Whole Blood CETSA®: Translating Target Engagement into Actionable Pharmacology
Abstract
Translational failure in clinical drug development is frequently attributed to inadequate target engagement (TE) validation in physiologically relevant systems. Pelago’s proprietary Cellular Thermal Shift Assay (CETSA®), particularly when applied to whole blood, offers a robust, label-free methodology for quantifying intracellular TE under systemic conditions. This article outlines the methodological foundations of whole blood CETSA®, examines its current adoption in the pharmaceutical industry, and its utility in bridging preclinical and clinical decision-making. Case studies from AbbVie, Takeda, and a landmark study published in Nature Biotechnology are highlighted to demonstrate feasibility and translational relevance.
Introduction
Target engagement in native biological matrices is a prerequisite for accurate pharmacological interpretation of in vivo efficacy. Despite this, many discovery pipelines continue to rely on reductionist systems to evaluate TE. This includes overexpressed targets in immortalised cell lines or biochemical assays in buffer. CETSA® bypasses such systemic limitations by enabling direct detection of protein-ligand interactions via thermal stabilisation of endogenous proteins. Its application in whole blood extends this capability to a matrix that mirrors systemic circulation, where exposure, distribution, metabolism, and molecular competition converge.
Whole blood CETSA® is uniquely suited to address early decision points and to inform clinical pharmacology by linking compound exposure to intracellular binding behaviour. The assay preserves cell integrity and physiological protein context while avoiding compound modification, target tagging, or recombinant overexpression.
Assay Overview and Implementation
The CETSA® workflow in whole blood involves compound incubation, controlled heat shock, and post-thermal separation of soluble versus aggregated protein fractions. Detection is achieved through immunoassay (e.g., AlphaLISA™, MSD®), western blot, or mass spectrometry. The choice of detection modality depends on target abundance, throughput requirements, and downstream application.
Pelago Bioscience has developed validated protocols supporting fresh and frozen blood samples, enabling streamlined integration into preclinical and clinical trial designs. The protocols account for matrix effects such as haemoglobin interference and inter-donor variability. Blood dilution, lysis buffer optimisation, and advanced normalisation strategies are incorporated to maintain signal fidelity.
Case Study 1: AbbVie – RIPK1 Engagement in Human Whole Blood
In a collaboration with Pelago Bioscience, AbbVie applied CETSA® to quantify TE of RIPK1 inhibitors in human whole blood. The study utilized AlphaLISA™ and MSD® platforms to monitor target stabilization, both in fresh and frozen blood samples. Crucially, the assay eliminated the need for PBMC isolation, allowing the detection of intracellular target engagement directly in unprocessed blood. The data supported PK/PD modelling and dose optimisation efforts in preclinical programs and were designed with clinical assay transfer in mind.
SLAS Discovery, 2023, AbbVie & Pelago Study
Case Study 2: Takeda – CETSA® for Kinase Inhibitor Profiling
Takeda integrated whole blood CETSA® into developing kinase inhibitors, including applications for Akt. The team observed significant inter-individual variation in thermal shift profiles, with baseline Tm values ranging from 5.0°C to 46.6°C across donors. This highlighted the assay’s utility for patient stratification and adaptive trial design. Takeda’s internal validations included fresh and cryopreserved samples, confirming the assay’s robustness across logistics workflows common in multicenter clinical trials.
Sci Rep, 2017, CETSA® Validation for RIPK1 in Tissues and Blood
Case Study 3: Perrin et al. – Identifying drug targets in tissues and whole blood with thermal-shift profiling
A landmark study by Perrin et al. in Nature Biotechnology demonstrated whole blood CETSA® with proteome-wide mass spectrometry (TPP). The team profiled panobinostat and JQ1 across thousands of proteins, detecting stabilization of known targets (e.g., HDACs, BRD2/3/4) and off-targets like ZNF512. The method enabled quantitative assessment of drug engagement in both human and murine whole blood, providing a framework for real-time clinical TE profiling.
Practical Considerations
CETSA® in whole blood introduces logistical and analytical challenges. Red blood cell lysis must be controlled to prevent hemoglobin interference. Anticoagulant selection (e.g., EDTA vs. heparin) impacts cellular integrity and background signal. Normalization methods must account for donor-specific protein expression and intrinsic thermal stability. Despite these, the benefits in translational confidence and system fidelity make the approach attractive for programs with narrow therapeutic indices or high efficacy risks.
Conclusion
CETSA® in whole blood represents a mature, implementable solution for capturing TE in physiologically intact systems. The method aligns with regulatory shifts emphasizing translational data, including Project Optimus. Early adopters such as AbbVie and Takeda have demonstrated its utility in both preclinical and clinical applications. Proteome-wide adaptations, as shown by Perrin et al., expand its scope to selectivity profiling and off-target discovery.
As Japanese pharmaceutical R&D increasingly prioritizes de-risked, mechanism-informed development, whole blood CETSA® offers a high-confidence, technically validated platform for accelerating discovery without compromising biological realism.
References
Ishii, T., et al. (2017). CETSA quantitatively verifies in vivo target engagement of novel RIPK1 inhibitors in various biospecimens. Scientific Reports, 7, 13000. https://doi.org/10.1038/s41598-017-12513-1
Perrin, J., Werner, T., Kurzawa, N., et al. (2020). Identifying drug targets in tissues and whole blood with thermal-shift profiling. Nature Biotechnology, 38, 303–308. https://www.nature.com/articles/s41587-019-0388-4
Martinez Molina, D., et al. (2013). Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science, 341(6141), 84–87. https://doi.org/10.1126/science.1233606
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