Biomarkers are rapidly gaining importance in biological research and drug discovery and development. A biomarker, which is measurable by minimally invasive means, reveals important physiologic information about an organism. Most people associate biomarkers with serum protein diagnostics. Biomarkers can, however, also be genetic, epigenetic, small molecule, or even an imaging marker.
As often as they are sought in diagnostics, they are also of interest for prognostic prediction, therapeutic monitoring, surrogate endpoints in clinical studies, and as companion diagnostics in drug development. All major pharmaceutical companies now have biomarker programs that complement their drug discovery programs from the earliest stages.
Protein biomarkers in human blood are some of the most desired because of their obvious clinical utility. Estimates of the number of proteins in the human plasma proteome start at around 50,000 and trend upward, depending on how one counts isomers, post-translational modifications, and other variations.
The range of abundance of proteins in blood or plasma covers about 12 orders of magnitude. Albumin is by far the most abundant protein in blood, and must usually be removed before studying low-abundance proteins. Devising a platform for protein biomarker discovery in the blood is, therefore, challenging.
Mass spectrometry is one of the most powerful methods for analyzing protein biomarkers, and Monarch Life Sciences uses it for preclinical and clinical biomarker sample testing, and biomarker discovery and validation. An assay based on mass spectrometry can be much more sensitive and specific in distinguishing isoforms or protein modifications.
In a case study presented by Monarch scientists at CHI’s “Biomarker World Congress” earlier this year, procollagen-1 n-terminal peptide (P1NP), was detected in rat plasma by selected reaction monitoring, a technique that measures the abundance of a target molecule based on peptide fragments from a trypsin digest. P1NP is an indicator of bone growth or other bone-related changes and has recently been implicated in some other diseases and conditions.
A critical part of assay development is establishing a linear working range for protein concentration. For P1NP, the effective range was from about 2 nM to 200 nM, which covers the effective physiologic range of the protein. By changing individual amino acids in the peptide targeted by mass spec, the assay can follow a therapeutic program from preclinical to clinical stages, progressing from rat, through dog and monkey, to human subjects.
“The hardest thing is the dynamic range of the proteome,” says Mu Wang, Ph.D., vp of research. Efforts are ongoing at Monarch to expand the effective dynamic range of its assays. In spite of this drawback, the mass spectrometry based approach has some major advantages over immunoassay-based methods, not least of which is that there is no need for antibodies and the development time can be as short as a few weeks.
Where antibodies are available, however, an immunoassay is an effective way to monitor a biomarker without investment in a mass spectrometer. Caspase-cleaved cytokeratin 18 (ccCK18) is a marker for apoptosis that is useful for monitoring the effects of cancer therapy. An immunoassay for ccCK18 (M30-Apoptosense) has been developed by Peviva in collaboration with Stig Linder, Ph.D., at the Karolinska Institute. The company also has an assay for total epithelial cell death (M65 ELISA). The combined use of these assays facilitates the determination of cell-death mode (i.e., distinguishing apoptosis from necrosis).
If a therapeutic agent is effective, cancer cells will begin to undergo apoptosis and release ccCK18 into the blood where it can be detected within a couple of days of treatment. Such early detection of therapeutic response is highly desirable—patients may, otherwise, endure weeks or months of chemotherapy before tumor response can be measured.
Because CK18 is expressed in epithelial cells, and most tumors (e.g., breast, prostate, lung, colon, liver, ovarian) are derived from epithelial cells, the assay can be very specific. “Most cancer therapeutical agents,” says Dr. Linder, “are toxic to white blood cells” which, he notes, do not express cytokeratins. “If we see an increased signal in patient blood using the M30-Apoptosense ELISA, we know that it is derived from an apoptosis product formed in epithelially derived cells (and not from white cells).” Dr. Linder will talk about his work at the upcoming Select Biosciences “European Biomarker Summit” in Barcelona.