All the side effects of chemotherapy arise from the drugs targeting normal as well as cancerous cells. If the targeting mechanisms were improved, wouldn’t the side effects decrease?
Cancer, characterized by abnormal cell growth in the body, has varied manifestations. Leukemia is one of them, affecting the bone marrow, which, being a major site of blood cell production, results in a large number of abnormal white blood cells entering the bloodstream. The most effective way of treating leukemia at present is chemotherapy, either by itself – with all its adverse side effects – or together with bone marrow or stem cell transplants.
Professor Rama S. Verma, and his team at the Department of Biotechnology, IIT Madras, are researching a novel treatment that uses a new class of molecules called immunotoxins, that could eliminate the side effects that cancer patients have to endure during their treatment.
Leukemia is a cancer of the blood, and is therefore distributed throughout the body. As a result, radiation therapy is not a feasible method of treatment – it can only target localized tumors. Hence, treatment of both these forms of leukemia is traditionally via chemotherapy. As cancer is basically unchecked and rapid cell division, the drugs used in chemotherapy target these cells by interrupting the process of cell division – or mitosis – and killing the cells in the process.
Unfortunately, other cells, such as those in bone marrow – which is where red and white blood cells are produced – hair follicles and the lining of the digestive tract, also divide at high rates. Consequently, the sideeffects of chemotherapy include a decreased production of white blood cells (and a corresponding decrease in immunity), anemia (because of a decrease in production of red blood cells), hair loss, nausea, and inflammation of the digestive tract. More permanent effects may also occur, including infertility, nerve damage and cognitive impairment. Although fraught with so many side-effects, chemotherapy is still the mainstream treatment simply because it is the only effective method of treatment there is.
All the side effects of chemotherapy arise from the drugs targeting normal as well as cancerous cells. If the targeting mechanisms were improved, wouldn’t the side effects decrease? It was this line of thought that inspired research on immunotoxins.
Immunotoxins are artificial proteins that consist of a toxin protein attached to a cell-selective targeting protein. They belong to a class of molecules called chimeric proteins, drawing a comparison between their hybrid nature and the ancient Greek monster said to have a lion’s head, a goat’s body and a snake for its tail.
The membranes of cells in our bodies have numerous chemical structures, called receptors, embedded in them. Hormones and other chemicals that are naturally secreted in the body, target and bind to these receptors all the time as part of the body’s natural processes. Each hormone binds only to a single receptor, like a lock and key. This receptor-specific binding is carried out by a part of the chemical, the targeting portion. If a receptor that is more prominently present in cancerous cells can be found, then engineering an immunotoxin to target only tumour cells, with very few normal cells being affected, becomes feasible. “The basic principle behind immunotoxins is that cancer cells overexpress some receptor molecules. We can then use these overexpressed molecules as targets,” says Prof. Verma.
I ask Prof. Verma what inspired him to work on immunotoxins, and more generally, work in the field of biochemistry. His face splits into a smile as he begins to reminisce. “I was always interested in medicine and biology. However, I didn’t clear the entrance exam we had in U.P. for medical school, and hence I did my B. Sc. in biology. I then specialized in biochemistry during my Master’s and Ph.D., as it was related closely with medicine.” After working in the USA for 13 years, including stints at the University of Pennsylvania, the National Cancer Institute and Indiana University, Prof. Verma returned to India and joined the industry, where he headed R&D. “I had read journals and other literature, and I found immunotoxins very interesting. But the industry didn’t want me to work on those compounds. They were more concerned with compounds whose patents were expiring, so that they could synthesize similar compounds and file for patents themselves,” says Prof. Verma.
“The basic principle behind immunotoxins is that cancer cells overexpress some receptor molecules. We can then use these overexpressed molecules as targets”
In search of opportunities to work on research problems that interested him, Prof. Verma then joined IIT Madras. “I applied for a grant, got it immediately, and started to work on the immunotoxins.”
But where do we get immunotoxins from? The toxin part of the immunotoxin is traditionally derived from one of many sources – plant (ricin, for example), bacteria (diphtheria), fungus or yeast. While immunotoxins have been around for four decades, what puts them at the cutting edge of research now is the choice of toxin molecules – instead of animal – or plant-based toxins, toxins produced by the human body itself are being used.
Human toxins are being researched because when plant or animal-derived toxins are used, our immune system recognizes that they are not part of our body. It then reacts the same way it does to any foreign chemical introduced into the bloodstream – it produces antibodies that neutralize the toxin, thus reducing the efficacy of the treatment. “They are, after all, foreign chemicals being introduced into the bloodstream,” says Prof. Verma.
If the toxins were from our own body, no immune response would be triggered, and the potency of the treatment would not decrease. But what exactly are these toxins that our body produces?
Programmed cell death, a natural part of the working of our bodies, is known as apoptosis, as opposed to necrosis, which is cell death due to trauma sustained by the cell. Between 50 and 70 billion cells die everyday due to apoptosis in the average human adult.
Apoptosis occurs when certain pathways are blocked in the normal functioning of the cell. And this is where Prof. Verma saw an opportunity. “The toxins we use are the chemicals that cause natural cell death, or apoptosis, in our bodies,” he says. With the aim of creating a compound that “minimally touches and affects normal cells, these chemicals were isolated, cloned and tagged with the targeting molecules. Prof. Verma and his team have synthesized seven new immunotoxins, with variations in the targeting portion.
The biggest challenge he faced on the research front was manufacturing the human proteins in bacteria. The combined molecule – the targeting portion and the toxin – is produced in genetically reengineered bacteria. The recipe that every cell uses to manufacture proteins, is DNA. Different portions in a strand of DNA correspond to different proteins. The beginning and end of each portion is denoted by a codon, a triplet of nucleotides, the building blocks of DNA. Ribosomes typically use cDNA (complementary DNA) – a copy of DNA that is synthesized from the original DNA present in the nucleus, to manufacture proteins. To produce chimeric proteins, the stop codon is removed from the cDNA corresponding to the first protein, and the cDNA corresponding to the second is chemically attached to it. When reinserted into a bacterial cell for protein manufacture, the chimeric protein will be produced in clumps, called inclusion bodies.
The extraction of proteins from these inclusion bodies involves two steps – extracting the inclusion bodies from the bacteria, and dissolving them to get a solution of the immunotoxins. “The proteins often get denatured, or deformed in the process. If the shape of the protein changes, it can no longer bind to the cell receptors. Reactivating them was a tedious process,” says Prof. Verma.
Humanized immunotoxins were not used before because knowledge of human DNA was not sufficient to map out which portions of it governed the production of the necessary toxins. It was not until the 1990s, when the human genome was mapped extensively, that their use became feasible.
When asked about the major obstacles he faced outside of the lab, Prof Verma has an immediate and emphatic answer: “Funding. Very few labs in India are working on immunotoxins, so it’s hard to convince the powers-that-be that I’m working on something novel and with immense scope. Further, IITs don’t have research funds on par with those in universities outside India, so I have to work with what I get. It can get very hard to manage.”
The next step, he says, is clinical studies – producing the immunotoxins in large amounts, and testing it on animals, in order to determine the maximum dosage of each immunotoxin that can be administered without causing harm. “We look at the absorption, distribution, metabolism, and excretion of the drug in the animal, and the extrapolate the amount required for a human,” he says. Prof. Verma and his team are waiting for another grant to be cleared so that they can begin mass production and clinical trials, which he expects will take 2-3 years to complete. “We’ll do patient samples – we draw blood from patients with chronic myeloid leukemia and acute myeloid leukemia – and test the immunotoxins on those,” he says.