Bioassays for Bispecific Antibodies (BiTEs) Bioassays 2010 Meeting November 8, 2010 Hyun Jun Kim Analytical Biochemistry

Transcription

1 Bioassays for Bispecific Antibodies (BiTEs) Bioassays 2010 Meeting November 8, 2010 Hyun Jun Kim Analytical Biochemistry

2 Outline Introduction BiTE MoA Bioassay Development Assay Performance Assay Specificity & Ruggedness Summary 2

3 Introduction: The BiTE Antibody Principle Bispecific T cell Engager (BiTE) Antibody Baeuerle P, Cancer Res 2009; 69(12). June

4 Mechanism of Action: BiTE-mediated Cytotoxicity 4

5 Research Bioassay Method Day 0: Purify CD3+T cells from Healthy Donor Time: 2-3 h Stimulate the T cells with hil-2 overnight (37 ºC) 24 h Day 1: Assay Setup 1-2 h Label Target cells Stimulated T cells BiTE Antibody Day 3: Incubation at 37 ºC for 48 h FACS Read-out 48 h h Total assay time: >3 days 5

6 Bioassay Development: Bioassay Optimization Bioassay Optimization: Identify assay critical components Considerations Target and Effector Cell Lines Detection Method Interaction-dependent assay factors Cell Density/Proximity limits Effector to Target Cell Ratio Assay tolerance/robustness parameter range Assay cell density Assay incubation time Assay read time 6

7 Bioassay Development: Characterization of Target & Effector Cells Identification of Cells: Target Cell-1 Target Cell-2 Effector T cell 91.26% 96.57% CD % Target - FITC Target - FITC 7

8 Bioassay Development: Comparison of Detection Methods Detection Methods Signal to Noise Ratio Calcein AM Release LDH Release <1.0 Caspase Activation Europium Release n/a (no response) Caspase Activation is optimal detection method 8

9 Response Bioassay Development: Interaction Dependent Assay Factors E:T Ratio vs S/N Ratio Protein Concentration E:T=1:3 E:T=1:2 E:T=1:1 E:T=2:1 E:T=3:1 S/N Ratio

10 Response Bioassay Development: Interaction Dependent Assay Response Cell Density vs S/N Ratio Graph#1 1.13e7 1.03e7 9.3e6 8.3e6 7.3e6 6.3e6 5.3e6 4.3e6 Total Cell Number/well S/N Ratio 15K K K K e6 2.3e6 1e Protein Concentration x axis 4-P Fit: y = (A - D)/( 1 + (x/c)^b ) + D: A B C D R^2 15k (15k cells@experiment#1: Concentration vs Mea e e k (20k cells@experiment#1: Concentration vs Mea e e k (25k cells@experiment#1: Concentration vs Mea e e k (30k cells@experiment#1: Concentration vs Mea e e7 1 Curve Fit Option - Fixed Weight Value: 1 10

11 Bioassay Development: New Bioassay Conditions Caspase activation: Detection method Cell seeding number: 25K cells/well ET ratio: 1:3.5 No need for pre-incubation Incubation time is h 11

12 Robustness DoE Target Bioassay Development: Robustness DoE Identify DoE testing range of assay performance parameter Demonstrate acceptable assay robustness tolerance Assay tolerance parameter should account for normal performance variability of 15-20% Conditions Cell Plating Density/Ratio (cells/well) Assay Incubation Time (h) Full-factorial robustness DoE (N=2) 12

13 Bioassay Development: Protocol Comparison Day 0: Purify CD3+ T cells from healthy donor + Day 1: Day 3: Research Protocol Stimulate the T cells with human IL-2 Prep. BiTE dilutions + Label the Target Cells + CD3 enriched T cells 96-U Plate, 37 C, 5% CO2 incubator (48 h) Day 0: Day 1: Optimized Protocol Prep. BiTE dilutions + Effector cells + Target cells (E:T=1:3.5) mix 96-W Plate, 37 C, 5% CO 2 incubator (23 h) After 23 h incubation, add Caspase-Glo for h Luminometer Reader Trypsinize the cells for 5 min and then neutralize the trypsin with FACS buffer Add PI LSR FACS reader 13

14 Response Bioassay Development: Accuracy & Intermediate Precision Protein Concentration 14

15 Bioassay Development: Repeatability Repeatability (N=3) Expected Relative Potency (%) 100% 100% 100% 100% 100% Observed Relative Potency (%) Assay Assay Assay % Mean Potency % CV Overall % CV

16 % Potency (Observed) Bioassay Development: Linearity y = 0.992x R 2 = % Potency (Expected) 16

17 % of Relative Potentcy Bioassay Development: Stability-indicating Ability 120 Heat Stress Study Time=0 3 Days 6 Days 13 Days Incubation 50 C 17

18 Highlights of Optimized Assay : More sensitive Fewer cells Fewer steps Faster Homogenous endpoint readout Robust Stability-indicating QC friendly Bioassay Development: Performance Summary 18

19 Response Response Bioassay MoA Characterization: Specificity Ref Std Spike alone Spike + -Target -Target alone Protein Concentration Ref Std Spike alone Spike + -CD3 -CD3 alone Protein Concentration 19

20 Response Response Assay Ruggedness: Target & Effector Cell Passage Target Cell Passage Number Effector T Cell Passage Number P55 P51 P3 P3 Protein Concentration Protein Concentration Target Cell P3 P55 Effector Cell P3 P55 S/N Ratio S/N Ratio

21 Summary A simple one-day homogeneous bioassay was developed and optimized for BiTE. Assay is reflective of the MoA by directly measuring T cell mediated target cell death. Assay is suitable for use in a QC environment. 21

22 Acknowledgements Tianmeng Shao Michael Doh Xu-Rong Jiang Robert Strouse Michael Washabaugh Mark Schenerman 22