Antibodies serve as markers, seeking out and binding to antigens, or foreign proteins including bacteria and viruses, so that other immune system cells called phagocytes can find and destroy them, ridding the body of the intruders. The antibodies do their work by binding themselves to the part of the antigen known as the epitope, and it proves to be significant for monoclonal antibody therapies that the binding takes place even when the entire antigen molecule is not present. (Cancer is exempt from this immunological defense, because the immune cells do their job by differentiating between the self and the non-self, and tumors count as home-grown cells.)
Another critical fact is that each antibody, like the B cell that produced it, is specific to one particular antigen—it only binds to the epitope it was meant to find. That means that our bodies are populated by vast numbers of B cells and the highly specialized antibodies they produce in response to the various antigens we have been exposed to.
For science, that understanding was both an opportunity and an obstacle until Kohler and Milstein made their breakthrough. If a specific antibody could be aimed at its intended epitope through clinical means, it could perform several key functions in diagnosis and treatment, including helping to deliver toxins to cancer cells without attacking normal cells.
The rub, however, was that to turn that knowledge into a useful tool required significant amounts of a single antibody from a single ancestor cell. Several factors combine to make that impossible in the normal course of cell development. First, a single B cell must be cultured to produce a population of identical B cells, each of which would spawn the desired antibodies—monoclonal antibodies. The quest for that ancestor cell from a human, or other organism, was stymied by the immune system’s response to any antigen, because the system invariably manufactures a wide range of antibodies with different structures, resulting in polyclonal antibodies.
Beyond that, the B cell’s own life cycle means that, even if a single antibody-producing cell is isolated and cultured, the population of antibodies it will produce is too limited for use in human therapy, because it would die out after a few generations in the lab.
To overcome these obstacles Kohler and Milstein immunized mice with specific antigens to trigger B cells to produce specific antibodies. They next harvested the antibody-secreting cells from the spleens of the mice, and fused them with myeloma cells, producing what are known as hybridoma.
The process built on the fact that the rapidly proliferating myeloma cells are, in scientific terms, immortal—and therefore would overcome the self-limiting life span of normal B cells—and the fact that the spleen cells contain a critical enzyme called HGPRT that the myeloma cells lacked, but need for growth. That meant that the hybridoma cells could grow indefinitely and produce an infinite number of monoclonal antibodies, because the spleen cell provides the HGPRT and the myeloma cell supplies the immortality.
For the first time, monoclonal antibodies (or, MAb in medical shorthand) could be created in unlimited supplies and put to work fighting an array of diseases.
The discovery, however, may have generated more excitement than results in the procedure’s early years. The biggest problem was that the antibodies the process yielded were mouse antibodies (murine), and the human immune system frequently reacted against them, as with any other foreign substance entering the body. That reaction produced problems ranging from rashes to swelling of the joints to kidney failure and death; it also destroyed the antibodies and defeated the mission.
Even so, scientists went back to the labs and found a number of ways to address the problem successfully, largely by finding a variety of ways to make the antibodies more human and less murine. One technique is to create “chimeric” antibodies by replacing some of the problematic regions of mouse content with human protein in a fusion process that results in antibodies that are about 65 percent human. At least four monoclonals now on the market in the U.S. are chimerics, including a drug called ReoPro, which binds to platelets to prevent blood clots.
Another approach has been a grafting process that produces what are known as “humanized MAbs,” wherein some 95 percent of the resulting molecule is human in origin. This technique is employed in such drugs as Herceptin, a monoclonal antibody used to target breast cancer.
Meanwhile, other researchers have developed techniques to create hybridomas that produce fully human MAbs, including at least one successful effort in England to fuse fully human B cells and immortalized cells, although scientists say it remains unclear what the long term implications and efficacy of the technology might be.
Other researchers have found ways to genetically alter the mice themselves to produce fully human antibodies, by causing the animals to contain human antibody genes. Still others are experimenting with an altogether different process that dispenses with the mice. “Phage display” (the word is an abbreviated form of bacteriophage, a virus that infests bacteria) involves inserting DNA from B cells into bacteria and then allowing phages to infect the bacteria. As the phages replicate, they also recreate the proteins from the antibody genes, which can be cultured.