Angel, 32 years old, has just learned she has breast cancer, the same disease that took her mother’s life when Angel was a teenager.
Recalling the pain, weariness, and hair loss that her mother went through while under doxorubicin chemotherapy, Angel asked her doctor whether there might be another treatment modality with less unpleasant side effects.
Fortunately, there was. Even better, there’s a slight possibility that the new drug might be more effective in stopping the cancer altogether than the other treatment modalities, and an even greater chance that Angel will not have to go through all of her mother’s painful experiences.
Although the new drug is also a doxorubicin, it is unlike the one used by Angel’s mother as it is locked inside a fatty covering called liposome, a smart bomb that hits the tumor and almost avoids everything else. The product’s name: Caelyx.
To better understand why Caelyx is better than free doxorubicin, we need to know that administering a drug to the body is like putting water I a bucket with a hole at the bottom since the natural response of the body is to eliminate foreign substances.
Compared with free drugs, liposomes are more slowly eliminated. Liposomes are hollow nanoparticles. At about 60 to 100 nanometers, they are smaller than most cells. It is harder for them to get out of the blood vessels in most tissues, thus are more able to escape the elimination mechanisms in the liver and the kidney. Furthermore, the polyethylene glycol molecules on their surface hide the liposomes from white blood cells tasked with gobbling up foreign materials in the blood. Thus, by enclosing the doxorubicin inside the liposomes, the body is tricked into thinking that it is dealing with liposomes, not doxorubicin.
Solid tumors have leaky blood vessels, all the better to grab nutrients from the blood. Liposomes are more easily able to leak through the gaps whenever they circulate around the tumor. Since the liposomes are able to stay in circulation for a long time, more opportunities are afforded for them to concentrate at the tumor site. There, the liposomes eventually degrade or are taken up by the tumor cells, in the process releasing the doxorubicin.
As liposomal doxorubicin concentrates on tumors, less of it goes to the rest of the body. The result: a higher, deadlier dose at the tumor. Of course, some of the doxorubicin still leak into the blood stream, but many studies on Caelyx have demonstrated decreased toxicity and a slight increase in patient’s survival time.
The Antibody and Molecular Oncology Research Program asked whether it would be possible to further enhance the concentration at the tumor if the liposomes were coated with antibodies that were specific to surface proteins uniquely present in tumor cells. In fact, human breast cancer cells and a few other tumor types express a surface antigen called TAG-72 that nearly all other normal cells do not express. We hypothesized that not only will the immonoliposomes – so called because they are coated with antibodies – concentrate at the tumor site: they will stick to the tumor cells themselves and will subsequently be ingested by them.
What antibody could we use? There is a monoclonal antibody specific to TAG-72, called CC49. Like most monoclomal antibodies, this was produced in mouse hybridoma cells. The problem with mouse antibodies, however, is that they are recognized as foreign by the human body and so are rapidly eliminated, or cause adverse side effects.
Dr. Eduardo Padlan, a Filipino scientist based in the United States, found the gene for CC49 and engineered it in such a way as to replace those sections of the antibody that were specifically mouse-like with sections that were human. The gene could be inserted into a plasmid and the antibody produced in any compatible cell, such as yeast, bacteria, or insect cells. It is not exactly the antibody itself that is produced – antibodies are very large molecules. Rather, only a much smaller part of the antibody is needed, the part called a single chain Fv fragment, which recognizes and binds with the TAG-72.
Plasmids and genes, however, are incompatible with each other: you can’t run the same plasmids and genes in all cells and expect the cells to produce the gene products in the same way. We thus re-engineered, in a process called codon optimization, the genetic code for the fragment from being a yeat compatible into a bacteria-compatible gene. Having succeeded in producing much as 6 mg/mL of fragment per liter of bacterial culture, we now have enough material to make lots of liposomes.
Making a liposome and loading it with a drug is difficult, but when we got the hang of it, we began to regularly produce doxorubicin-containing liposomes that were chemically and physically indistinguishable from Caelyx.
Using simple chemistry, we coated the surface of the liposomes with the antibody fragments. We then tested the product in nude mice, a special strain that had a defective immune system that made it unable to reject transplanted human cells. We gave the mice human breast cancer cells.
We found that doxorubicin-containing liposomes has a superior tumor reduction capability at a lower dose than free doxorubicin at a higher dose. However, there was no clear evidence that immunoliposomes were concentrating more at tumors compared to liposomes that did not have the antibodies. There was also no significant difference in the tumor-reducing capabilities of these two kinds of liposomes. Perhaps the effect of simply having a more leaky vasculature outweighs the effect of having a unique surface antigen. At least in mice.
What is the futureof the project? Since Caelyx and doxorubicin are already in the market, it should not take much too long to evacuate the safety and effectiveness of the immunoliposomes. What we need to do now to bridge our achievement with doing the first tests in human is to ensure that pure liposomes can be produced in large amounts.
Jose Enrico H. Lazaro
University of the Philippines Diliman
Department of Science and Technology-Science and Technology Information Institute (DOST-STII)