In 1975 George Kohler and Cesar Milstein published a paper describing
their method for producing monoclonal antibodies (MABs). Their work
included the observations that "the manufacture of predefined specific
antibodies by means of permanent tissue culture cell lines is of general
interest" and "such cells can be grown in vitro in massive cultures."
Although the original research was principally an in vitro technique,
monoclonal antibodies can be produced by injecting the hybridoma cells
into the abdominal cavities of rodents. This was the initial use of the
ascites method. Since MABs were easily made in any laboratory and the
ascites process was introduced early, its use rapidly expanded.
Unfortunately, the original possibility of MAB production replacing uses
of laboratory animals was and often continues to be overlooked or
ignored. In the decades that followed the original discovery, tens of
millions of animals suffered and died despite the availability of more
humane alternatives.
During this same period of time the appropriateness of using the
ascites method was increasingly questioned in Europe. Milstein noted
that "in later years, both on practical and humane grounds, I became
concerned with the use of ascitic fluids," (Pers. com., 1997). As new in
vitro alternative techniques were developed and validated, it became
more difficult to justify the suffering associated with the use of
ascites.
There are three principal stages in the production of monoclonal
antibodies: immunization (in vitro or in vivo), hybridoma formation (in
vitro), and MAB production (in vitro or in vivo). If done in vivo, the
first and last procedures are routinely associated with animal suffering
and therefore are prime candidates for replacement alternatives.
Support for the continued use of ascites methods are usually based on
claims of: (1) more rapid production and high yields of concentrated
MABs; (2) minimal requirements for materials, labor, and technical
expertise; (3) most hybridomas will grow in mice; (4) supposedly less
expensive; and (5) either a generic resistance to or lack of familiarity
with in vitro methods.
With increasing expenses of caring for laboratory animals and
decreasing costs for in vitro alternatives, the differential (if it
exists) between animal and non-animal-based methods is rapidly
disappearing.
The most compelling arguments against the use of ascites are derived
from the suffering of the animals. In human clinical experience the
growth of abdominal tumors is very painful. Ascites fluid accumulation
in human patients causes discomfort and pain, while never allowed to
progress to advanced stages routinely seen during ascites production in
animals. Such procedures in the United States involve 500,000 to 1
million animals per year.
Animals utilized for ascites frequently exhibit a spectrum of
clinical symptoms ranging from anorexia and dehydration to difficulty
walking, respiratory distress, circulatory shock and classical
peritonitis. These symptoms become increasingly severe the longer the
animals are allowed to survive (Jackson et al., 1996; Anon., 1996.).
Pathological changes associated with ascites are known for each step
in the process. Use of adjuvants produces mild to severe peritonitis and
inflammation. Fluid removal may cause hemorrhage, edema, and death. As
expected, growth of the ascitic tumors creates: (1) adhesions throughout
the abdomen; (2) lymphatic obstruction; (3) tumors with extensive
hemorrhagic and necrotic areas; (4) centro-lobular liver necrosis; and
other pathologies. (Anon., 1989; Jackson, et al. 1996).
The significance of the list of abnormalities associated with ascites
production is further emphasized by observations that the animals may
experience severe pathologic changes in their abdomens and chests, but
appear to be clinically normal.
From all of the above, it is apparent that animals used for ascites
production of monoclonal antibodies are routinely subjected to chronic
pain and distress. Simple common sense dictates that it is not possible
to humanely produce MABs using rodent ascites.
The most appropriate type of in vitro alternative for each research
or diagnostic situation depends largely on the quantity and purity of
monoclonal antibody needed. There are, however, some general criteria
for rating each system. The ideal method (1) should have expendable
material costs similar to that of a mouse; (2) be a simple technique;
(3) not require prior adaptation of the hybridomas or special culture
conditions; (4) have significantly higher concentration and quality of
MABs than for simple stationery cultures; (5) be a closed, reusable
system; (6) be affordable to all laboratories; and (7) produce adequate
quantities of MABs in a reasonable period of time (Falkenberg, 1995).
Only a few of the available in vitro methods meet all of these
criteria, but there are a wide variety of options available to
investigators, ranging from simple, individual cell culture containers
to giant, commercial bioreactors (Kamp & de Leeuw, 1996).
In contrast, ascites is subject to criticism on technical criteria,
including: (1) ascitic fluids contaminated with rodent plasma proteins,
immunoglobulins, infectious agents and bioreactive cytokines; (2) the
need for extensive animal facilities, associated support services with
individual animals requiring daily monitoring; (3) some hybridomas are
difficult to grow in rodents; (4) rodents only produce MABs for a few
days; (5) from 60 to 80% of mice may not produce ascites due to
premature death, development of solid tumors, or failure to establish
hybridoma growth; and (6) individual batches of ascites may vary
significantly in quality and quantity.
Because in vivo production of monoclonal antibodies involves the
largest number of animals and greatest degree of suffering, it receives
the most attention for the development of humane replacement
alternatives. There are, however, three additional aspects of the
process for which alternatives are appropriate and needed.
In vitro immunization needs further development so it can be applied
to the broad range of MAB needs. Due to serious humane concerns related
to its production, the use of animal serum represents another candidate
for appropriate replacement in all in vitro alternatives. Finally, there
is a need to replace the use of animal cells with those derived from
humans.
Although ascites production can be refined (limiting the weight gain,
number of animals and tappings, requiring anesthetics, changing the
types of adjuvants, etc.), there really is no need to do so. Replacement
is the preferred and available option.
The issues discussed in this workshop have already been addressed in
considerable detail in Europe. In June 1988, the Dutch Society for
Laboratory Animal Science, the Animal Experimentation Department of the
Veterinary Chief Inspectorate and the Dutch Societies for Biochemistry,
Immunology, Microbiology, Pathology and Toxicology held an inquiry,
which determined that ascites involved "considerable distress for the
animals."
As a result of this meeting, in 1989 The Netherlands government
introduced a Code of Practice for the Production of Monoclonal
Antibodies (Anon., 1989), which placed reasonable restrictions on the
use of ascites. The resulting increased humane awareness among Dutch
researchers provided further encouragement for adoption of in vitro
approaches to MAB production. In November 1995, a symposium held in
Bilthoven, The Netherlands, concluded that progress in the development
of such alternatives (both in efficacy and cost) was sufficient that the
use of ascites could no longer be justified (Hendriksen et al., 1996).
The resulting prohibition of animal-based MAB production caused no
serious difficulties within the Dutch biomedical research community.
Despite initial academic resistance, bans on ascites in Germany and
Switzerland experienced similar results, as did the restrictions placed
on ascites use in Sweden and The United Kingdom.
Scientist representatives from several member states of the European
Union met in November 1996 at the European Center for the Validation of
Alternative Methods to discuss the current status of in vitro and in
vivo methods of monoclonal antibody production. After careful
consideration of the research and commercial needs for MABs and the
available production options, they concluded "for all levels of MAB
production, there are one or more in vitro methods which are not only
scientifically acceptable, but are also reasonably and practically
available; and as a consequence, in vivo production can no longer be
justified and should cease." (Marx et al. 1997.) The group further
called for a Europe-wide prohibition on the routine use of ascites
methods.
In the United States another important issue is the responsibilities
of the Institutional Animal Care and Use Committees (IACUCs) in relation
to alternatives generally, and rodent ascites specifically. IACUCs have
a statutory and moral responsibility to require investigators to search
for suitable alternatives to painful procedures and to require their
use, if found. Since multiple alternatives to ascites are readily
available for all routine MAB production needs, no IACUC should approve
an ascites protocol. Investigators should be aware of these alternatives
and be required to use them. Failure to do so suggests an incomplete or
non-existent search for appropriate alternatives.
It is important to stress that a requirement to use alternatives to
ascites has no more to do with experimental design, than does a
requirement to use a particular anesthetic or veterinary procedure.
If current trends continue, the use of animals to produce monoclonal
antibodies will be replaced with in vitro alternatives.
More than 20 years ago a new, in vitro alternative was developed for
the production of monoclonal antibodies. Because of its simplicity and
importance, this technique was widely adopted. For similar reasons, a
new form of laboratory animal cruelty and suffering was also created --
the use of ascites.
For the last decade researchers in Europe and the United States
systematically developed and adopted multiple in vitro replacements for
the use of rodent ascites. This process has now progressed to the point
that it is possible to prohibit the use of ascites in all but the most
unusual and rare circumstances.
The American Anti-Vivisection Society decided it was time for
researchers and regulatory bodies in the United States to join their
European colleagues in ending such widespread, systematic and
unnecessary animal suffering. The Antibodies Without Animals Campaign
was initiated to finally implement the humane possibilities of Kohler
and Milstein's original work and to initiate a new perspective among
U.S. regulatory agencies regarding the promotion and use of alternatives
to traditional forms of laboratory animal usage. These petitions are
good for the animals. good for promoting alternatives, and good for
biomedical sciences. Further, they represent an ideal opportunity for
the animal advocacy and biomedical research communities to work
together. The case for replacement of ascites is so strong, that this
should be a win-win situation for both groups (McArdle, 1997).
Alternatives have been described as the gentle hand of science. The
topics of this workshop represent a perfect opportunity to promote that
perception.
References
1. Anon (1989) Code of Practice for the Production of Monoclonal
Antibodies. Rijswijk, The Netherlands: Veterinary Health Inspectorate.
2. Hendriksen, C., J. Rozing. M. der Kamp and W. de Leeuw (1996) The
production of monoclonal antibodies: Are animals still needed? ATLA. 24.
109-110.
3. Jackson, L.R., L.J. Trudel, J.G. Fox and N.S. Lipman (1996)
Clinicopathologic features and production parameters of monoclonal
antibody production in murine ascites. (In preparation.)
4. Kamp, M. and W. de Leeuw (1996) Short review of in vitro production
methods for monoclonal antibodies. NCA Newsletter. 3. 10-11.
5. Kohler, G. and C. Milstein (1975) Continuous cultures of fused cells
secreting antibody of predefined specificity. Nature. 256. 495-497.
6. Marx, U., M.J. Embleton, R. Fisher, F.P. Gruber, U. Hansson, J. Heuer,
W.A. de Leeuw, T. Logtenberg, W. Merz, D. Portetelle, J-L Romette and
D.W. Straughan (1997) Monoclonal antibody Production; the report and
recommendations of ECVAM Workshop 23. ATLA. 25. 121-137.
7. McArdle, J.E. (1997) Alternatives to ascites production of monoclonal
antibodies. AWIC Newsletter. In press.
