A team of researchers from the Indian Institute of Technology (IIT) Madras has developed a cheaper yet reliable alternative for diagnosing leukaemia and colorectal cancer. Like monoclonal antibodies which are currently used for cancer diagnostics, the fusion protein developed by the researchers has high specificity and sensitivity. The results were recently published in the journal Molecular Diagnosis & Therapy.
The researchers designed recombinant fusions of a ligand (stem cell factor) to a protein tag (SNAP-tag). The ligand binds to a particular receptor (c-kit) that is present in more than normal numbers (overexpressed) on some cancer cells.
To quantitatively determine the amount of ligand that is bound to the receptors, the researchers used a commercially available fluorescent material (O6-Benzylguanine) that the SNAP-tag binds to. The bound protein with the fluorescent derivative can be detected using a fluorescent microscope or flow cytometry.
“We report the first evidence that SNAP-tag could be used for ex vivo [outside the body] detection of enriched biological markers using SCF/c-kit as the target receptor system. The c-kit receptor is a pathological and diagnostic marker for a variety of cancers,” Dr. Swati Choudhary from the Department of Biotechnology, IIT Madras and the first author of the paper says in an email. “It is a proof-of-concept study.”
Using c-kit positive and negative cell lines, the researchers first tested the capacity of the protein tag to bind specifically to the c-kit receptors. “The specificity was comparable to the currently used monoclonal antibodies,” she says. “We then carried out a pilot study to test whether these proteins could be used for diagnostic purposes through ex vivo immunophenotyping of the c-kit receptor. We tested it on 16 peripheral blood samples from leukaemia patients and four colorectal biopsy specimens.”
“The sensitivity is as good as commercially available monoclonal antibodies. If sensitivity and specificity are high in large-scale studies, we could in future replace monoclonal antibody with SNAP-tag fusions to select ligands for diagnostic applications. It will also be much cheaper,” says Prof. Rama S. Verma from the Department of Biotechnology, IIT Madras, and one of the corresponding authors of the paper.
“In the long term, these probes could potentially be used for diagnosing and staging of cancer and in the follow-up management of the disease,” Dr. Choudhary says. According to Prof. Verma, it would even be possible to find out early stages of cancer as the technique has high sensitivity.
Since c-kit receptor is overexpressed in other cancers such as gastrointestinal stromal tumours, small cell lung cancer, ovarian cancer, breast cancer and melanoma, the SNAP-labelled protein could theoretically be used for diagnosing these cancers as well. Further studies are needed to confirm this.
“By replacing the stem cell factor with different ligands that targets other cancer cells, the technique can potentially be used for identifying other cancers as well,” Dr. Choudhary says.
As a DAAD fellow, Dr. Choudhary carried out a part of the study at Fraunhofer Institute of Molecular Biology, Aachen, Germany.