Development and validation of a phosphorylated SMAD ex vivo stimulation assay.
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.
Farrington
D L
DL
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA. farrington_daphne_l@lilly.com
Yingling
J M
JM
Fill
J A
JA
Yan
L
L
Qian
Y-W
YW
Shou
J
J
Wang
X
X
Ehsani
M E
ME
Cleverly
A L
AL
Daly
T M
TM
Lahn
M
M
Konrad
R J
RJ
Ray
C A
CA
D030201:Activin Receptors, Type I / Q000037:antagonists & inhibitors*; D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003627:Data Interpretation, Statistical; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D011916:Rats, Inbred F344; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D051785:Smad Proteins / Q000032:analysis / Q000378:metabolism*; D051899:Smad2 Protein / Q000032:analysis / Q000378:metabolism; D051900:Smad3 Protein / Q000032:analysis / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays / Q000379:methods
D015415:Biomarkers; D047428:Protein Kinase Inhibitors; D018125:Receptors, Transforming Growth Factor beta; D051785:Smad Proteins; D051899:Smad2 Protein; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625843:Tgfbr1 protein, rat