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By Maureen C. Carroll, DVM, DACVIM and Megan Reilly, BS
Antibodies have become a clinically significant intervention in human medicine for over 30 years and fall under the umbrella of biotherapeutics. Hundreds of antibodies are currently in clinical development for diseases and conditions ranging from inflammation to cancer. Since 2015, 23% of drugs approved by the FDA have been antibodies and on average four monoclonal antibodies (mAbs) enter the drug market annually. MAbs in particular are developed to selectively target cellular receptors and molecules designed to prevent and treat disease.
Figure 1. From Microbials for the production of monoclonal antibodies and antibody fragments. Spadiut et al. 2013
Antibodies, produced by B-lymphocytes (B cells, plasma cells), are glycoproteins that consist of two heavy and two light chains (see figure 1 diagram). Each heavy and each light chain has a variable region. The Fab region is involved in antigen recognition and the Fc region binds to immune cells. In response to a ‘foreign invader’ the natural immune response involves the production of targeted antibodies to foreign molecules on the surface of these invaders. These molecules include proteins expressed on cancer cells, cytokines, and self-proteins like those produced in autoimmune disease. Typically the response to such an antigen is a polyclonal response, where many, many B cells produce thousands of antibodies directed at multiple components of the target. The important point here is that EACH clone of a particular plasma cell produces a single antibody, targeted at one surface protein. Hence these antibodies are deemed monoclonal antibodies. In summary, monoclonal antibodies are antibodies that are made by identical immune cells that are all clones of a unique parent cell. In contrast, polyclonal antibodies bind to multiple epitopes and are usually made by several different plasma cell lineages.
Figure 2. From Origin of mAb therapy. Kohler & Milstein 1975
In 1970, the first monoclonal antibodies were developed in mice. The mouse was injected with an antigen of interest, for example, human tumor necrosis factor (TNF). The plasma cells of the mouse began to make antibodies against human TNF. These plasma cells, clones making the same antibody, were isolated. Once isolated, they were fused into mouse myeloma cells (in vitro) so they could live and replicate indefinitely. The monoclonal antibodies made by the now myeloma, were isolated. Recombinant DNA technology is then applied to ‘humanize’ the antibody, so as to prevent rejection of mouse protein by the human immune system. Once injected into a human, these antibodies would target TNF and inactivate or eliminate it. Similarly antibodies can be ‘caninized’ or ‘felinized’ for use in dogs and cats.
Fast forward to today, where monoclonal antibodies are made in other mammalian cells (i.e. CHO or Chinese hamster ovary cells) as well as in bacteria (E. Coli) and yeasts. By far, most mAbs are of the IgG class. Hybrid antibodies (chimeras) have been created which reduces the immunogenicity of the mAb while maintaining specificity. It should be noted that an important objective in designing any mAb is to ensure that the therapy avoids abolishing normal and important physiologic and immune responses.
The mechanisms of action of mAbs are outlined below (see figure 2).
Ligand blockade: mAb binds to ligands and prevents activation of the receptor
Receptor blockade: mAb binds to the receptor, and blocks the ligand from binding.
Receptor downregulation: mAb binds to cell-surface receptors, downregulates production of the receptors.
Depletion: mAb binds to antigen-bearing cells, which then causes the destruction of the cells.
Signaling induction: mAb bind to cell-surface and transmits intracellular signals.
As mentioned earlier, the targets of the mAb can be a cytokine, a target on a cell surface to block activation, or a target on a cell surface that results in antibody-dependent destruction.
The route of administration of antibody therapy is either via intramuscular or subcutaneous injection. Most proteins, when taken orally, would be broken down by stomach acid, therefore parenteral routes are necessitated. The half-life of each specific antibody will define the dosing schedule and mode of delivery; the half-lives of most antibodies are several weeks in duration. Some antibodies are infused every 2 to 4 weeks, while others are infused every 48 weeks. Subcutaneous administration is the most convenient for human patients particularly those with chronic diseases requiring long-term therapy. Visits to the doctor’s office for infusions are not required for these particular antibodies.
Because monoclonal antibodies don’t have intracellular activity, they are usually well-tolerated in both animals and people. Common self-limiting side effects include vomiting, diarrhea, and lethargy. However, although the development of chimeric monoclonal antibodies has decreased the incidence of immune responses against the antibody itself, antibody synthesis directed against the therapeutic monoclonal antibody can occur; this is known as immunogenicity. As a result the half-life and clinical effectiveness of the mAb can be diminished and some patients experience infusion-related anaphylaxis. The incidence of immunogenicity can be attenuated by co-administration of immunosuppressive agents in these patients.
Monoclonal antibody therapy in human medicine is used in the fields of oncology, inflammation, and autoimmunity which includes conditions like rhinosinusitis, rheumatoid arthritis and inflammatory conditions of the gastrointestinal tract, such as Crohn’s disease. The first therapeutic antibody for the treatment of inflammatory diseases was infliximab, or Remicade, which was developed in 1998 for the treatment of Crohn’s disease and rheumatoid arthritis. Infliximab is a chimeric antibody with mouse variable domains and human constant domains which binds both soluble and membrane associated tumor necrosis factor or TNF. The TNF antagonists are presently the most successful class of biological drug for inflammatory diseases with a total worldwide sales of 16.4 billion in 2008.
In veterinary medicine, one of the first monoclonal antibodies was developed in the 1990s for the use and treatment of canine lymphoma. Monoclonal Ab231 was the first antibody licensed for cancer treatment, but unfortunately was discontinued in 1996 due to lack of market demand. The most popular and commonly used monoclonal antibody on the market today in veterinary medicine is Lokivetab, trade name Cytopoint. This mAb is an anti-canine IL-31 monoclonal antibody that binds circulating IL-31. IL-31 is a cytokine involved in the development of pruritus and atopic dermatitis. In a recent study up to 57% of dogs experienced treatment success for 28 days post-injection.
Nerve growth factor (NGF) is yet another target for monoclonal antibody therapy in both human and veterinary patients. NGF is a cytokine produced and released by peripheral tissues before it binds to tropomyosin receptor kinase A (trkA). This receptor has been found to play a critical role in nociception in acute and chronic pain conditions. Recently a species-specific anti-NGF monoclonal antibody for the management of osteoarthritis-associated pain in dogs and cats has been developed and is in early clinical trials. The results thus far appear to be very promising as a novel therapy against chronic pain in dogs and cats. Nexvet has a proprietary method of creating fully caninized and fully felinized anti-NGF monoclonal antibodies, ranevetmab and frunevetmab, respectively. Preliminary studies are very promising and also include control of fracture pain, cancer pain, and pancreatic pain. In nearly all of the conditions tested the average pain is reduced by 30-50%.
The outlook is promising as it pertains to the development of monoclonal antibody therapy in veterinary medicine. We have already developed mAbs for lymphoma, allergy, and pain. On the horizon would be treatment for autoimmune disease such as IMHA, ITP, and myasthenia, as well as other cancers. Biotherapeutics and targeted pharmacology are the wave of the future in the treatment of disease in veterinary medicine.
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