Abstract
Abstract Background: Regulators of apoptotic cell death are high interest targets for cancer therapy, and several mechanism-based investigational agents in clinical development directly target the molecular components of apoptosis pathways. We have undertaken development of a multiplex assay of candidate biomarkers of this mechanism for quantifying the commitment, onset, and induction of apoptosis by both intrinsic and extrinsic pathways. Quantitation of these pharmacodynamic biomarkers in early clinical trials will assist hypothesis testing and confirm the intended mechanism of action of investigational agents. The panel may also be adaptable to diagnostic applications to identify patients most likely to respond to a particular apoptosis inducer. Methods: The panel being developed includes quantitative determinations of Bcl-xl, Mcl-1, Survivin, Smac dimer, cleaved Lamin-B, active/total Caspase-3, BIM, and six oligomeric forms of Bax and Bak with Bcl2 family proteins in lysates of tumor biopsies. Calibrators for oligomeric proteins, which are formed in vivo by the non-covalent association of BH3 domain proteins, are being developed as recombinant fusion proteins. The housekeeping gene GAPDH is used as an internal control. The multiplex, sandwich immunoassay is built on the Luminex® xMAP technology platform using MagPlex bead, which allows for significant automation in the detection of multiple proteins in a 96-well plate format, with increased sensitivity, smaller sample volumes, and less preparation time. Results: Suitable, high quality capture antibodies against proteins in the panel as well as detection antibodies labeled with fluorescent dyes have been identified and tested for sandwich formation using recombinant calibrators and cell lysates. Capture and reporter antibody pairings are currently standardized for optimal assay performance. The feasibility of developing several oligomeric calibrators (among Bak-Bcl-xl, Bak-Mcl-1, Bak-Bak, Bak-Bax, Bax-Bax, and Bax-Bcl2) as recombinant fusion proteins has been established. Conclusions: The proposed multiplex panel represents the most comprehensive selection of putative biomarkers for the apoptosis pathway, to date. Our strategy to quantify oligomeric forms of Bcl-2 family proteins in tumor lysates represents a novel approach since it evaluates a pathway based on alterations in protein dynamics, instead of the levels of various proteins. Once developed, validated, and fit to purpose, the panel will facilitate drug discovery and the real-time pre-clinical and clinical evaluation of drugs targeting apoptosis. Funded by NCI Contract No HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-8.
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