Quantification of molecular interactions across the matrix spectrum enables cancer research.

Project Summary We propose to further develop, refine, and validate our emerging free-solution assay (FSA) technology and our relatively new compensated Interferometric reader (CIR).1,2 The development of FSA-CIR addresses a significant void, a blind spot in cancer research because it represents the only label-free, solution-phase, ultra- sensitive, enzyme-free, technology compatible with essentially any matrix. Unlike existing tools, FSA-CIR has been shown to be useful for; a) mechanism of Action (MOA) studies on unadulterated/unmodified targets and probes with no relative mass sensitivity, b) full-length membrane protein interaction studies in native matrix, c) defining allosteric modulation and weak protein-protein interactions, d) accelerating quantitative assay development, e) potentially addressing biomarker discovery/validation bottleneck, f) performing quantitative interactions across the matrix spectrum on a single platform and g) enabling ex-vivo measurements to guide first-in-human dose determinations (FIHD) (see Pfizer letter). FSA-CIR is a paradigm shifting technology based on a novel molecular interaction transduction method with fluorescence-level sensitivity, and capabilities for targeting, probing, and assessing molecular and cellular features of cancer biology, as well as improving early detection and screening, clinical diagnosis. FSA is mix-and-read, agnostic to the molecular interaction pair and compatible with complex matrices, making it uniquely applicable in both the basic and clinical cancer research arenas. CIR represents a major advancement in interferometric sensing, due to an unprecedented level of sample-reference compensation CIR is operated without external thermal control, a unique feature for a refractive index (RI) sensor with <10-7 RIU sensitivity. The optical engine in CIR is unique, patented and quite simple, consisting of a diode laser, capillary tube, mirror and detector. When combining the interferometer with a droplet generator for sample introduction, CIR facilitates quantification of molecular interactions without relative mass dependence, at picomolar sensitivity and allows good sample throughput (50 serum sample-reference pairs run in quintuplet, [5 replicate droplet pairs], plus calibrations in a day. Feasibility of our assay methodology is demonstrated for mechanism-of-action (MOA) studies, quantification of drug target engagement as needed in theranostics and ultrasensitive, volume constrained, biomarker assay development, and target quantification. Data indicate FSA-CIR has the potential for widespread applicability and adoption throughout the scientific community and is mature enough to be an R-33 project. At project completion we aim to provide the scientific and medical community with a user-friendly platform technology for biochemical mechanism of action studies, to aid in improving cancer prognostics, and the ability to measure properties such as molecular and/or cellular mechanisms important in cancer.