ADCC Reporter Bioassays - V and F Variants:

ADCC Reporter Bioassays - V and F Variants: Novel, Bioluminescent Cell-Based Assays for Quantifying Fc Effector Function of Antibodies Promega Corporation April 2014 Rev 01

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 Image source: www.iconplc.com F Variant ADCC assay performance Assay optimization, qualification, potency & stability Indicating Commercial Formats Kits & Cell Propagation Model, custom materials Promega Corporation 2

An Ideal Bioassay Is 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) On the following pages, we demonstrate how the novel ADCC Reporter Bioassay fulfills each of these parameters Promega Corporation 3

Fold of Induction Fold of Induction Characteristics of a Valid Bioassay Are Validation of Analytical Procedures: Accuracy Precision: Specificity Linearity Range Robustness Repeatability (intra-assay precision) Intermediate precision (day to day, analyst to analyst) Reproducibility (lab to lab) - ICH Guideline Q2 [R1] Repeatability 35 30 25 20 15 10 5 Design: Two analysts Three days 30 25 20 Four plates per day 50% 15 100% vs 50% 10 100% 100% vs 75% 100% vs 125% 100% vs 150% 5 0-10 -9-8 -7-6 150% -5 plate1 plate2 plate3 plate4 0-10 -9-8 -7-6 -5 Log 10 [B1 antibody], g/ml Log [control antibody], g/ml Relative Potency 35 Log 10 [B1 antibody], g/ml Log [control antibody], g/ml Linearity Y=1.026X-5.126 R2=0.995 Promega Corporation 4

The Problem with Classic ADCC Assays Promega Corporation April 2014

Y Classic ADCC Assays Are Limited by The performance of Classic assay principle Effector cells: PBMCs (peripheral blood mononuclear cells) Target cell FcgRIIIa Antibody Primary NK effector cell NK from PBMCs Cell lysis 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 Promega Corporation 6

Solution: A Better ADCC Bioassay Introducing ADCC Reporter Bioassay Promega Corporation April 2014

Scientific Basis of ADCC Reporter Bioassay Target-cell bound Ab binds to FcgRIIIa (CD16) on effector cell activating pathway Luciferase reporter is readout of pathway activation state New reporter-based bioassay measures a step earlier in the pathway Image source: Leibson-PJ, Immunity (1997) 6(6): 655-661; doi: 10.1016/S1074-7613(00)80441-0 Promega Corporation 8

Y Y Improving Upon the Classic ADCC Bioassay Classic ADCC bioassay Cell lysis Target cell FcgRIIIa Antibody Primary NK effector cell Reporter-based ADCC bioassay Target cell FcgRIIIa (V158 or F158) Antibody Effector cell (engineered Jurkat) Glo = NFAT-RE-luc 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 Parekh, BS et al (2012). mabs, 4(3), 310-318; doi: 10.4161/mabs.19873 Promega Corporation 9

Why an F Variant Version of the Assay is Needed Polymorphism in the FcgRIIIa Receptor Impacts ADCC Response 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. V158 or F158 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. Promega Corporation 10 See the section beginning on page 34, Polymorphism in the FcgRIIIa Receptor for information on our newly developed F Variant ADCC Reporter Bioassay. 158F/V or F/F FcyRIIIa 158 V/V >85% population ~10-15% population Less efficient Ab binding and ADCC More efficient Ab binding and ADCC

Cells As Critical Reagents Promega Corporation April 2014

Cells as Critical Reagents Traditional cell-based assay using fresh cells from cell culture (1-2 weeks) Time: 1-2 weeks Thaw for culture Cell culture Cell count, harvest, prepare for assay 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: <24hr See pages 50-51 for information on a convenient, low cost luminometer from Promega: GloMax Discover Promega Corporation 12

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 Thaw-and-Use cells + 3. Minimizes pre-assay planning, time & labor - Ample cell banks provide long-term supply means no cell culture required Promega Corporation 13

Biological Performance Equivalent to Fresh Cells Fresh cells from continuous culture Frozen, thaw-and-use cells EC 50 =3.5ng/ml FI=34-fold EC 50 =2.9ng/ml FI=41-fold Promega Corporation 14 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.

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 EC 50 response using frozen, thaw-and-use WIL2-S cells was 16.8ng/ml for Control Ab, Anti-CD20. For Rituximab, 1.94ng/ml. Promega Corporation 15 Assay specifics: E:T ratio = 6:1; 6hr induction; Bio-Glo reagent

Frozen, Thaw-and-Use Raji Target Cells, Too Kit Control Ab Rituximab ADCC Reporter Bioassay response to ADCC Bioassay Control Antibody (left) or Rituximab (right). The EC 50 response using frozen, thaw-and-use Raji cells was 59.7ng/ml for Control Ab, Anti-CD20. For Rituximab, 17.0ng/ml. Promega Corporation 16 Assay specifics: E:T ratio = 6:1; 6hr induction; Bio-Glo Reagent

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 (EC 50 and fold induction) Promega Corporation 17

Performance of ADCC Reporter Bioassay (V Variant) Promega Corporation April 2014

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 Promega Corporation 19

Bioassay Development Using DOE Variables: Induction time Target/Ab pre-incubation Effector cell number Target cell number 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 Outputs & Results: Good response (fold induction) = 19-27 Good (low) L-term* values = 0.1-0.2 Promega Corporation 20 *L-term is a measure of assay precision around the EC 50 determination (log width of the 95% confidence interval around logec 50 )

Useful and Effective ADCC Bioassay Demonstrated Qualification Studies: 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 Promega Corporation 21

V Variant: Able to Measure Potencies of On-Market mab Biologic Drugs Three best-selling, on-market mab biologic drugs that posses ADCC as main MOA 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. Rituximab tests performed using ADCC Reporter Bioassay, Complete Kit; Trastuzumab and Cetuximab using Core kit. Both V Variant. Promega Corporation 22

Y Able to Measure Fc Effector Function in ADCC for TNF Blockers An engineered CHO-K1 cell line expressing membrane-bound TNF was established as target cells. Infliximab mtnf mtnf CHO-K1 Target cells FcgRIIIa anti-tnf engineered Jurkat effector cells = NFAT-RE-luc Adalimumab Glo 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 Promega Corporation 23

Response Understanding Potency Determinations Using Quantitative Bioassays Relative Potency: Use a bioassay to establish potency activity of unknown biologic relative to a reference standard 4-parameter logistic curve fit and potency determination upper asymptote a - d y = d + 1 + (conc/c) b d slope b c EC50 Reference Potency (% of Reference) lower a Test sample asymptote Concentration 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 EC 50 s differ Relative potency calculation: EC 50 Test Sample EC 50 Reference Sample Promega Corporation 24

Assay Qualification Results with WIL2-S Cells Bioassay uses frozen, thaw-and-use cells for both effector & WIL2-S target cells Design: Two analysts Three days Four plates per day 100% vs 50% 100% vs 75% 100% vs 125% 100% vs 150% Representative plate layout 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 Repeatability Precision = average of RSD (%) = 7.3% Accuracy = average of Recovery (%) = 95.8% Linearity Promega Corporation 25 Measured Potency (%) Mean Potency (%) SD % Recovery (%) Antibody Sample 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 Y=1.026X-5.126 R2=0.995 Good repeatability, accuracy, precision and linearity were obtained

Assay Qualification Results with Raji Cells Analyst 1: Analyst 2: Day Measured Antibody Potency Sample (%) 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 Linearity: 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 Promega Corporation 26

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 Promega Corporation 27

Fold of Induction Fold of Induction ADCC Reporter Bioassay is Robust 30 20 10 1 Time of induction 1 6 7 14 Run Induction time EC 50 1 6.0hr 3.15x10-8 g/ml 2 5.5hr 3.83x10-8 g/ml 0-10 -9-8 -7-6 -5 Log 10 [B1 antibody], g/ml 30 20 10 E:T ratio and cell # per well 1 7 13 16 Run E:T ratio E cell # T cell # EC 50 1 7.5:1 75k 10k 3.09x10-8 g/ml 2 6:1 90k 15k 3.83x10-8 g/ml 0-10 -9-8 -7-6 -5 Log 10 [B1 antibody], g/ml Promega Corporation 28

Stability Indicating for Fc Effector Function Rituximab: EC 50 = 5.77ng/ml Activity following heat-treatment of antibody drugs Trastuzumab: Tositumomab: EC 50 = 31.0ng/ml Promega Corporation 29

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 Promega Corporation 30

Antibody Variants, Glycosylation and Benchmarking with Classic ADCC Assay Promega Corporation April 2014

RLU RLU RLU RLU RLU RLU Sensitive to Detect Differences in Glycosylation 2 10 06 Deglycosylated Herceptin unt 50%unt/ 50% degly 2 10 06 Deglycosylated Herceptin unt 40%unt/ 60% degly 2 10 06 Deglycosylated Herceptin unt 30%unt/ 70% degly 1 10 06 1 10 06 1 10 06 5 10 05 5 10 05 5 10 05 0-12 -10-8 -6-4 log [ab], (g/ml) 0-12 -10-8 -6-4 log [ab], (g/ml) 0-12 -10-8 -6-4 log [ab], (g/ml) 2 10 06 1 10 06 EC50 Deglycosylated Herceptin unt unt 2.110e-008 20%unt/80% degly 50%unt/ 50% degly 2.990e-008 2 10 06 1 10 06 EC50 Deglycosylated Herceptin unt unt 2.082e-008 10%unt/ 90% degly 40%unt/ 60% degly 3.486e-008 2 10 06 1 10 06 EC50 unt 2.002e-008 Deglycosylated Herceptin unt 100% degly 30%unt/ 70% degly 4.153e-008 5 10 05 5 10 05 5 10 05 0-12 -10-8 -6-4 log [ab], (g/ml) 0-12 -10-8 -6-4 log [ab], (g/ml) 0-12 -10-8 -6-4 log [ab], (g/ml) EC50 unt 1.720e-008 20%unt/80% degly 4.626e-008 EC50 unt 2.037e-008 10%unt/ 90% degly 7.174e-008 EC50 unt 1.988e-008 100% degly 3.202e-008 Target cells: SKBR3; Unt = 100% glycosylated Promega Corporation 32

Relative activity in reporter ADCC Relative activity in reporter ADCC Activity Correlates with Amount of Antibody N-Glycosylation Linear correlation obtained between percentage of N-glycosylated antibody in blended antibody samples and relative luciferase reporter activity in ADCC Reporter Bioassay 0.700 0.600 0.500 0.400 y = 0.0125x - 0.0095 R² = 0.9926 Rituximab 0.300 Trastuzumab 0.200 0.700 0.100 0.600 0.000 0.500 0.400 0.300 y = 0.0127x - 0.0314 R² = 0.966 0 10 20 30 40 50 60 Percent N-glycosylated sample 0.200 0.100 0.000-0.100 0 10 20 30 40 50 60 Percent N-glycosylated sample Small differences in Fc effector activity in ADCC pathway activation are easily distinguished in the ADCC Reporter Bioassay Promega Corporation 33

Benchmarking Study: Good Correlation with Lytic LDH Release Assay ADCC Reporter Bioassay, V Variant ADCC cytotoxicity bioassay (PBMCs from VV allele blood donors) A series of trastuzumab glycosylation blend mixtures were prepared by blending PNGase F treated (deglycosylated) and untreated trastuzumab (N-glycosylated). The antibody samples were tested with SK-BR-3 target cells, using untreated antibody as the 100% activity reference. Promega Corporation 34

Polymorphism in the FcgRIIIa Receptor Optimizing the F Variant of the ADCC Reporter Bioassay Promega Corporation April 2014

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 Promega Corporation 36

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 Promega Corporation 37

Bioassay Development Using DOE DOE = Design of Experiments Allow understanding of the interactions between critical assay factors Minimum amount of work needed to develop robust assays Factors: Target cell density Target cell/antibody incubation time Effector cell density Induction time Outputs: Fold of induction EC 50 EC 50 difference between F and V variant Jurkat effector cells ADCC Reporter Bioassay, F Variant Promega Corporation 38

The F Variant Bioassay is Specific No target cells FcgRIIIa blocked No Ab or wrong Ab Assay signal is specifically dependent on: Target cells expressing Ab-targeted antigen Specific antibody Effector cells expressing FcgRIIIa receptor No FcgRIIIa Promega Corporation 39

F Variant: Able to Measure Potencies of On-Market mab Biologic Drugs Three best-selling, on-market mab biologic drugs that posses ADCC as main MOA 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 Tests performed using ADCC Reporter Bioassay, Core Kit (F Variant) Promega Corporation 40

Antibody IgG-Isotype Specificity 2 1 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 Order of response: hu IgG1, IgG3 > mouse IgG2a >> hu IgG2, IgG4, mouse IgG1 Data generated using the ADCC Reporter Bioassay, F Variant Promega Corporation 41

Detects Loss of Activity Due to Heat-Stress EC 50 increases (right shift) Fold induction decreases Promega Corporation 42 Reporter bioassay exhibits stability-indicating capability Can potentially be used in stability studies for therapeutic antibody ADCC Reporter Bioassay, F Variant

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. Promega Corporation 43

Product Formats Promega Corporation April 2014

Product Configurations: ADCC Reporter Bioassay, V Variant Multiple formats flexible to your research needs: 1. Core Kits: 1X kit Cat.# G7010 5X kit Cat.# G7018 Engineered Jurkat cells Bio-Glo Reagent Core Kit 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 Promega Corporation 45 Medium Target WIL2-S or Raji Cells + Serum Control Antibody Engineered Jurkat cells, V variant Complete Kits Target Kits Propagation Model

Custom Product Configurations for ADCC Reporter Bioassay, F Variant 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 Engineered Jurkat cells, F variant Medium + Bio-Glo Reagent Serum Core Kit 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 Target WIL2-S or Raji Cells Control Antibody Jurkat effector cells, F variant Target Kits Catalog products Propagation Model Promega Corporation 46

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 Reporter Bioassays (F Variant) Pre-commercial materials are available now through our Custom Assay Services group. Simply e-mail CAS at the address below. Product ADCC Reporter Bioassay, F Variant, Core Kit ADCC Bioassay Effector Cells, F Variant, Propagation Model Contact CAS@promega.com Cat. Pls enquire Pls enquire ADCC Bioassay Effector Cells, Propagation Model 1ea Pls enquire Contact: COD@promega.com Bio-Glo Luciferase Assay System 100ml 10ml G7940 G7941 Promega Corporation 47

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 Promega Corporation 48

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 Promega Corporation 49

Simple, Convenient Luminometer Tested with ADCC Reporter Bioassay Promega Corporation April 2014

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 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 Product Size Cat. GloMax Discover Multimode Detection System 1ea GM3000 Promega Corporation 51

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. 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. 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. Quality IQ/OQ Service available. Provides required technical elements of a 21CFR 11 compliant system to be used with the appropriate laboratory workflow. Compatible for 3rd party automation control. Promega Corporation 52

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 Promega Corporation 53