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Updated: Jul 15, 2026
Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells
Published on: September 9, 2021
Development and validation of a phosphorylated SMAD ex vivo stimulation assay
D L Farrington1, J M Yingling, J A Fill
1Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA. farrington_daphne_l@lilly.com
Abstract:
Assessing the pharmacodynamics (PD) of a potential therapeutic through the use of a downstream biomarker is essential. This is traditionally performed in the target tissue but limited volume and invasiveness of sampling pose challenges with solid tumours. Currently, there are several small molecule receptor kinase inhibitors and large molecule therapeutic antibodies in clinical trials that interfere with TGFbeta signalling to treat various forms of cancer. With the advent of these new therapies, there is a need for a surrogate tissue that is easily accessible and indicative of tumour response. We propose the use of an ex vivo TGFbeta1 stimulation of peripheral blood mononuclear cells (PBMCs) coupled with the measurement of phosphorylated SMAD2 (Sma/Mothers Against dpp, a downstream transcriptional activator) using a sandwich ELISA. TGFbeta is involved in many different cellular responses, such as proliferation, angiogenesis, migration, invasion and immunomodulation. SMAD2 and SMAD3 are phosphorylated as a result of the canonical cascade through ligand binding and receptor kinase activation. These phosphorylated SMADs (pSMAD) associate with SMAD4, a co-SMAD, and transcriptionally activate TGFbeta-mediated genes. This paper describes the novel method for measuring the downstream effects of inhibiting canonical TGFbeta signalling using ex vivo stimulation of surrogate tissue to predict tumour response. In addition, we present the assay validation rationale and data. This novel, validated assay can be used to gain insight into clinical trials regarding TGFbeta signal modulation by multiple inhibitor platforms for both large and small molecules.
Insights
This study introduces a novel method using peripheral blood mononuclear cells (PBMCs) to assess the pharmacodynamics of TGFbeta-targeting cancer therapies. The validated assay measures phosphorylated SMAD2, offering a surrogate tissue approach for monitoring treatment response in clinical trials.
Area of Science:
- Oncology
- Pharmacology
- Biochemistry
Background:
- Assessing cancer therapeutic pharmacodynamics (PD) traditionally requires invasive tissue sampling, posing challenges for solid tumors.
- Transforming growth factor-beta (TGFbeta) signaling is a key target for novel cancer therapies, including small molecule inhibitors and therapeutic antibodies.
- There is a critical need for accessible surrogate tissues to evaluate the efficacy of TGFbeta-targeting agents in clinical trials.
Purpose of the Study:
- To develop and validate a novel method for assessing the pharmacodynamics of TGFbeta pathway inhibitors.
- To establish peripheral blood mononuclear cells (PBMCs) as a surrogate tissue for monitoring TGFbeta signaling in cancer patients.
- To provide an accessible assay for evaluating tumor response to TGFbeta-targeted therapies.
Main Methods:
- Ex vivo stimulation of PBMCs with TGFbeta1.
- Measurement of phosphorylated SMAD2 (pSMAD2) using a validated sandwich ELISA assay.
- Utilizing pSMAD2 as a downstream biomarker for canonical TGFbeta pathway activity.
Main Results:
- A novel, validated method for measuring the downstream effects of TGFbeta signaling inhibition in PBMCs was established.
- The assay demonstrated utility in assessing TGFbeta pathway modulation by various inhibitor platforms.
- The developed assay provides a surrogate tissue approach for evaluating therapeutic response.
Conclusions:
- Ex vivo stimulation of PBMCs offers a viable and accessible surrogate tissue for assessing TGFbeta pathway pharmacodynamics.
- This validated assay can provide crucial insights into clinical trials targeting TGFbeta signaling.
- The method supports the evaluation of both small molecule and large molecule inhibitors across different cancer types.
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