I guess this description was inevitable, but here goes.
You have probably heard the term thrown around a lot lately and you will certainly hear it more and more as the new serologic tests and plasma treatments become more common (sorta neat huh, that you now know what a “serologic” test is and what “plasma” is?).
Anyway, most stories start at Genesis and go on to finish at Deuteronomy. This time I am going to tell the story in reverse. It’s more fun that way, and I think it may actually make more sense. The story has a number of interesting elements to it, not only about the current tests and treatment, but about a whole range of drugs that you know, take or have seen advertised on TV. It also is a great window into understanding the immune system itself, and it is a fantastic tale of how basic research, research that would never have been funded by for-profit companies, research that wouldn’t have been funded by charitable organizations interested in treating their pet diseases (as opposed to the diseases of their pets), but had to have been funded by governmental sources, led to a revolution in medicine.
So, let’s start at today and then go backwards.
We have talked about ELISA and RIA tests and how you need to have antibodies that recognize the virus in order to use those tests. (As an aside, the new point-of-care serologic tests are very similar to ELISA tests, but reduced to a form that can be done on a strip of paper, or a plastic test strip. It isn’t necessary to go into detail about the technology, but it is, at its core the same thing as an ELISA test.)
We have also talked about how the immune systems builds a large armamentarium of antibodies, randomly changing the “locks” on the ends of the antibodies, looking to find new “keys” on foreign things in the body like viruses; the immune system may produce millions of different antibodies whose locks fit on that virus, binding to different proteins on the virus surface or different places on the same protein. There may be some locks that are so-so fits to the keys, some that are pretty good and some that are great. When the locks on the pretty good ones and the great ones engage with the keys on the virus, the locks are opened, and the cell is told “go forth and multiply”. The open lock flips a switch on the cell, which then begins to divide. It secretes lots of antibodies into the blood and it signals other parts of the immune system that have the same locks on them but perform different tasks than just making antibodies, that they have to start dividing also.
The plasma of a patient who has had such an immune response is, therefore, filled with a wide variety of antibodies that can bind to the virus, as well as even more antibodies against lots of other things. Some of these antibodies bind to parts of the virus that actually stop it from binding to the cells in the body, some just bind to the virus without really causing a “neutralizing”, or protective effect. By giving this plasma to another sick patient, the hope is that the antibodies in that plasma will act on the virus in the same way that they acted in the donors blood, protecting the patient from disease, and giving that patient some time to develop their own immune response. But there is also a risk that the other antibodies, proteins, hormones and substances in the donor’s blood might also affect the patient’s health.
But, don’t you think that it would be even better if, rather than using these millions of different antibodies, with millions of different ways to “recognize” the virus, we were able to separate ONE, very specific antibody, with the best lock, recognizing the most perfect key on the virus, the one that shuts down the virus and prevents it from infecting a cell? That would be a great protein to treat the patient, and it would be the perfect reagent to use in the ELISA tests, or in the quick point-of-care tests.
So, how do you single out the one antibody from the millions in the plasma of a patient, and then, how do you make lots and lots of it so that it can be shipped around the country and the world for use?
The answer is “monoclonal antibodies”. The term can be read: “mono”, meaning “single”, clonal, meaning “a group of cells that are all exactly the same”, and antibodies meaning, well I think we all know that one by now. So Monoclonal Antibodies (generally abbreviated as MAB), are the proteins that we want to use for plasma treatment and for assay tests.
ARE MABs NEW?
No. The pharmaceutical use of Monoclonal Antibodies has been around since 1986 when the first one was used to help in transplantation. This MAB was specific for a receptor on a class of immune cells that are responsible for tissue rejection, and therefore, by stopping rejection it was very useful for organ transplant patients who were resistant to steroids, chemicals traditionally used to suppress the immune response.
Today, there are many MAB drugs on the market for a wide variety of diseases. The next time you see a drug commercial on TV, take a look at the name of the drug in parentheses below the brand name. If that name ends in the letters “mab”, you can be certain that the drug is a Monoclonal Antibody. Since these drugs are proteins, not small chemical molecules, if you were to take them through an oral pill, they would be digested in your stomach and intestines before they ever got into your blood stream, so they all have to be given intravenously.
Some of the common drugs of this type include:
HUMIRA (adalimumab)
BENLYSTA (belimumab)
RAPTIVA (efalizumab)
REMICADE (inflectra)
OPDIVO (nivolumab)
COSENTYX (secukinumab)
HOW DO YOU MAKE AN MAB?
So, now we need to jump back to the beginning of the story.
In 1973 an Argentinean émigré named Cesar Milstein who had joined the faculty at Cambridge University in England was working at the Basel Institute of Immunology in Switzerland. There he met a graduate student from Germany named Georges Kohler. Kohler joined Milstein’s lab as a post-doctoral fellow in 1974.
These scientists, as well as a number of others in that department at Cambridge were interested in trying to figure out a basic question in immunology. That question was maybe the same question that you have been wondering about having read my posts. Namely, how is it that the immune system is able to generate so many different types of antibodies? The small variations right at the tips of the antibodies where the “locks” are, are mutated constantly in order to create those vast libraries of different antibodies (referred to as the antibody “specificities”). How does this happen? What is the way the cell manufacturing machinery does this in these cells when in other cells, the proteins that they make are always exactly the same over and over? For example, the islet cells in your pancreas all make the same molecule of insulin (or at least in some of us, they used to).
This is a very basic scientific question. It doesn’t pertain to a disease particularly; it doesn’t ask any question about a cure or a treatment; it doesn’t answer any question about how to make a product that can be sold. It is just a basic question that, to those involved in the field is particularly interesting and difficult to understand. This is what is referred to when people talk about “Basic Research”. It will never be funded by any commercial enterprise, but it can yield, as we will see, the MOST important advances.
On August 1, 1975, Kohler and Milstein published a very short (only three pages) paper in Nature, perhaps the single most highly respected scientific journal in biology in the world. The purpose of their research was to “understand the expression and interactions of the Ig chains from” parental cells. In order to try to study this question, they had developed a tool to use for the study.
They knew at the time, that there were certain cancer cells, called myelomas, that are cancers of the antibody producing cells (knows as “B-Cells”). A cancer is basically one cell that has lost the ability to STOP dividing. Because it continues to divide and grow, it takes up more and more space in the organ or your body, draining nutrients, destroying neighboring tissue, etc. In this case the cancer is a B-cell that keeps dividing over and over and making its single antibody over and over again. The problem is that you can’t control what antibody is produced or what it recognizes, so it is difficult to assay the binding site (the “lock”).
There are several myelomas that have been adapted to be able to grow in tissue culture (in flasks filled with a nutrient solution) and maintained constantly for laboratory study. These myeloma tissue culture lines can be derived from different animals. In the case of this work, and even with the drugs mentioned above, the myelomas were derived from mice.
Now, Kohler and Milstein discovered a way to take other B-Cells from mice and “fuse” them with the cells of one of these mouse myeloma lines. They announced in that Nature article the way they intended to use the tool and their technique.
They immunized mice with sheep red blood cells.
So, I hear you. SHEEP RED BLOOD CELLS? REALLY? This might seem a strange thing to use, but actually it was a very common item used to study immunity. Two of the reasons are that, first, it produces a nice strong immune reaction in mice and second, it is very easy to determine an immune response to sheep RBCs. If you put fresh sheep RBCs in a solution, add some serum that you suspect has antibodies against them, add some “complement” (these are a group of chemicals present in your blood all the times, that are responsible for destroying foreign cells like bacteria in the presence of antibodies), the red blood cells will be broken open and the red stuff on the inside will be released into the solution. So, if you use one of those 96 well plates that we discussed with ELISA, you can put Sheep RBCs and complement in each well, add suspected serum from mice who were immunized and see what happens. Those cells that actually have antibody will become red, while those that don’t will still be clear with a small red button of Sheep RBCs settled in the bottom of the well. This is a very quick and easy assay, and you can determine the amount of antibody in the serum by diluting it sequentially in those wells until it no longer has sufficient amount to break apart the red blood cells.
After waiting sufficient time for the immune response, the spleens from the immunized mice were removed (the spleen is an organ where B-Cells concentrate). The spleen was then ground up and using the technique invented by Kohler and Milstein, cells from those spleens were fused with cells from one of the mouse myeloma cell lines. The new cells resulting from the merging of the two cells were then isolated and individual cells were grown into their own new cell lines, each one secreting only one antibody, one of the antibodies from one of the B-Cells from the mouse spleen.
Using the assay that I described above, they were then able to choose the new cell line that had the best, strongest and highest concentration of the antibody that can perform best in that test.
That allowed them to create and grow new cell lines with a continuous source of one, very specific, very well understood antibody.
Because this technique merged two cells to create a new myeloma line, Kohler and Milstein called these new cells “hybridomas”. We now call them Monoclonal Antibodies.
The paper went reasonably unnoticed for 2 years until an editorial in the Lancet (the most prestigious British medical journal) suggested that these new hybridomas could have “profound implications for medical practice”.
In 1984, Cesar Kohler and Georges Milstein were awarded the Nobel Prize in Medicine for this three-page paper and the subsequent effect on medical research.
Subsequently, mice have been immunized against a wide variety of things. In the laboratory where I worked, we made MABs against influenza to better understand how antibodies recognize viruses.
The pharmaceutical companies began to use the technique to treat disease, but they never would have even attempted something like that without the work of Kohler and Milstein and the governmental support for that basic research.
Now, I assume, labs are working on developing new MABs that can specifically recognize COVID-19 for ELIZA and point-of-care antibody tests. Those same antibodies will eventually be able to be used for direct virus testing and, potentially for therapeutic treatment. However, it takes time to generate these types of antibodies, select the right ones for use, and then ramp up production of them.
I know this post has been quite long, and I don’t know how many of you have made it through. I do hope that I have been able to explain the science so that you can better understand what the medical experts have been and will be talking about.