Photocleavage Technology for Improved Serum-based Multi-Biomarker Cancer Assays

Period of Performance: 08/01/2016 - 07/31/2017

$225K

Phase 1 SBIR

Recipient Firm

Ambergen, Inc.
WATERTOWN, MA 02472
Principal Investigator

Abstract

SUMMARY/ABSTRACTBlood-based multi-biomarker panels hold great promise for effective early cancer detection and population-widescreening. For example, panels consisting of three or more biomarkers which exhibit high sensitivity and specificityhave been reported for several different cancers including ovarian, lung, breast and colorectal cancer. A variety ofminiaturized solid-phase immunoassay platforms have also been developed to perform high throughput and low-cost multiplex biomarker detection and quantification. These include instruments based on microarrays,microfluidics and micro-bead technology.A high priority is now to transition promising blood-based multi-biomarker cancer panels to multiplex platformsfor large-scale biomarker validation and ultimate use in the clinic. Multiplex assay technologies are especiallyimportant to provide the cost-effectiveness and high throughput capacity necessary for population-wide screening.However, a major problem with multiplex immunoassays is the so-called ?matrix effect?. Compared to mostconventional single-plex assays such as ELISA, miniaturized multiplex assays are highly susceptible to interferencecaused by the presence of the more abundant, non-target agents in blood. Such interference can originate from avariety of mechanisms including: i) low specificity heterophile antibodies; ii) matrix-induced bead aggregation (e.g.in Luminex® assays) and iii) specific or non-specific binding of non-target matrix components to any componentof the biomarker assay. In addition, high viscosity of the sample matrix (e.g. from high total protein concentration)can interfere with the microfluidics commonly used for multiplex assays. Importantly, the matrix effect not onlylimits assay sensitivity, but reduces linearity and quantitative accuracy.During Phase I we will evaluate a new approach to multiplex serological cancer assays termed PC-PURE? which isdesigned to eliminate the matrix effect. This technology is based on the use of novel photocleavable (PC) linkersdeveloped by AmberGen which are incorporated into affinity capture agents such as aptamers or antibodies. Thephotocleavable capture agents are then tethered to micro-beads, affinity resins or other surfaces and used to isolatethe target biomarker. The biomarker-[capture agent] complexes are gently and rapidly photo-released in minutesunder non-denaturing conditions by low-intensity near-UV light into a well-defined buffer, enabling simultaneouspre-purification and concentration of the target biomarkers prior to multiplex immunoassay. Unlike conventionalapproaches using blocking buffers, diluents and selected depletion, specific to particular matrix components, PC-PURE? eliminates all matrix effects by rapidly pre-purifying the biomarkers of interest.In Phase I we will evaluate the application of PC-PURE? to improve the multiplex detection of blood-based panelsof tumor-shed protein biomarkers. Tumor-shed biomarkers have great potential for high cancer specificity but arefound at extremely low abundance in the blood and hence suffer most from the matrix effect. A model 5-biomarkerprotein panel for ovarian cancer diagnosis will be tested. Both spike-in samples with known concentrations of thebiomarkers and ovarian cancer patient blood samples will be analyzed on a multiplex Luminex® MagPix® platformand in Phase II on a Bio-Plex 2200 platform designed for high-throughput clinical testing.This research will be conducted in collaboration with Dr. Gheorghe Doros, Associate Professor of Biostatistics andDirector of the Biostatistics Consulting Group at the Boston University School of Public Health, who will provideexpert guidance for statistical analysis of the data. We will also work closely with Dr. Bill Jackson, Founder and CSOof Base Pair Biotechnologies, a leading expert on aptamers. To accelerate commercialization of PC-PURE?, we willwork closely with Luminex®, one of the leading manufacturers of multiplex assay platforms (see letters of support).