Shifting Away from Animal-Derived Antibodies in Medical Research
Antibody production is a massive industry as antibodies serve as fundamental tools in biomedical research. The vast majority of research antibodies are produced by live animals using procedures that are extremely painful and ultimately lethal. Physicians Committee scientists are working to prove that antibodies can be made without harming animals. Our goal is to demonstrate the viability of fully in vitro antibodies, promote their acceptance by researchers, and force a change in the funding requirements of the National Institutes of Health.
Antibodies are a class of specialized molecules produced by the immune system of an organism. They are Y-shaped proteins belonging to the immunologlobulin (Ig) superfamily of proteins, which are generated and secreted by a type of white blood cell known as B lymphocytes. Their biological function is to fight disease, to attack and help remove bacteria, viruses, and other foreign particles from the body.
However, scientists have found that antibodies can be used as a tool in laboratory applications. They have exploited the body’s innate biological immune response to generate antibodies against virtually any foreign material of interest (proteins, DNA fragments, hormones, lipids, etc.) in order to study how diseases progress and develop. Antibodies are now widely used as essential tools to detect, isolate, track, and quantify various biological molecules in medical research. For example, scientists can use an antibody generated against insulin to monitor how insulin levels change in diabetes.
Figure 1: Animals used for research antibody production
Currently, the vast majority of antibodies available for research purposes are produced by live animals through procedures that are of great concern from both animal welfare and scientific standpoints. Animal-derived research antibodies are produced using methods that are inherently cruel. Millions of animals suffer for prolonged periods of time to produce antibodies used in research.
Two methods are used to produce the two types of antibodies that are most commonly used in research: monoclonal antibodies (primarily generated in mice) and polyclonal antibodies (primarily generated in larger animals, such as rabbits and goats). As you can see from Figure 1, scientists have not left many species behind when it comes to antibody production.
In order to use an animal as an antibody factory, researchers first inject the animal with a substance against which an antibody is desired (e.g., proteins, DNA fragments, hormones). The animal then receives booster immunizations and other chemicals to heighten the immune response. The animal’s immune response (i.e., the creation and presence of antibodies in the blood) is then monitored by extracting blood.
Mouse Monoclonal Ascites Method
In the mouse monoclonal ascites method, once the animal begins making the desired antibody, the mice are killed and their spleens are harvested to isolate the antibody-producing B lymphocytes. These cells are then fused with cancerous cells to generate a hybrid cell type known as a “hybridoma,” which will multiply indefinitely.
These hybridomas are injected into the abdominal cavities of mice where they multiply continually. This causes relentless painful fluid accumulation (ascites fluid) in the sac surrounding the abdomen. The ascites fluid contains high concentrations of antibodies specific to the hybridoma clone used. Researchers periodically extract this fluid to collect the antibodies (Figure 2).
Potential side effects of these procedures include hemorrhage, swelling, and death. The ascites method causes excruciating pain, as the ascites fluid accumulates in the abdomen. Mice used in ascites frequently suffer from anorexia, dehydration, difficulty walking, respiratory distress, circulatory shock, and peritonitis (an extremely painful inflammation of the thin tissue that lines the inner wall of the abdomen and covers most of the abdominal organs). The longer the process lasts, the more the animal suffers.
Mice used for ascites production are usually given a “priming” agent before the hybridoma is injected into them. This agent prepares their abdominal cavities for ascites fluid growth. Priming agents such as pristane, a carcinogen, causes peritonitis (inflammation of the abdominal lining), arthritis, inflammation of the kidneys, and other painful pathological conditions.
The ascites method has been deemed so cruel that it has been banned (either fully or partially) in Belgium, Germany, the Netherlands, Switzerland, the United Kingdom, Sweden, and Australia. There is no sign of progress towards its prohibition here in the United States.
Figure 2: Monoclonal and polyclonal antibody production methods
Polyclonal Antibody Production
In polyclonal antibody generation, animals such as rabbits, goats, and sheep are immunized with a specific substance against which an antibody is desired (e.g., proteins, DNA fragments, hormones). They are given booster shots at certain intervals (usually weekly) to produce more of the desired antibody.
The animals are then bled to confirm that the desired antibody is in their blood serum. Researchers inject the animals with more booster shots if they want to increase their immune response. Researchers perform further test bleeds to ensure antibodies specific to the target are being generated in high concentrations.
Once the animal is producing antibodies specific to the target of interest, researchers extract large volumes of blood at various intervals to isolate large quantities of antibodies. Bleedings can also be painful and distressing, and bleeding an animal frequently can lead to anemia, resulting in loss of energy.
Researchers bleed animals for a few months and finally bleed them to death. Animals suffer through these procedures without appropriate painkillers, which are costly and can interfere with the immune response. They also endure repeated invasive procedures for long durations of time, ranging from several weeks in a mouse to several months in large animals.
Animal welfare concerns regarding the ascites method has prompted an inquiry by the NIH to the National Academy of Sciences. The inherent cruelty involving hybridoma production for monoclonal antibodies has received considerable attention. The cruelty involved in the production of polyclonal antibodies is also intense, but receives less scrutiny.
Figure 3: Animal welfare violations at SCBT
Although U.S. suppliers are required to adhere to the minimal animal welfare standards of the Animal Welfare Act (AWA), scant federal oversight invites shortcuts, as evidenced by United States Department of Agriculture (USDA) inspections of Santa Cruz Biotechnology (SCBT). SCBT, the second-largest suppliers of research antibodies in the United States, has repeatedly violated the AWA. Between 2007 and 2012, the UDSA cited SCBT numerous times (with nine separate inspections in 2012, alone) for negligence and for leaving animals with injuries and serious medical conditions without veterinary care (Figure 3).
In addition to the animal welfare concerns, commercial antibodies made with animals commonly lack two important characteristics, specificity and affinity. That is, if antibodies were torpedoes, ideally they would attack only a certain type of target (specificity) and would attack voraciously (affinity). Specificity and affinity are the two hallmark features that make research antibodies especially useful for scientists, and the current lack of specificity and affinity that result from animal-derived antibodies wastes considerable time, intellectual effort, and money and hinders research progress.
Lack of specificity in antibodies is a major issue that arises due to the inherent unpredictability of animals’ immune systems. Researchers know that when it comes to commercial antibodies, what is on the label does not necessarily correspond to what is in the tube. There are numerous documented examples of nonspecific, low-affinity antibodies, and of antibodies that completely fail to recognize their targets. Some scientific journals have implemented strict measures to require that details of specificity validation be included in manuscripts publishing antibody-based studies. In all of these cases, researchers are referring to animal-derived antibodies.
Developing Nonanimal Antibodies
Technologies already exist to make highly specific, high-affinity, antibodies without using animals. These are called recombinant antibodies.
However, without a market demand, biotechnology companies have no incentive to change their manufacturing practices. Market demand has to be driven by researchers and major research funding agencies.
Unfortunately, most researchers are either unaware that the technology exists or are skeptical about the quality of the end products because they have not had the opportunity to use recombinant antibodies in their own research. Of the catalogued 350,000+ animal-derived antibodies, only around 300 are recombinant antibodies.
Even if researchers recognize the advantages of recombinant antibodies over those derived from animals, they are limited by the quality of the antibodies supplied to them by the industry, which is almost exclusively animal-derived. The NIH last reviewed the methods of monoclonal antibody production in 1999 and places no restrictions on how antibodies used in federally funded research are sourced.
Even in countries that have banned the monoclonal ascites method, they still use animals in the first stage of antibody production until they make “hybrid cells” (immunization, bleeding, and harvesting of spleen to isolate antibody-producing cells, see Figure 2), which are then grown using cell culture methods to generate a continuous supply of antibodies. There are no alternatives to the use of animals in polyclonal antibody production. Therefore, the goal of our project is to demonstrate that highly specific, high-affinity, fully in vitro recombinant antibodies obtained without the use of animals at any stage of antibody production can replace nonspecific, low-affinity, animal-derived antibodies in research.
Once we show this, we will promote the widespread acceptance and adoption of those antibodies for research purposes. As long as animals are used at any stage of antibody production, animal cruelty is likely to continue. The onus is on the researchers, producers, and government, collectively, to advance cruelty-free antibody production methods.