridomas in capillary dialysis membranes and spent several months trying to make this technology function to produce good monoclonal antibodies. This approach requires very large amounts of tissue culture medium, which contains 10% fetal calf serum. So the expense was quite high. The antibody production never came near matching what we could get from a few mice. Moreover, the antibodies recovered from this system were proteolytically damaged, something we have not seen with antibodies derived from ascites fluid. After several months of frustration with batches of monoclonal antibodies that did not retain their normal binding capacity, we had to abandon this approach. When I find that the technology for in vitro production of monoclonal antibodies is available, I plan to put it to use. At present, I see no affordable technology that is adequate for our purposes. For the isolation of new hybridomas, in vitro technologies are not yet adequate. Even in vitro methods require sacrificing animals to obtain spleen or lymph node cells. In vitro immunization of these cells does not lead to hybridomas that secrete high affinity monoclonal antibodies to most antigens. To my knowledge, the only method that is successful for getting high affinity monoclonal antibodies to most antigens involves repeated immunization of animals, followed by generation of hybridomas. Our lab is a supplier of monoclonal antibodies (free of charge) to many other labs, well over 100 in the past 5 years. Because of the economy of obtaining monoclonal antibodies through live animal techniques, we are able to make these available to all of these other labs. Scaling back production to meet just my own lab's needs without use of live animals would force us to curtail this service to the scientific community."

In conclusion, I would like to say that the recommendations made to investigators by our IACUC will continue to change and evolve as new information and techniques become available; and these recommendations will take into consideration feedback from monoclonal antibody users regarding their experience with in vivo and in vitro methods. The composite of published information and investigator feedback assists the IACUC in making realistic and humane recommendations concerning in vivo monoclonal antibody production.

References

Amyx, H.L.(1987) Control of animal pain and distress in antibody production and infectious disease studies. JAVMA 191: 1287-1289.

CFR (Code of Federal Regulations) (1985) Title 9 (Animals and Animal Products), Subchapter A (Animal Welfare). Washington, D.C.: Office of the Federal Register.

Guide (Guide for the Care and Use of Laboratory Animals) (1996) Institute of Laboratory Animal Resources, Washington, D.C.: National Academy Press.

IRAC (Interagency Research Animal Committee) (1985)U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. Federal Register, May 20, 1985. Washington, D.C.:Office of Science and Technology Policy.

Jackson, L.R, and Fox, J.G.(1995) Institutional Policies and Guidelines on Adjuvants and Antibody Production. ILAR J. 37: 141-152.

McGuill, M.W.,and Rowan, A.N.(1989) Refinement of monoclonal antibody production and animal well-being. ILAR News 31: 7-11.

PHS (Public Health Service) (1996) Public Health Service Policy on the Humane Care and Use of Laboratory Animals. Washington, D.C.: U.S. Department of Health and Human Services, 28 pp.

Russell, W.M., and Burch, R.L. (1959) The Principles of Humane Experimental Techniques. Charles C. Thomas, Springfield, Ill.

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