Development and validation of a highly sensitive ELISA method for the quantitation of Hsp70 -a tumor biomarker associated with Hsp90 inhibitor therapy

A121

Background: Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of oncogenic signaling proteins including HER2, AKT, BCR-ABL and mutant p53. Hsp90 inhibitors induce proteosomal degradation of Hsp90 client proteins and are being tested in phaseI/II clinical trials for oncology indications. Current methods to detect bioactivity of Hsp90 inhibitor compounds involve a western blot assay from either tumor biopsies or hPBMCs, a method that is laborious and semi-quantitative. In an attempt to develop more quantitative and robust methods that would ultimately help to determine optimal biological dose and facilitate starting dose selection for Hsp90 inhibitor phase II clinical trials, we evaluated alternative ELISA based methods for detection and quantitation of secreted and intracellular forms of Hsp70 in hPBMC treated with the Hsp90 inhibitors ex vivo. Methods: Human PBMCs and SCLC cell lines were cultured with different doses of HSP90 inhibitor drugs. Cell pellets and supernatants were collected after 24, 48 and 72 hours. Intracellular HSP70 levels were quantified by Western blot and a highly sensitive ELISA method on an electrochemiluminescent (ECL) platform. Secreted Hsp70 levels were assayed by ECL based ELISA. Results: The time for maximal detection of intracellular Hsp70 was between 24h and 48h. Intracellular Hsp70 levels detected by ELISA in drug treated cells were far higher than the Western blot assay (3 fold up by Western blot versus 23-fold up by ELISA). In addition, detection by ELISA format offered a superior dynamic range, was more quantitative and sensitive than the Western blot assay. Furthermore, about 30-fold less total cell protein was required to quantify Hsp70 levels by ELISA compared with Western blotting. Recent studies have demonstrated that Hsp70 is also secreted into the serum of cancer patients by transit via an endolysosomal compartment. Using the ECL based ELISA system to quantify secreted Hsp70 we detected a 10 to 25-fold increase in the secretion of Hsp70 by hPBMC and tumor lines treated with various Hsp90 inhibitors. The time for the maximal detection of secreted Hsp70 was between 48h and 72h. Furthermore, a good correlation (R² > 0.9) was found in the levels of intracellular and secreted forms of Hsp70 following treatment of cell lines with different doses of Hsp90 inhibitors. Additionally, using this platform we have demonstrated the ability to detect basal levels (2-5 ng/mL) of Hsp70 in the serum from cancer patients and normal donors. This method for the detection of secreted Hsp70 was validated for clinical use by assessing for accuracy, precision, matrix effects and stability. Conclusions: We report development and validation of a highly sensitive, quantitative and robust method for the accurate determination of intracellular and secreted forms of Hsp70. The validated assays should be useful in dose selection and monitoring the biological effects of investigational Hsp90 inhibitor therapy for cancer patients. Furthermore, the ability to quantify Hsp70 in serum is a good alternative to the currently used intracellular Hsp70 assessments as it is challenging to implement the latter protocol in a clinical setting.