Novel Reporter Gene Bioassays for Immunotherapy Drug Research and Development Jey Cheng, PhD Sr Research Scientist Promega Corporation

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1 Novel Reporter Gene Bioassays for Immunotherapy Drug Research and Development Jey Cheng, PhD Sr Research Scientist 215

2 Outline Immune Checkpoint and Combination Therapies Reporter Gene Bioassays: Design, Optimization and Qualification Co-inhibitory Bioassays Co-stimulatory Bioassays Combination Bioassays Custom Assay Services Summary

3 Immune Checkpoint Modulation antigen presenting cell T cell Co-stimulatory Receptors GITR 4-1BB (CD137) OX4 CD4 HVEM CD27 ICOS Co-inhibitory Receptors PD-1 CTLA-4 TIGIT LAG-3 BTLA Combination Targets PD-1 + TIGIT PD-1 + CTLA-4 PD-1 + LAG Mahoney, Rennert, Freeman et al. Nature Rev Drug Discovery (215) 3

4 Immune Checkpoint Therapy Releasing the Brakes, Hitting the Gas Strategy Blocking Ab for immune coinhibitory receptors (PD-1, CTLA-4, TIGIT, LAG3), release the brakes Agonist Ab for immune costimulatory receptors (GITR, OX4, CD27, CD137), hit the gas T effector cells are activated

5 Reporter Gene Bioassay: Design, Optimization, and Qualification 215.

6 Building Bioluminescent Reporter-Gene Bioassay Platform Performance capability: Reflect MOA; fast, highly sensitive, quantitative and reproducible; more stability-indicating than cell-free system Trainability/Transferability: Simple protocol, minimal effort needed for assay validation, analyst training and global transfer Standard equipment and reagents: Luminometer and luciferase reagents are available and commonly used in most cell biology labs

7 Reporter Gene Bioassay Development Workflow Phase 1: Assay design and POC study Phase 2: Stable cell line generation and characterization Phase 3: Make thaw-anduse cells Phase 4: Assay optimization, qualification, application MOA reflecting Choose relevant cell background Select appropriate reporter gene vector Transient transfection/proof of concept Assay specificity Endogenous or exogenous proteins Transfection FACS sorting Single cell cloning Clone morphology, growth rate & stability Functional performance Cell growth Cell freezing Cell storage Equivalent assay performance Assay optimization: single factor, DOE Assay qualification Assay application: 384-format, stability study, human serum tolerance

8 Immune Co-inhibitory Bioassays Case study: CTLA-4 Blockade Bioassay Other assays: PD-1, LAG3, TIGIT, BTLA 215.

9 General Assay Design for Co-Inhibitory Bioassay Co-inhibitory receptor ligand PD-L1/L2 CD8/CD86 CD155 MHC-II HVEM antigen presenting cell Ligand receptors TCR activator TCR T cell Inhibitory receptors RE Immune Co-inhibitory receptor Luciferase PD-1 CTLA-4 TIGIT LAG-3 BTLA 215. Ligand receptors aapc cells Co-stimulatory receptors (CD28, CD226) T Effector cells 9

10 Case Study: CTLA-4 Bioassay Development Stable Cell Generation and Characterization Transfection FACS Sorting Single Cell Cloning Cell line 1: CTLA-4 Effector cells CTLA-4 Luciferase reporter Receptor Expression Functional Tests Clone Stability Cell line 2: aapc/raji cells TCR activator protein

11 Case Study: CTLA-4 Bioassay Development Stable Cell Generation and Characterization Transfection FACS Sorting Single Cell Cloning Cell line 1: CTLA-4 Effector cells CTLA-4 Luciferase reporter Count Plot P5, ungated control anti-ctla Red Fluorescence (RED-HLog) Receptor Expression Functional Tests Clone Stability Cell line 2: aapc/raji cells TCR activator protein anti-tcr activator control

12 Case Study: CTLA-4 Bioassay Development Stable Cell Generation and Characterization Transfection FACS Sorting Single Cell Cloning Cell line 1: CTLA-4 Effector cells CTLA-4 Luciferase reporter Biolum inescence (RLU) passage# for CTLA-4 E ffe c to r c e lls P14 P26 P39 P53 P Log[ipilim um ab], ug/m l EC5 P P P P P Receptor Expression Functional Tests Clone Stability Cell line 2: aapc/raji cells TCR activator protein Biolum inescence (RLU) passage# for aapc/raji cells P13 P26 P44 P59 EC5 P P P P Log[ipilim um ab], ug/m l

13 Proposed Assay Protocol Prototype Assay Procedure: 1. Make assay buffer Buffer components 2. Plate CTLA-4 Effector cells Cell number/well 3. Add anti-ctla-4 Ab 4. Plate aapc/raji cells Cell number/well 5. Induction Induction time 6. Add Bio-Glo Reagent, read plates Add anti- CTLA-4 Ab plate CTLA-4 Effector Cells Effector Cell number Assay buffer components aapc Cell number Plate aapc/raji Cells Add/Read Induction Induction time Add Bio-Glo Reagent Measure Luminescence

14 Assay Optimization Cell Plating Density & E:T Ratio Two Factors 1. # Effector Cells 1K 15K 2. # aapc Cells 5K 1K 15K 2K Output 1. Uninduced RLU 2. EC 5 3. Fold induction Bioluminescence (RLU) Log[ipilimumab], ug/ml E : T ratio 1K : 5K 1K : 1K 1K : 15K 1K : 2K 15K : 5K 15K : 1K 15K : 15K 15K : 2K 17 hour induction 1% FBS in assay buffer Test factors/parameters Outputs run Effector Cell# aapc Cell# uninduced RLU EC 5, ug/ml Fold induction 1 1K 5K K 1K K 15K K 2K K 5K K 1K K 15K K 2K

15 DOE for Assay Optimization Two factors: Induction Time & Assay Buffer Two Factors 1. Induction time: 6 hours 16 hours 24 hours 2. % FBS in assay buffer: 1%- 1% FBS Output 1. Uninduced RLU 2. EC 5 3. Fold induction E: T= 1K : 2K Bioluminescence (RLU) induction time/assay buffer 6 hour/1% FBS 6 hour/5%fbs 6hour/2% FBS 6hour/1% FBS 6 hours 1 16 hours 23 hours Bioluminescence (RLU) 3 induction time/assay buffer 16 hour/1% FBS 16 hour/1% FBS 2 23 hour/1% FBS 23 hour/1% FBS Log[ipilimumab], ug/ml Log[ipilimuab], ug/ml Test factors/parameters Output run Induction time Assay Buffer uninduced RLU EC 5, ug/ml Fold induction 1 6 hour 1% FBS hour 2% FBS hour 5% FBS hour 1% FBS hour 1% FBS hour 1% FBS hour 1% FBS hour 1% FBS

16 DOE for Assay Optimization Two Factors: Induction Time & # aapc/raji Cells Two factors 1. Induction time: 6 hours 16 hours 2. # aapc/raji cells/well: 1K 5K 2K Output 1. Uninduced RLU 2. EC 5 3. Fold induction Bioluminescence (RLU) aapc/raji cells/well 1K 5K 2K 6 hours Log[ipilimumab], ug/ml Bioluminescence (RLU) k effector cells, 1% FBS in assay buffer aa P C /R aji cells/w ell 1K 5K 2K 16 hours Log [ipilimumab], (ug/ml) Test factors/parameters Outputs run Induction time aapc/raji cells/well uninduced RLU EC 5, ug/ml Fold induction 1 6 hour 1K hour 5K hour 2K hour 1K hour 5K hour 2K

17 Final Assay Protocol Assay Procedure: 1. Make assay buffer containing 1% FBS 2. Plate CTLA-4 Effector cells at 1K/well 3. Add anti-ctla-4 Ab 4. Plate aapc/raji cells at 5K/well 5. Induction for 6 hours or 16 hours depending on plate reader and Ab binding kinetics 6. Add Bio-Glo Reagent, read plates Add anti- CTLA-4 Ab Plate CTLA-4 Effector Cells Plate aapc/raji Cells Add/Read Induction Add Bio-Glo Reagent Features: No need for primary cells Simple, homogenous Sensitive, highly reproducible Short assay time Measure Luminescence

18 CTLA-4 Bioassay Qualification Qualification Parameters: Specificity Accuracy Precision: Repeatability (intra-assay precision) Intermediate precision (day to day, analyst to analyst) Reproducibility (lab to lab) Linearity Range Robustness - ICH Guideline Q2 [R1] - USP <133> Biological Assay Validation Bioluminescence (RLU) Assay Qualification Design: Two analysts Three days Four plates per day 1. 1% vs 5% 2. 1% vs 7% 3. 1% vs 13% 4. 1% vs 15% 5% 7% 1% 13% 15% Relative Potency Log 1 [ipilim um ab], ug/m l

19 Precision, Accuracy and Linearity Parameter Results % Expected Relative Potency % Recovery Accuracy Repeatability (%CV) 1% (Reference) 6.66 Intermediate Precision (% CV) 1.22 Linearity (r 2 ).991 Linearity (y = mx + b) y = x Measured Relative Potency (%) Linearity Y=1.175X R2= Expected Relative Potency (%) A 5 15% theoretical potency series of ipilimumab was analyzed in triplicate in three independent experiments performed on three days by two analysts Data were analyzed and relative potencies calculated after parallelism determination using JMP software Data were generated using thaw-and-use cells

20 Assay Specificity anti-ctla-4, ipilimumab, human IgG1 Bioluminescence (RLU) anti-pd-1, nivolumab, human IgG4 anti-tigit, mouse IgG1 anti-her2, trastuzumab, human IgG Log 1 [test antibody], ug/ml

21 Assay is Stability-Indicating Heat treatment Bioluminescence (RLU) o C Control 65 o C 8hr 65 o C 16hr 65 o C 24hr EC 5, µg/ml 4 o C Control o C 8h o C 16h NA 65 o C 24h NA Log 1 [ipilim um ab], ug/m l

22 Combine with ADCC Reporter Bioassay for Fc Effector Function of anti-ctla-4 Antibody CTLA-4 CTLA-4 Effector FcγRIIIa Cells serve as Target anti-ctla4 cells Y anti-ctla-4 Ab ipilimumab, human IgG1 ADCC Effector Cells = NFAT-RE-luc Glo Assay specifics: Effector cells: ADCC Bioassay Effector Cells, V Variant Target cells: CTLA-4 Effector Cells Bioluminescence (RLU) research grade anti-ctla-4 Ab: Control Ab, human IgG1 Ab 2, human IgG4 Ab 3, mouse IgG1 E:T ratio: 6:1 6 hour induction Log 1 [anti-ctla -4 Ab], ug/m l

23 Immune Co-inhibitory Bioassay Portfolio PD-1/PD-L1/L2 Blockade Assay A Pair of PD-1 Blockade Bioassays Specific for Individual PD-1 Ligand Interaction Brief protocol: 1. Plate aapc cells 2. Add blocking Ab 3. Plate Effector cells 4. Induction; add Bio-Glo Reagent and read plates Bioluminescence (RLU) A. PD-1/PD-L1 Bioassay B. PD-1/PD-L2 Bioassay PD-1 Ab PD-L1 Ab PD-L2 Ab Log [research grade antibody], g/ml Bioluminescence (RLU) PD-1 Ab PD-L1 Ab PD-L2 Ab Log [research grade antibody], g/ml

24 Immune Co-inhibitory Bioassay Portfolio (cont.) Brief protocol: 1. Plate aapc cells 2. Add blocking Ab 3. Plate Effector cells 4. Induction; add Bio-Glo Reagent and read plates TIGIT A. TIGIT/CD155 Bioassay B. LAG-3/MHC-II Bioassay C.BTLA/HVEM Bioassay Bioluminescence (RLU) A nti- TIG IT A nti- PD -1 Biolum inescence (RLU) Biolum inescence (RLU) Log[test antibody], µ g/ml Log[anti-LAG-3] µ g /m l L o g [a n ti-h V E M ], g /m l

25 Immune Co-Stimulatory Bioassays Case study: OX4 Bioassays Other bioassays: GITR, 4-1BB, CD4, HVEM, CD

26 Immune Co-stimulatory Receptors Expressed on T cells, B cells, NK cells and antigen presenting cells I. List of immune co-stimulatory receptors Belong to the TNFR superfamily; activate NF-κB pathway II. Immune co-stimulatory receptor signaling Play important roles in the amplification of T cell-mediated immune responses 215. Chen and Flies, Nature Reviews Immunology 13 (213) 26

27 General Assay Design for Co-stimulatory Bioassays Ligand (Soluble or cell bound) Agonist Ab Co-stimulatory Receptors: OX4 GITR 4-1BB CD4 HVEM CD27 RE Co-stimulatory Receptor Luciferase Co-stimulatory Receptor-expressing Effector Cell

28 Case study: OX4 Bioassay Development Cell Line Stability and Characterization Transfection OX4 Effector cells 1. OX4 2. Luciferase reporter FACS Sorting Single Clone Selection Receptor Expression & Functional Tests Clone Stability

29 Case study: OX4 Bioassay Development Cell Line Stability and Characterization Transfection FACS Sorting Single Clone Selection Receptor Expression & Functional Tests Clone Stability Count OX4 Effector cells 1. OX4 2. Luciferase reporter Plot P3, gated on P1.R1 control anti-ox4 R3 R Yellow Fluorescence (YEL-HLog)

30 Case study: OX4 Bioassay Development Cell Line Stability and Characterization Transfection OX4 Effector cells 1. OX4 2. Luciferase reporter FACS Sorting Single Clone Selection 12 p22 Receptor Expression & Functional Tests Fold of Induction 8 4 p4 p53 Clone Stability Log[OX4L], ng/m l

31 Proposed Assay Protocol Prototype Assay Procedure: 1. Make assay buffer Buffer components 2. Plate OX4 Effector cells Cell number/well 3. Add OX4L or anti-ox4 Ab 4. Induction Induction time 5. Add Bio-Glo Reagent, read plates Plate OX4 Effector Cells Add OX4L or anti-ox4 Ab or Add/Read Induction Induction time Add Bio-Glo Reagent Cell number Assay buffer components Measure Luminescence

32 Assay Optimization FBS in Assay Buffer Thaw-and-Use Cells Fresh-from-Culture Cells Biolum inescence (RLU) 3 1% FBS 5% FBS 2 1% FBS Log[OX4L], g/m l Biolum inescence (RLU) 4 1% FBS 5% FBS 3 1% FBS Log[OX4L], g/m l EC5 1% FBS 2.555e-8 5% FBS 2.89e-8 1%FBS 2.86e-8 EC5 1% FBS ~ 2.437e-24 5%FBS 1.981e-8 1% FBS 1.49e-8 Summary: 5% FBS in assay buffer is optimal for assays with thaw-and-use cells and fresh cells from culture

33 Assay Optimization Induction Time & Cell Number Induction Time Cell Number Biolum inescence (RLU) 25 3hr 4hr 2 5hr 6hr 15 7hr 24hr Log[O X4L], ng/m L Biolum inescence (RLU) 3 5K 1k 2 15k Log[O X4L], ng/m L Induction time EC 5, ng/ml Fold Cell#/well EC 5, ng/ml Fold 3 hour K hour hour hour K K hour hour NA ~

34 Final Assay Protocol Assay Procedure: 1. Make assay buffer containing 5% FBS 2. Plate OX4 Effector cells at 5K/well 3. Add OX4L or anti-ox4 Ab 4. Induction for 5 hours 5. Add Bio-Glo Reagent, read plates Plate OX4 Effector Cells Add OX4L or anti-ox4 Ab or Add/Read Induction Add Bio-Glo Reagent Measure Luminescence

35 OX4 Bioassay Response to soluble ligand and agonist antibody OX4 Ligand OX4 Agonist Antibody Biolum inescence (RLU) EC5=8.1 ng/ml Fold Induction= Biolum inescence (RLU) EC5 = 14.7 ng/ml Fold Induction= Log[OX4L], ng/m l Log[anti-OX4 Ab], ng/m l

36 GITR Bioassay and 4-1BB (CD137) Bioassay Brief protocol: 1. Plate Effector cells 2. Add ligand or agonist Ab 3. Induction 4. Add Bio-Glo Reagent and read plates Biolum inescence (RLU) Biolum inescence (RLU) GITR Ligand Crosslinked G ITRL EC5=29.1 ng/ml Fold Induction=33 G IT R L EC5=9.1 ng/ml Fold Induction= GITR Agonist Antibody EC5=185 ng/ml Fold Induction=3 Log[GITRL], ng/m l Log[anti-GITR Ab], ng/m l Biolum inescence (RLU) Biolum inescence (RLU) BB Ligand crosslinked 4-1BBL EC5=12.5 ng/ml Fold Induction= B B L EC5=17.9 ng/ml Fold Induction= Log[4-1BBL], ng/m l 4-1BB Agonist Antibody EC5=32.9 ng/ml Fold Induction= Log[anti-4-1BB Ab], ng/m l

37 CD27 Bioassay CD27 activation by CD7 expressing cells Block CD27/CD7 interaction by anti-cd27 antibody Bioluminescence (RLU) Biolum inescence (RLU) log cells/well (CHO-CD7) lo g [a n ti-c D 2 7 ], g /m l Brief protocol: 1. Plate CD7 expressing cells 2. Add CD27 Effector cells 3. Induction; add Bio-Glo Reagent and read plates Brief protocol: 1. Plate CD7 expressing cells 2. Add CD27 Effector cells 3. Add anti-cd27 blocking Ab 4. Induction; add Bio-Glo Reagent and read plates

38 HVEM/LIGHT Bioassay HVEM activation by soluble LIGHT Block HVEM/LIGHT interaction by anti-hvem antibody Biolum inescence (RLU) L IG H T lig a n d a n ti-h V E M b lo c k in g A b Log [test agents], g/m L Bioluminescence (RLU) HVEM activation by LIGHT-expressing cells log [LIGHT Target Cells/well] Brief protocol: 1. Plate HVEM Effector cells 2. Add soluble LIGHT (left, blue line), or LIGHT expressing cells (right) 3. Add anti-hvem antibody (left, red line) 4. Induction; add Bio-Glo Reagent and read plates

39 Determining Antibody Specificity HVEM/LIGHT vs BTLA/HVEM Bioassays HVEM/LIGHT Bioassay BTLA/HVEM Blockade Bioassay Biolum inescence (RLU) Ab 1, m onoclonal Ab Ab 2, polyclonal Ab Biolum inescence (RLU) Ab 1, m onoclonal Ab Ab 2, polyclonal Ab L o g [a n ti-h V E M ], g /m l L o g [a n ti-h V E M ], g /m l EC5 Ab 1, monoclonal Ab 2.934e-8 Ab 2, polyclonal Ab 2.64e-8 EC5 Ab 2, polyclonal Ab 3.967e

40 CD4 Bioassay CD4 activation by CD4L Blocking CD4/CD4L interaction with anti-cd4l antibody CD4 activation by CD4 Agonist Ab Biolum inescence (RLU) CD154 (CD4L) anti-cd4l blocking Ab Biolum inescence (RLU) L o g [te s t a g e n ts ], g /m l L o g [a n ti-c D 4 ], g /m l Brief protocol: 1. Plate CD4 Effector cells 2. Add soluble CD4 ligand (left, blue line), or CD4 agonist Ab (right) 3. Add anti-cd4l antibody (left, red line) 4. Induction; add Bio-Glo Reagent and read plates

41 Crosslinking of Agonist Antibody by FcγRIIb Promotes Downstream Signaling Assay Design FcγRIIb expressing cells Wilson et al. Cancer Cell (211) Crosslinking of antibodies bound to target by FcγR-expressing cells can promote receptor clustering and increase downstream signaling FcγRIIb crosslinking is important for anti-tnfr receptor antibodies (anti-cd4, anti-trail) Antibodies can be engineered for higher or lower FcgRIIb binding anti-cd4 Agonist Ab Luc CD4 Effector Cells

42 CD4 Bioassay can be used to Detect FcγRIIb-dependent Ab Bioactivities Ab characterization in primary cells: same Fab, different Fc CD23 B Cell FcγR Isotype upregulation Proliferation Dependency No1 + + No higg2 No2 + + Yes migg1 FcγRIIb-independent FcγRIIb-dependent 6 T e s t A b 1 (+ C H O w.t.) 6 T e s t A b 2 (+ C H O w.t.) Fold Induction 4 2 Test Ab 1 (+ CHO Fcγ R IIb ) Fold Induction 4 2 Test Ab 2 (+ CHO Fcγ R IIb ) lo g [te s t A b ], g /m l lo g [te s t A b ], g /m l

43 Immune Combination Bioassays PD-1+TIGIT PD-1+LAG3 215.

44 General Assay Design for Dual-inhibitory Combination Bioassays Two co-inhibitory receptor ligands PD-L1+CD155 PD-L1+MHC-II Two Ligand receptors Two immune co-inhibitory receptors Two Inhibitory receptors PD-1+TIGIT PD-1+LAG3 TCR activator TCR RE Luciferase Ligand receptors aapc cells Co-stimulatory receptors (CD28, CD226) T Effector cells

45 Combination Bioassays PD-1+TIGIT, PD-1+LAG3 A. PD-1+TIGIT Combo Bioassay B. PD-1+LAG-3 Combo Bioassay Bioluminescence (RLU) anti-pd -1 anti-tig IT anti-pd-1 + anti-tig IT Log[test antibody], µ g/m l Biolum inescence (RLU) a n ti-p D an ti-l A G -3 a n ti-p D -1 + an ti-l A G Log[test antibody], µ g /m l

46 Promega Bioassay Portfolio Fc Functions Fc Effector Bioassays FcγRIIIa (V/F) FcγRIIa (H/R) FcγRI madcc Immune Target Bioassays Immune Checkpoint Bioassays Co-inhibitory: PD-1, CTLA-4, TIGIT, LAG-3, BTLA Co-stimulatory: OX4, GITR, 4-1BB, CD4, HVEM, CD27 Combination Bioassays Target Based Immunotherapy T Cell Activation Bioassays (BiTE, CAR) Other Biologics Cytokine Bioassays IL-2, IL-6, IL-12/23 EGF, VEGF IFNα, IFNβ, IFNγ

47 Promega Custom Bioassays Promega Custom Assay Services (CAS): Builds custom bioassays to meet your specific needs Projects are milestone based and payments are made upon completion of each milestone Example Project Outline: Feasibility study Stable cell line generation Assay optimization Assay qualification Transient transfection Proof of concept Assay reproducibility Stable pool generation Single clone selection Cell number/well Agonist concentration Luciferase induction time Antibody concentration Accuracy Precision Specificity Linearity

48 Reporter Gene Bioassays Offer A MOA-based functional bioassay for biologics drug development Consistent and reliable measure of biologics potency and stability o Demonstrated precision, accuracy, reproducibility and robustness o Simple thaw-and-use format o Kits include all required reagents in a standardized format Easy-to-implement o Rapid and convenient workflow o Compatible with 96- and 384-well assay formats Building blocks for combination immunotherapies

49 Thank You! Jey Cheng, PhD R&D Group Leader, Bioassay Development Gopal Krishnan PhD Global Product Manager Vanessa Ott, PhD Global Strategic Marketing Manager Custom Assay Services

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