Development and validation of a phosphorylated SMAD ex vivo stimulation assay

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 TGFβ 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 TGFβ₁ 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. TGFβ 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 TGFβ-mediated genes. This paper describes the novel method for measuring the downstream effects of inhibiting canonical TGFβ 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 TGFβ signal modulation by multiple inhibitor platforms for both large and small molecules.

Keywords: :

• pSMAD
• tSMAD
• TGFβ receptor I kinase inhibitor
• cancer
• ex vivo stimulation
• LY2157299

Acknowledgements

We would like to thank Bill Alborn and Jim Bourdage for their constructive comments and Megan Freeman for execution of the validation experiments. We also thank Drs Jose Baselga, Mace L. Rothenberg, Josep Tabernero, Joan Seoane, Carlos Arteaga, Lisa Anne Wallace and Michael Carducci.