Most of us have had to take medicine at some point in our lives, but very few of us ever stop to think about where our medicine comes from or the technology used to produce it. The technology discovered allowing production of monoclonal antibodies has been critical in finding new life saving medicines over the last few decades. Antibodies are produced by B lymphocytes, as part of our immune system, and they act as identifiers and bind to foreign proteins in the body called antigens. Once bound to an antibody, the antigen can be recognized and eliminated by phagocytes in the body, thus protecting us from foreign invaders.
Moving production of antibodies from the human body to the lab was heavily researched in the early 1970’s. In 1975 two researchers, Georges Kohler and Caesar Milstein, joined together and discovered the technology for producing a monoclonal antibody using hybridoma technology. In another laboratory around the same time, Niels Jerne had discovered a plaque assay to show a visual representation of antibody producing B cells. These three researchers changed medicine forever and went on to share the Nobel Prize in medicine in in 1984. Because Milstein and Kohler never patented their discovery, it opened the door for new monoclonal antibody medicines to be discovered over the next thirty years.
The technology for creating a hybridoma to produce monoclonal antibodies involves creating a hybrid cell (two cells fused together). The fused cells are a B lymphocyte and a myeloma cancer cell. The B lymphocyte cell carries the specific antibody production capability and the myeloma cell has the ability to grow indefinitely at a faster rate than normal cells. The B lymphocyte cells have a short lifespan, so they need to be fused to an immortal myeloma cell and there is obvious benefit to using a cell for discovery that grows fast. Researchers get the antibody producing B lymphocyte by using a lab animal, usually a mouse that has been exposed to an antigen that the researcher is interested in producing an antibody against. The selected antigen is then injected into the mouse to trigger an immune response. The mouse’s immune system begins creating antibodies to the antigen and then it’s B cells are then retrieved from the mouse. The B cells are then fused to the myeloma cells, put into selection media to eliminate any unfused cells, and then the antibodies are screened to check for antibody production and specificity. If researchers find an antibody of interest, they can clone the specific hybridoma cell then grow these cells in culture to produce monoclonal antibodies at larger scale.
Utilizing hybridoma technology, the first monoclonal antibody was approved by the FDA in June 1986. The drug, Orthoclone OKT3, was approved as an immune suppressant to reduce rejection in organ transplant patients. The problem with murine (mouse) antibodies is that they are seen as foreign by the human body and in some patients can cause side effects similar to a severe allergic response. In addition, because they are seen as foreign, the body acts quickly to remove them from the blood stream meaning frequent dosing and an overall weaker immune response to the antigen.
As the technology has evolved, the goal has been to make a more human antibody by using genetic engineering. Using genetic engineering, researchers can obtain the sequence for the antibody and replace some of the mouse DNA code with human DNA code. The result is called a chimeric antibody, which is usually about 33% mouse and 67% human. There can still be some negative immune response, but much reduced from what is found with murine antibodies. The first FDA approved chimeric antibody was Reopro, an antithrombotic therapy, approved in 1994.
In an effort to continue to make these antibodies more human-like, researchers were able to use genetic engineering to replace more of the mouse DNA with human and create humanized antibodies. Now these antibodies are generally 5-10% mouse and 90-95% human. With these antibodies there is minimal negative immune response. The first humanized antibody approved by the FDA was Zenapax in 1997. Zenapax is used to prevent rejection of kidney transplants.
The next phase of development was to produce a fully human antibody with no mouse portions. This is accomplished by using a human antibody sequence or by using a transgenic mouse that carries human antibody genes. In 2002, the FDA approved the first fully human antibody, Humira, to treat rheumatoid arthritis. The antibody used in Humira was discovered using the sequence found in a human antibody library. In 2006, the FDA approved the first human antibody derived from a transgenic mouse, Vectbix, approved for treatment of colorectal cancer.
Another evolution for hybridoma technology has been the media in which the cells are grown. In the beginning, hybridoma media usually consisted of base media plus serum. Serum and other animal derived ingredients are undesirable because they are undefined and have high batch-to-batch variation that leads to unpredictable results. Serum also contains contaminating IgG, which makes purification of the antibody difficult. These issues have caused a migration toward serum-free hybridoma culture. To assist in the endeavor to become animal-free, animal components can now be replaced by utilizing innovative animal-free supplements, such as recombinant albumin and recombinant transferrin.
Twenty-two monoclonal antibodies have been approved by the FDA since the first in 1986 and there are many more in the pipeline. The technology has evolved dramatically since being discovered in 1975 and one can only wonder where monoclonal antibody technology will take us in the future and what kinds of treatments will be developed as a result.