Promega CorporationPromega Corporation
©2013 Promega Corporation.
March 2014
ADCC Reporter Bioassays -
V and F Variants:
N...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 2
Topics Presented
 Introduction to ADCC
– Problem with...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 3
 Reflective of the mechanism of action (MOA) of the
b...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 4
Characteristics of a Valid Bioassay Are…
-10 -9 -8 -7 ...
Promega CorporationPromega Corporation
©2013 Promega Corporation.
March 2014
The Problem with Classic ADCC
Assays
©2013 Promega Corporation.
Promega CorporationPromega Corporation 6
Classic ADCC Assays Are Limited by
The performance of…...
Promega CorporationPromega Corporation
©2013 Promega Corporation.
March 2014
Solution: A Better ADCC Bioassay
Introducing ...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 8
Image source: Leibson-PJ, Immunity (1997) 6(6): 655-66...
©2013 Promega Corporation.
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Target
cell
Y
= NFAT-RE-luc
FcgRIIIa
(V158 or
F158)
An...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 10
Why an F Variant Version of the Assay is Needed
Polym...
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©2013 Promega Corporation.
March 2014
“Cells As Critical Reagents”
©2013 Promega Corporation.
Promega CorporationPromega Corporation 12
Cells as Critical Reagents
Traditional cell-based ass...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 13
No Cell Culture Required
ADCC Reporter Bioassay featu...
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Promega CorporationPromega Corporation 14
Biological Performance Equivalent to Fresh Cells
Fres...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 15
Frozen, Thaw-and-Use WIL2-S Target Cells Provide
Conv...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 16
Frozen, Thaw-and-Use Raji Target Cells, Too…
ADCC Rep...
©2013 Promega Corporation.
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Complete QC on Cells Ensures Consistent Results
Produ...
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©2013 Promega Corporation.
March 2014
Performance of ADCC Reporter
Bioassay (V Vari...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 19
Assessing Critical Assay Parameters
Induction time E:...
©2013 Promega Corporation.
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Bioassay Development Using DOE
Target cell / Ab Jurka...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 21
 Parallelism and measurement of Potency relative to ...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 22
V Variant: Able to Measure Potencies of On-Market
mAb...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 23
Able to Measure Fc Effector Function in ADCC for
TNF...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 24
Understanding Potency Determinations Using
Quantitati...
©2013 Promega Corporation.
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Assay Qualification Results with WIL2-S Cells
Design:...
©2013 Promega Corporation.
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Assay Qualification Results with Raji Cells
Day
Antib...
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ADCC Reporter Bioassay (V Variant) is Specific
Assay ...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 28
ADCC Reporter Bioassay is Robust
-10 -9 -8 -7 -6 -5
0...
©2013 Promega Corporation.
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Stability Indicating for Fc Effector Function
EC50 = ...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 30
Miniaturizes to 384-Well Format
WIL2-S target cells R...
Promega CorporationPromega Corporation
©2013 Promega Corporation.
March 2014
Antibody Variants, Glycosylation and
Benchmar...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 32
Deglycosylated Herceptin
-12 -10 -8 -6 -4
0
5100 5
1...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 33
Linear correlation obtained between
percentage of N-g...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 34
Sample set: Blended mixes of fully fucosylated and af...
Promega CorporationPromega Corporation
©2013 Promega Corporation.
March 2014
Polymorphism in the FcgRIIIa Receptor
Optimiz...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 36
Effector:Target (E:T) Ratio
Suspension target cells A...
©2013 Promega Corporation.
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Induction Time
Fresh-from-culture effector cells Froz...
©2013 Promega Corporation.
Promega CorporationPromega Corporation 38
Bioassay Development Using DOE
Factors:
 Target cell...
©2013 Promega Corporation.
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The F Variant Bioassay is Specific
Assay signal is sp...
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F Variant: Able to Measure Potencies of On-Market
mAb...
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Antibody IgG-Isotype Specificity
Order of response: h...
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Detects Loss of Activity Due to Heat-Stress
EC50 incr...
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Polymorphism in the FcgRIIIa Receptor Impacts
ADCC Re...
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©2013 Promega Corporation.
March 2014
Product Formats
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Product Configurations: ADCC Reporter Bioassay,
V Var...
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Custom Product Configurations for ADCC
Reporter Bioas...
©2013 Promega Corporation.
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Ordering Information
ADCC Reporter Bioassays (V Varia...
©2013 Promega Corporation.
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Adopted by the Contract Services Industry
 Approved ...
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Highlights of the ADCC Reporter Bioassay…
Features
 ...
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©2013 Promega Corporation.
March 2014
Simple, Convenient Luminometer
Tested with AD...
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Easy Reporter Assay Detection
GloMax® Discover System...
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GloMax® Discover System
Easy-To-Use
 Choose from pre...
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For More Information
Neal Cosby, PhD
Strategic Market...
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ADCC Reporter Bioassay - V and F Variants: Novel, Bioluminescent Cell-Based Assays for Quantifying Fc Effector Function of Antibodies

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The ADCC Reporter Bioassay from Promega is now available in both V158 and F158 variant formats. Use this functional, sister bioassay approach to better characterize Ab drugs with MOA directed through FcgammaRIIIa (CD16a) receptor. Not yet familiar with this novel bioassay that is becoming the industry standard? This slidedoc introduces the reporter-based ADCC technology. Contact me for any questions.

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ADCC Reporter Bioassay - V and F Variants: Novel, Bioluminescent Cell-Based Assays for Quantifying Fc Effector Function of Antibodies

  1. 1. Promega CorporationPromega Corporation ©2013 Promega Corporation. March 2014 ADCC Reporter Bioassays - V and F Variants: Novel, Bioluminescent Cell-Based Assays for Quantifying Fc Effector Function of Antibodies Rev 01
  2. 2. ©2013 Promega Corporation. Promega CorporationPromega Corporation 2 Topics Presented  Introduction to ADCC – Problem with classic ADCC assays  Solution: Designing a Better ADCC Bioassay – Introducing ADCC Reporter Bioassay  “Cells as Critical Reagents” – Cell selection, frozen, thaw-and-use format  V Variant ADCC assay performance – Assay optimization, qualification, potency & stability indicating, benchmarking  F Variant ADCC assay performance – Assay optimization, qualification, potency & stability Indicating  Commercial Formats – Kits & Cell Propagation Model, custom materials Image source: www.iconplc.com
  3. 3. ©2013 Promega Corporation. Promega CorporationPromega Corporation 3  Reflective of the mechanism of action (MOA) of the biological product  Well controlled (precise, accurate, robust, reproducible)  Stability-indicating  Usable as a QC lot-release assay Modified from Chana Fuchs (DMA/CDER) An Ideal Bioassay Is… On the following pages, we demonstrate how the novel ADCC Reporter Bioassay fulfills each of these parameters
  4. 4. ©2013 Promega Corporation. Promega CorporationPromega Corporation 4 Characteristics of a Valid Bioassay Are… -10 -9 -8 -7 -6 -5 0 5 10 15 20 25 30 35 plate1 plate2 plate3 plate4 Log10 [B1 antibody], g/ml FoldofInduction Repeatability Design:  Two analysts  Three days  Four plates per day 100% vs 50% 100% vs 75% 100% vs 125% 100% vs 150% -10 -9 -8 -7 -6 -5 0 5 10 15 20 25 30 35 100% 50% 150% Log10 [B1 antibody], g/ml FoldofInduction Y=1.026X-5.126 R2=0.995 Relative Potency Linearity Log [control antibody], g/ml Log [control antibody], g/ml Validation of Analytical Procedures:  Accuracy  Precision:  Repeatability (intra-assay precision)  Intermediate precision (day to day, analyst to analyst)  Reproducibility (lab to lab)  Specificity  Linearity  Range  Robustness - ICH Guideline Q2 [R1]
  5. 5. Promega CorporationPromega Corporation ©2013 Promega Corporation. March 2014 The Problem with Classic ADCC Assays
  6. 6. ©2013 Promega Corporation. Promega CorporationPromega Corporation 6 Classic ADCC Assays Are Limited by The performance of… Effector cells:  PBMCs (peripheral blood mononuclear cells)  NK from PBMCs  NK cell lines Target cells:  Load with chromium-51 or Eu  Monitor cell lysis (LDH, Calcein AM, GAPDH, CytoTox-Glo™ Assay) CytoTox-Glo™ Cytotoxicity Assay Target cell Y Primary NK effector cellAntibody FcgRIIIa Classic assay principle Cell lysis
  7. 7. Promega CorporationPromega Corporation ©2013 Promega Corporation. March 2014 Solution: A Better ADCC Bioassay Introducing ADCC Reporter Bioassay
  8. 8. ©2013 Promega Corporation. Promega CorporationPromega Corporation 8 Image source: Leibson-PJ, Immunity (1997) 6(6): 655-661; doi: 10.1016/S1074-7613(00)80441-0 Luciferase reporter is readout of pathway activation state Target-cell bound Ab binds to FcgRIIIa (CD16) on effector cell – activating pathway Scientific Basis of ADCC Reporter Bioassay New reporter-based bioassay measures a step earlier in the pathway
  9. 9. ©2013 Promega Corporation. Promega CorporationPromega Corporation 9 Target cell Y = NFAT-RE-luc FcgRIIIa (V158 or F158) Antibody Improving Upon the Classic ADCC Bioassay Effector cell (engineered Jurkat) Reporter-based ADCC bioassay Glo Target cell Y Primary NK effector cellAntibody FcgRIIIa Classic ADCC bioassay Specific signal is from target cell:  High variability of assay results – mainly due to primary NK cells  Spontaneous lysis of target & effector cells results in high background  Complex & tedious to perform Signal is from effector cell:  Reduced variability - replace NK cells with genetically engineered stable cell line  ADCC MOA-based bioassay  Simple & easy to perform  Improved bioassay performance with robust reagents and assay design Cell lysis Parekh, BS et al (2012). mAbs, 4(3), 310-318; doi: 10.4161/mabs.19873
  10. 10. ©2013 Promega Corporation. Promega CorporationPromega Corporation 10 Why an F Variant Version of the Assay is Needed Polymorphism in the FcgRIIIa Receptor Impacts ADCC Response FcyRIIIa 158F/V or F/F 158 V/V >85% population ~10-15% population Less efficient Ab binding and ADCC More efficient Ab binding and ADCC  The FcgRIIIa receptor has a polymorphism at amino acid 158 with human genotypes of VV, FV and FF. The V variant receptor has higher affinity for Abs. Patients with FV or FF genotype respond less well to Ab drugs having ADCC MOA.  Many new generation mAb drugs are being designed to improve Ab affinity for the F variant of the receptor, to improve patient response.  Drug developers need a low variability ADCC bioassay specifically to measure the new drug potency via the F158 variant of FcgRIIIa, and regulatory agencies are asking for this information. See the section beginning on page 34, “Polymorphism in the FcgRIIIa Receptor” for information on our newly developed F Variant ADCC Reporter Bioassay. V158 or F158
  11. 11. Promega CorporationPromega Corporation ©2013 Promega Corporation. March 2014 “Cells As Critical Reagents”
  12. 12. ©2013 Promega Corporation. Promega CorporationPromega Corporation 12 Cells as Critical Reagents Traditional cell-based assay using fresh cells from cell culture (1-2 weeks) Cell culture Cell count, harvest, prepare for assay Thaw for culture Read plates New assay format using frozen, thaw-and-use cells: convenience and improved run-to-run reproducibility Thaw-and-Use Resuspend in assay buffer, plate for assay Read plates Time: 1-2 weeks Time: <24hr See pages 50-51 for information on a convenient, low cost luminometer from Promega: GloMax® Discover
  13. 13. ©2013 Promega Corporation. Promega CorporationPromega Corporation 13 No Cell Culture Required ADCC Reporter Bioassay features Frozen, Thaw-and-Use Cells 1. Human cell lines (Jurkat, WIL2-S, Raji) - Developed for immediate thaw-and-use in bioassay - Designed for good recovery and robust response upon thawing 2. Thaw-and-Use format - Cell propagation conditions & defined freezing protocol control assay performance for a consistent bioassay response - No pre-culturing prior to assay means less variability introduced - Indefinite storage - Identical cells in bioassay, day to day 3. Minimizes pre-assay planning, time & labor - Ample cell banks provide long-term supply means no cell culture required Thaw-and-Use cells +
  14. 14. ©2013 Promega Corporation. Promega CorporationPromega Corporation 14 Biological Performance Equivalent to Fresh Cells Fresh cells from continuous culture Frozen, thaw-and-use cells EC50=3.5ng/ml FI=34-fold EC50=2.9ng/ml FI=41-fold Assay specifics: E:T ratio = 6:1; fresh WIL2-S cells as target cells; Bio-Glo™ reagent; 20hr induction for fresh Jurkat cells assay; 6hr induction for frozen Jurkat cells assay.
  15. 15. ©2013 Promega Corporation. Promega CorporationPromega Corporation 15 Frozen, Thaw-and-Use WIL2-S Target Cells Provide Convenience Kit Control Ab Rituximab ADCC Reporter Bioassay response to ADCC Bioassay Control Antibody (left) or Rituximab (right). The EC50 response using frozen, thaw-and-use WIL2-S cells was 16.8ng/ml for Control Ab, Anti-CD20. For Rituximab, 1.94ng/ml. Assay specifics: E:T ratio = 6:1; 6hr induction; Bio-Glo™ reagent
  16. 16. ©2013 Promega Corporation. Promega CorporationPromega Corporation 16 Frozen, Thaw-and-Use Raji Target Cells, Too… ADCC Reporter Bioassay response to ADCC Bioassay Control Antibody (left) or Rituximab (right). The EC50 response using frozen, thaw-and-use Raji cells was 59.7ng/ml for Control Ab, Anti-CD20. For Rituximab, 17.0ng/ml. Kit Control Ab Rituximab Assay specifics: E:T ratio = 6:1; 6hr induction; Bio-Glo™ Reagent
  17. 17. ©2013 Promega Corporation. Promega CorporationPromega Corporation 17 Complete QC on Cells Ensures Consistent Results Production cell batches are rigorously tested:  STR analysis – cell ID profile (human)  CO1 analysis (cytochrome oxidase) – test for presence of species (human and other potential contaminants)  Cell doubling time under propagation conditions  Mycoplasma (Hoechst and direct culture)  Sterility  Cell density  Cell viability after thaw  Fill volume  ADCC Reporter Bioassay (EC50 and fold induction)
  18. 18. Promega CorporationPromega Corporation ©2013 Promega Corporation. March 2014 Performance of ADCC Reporter Bioassay (V Variant)
  19. 19. ©2013 Promega Corporation. Promega CorporationPromega Corporation 19 Assessing Critical Assay Parameters Induction time E:T ratio (constant Effector cell #) Other parameters tested:  Assay buffer: serum concentration, use of low IgG serum  Cell numbers per well  Pre-plating and incubation time: target cell plating, antibody/target cells incubation  White flat-, V- or U-bottom assay plates
  20. 20. ©2013 Promega Corporation. Promega CorporationPromega Corporation 20 Bioassay Development Using DOE Target cell / Ab Jurkat cell Target cell run induction time hr incubation time(mins) plating number (K) plating number (K) 1 5.5 30 75 10 2 5.5 30 75 12.5 3 5.5 30 90 12 4 5.5 30 90 15 5 5.5 45 75 10 6 5.5 45 75 12.5 7 5.5 45 90 12 8 5.5 45 90 15 9 6 30 75 10 10 6 30 75 12.5 11 6 30 90 12 12 6 30 90 15 13 6 45 75 10 14 6 45 75 12.5 15 6 45 90 12 16 6 45 90 15 Variables:  Induction time  Target/Ab pre-incubation  Effector cell number  Target cell number Outputs & Results: Good response (fold induction) = 19-27 Good (low) L-term* values = 0.1-0.2 *L-term is a measure of assay precision around the EC50 determination (log width of the 95% confidence interval around logEC50)
  21. 21. ©2013 Promega Corporation. Promega CorporationPromega Corporation 21  Parallelism and measurement of Potency relative to the reference antibody  Linearity & Accuracy of observed versus expected potencies across the desired working range of potencies  Precision - intra-assay - intermediate (inter-assay) precision  Specificity to show response is dependent on specific antibody and the presence of target cells and FcgRIIIa on effector cells, and not other components  Stability-indicating to show the bioassay is capable of detecting loss of structural integrity of an antibody These qualification studies are critical to demonstrate a useful and effective ADCC bioassay Useful and Effective ADCC Bioassay Demonstrated Qualification Studies:
  22. 22. ©2013 Promega Corporation. Promega CorporationPromega Corporation 22 V Variant: Able to Measure Potencies of On-Market mAb Biologic Drugs Rituximab, anti-CD20, chimeric IgG1. Approved to treat B-cell non-Hodgkin lymphoma and chronic lymphocytic leukemia. Trastuzumab, anti-HER2, humanized IgG1. approved to treat HER2-positive breast cancer and stomach cancer. Cetuximab, anti-EGFR, chimeric IgG1. Approved to treat colorectal cancer and certain types of head and neck cancer. Panitumumab, anti-EGFR, human IgG2 with NO ADCC. Three best-selling, on-market mAb biologic drugs that posses ADCC as main MOA Rituximab tests performed using ADCC Reporter Bioassay, Complete Kit; Trastuzumab and Cetuximab using Core kit. Both V Variant.
  23. 23. ©2013 Promega Corporation. Promega CorporationPromega Corporation 23 Able to Measure Fc Effector Function in ADCC for TNF Blockers mTNF CHO-K1 Target cells Y = NFAT-RE-luc FcgRIIIa anti-TNF engineered Jurkat effector cells Glo mTNF Assay specifics:  Effector cells: ADCC Bioassay Effector Cells, (V Variant) frozen, thaw-and-use  Target cells: mTNF CHO-K1 target cells  E:T ratio: 7.5:1 An engineered CHO-K1 cell line expressing membrane-bound TNF was established as target cells. Infliximab Adalimumab
  24. 24. ©2013 Promega Corporation. Promega CorporationPromega Corporation 24 Understanding Potency Determinations Using Quantitative Bioassays Relative Potency: Use a bioassay to establish potency activity of unknown biologic relative to a reference standard Relative potency can only be determined when:  The upper and lower asymptotes as well as the slopes of the curves are not significantly different. Hence the curves are parallel  Only the EC50s differ  Relative potency calculation: __EC50 Test Sample___ EC50 Reference Sample y = d + a - d 1 + (conc/c)b a c b d Concentration Response Potency (% of Reference) 4-parameter logistic curve fit and potency determination Reference Test sample upper asymptote slope lower asymptote EC50
  25. 25. ©2013 Promega Corporation. Promega CorporationPromega Corporation 25 Assay Qualification Results with WIL2-S Cells Design: •Two analysts • Three days • Four plates per day 100% vs 50% 100% vs 75% 100% vs 125% 100% vs 150% Linearity Representative plate layout Repeatability Y=1.026X-5.126 R2=0.995 1 2 3 4 5 6 7 8 9 10 11 12 Plate1 A B no Ab dilu9 dilu8 dilu7 dilu6 dilu5 dilu4 dilu3 dilu2 dilu1 100% C no Ab dilu9 dilu8 dilu7 dilu6 dilu5 dilu4 dilu3 dilu2 dilu1 50% D no Ab dilu9 dilu8 dilu7 dilu6 dilu5 dilu4 dilu3 dilu2 dilu1 100% E no Ab dilu9 dilu8 dilu7 dilu6 dilu5 dilu4 dilu3 dilu2 dilu1 50% F no Ab dilu9 dilu8 dilu7 dilu6 dilu5 dilu4 dilu3 dilu2 dilu1 100% G no Ab dilu9 dilu8 dilu7 dilu6 dilu5 dilu4 dilu3 dilu2 dilu1 50% H Precision =average of RSD (%) = 7.3% Accuracy= average of Recovery(%) = 95.8% Antibody Sample Measured Potency (%) Mean Potency (%) SD % Recovery (%) RSD (%) day 1 48.5 day 2 50% 45.2 48.9 3.9 97.7 7.9 day 3 52.9 day 1 63.1 day 2 75% 62.9 66.4 5.9 88.5 8.9 day 3 73.2 day 1 112.1 day 2 125% 136.3 123.0 12.3 98.4 10.0 day 3 120.5 day 1 148.8 day 2 150% 150.4 147.6 3.6 98.4 2.4 day 3 143.6 Bioassay uses frozen, thaw-and-use cells for both effector & WIL2-S target cells Good repeatability, accuracy, precision and linearity were obtained
  26. 26. ©2013 Promega Corporation. Promega CorporationPromega Corporation 26 Assay Qualification Results with Raji Cells Day Antibody Sample Measured Potency (%) Mean Potency (%) SD (%) Recovery (%) CV (%) 1 49.9 2 50% 51.3 51 0.7 102 1.4 3 50.5 1 78.9 2 75% 78.8 76 5.11 101 6.7 3 70 1 118.6 2 125% 116.9 117 1.19 94 1 3 116.3 1 143.2 2 150% 142.5 145 3.91 97 2.7 3 149.6 Day Antibody Sample Measured Potency (%) Mean Potency (%) SD (%) Recovery (%) CV (%) 1 38.4 2 50% 47.2 41.8 7.2 83.5 17.2 3 33.2 4 48.2 1 59.6 2 75% 70.2 67.4 5.2 89.9 7.7 3 69.3 4 70.5 1 120 2 125% 132.3 129.7 7.5 103.7 5.8 3 137.8 4 128.6 1 160.2 2 150% 158.2 162.8 5.2 108.5 3.2 3 162.7 4 170 Precision: 2.95% Accuracy (recovery avg): 98.5% Linearity: y = 0.922x + 5.0 Precision: 8.47% Accuracy (recovery avg): 96.4% Linearity: y = 1.22x - 21.3 Analyst 1: Analyst 2: Linearity:
  27. 27. ©2013 Promega Corporation. Promega CorporationPromega Corporation 27 ADCC Reporter Bioassay (V Variant) is Specific Assay signal is specifically dependent on:  Target cells expressing Ab-targeted antigen  Specific antibody  Effector cells expressing FcgRIIIa receptor
  28. 28. ©2013 Promega Corporation. Promega CorporationPromega Corporation 28 ADCC Reporter Bioassay is Robust -10 -9 -8 -7 -6 -5 0 10 20 30 1 6 7 14 Log10 [B1 antibody], g/ml FoldofInduction -10 -9 -8 -7 -6 -5 0 10 20 30 1 1 7 13 16 Log10 [B1 antibody], g/ml FoldofInduction Time of induction E:T ratio and cell # per well Run Induction time EC50 1 6.0hr 3.15x10-8 g/ml 2 5.5hr 3.83x10-8 g/ml Run E:T ratio E cell # T cell # EC50 1 7.5:1 75k 10k 3.09x10-8 g/ml 2 6:1 90k 15k 3.83x10-8 g/ml
  29. 29. ©2013 Promega Corporation. Promega CorporationPromega Corporation 29 Stability Indicating for Fc Effector Function EC50 = 5.77ng/ml EC50 = 31.0ng/ml Rituximab: Tositumomab: Trastuzumab: Activity following heat-treatment of antibody drugs
  30. 30. ©2013 Promega Corporation. Promega CorporationPromega Corporation 30 Miniaturizes to 384-Well Format WIL2-S target cells Raji target cells Assay volume per well Target cells Antibody Effector cells Bio-Glo™ 96-well plate 25µl 25µl 25µl 75µl 384-well plate 5µl 5µl 5µl 15µl
  31. 31. Promega CorporationPromega Corporation ©2013 Promega Corporation. March 2014 Antibody Variants, Glycosylation and Benchmarking with Classic ADCC Assay
  32. 32. ©2013 Promega Corporation. Promega CorporationPromega Corporation 32 Deglycosylated Herceptin -12 -10 -8 -6 -4 0 5100 5 1100 6 2100 6 unt 10%unt/ 90% degly EC50 unt 2.037e-008 10%unt/ 90% degly 7.174e-008 log [ab], (g/ml) RLU Deglycosylated Herceptin -12 -10 -8 -6 -4 0 5100 5 1100 6 2100 6 unt 100% degly EC50 unt 1.988e-008 100% degly 3.202e-008 log [ab], (g/ml) RLU Deglycosylated Herceptin -12 -10 -8 -6 -4 0 5100 5 1100 6 2100 6 EC50 unt 1.720e-008 20%unt/80% degly 4.626e-008 unt 20%unt/80% degly log [ab], (g/ml) RLU Deglycosylated Herceptin -12 -10 -8 -6 -4 0 5100 5 1100 6 2100 6 unt 30%unt/ 70% degly EC50 unt 2.002e-008 30%unt/ 70% degly 4.153e-008 log [ab], (g/ml) RLU Deglycosylated Herceptin -12 -10 -8 -6 -4 0 5100 5 1100 6 2100 6 unt 40%unt/ 60% degly EC50 unt 2.082e-008 40%unt/ 60% degly 3.486e-008 log [ab], (g/ml)RLU Deglycosylated Herceptin -12 -10 -8 -6 -4 0 5100 5 1100 6 2100 6 unt 50%unt/ 50% degly EC50 unt 2.110e-008 50%unt/ 50% degly 2.990e-008 log [ab], (g/ml) RLU Target cells: SKBR3; Unt = 100% glycosylated Sensitive to Detect Differences in Glycosylation
  33. 33. ©2013 Promega Corporation. Promega CorporationPromega Corporation 33 Linear correlation obtained between percentage of N-glycosylated antibody in blended antibody samples and relative luciferase reporter activity in ADCC Reporter Bioassay y = 0.0127x - 0.0314 R² = 0.966 -0.100 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0 10 20 30 40 50 60 RelativeactivityinreporterADCC Percent N-glycosylated sample y = 0.0125x - 0.0095 R² = 0.9926 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0 10 20 30 40 50 60 RelativeactivityinreporterADCC Percent N-glycosylated sample Rituximab Trastuzumab _____________________________________ Small differences in Fc effector activity in ADCC pathway activation are easily distinguished in the ADCC Reporter Bioassay _____________________________________ Activity Correlates with Amount of Antibody N-Glycosylation
  34. 34. ©2013 Promega Corporation. Promega CorporationPromega Corporation 34 Sample set: Blended mixes of fully fucosylated and afucosylated Ab samples – provides a series with a range of ADCC potencies. Relative activity: Expressed relative to 100% fucosylated Ab (lowest potency of series). Both assays show increase in relative potency with increased % afucosylation of mAb WIL2-S target cells; 6hr assay. E:T 6:1LDH release assay; WIL2-S target cells; PBMCs as effector cells. 4hr assay. E:T 25:1 Benchmarking Study: Good Correlation with Lytic LDH Release Assay Classic ADCC assay(1) ADCC Reporter Bioassay(2) 1. Chung, S et al (2012). mAbs, 4(3), 326-340; doi: 10.4161/mabs.19941 2. Cheng, J et al (2012) AACR, poster #4606.
  35. 35. Promega CorporationPromega Corporation ©2013 Promega Corporation. March 2014 Polymorphism in the FcgRIIIa Receptor Optimizing the F Variant of the ADCC Reporter Bioassay
  36. 36. ©2013 Promega Corporation. Promega CorporationPromega Corporation 36 Effector:Target (E:T) Ratio Suspension target cells Adherent target cells 10:1 6:1 4:1 15:1 10:1 ADCC Reporter Bioassay, F Variant
  37. 37. ©2013 Promega Corporation. Promega CorporationPromega Corporation 37 Induction Time Fresh-from-culture effector cells Frozen, thaw-and-use effector cells 24hr 7hr 5hr 6hr 24hr Jurkat cell format ADCC Reporter Bioassay, F Variant
  38. 38. ©2013 Promega Corporation. Promega CorporationPromega Corporation 38 Bioassay Development Using DOE Factors:  Target cell density  Target cell/antibody incubation time  Effector cell density  Induction time Outputs:  Fold of induction  EC50  EC50 difference between F and V variant Jurkat effector cells DOE = Design of Experiments  Allow understanding of the interactions between critical assay factors  Minimum amount of work needed to develop robust assays ADCC Reporter Bioassay, F Variant
  39. 39. ©2013 Promega Corporation. Promega CorporationPromega Corporation 39 The F Variant Bioassay is Specific Assay signal is specifically dependent on:  Target cells expressing Ab-targeted antigen  Specific antibody  Effector cells expressing FcgRIIIa receptor No target cells No Ab or wrong Ab FcgRIIIa blocked No FcgRIIIa
  40. 40. ©2013 Promega Corporation. Promega CorporationPromega Corporation 40 F Variant: Able to Measure Potencies of On-Market mAb Biologic Drugs Rituximab, anti-CD20, chimeric IgG1. Approved to treat B-cell non-Hodgkin lymphoma and chronic lymphocytic leukemia. Trastuzumab, anti-HER2, humanized IgG1. Approved to treat HER2-positive breast cancer and stomach cancer. Cetuximab, anti-EGFR, chimeric IgG1. Approved to treat colorectal cancer and certain types of head and neck cancer. Target cell: SK-BR-3 Target cell: A431 Three best-selling, on-market mAb biologic drugs that posses ADCC as main MOA Tests performed using ADCC Reporter Bioassay, Core Kit (F Variant)
  41. 41. ©2013 Promega Corporation. Promega CorporationPromega Corporation 41 Antibody IgG-Isotype Specificity Order of response: hu IgG1, IgG3 > mouse IgG2a >> hu IgG2, IgG4, mouse IgG1 Expanding assay applications: 1. Confirm desired ADCC activity for IgG1 and IgG3-based mAbs 2. Identify non-designed ADCC activity for mAbs with non-ADCC MOA 2 1 Data generated using the ADCC Reporter Bioassay, F Variant
  42. 42. ©2013 Promega Corporation. Promega CorporationPromega Corporation 42 Detects Loss of Activity Due to Heat-Stress EC50 increases (right shift)  Reporter bioassay exhibits stability-indicating capability  Can potentially be used in stability studies for therapeutic antibody Fold induction decreases ADCC Reporter Bioassay, F Variant
  43. 43. ©2013 Promega Corporation. Promega CorporationPromega Corporation 43 Polymorphism in the FcgRIIIa Receptor Impacts ADCC Response Because many new generation mAb drugs are being designed to improve Ab affinity for the F variant of the receptor, drug developers need a low variability ADCC bioassay specifically to measure the new drug potency via the F158 variant of FcgRIIIa. Rituximab Trastuzumab The dual receptor bioassay approach of the Promega ADCC Reporter Bioassay, with both V and F variants of the FcgRIIIa allow researchers to better characterize their Ab drugs.
  44. 44. Promega CorporationPromega Corporation ©2013 Promega Corporation. March 2014 Product Formats
  45. 45. ©2013 Promega Corporation. Promega CorporationPromega Corporation 45 Product Configurations: ADCC Reporter Bioassay, V Variant Engineered Jurkat cells Bio-Glo™ Reagent Control Antibody Target WIL2-S or Raji Cells Core Kit Complete Kits +Medium Serum 1. Core Kits: 1X kit – Cat.# G7010 5X kit – Cat.# G7018 2. Complete Kits: WIL2-S Target Cells – Cat.# G7014 Raji Target Cells – Cat.# G7015 3. Target Kits: WIL2-S Target Cells – Cat.# G7013 Raji Target Cells – Cat.# G7016 4. Cell Propagation Model ADCC Bioassay Effector Cells, Propagation Model – Cat.# G7102 2 vials of Jurkat Effector cells: allows propagation and banking for use in ADCC via unique LULL Target Kits Multiple formats flexible to your research needs: Engineered Jurkat cells, V variant Propagation Model
  46. 46. ©2013 Promega Corporation. Promega CorporationPromega Corporation 46 Custom Product Configurations for ADCC Reporter Bioassay, F Variant Engineered Jurkat cells, F variant Bio-Glo™ Reagent Control Antibody Target WIL2-S or Raji Cells Core Kit + Medium Serum Target Kits Catalog products Jurkat effector cells, F variant Propagation Model 1. Core Kit (Part# CS1324F01) Components:  ADCC Bioassay Effector Cells, F Variant: in frozen, thaw-and-use format  RPMI Medium  Low IgG Serum  Bio-Glo™ Luciferase Assay System 2. Cell Propagation Model (Part# CS1324F08) Components:  ADCC Bioassay Effector Cells, F Variant, Propagation Model: allows propagation and banking for use in ADCC via unique LULL with Bio-Glo™ Luciferase Assay System
  47. 47. ©2013 Promega Corporation. Promega CorporationPromega Corporation 47 Ordering Information ADCC Reporter Bioassays (V Variant) Product Size Cat. ADCC Reporter Bioassay, Core Kit 1ea G7010 ADCC Reporter Bioassay, Core Kit 5X 1ea G7018 ADCC Reporter Bioassay, Complete Kit (WIL2-S) 1ea G7014 ADCC Reporter Bioassay, Complete Kit (Raji) 1ea G7015 ADCC Reporter Bioassay, Target Kit (WIL2-S) 1ea G7013 ADCC Reporter Bioassay, Target Kit (Raji) 1ea G7016 ADCC Bioassay Effector Cells, Propagation Model 1ea Pls enquire Contact: COD@promega.com Bio-Glo™ Luciferase Assay System 100ml 10ml G7940 G7941 Product Cat. ADCC Reporter Bioassay, F Variant, Core Kit Pls enquire ADCC Bioassay Effector Cells, F Variant, Propagation Model Pls enquire Contact CAS@promega.com ADCC Reporter Bioassays (F Variant) Pre-commercial materials are available now through our Custom Assay Services group. Simply e-mail CAS at the address below.
  48. 48. ©2013 Promega Corporation. Promega CorporationPromega Corporation 48 Adopted by the Contract Services Industry  Approved manufacturing cell line switch by a pharmaceutical company  Submitted in an IND filing by a pharmaceutical company  In development for lot-release testing by a pharmaceutical company  Adopted by multiple pharmaceutical companies globally  BioAgilytix, Catalent, Covance & Charles River Laboratories are providing ADCC Reporter Bioassay services
  49. 49. ©2013 Promega Corporation. Promega CorporationPromega Corporation 49 Highlights of the ADCC Reporter Bioassay… Features  Low variability  Engineered effector cells (Jurkat FcgRIIIa/NFAT-RE luc2) replace primary NK cells  “Cells as reagents” - frozen, thaw-and-use format  Simple & robust protocol  Broad applicability in use with suspension or adherent target cells  Good correlation with classic ADCC bioassay (cytolytic) Benefits  Demonstrates precision, accuracy, linearity, robustness, specificity  Can quantify potency and stability of therapeutic Ab drugs  Can differentiate biological activity of Fc effector function in ADCC MOA resulting from small changes in Ab glycosylation
  50. 50. Promega CorporationPromega Corporation ©2013 Promega Corporation. March 2014 Simple, Convenient Luminometer Tested with ADCC Reporter Bioassay
  51. 51. ©2013 Promega Corporation. Promega CorporationPromega Corporation 51 Easy Reporter Assay Detection GloMax® Discover System… Integrated for Promega’s ADCC Reporter Bioassay And ready to run any of the following reporter, cell health and protein interaction assays Product Size Cat. GloMax® Discover Multimode Detection System 1ea GM3000 Cell Health:  CellTiter-Glo® Assay  CellTox™ Green Assay  Caspase-Glo® Assay  BacTiter-Glo® Assay Luciferase Reporters:  Nano-Glo® Assay  ONE-Glo™ Assay  Dual-Glo® & DLR Assays  Bright-Glo™ Assay Protein Interaction Assays:  ELISAs  BRET  FRET See www.promega.com/glomax for more information
  52. 52. ©2013 Promega Corporation. Promega CorporationPromega Corporation 52 GloMax® Discover System Easy-To-Use  Choose from preloaded Promega protocols or customize your own.  Build custom protocols using the intuitive drag-and-drop interface.  Export data to a network, cloud or any drive desired. Superior Sensitivity  Plate mask (aperture control) for switching between 96-well and 384-well plates.  Low cross-talk between wells gives you better, more usable results.  Broad dynamic range of the instrument extends the linear range of your assay. Minimal Manual Intervention  Filter paddles and automatic filter switching allow easy two-color multiplexing assays or kinetic studies.  Quick read-speeds for high efficiencies in your laboratory.  Compatible for 3rd party automation control. Quality  IQ/OQ Service available.  Provides required technical elements of a 21CFR 11 compliant system to be used with the appropriate laboratory workflow.
  53. 53. ©2013 Promega Corporation. Promega CorporationPromega Corporation 53 For More Information Neal Cosby, PhD Strategic Marketing Manager Neal.cosby@promega.com Custom Order Department COD@promega.com Or see: www.promega.com/adcc

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