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Assay Development for Patient Response Measurement in Phase 0 Trials

This presentation discusses the development of assays to measure patient response in Phase 0 clinical trials. Topics include assay design parameters, assay validation, assay format, and application in preclinical modeling.

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Assay Development for Patient Response Measurement in Phase 0 Trials

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  1. Development of Assays for Use in Patient Response Measurement in Phase 0 Trials Robert Kinders Ralph Parchment Pharmacodynamic Assay Development and Implementation Section, and Laboratory of Human Toxicology & Pharmacology SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702 Funded by Contract N01-CO-12400

  2. Assay Design Parameters for Phase 0 • Quantitative assay readouts are essential to meet protocol endpoints • Accuracy by recovery, interfering substances, cross-reactivity and dilution linearity • LLQ, slope and dynamic range must be consistent with expected signal level in an 18-gauge needle biopsy or 2.5-10 E5 PBMC • Specimen turnaround in 24 hours • Assay must yield consistent results over at least a year of clinical testing • Controls and calibrators are critical • Calibrators should be available, stable, quantified, characterized • Controls should mimic specimen and be run in every assay • PAR assay uses cell extracts • Specific antibody-stained biopsies used in γ-H2AX assay • Reagent sources and an alternate vendor should be identified

  3. Tumor Extract Dilution Analysis and Recovery Validated PAR Immunoassay

  4. Assay Validation • Most Critical Aspect: Demonstrate that the assay is fit for purpose • An assay with a 20% interday/instrument/operator CV cannot detect a 20% modulation of signal • Must know accuracy and precision to assess performance • This does not capture biological variation, which is large • Analytical sensitivity yields real-world benefits: • Allows repeat determinations on the same specimen • Adapted for the size of the specimen to be used in the clinic: e.g., an 18-gauge needle biopsy • Robustness: Must be transferable to other labs

  5. PAR Immunoassay Development • Key changes made included: • Specimen treatment step added • Specimen diluent and dilution level • Specimen incubation time and temperature • Substrate incubation time and temperature • Other Changes • Use of a shaking step before the read • Blockers • Carrier protein • Read time • Instrument settings • Conjugate, conjugate concentration, and diluent [PAR] Total %CV Previous New 10 46.4 ? 30 30.2 8.1 100 24.6 7.4 300 19.5 6.9 1000 13.9 6.4

  6. Completion of a Validated Assay Format • Selection of assay format • Production of assay controls (QC samples) • Adequate instrumentation (calibrated and validated) • Selection of standards and the standards matrix • Will the standards matrix be a cell extract? • How will that affect stability of the standards? • Adequate assay sensitivity for multiple determinations on the same clinical specimen • Specific optimization of handling by specimen type • Validation issues: • Use common reagents (master lot) • Precision validation (operator, day, instrument) • Accuracy validation (dilution linearity, spike recovery) • Assay transfer between sites NOW WE CAN START!

  7. Now Start Application in Preclinical Modeling • Establish the dynamic range of response to treatment • Time window of sampling consistent with clinical realities • Tie the dose/response curve in tumor growth inhibition to marker modulation • What is the minimum detectable signal? • Is that in the microdose range? • Can the assay report whether an effective dose is delivered to the tumor?

  8. Vehicle Control Biopsies at 100x, All 4 Mice

  9. Intratumoral γ-H2AX Response to 4.7 mg/kg Topotecan +2 Hours, Mouse #22, All 3 Fields The biopsy assay counts pixels to determine the fraction of nuclear area that is γ-H2AX-positive over three 200x fields. It uses the DAPI signal to identify fields containing mostly live cells. It matters where the needle goes!

  10. Comparison of g-H2AX Response to Topotecan or Indenoisoquinoline Drug Dose, +2-Hour Timepoint • Plot is vehicle- corrected Ln/Ln with plateau response points omitted for topotecan (TPT) and NSC 725776 • Range of vehicle reads was the same across the 3 experiments; means & SDs overlapped

  11. Helpful Links and References • http://www.westgard.com/lesson.htm • http://www.nihs.go.jp/drug/validation/q2bwww.html • http://www.fda.gov/cder/guidance/4252fnl.htm • http://www.clinchem.org/info_ar/anal_meth.shtml • http://www.cbrlabs-inc.com/assay-validation.html • Validation of Immunoassays for Bioanalysis: a Pharmaceutical Industry Perspective. Findlay J., Smith W., Lee D., Nordblom G., Das I., DeSilva B., Khan M., and Bowsher R. Journal of Pharmaceutical and Biomedical Analysis. 2000; 21: 1249-1273. • Immunoassay, A Practical Guide. Ed. Dan Chan and Marie Pearlstein. Academic Press, 1987, 1992.

  12. Operations and Technical Support Contractor for The National Cancer Institute at Frederick

  13. The Next Speaker is:Dr. Susan Galbraith

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