Clinical translation of a NexGen platform for quantifying protein networks in human biospecimens

PROJECT SUMMARY In this competitive renewal for the Academic-Industrial Partnership (R01CA235575) between the Fred Hutchinson Cancer Center and AstraZeneca, we will continue to advance the clinical translation of multiple re- action monitoring mass spectrometry (MRM-MS) protein assays into the clinical trial setting. The MRM-MS tech- nology is agnostic to the patient population. In the first funding cycle, our AIP successfully (a) optimized the MRM assay technology for use on small clinical biospecimens; (b) developed and optimized reagents and SOPs for controlling sample processing variation, calibrating assay results across samples & labs, maximizing sensitivity of the platform for analysis of small volume clinical biospecimens, & improving methods for greater multiplexing; (c) developed novel methods to account for microheterogeneity in tissue biospecimens; (d) achieved analytical validation of MRM-based assay panels in our CLIA environment; (e) translated MRM-MS protein assays into clinical trials, & (f) licensed the technology for commercialization. Data that we generated during the first funding cycle uncovered an exciting opportunity and a new technological challenge. Specifically, some of the MRM- based assays that we translated into clinical trials enabled quantification of phosphosignaling in the DNA Dam- age Response (DDR) network. These assays are of high interest to our biopharmaceutical partners since the DDR network is a major target for cancer drugs. Excitingly, our data indicate that analyzing DDR network signal- ing through serial collections of peripheral blood mononuclear cells (PBMCs) before and after treatment can provide meaningful pharmacodynamic data via a simple blood draw (vs invasive tissue sampling). Yet, the chal- lenge lies in the low basal DDR signaling levels in PBMCs, hindering precise treatment effect quantification. To address this, we've formed a collaboration with Rules Based Medicine (RBM) to develop a prototype "DDR boost tube," an extension of RBM's TruCulture® immune activation blood tubes. DDR Boost tubes are blood culture tubes containing a DNA damaging agent. Our preliminary data demonstrate that incubating blood ex vivo in the DDR Boost tubes activates DDR signaling in PBMCs, boosting basal signaling levels, & improving pharmacody- namic measurements. In cycle 2 of funding, we will translate the integrated technologies (DDR Boost tubes + streamlined MRM assay) into clinical trials via the following Specific Aims: Aim 1: Optimize the design of DDR Boost tubes to maximize MRM signal-to-noise ratios & develop a streamlined DDR MRM assay panel that seam- lessly integrates with the tubes. Aim 2: Investigate biospecimen preanalytical variables affecting DDR measure- ments using the optimized DDR Boost tubes & develop an evidence-based Standard Operating Procedure for clinical trial laboratory manuals. Aim 3. Validate the DDR Boost tubes in a prospective clinical trial. Successful completion of this project will deliver a clinically validated, next-generation solution for pharmacodynamic, proof of mechanism, and correlative study measurements in the context of DDR-targeting therapies- and offer a blue- print for applying a comparable approach to additional cell signaling networks with therapeutic potential.