Development of Assays for Use in Patient Response Measurement in

Phase 0 Trials

Robert KindersRalph 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

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

Tumor Extract Dilution Analysis and Recovery Validated PAR Immunoassay

Human Tumor Extract Titration

0

100

200

300

400

500

600

700

800

900

0 2 4 6 8 10 12

Tumor lysate (μg)

PA

R (p

g/m

L)

D2

D3

D4

D5

D6

D7

D8

D9

D10

D15

B4

B7

Spiked Read = f(Std Curve)

0.E+00

1.E+05

2.E+05

3.E+05

4.E+05

0.E+00 1.E+05 2.E+05 3.E+05 4.E+05 5.E+05 6.E+05

PAR Standard Curve RLU

PA

R s

pike

d in

to e

xtra

ct, R

LU

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

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

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!

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?

Vehicle Control Biopsies at 100x, All 4 Mice

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 toidentify fields containing mostly live cells.

It matters where the needle goes!

Comparison of H2AX Response to Topotecan or Indenoisoquinoline Drug Dose, +2-Hour Timepoint

H2AX Dose Response

-0.5

0

0.5

1

1.5

2

2.5

-1 0 1 2 3 4 5

Ln Dose (mg/kg)

Ln

%N

AP

-V

TPT

724998725776

706744

• 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

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.

Operations and Technical Support Contractor for

The National Cancer Institute at Frederick

The Next Speaker is:

Dr. Susan Galbraith