Robust and Reproducible ADCC and T Cell Redirection with CD16-Engineered Effector Cells. Jane Lamerdin, Ph.D. Director, R&D, DiscoverX June 27, 2017

Transcription

1 Robust and Reproducible ADCC and T Cell Redirection with CD16-Engineered Effector Cells Jane Lamerdin, Ph.D. Director, R&D, DiscoverX June 27,

2 Bioassays and ADCC MOA reflective Bioassays are critical for development of biologics Lot release and stability testing Product characterization Must serve as a surrogate measure of the biological activity in vivo For antibodies with Fc-mediated effector functions, ADCC assays are required during development to quantify effector function Ideal ADCC assay for lot release: Reflective of drug MOA (e.g. measures immune cell mediated killing) Provide consistent performance from effector cells that eliminate donor variability and minimize lot-to-lot variability 2

3 Limitations of Use of Cytotoxicity Assays for Lot Release Need for consistent performance from human effector cells Primary cell-driven ADCC High donor-to-donor variability Logistical challenges with getting enough PBMCs or NKs ADCC assays often have low signal to background ratios Effector Cells Target Cells Engineered NK92 CD16 lines Challenging to grow Not compatible with many target cell types Require expensive license fees % ADCC ADCC with KILR ARH-77 donor a donor b donor c donor d donor e R ituxim ab [g/m l] 3 A reliable source of human effector cells could address these challenges

4 Single Donor-Derived Engineered Effector Cells KILR CD16 (V158) Effector Cells CD16 (V158) Primary human cytotoxic T lymphocytes (CTLs) Human CTL Engineered to stably express CD16 (V158) Uniformly manufactured to ensure lot-to-lot consistency CD8 CD8 CD8 CD4 CD3 CD16 4 Polyclonal population, 98% CD8 + 98% CD3+ / CD8 + Stable CD16 expression

5 KILR CD16 Effectors Mediate ADCC Via CD16 MHC I-independent killing of target cells ADCC KILR CD16 Effector Cell CD16 Antigen-specific Ab Cytokines 5 Target Cell

6 Why Use T Cells Rather Than NK Cells? Lower background lysis relative to NK92 CD16 or primary cells NK92 CD16 lines often used as effectors for ADCC assays in QC labs Disadvantages of NK92 lines: o Difficult to grow o High non-specific lysis with many cell types KILR CD16 Effector Cells mediate much lower background lysis SKOV3 PBMCs KILR CD16 NK92 CD16 ARH % Lysis

7 KILR CD16 Effector Cells for ADCC Assay Example: KILR ADCC assay Thaw and Rest KILR CD16 Effectors (24hr) - Plate Target Cells with Antibody - Add KILR CD16 Effector Cells Add KILR Detection Reagents Read Luminescence Target Cells: Assays: - KILR target cell lines/pools - KILR ADCC - Target cells generated using KILR retroparticles - In-house target cells / cell lines - Chromium 51 release - Calcein release 7

8 KILR CD16 Effector Cells Highlights Compatible with Different Cytotoxicity Assays (KILR, Chromium Release, Calcein Release, etc.) Eliminate Donor Variability - Primary effector cells from a single donor Easily Implement in Any Lab - Frozen ready-to-use cell Fit for Long-Term QC Testing - High lot-to-lot reproducibility Mediate Target Cell Death - Relevant measure of ADCC and TCR 8

9 Larger Assay Window Improves Analysis of Antibody Activity Consistent pharmacology with lower effector: target ratios K ILR A R H -77 C ell Line KILR SKOV-3 Cell Pool 60 KILR CD16 Effector Cells PBMCs 40 KILR CD16 Effector Cells PBMCs % C ytotoxicity % C ytotoxicity R ituxim ab [g/m l] Trastuzumab [g/ml] Sample EC 50, ng/ml S/B Max Cytotoxicity, % Sample EC 50, ng/ml S/B Max Cytotoxicity, % KILR CD16 cells (10:1) % KILR CD16 cells (12.5:1) % PBMCs (25:1) % PBMCs (25:1) % 9

10 Consistent Performance of KILR CD16 Cells Post-Thaw No reduction of killing capacity over 10 days in culture KILR Raji Cell Pool % C ytotoxicity Day 1 Day 3 Day 7 Day anti-cd20 (µ g/m L) Days Post- Thaw EC 50, ng/ml Max Cytotoxicity (%) Day Day Day Day % RSD= 4.5% We recommend thaw and rest of KILR CD16 effectors for at least 24hr prior to use (Day 1). 10

11 Reproducibility in KILR SKOV-3 ADCC Assay Lower inter-assay variability than for PBMCs (V/V donor) or NK92 CD % RSD Trastuzumab EC 50 (ng/ml) 9.6% RSD 42% RSD 11

12 Excellent Inter-Lot Reproducibility Lower lot-to-lot variability than typical inter-donor variability (PBMCs) % Cytotoxicity % Cytotoxicity (Mean +/- SD) Lot 1 Lot 2 Lot 3 Lot 4 KILR CD16 Effector Cells EC 50 (ng/ml) Lot Lot Lot Lot % RSD PBMCs EC 50 (ng/ml) Donor Donor Donor Donor % RSD Log [Anti-CD20 Ab] (ng/ml) Mean % Mean % Note: Data represent lots of KILR CD16 Effector Cells manufactured over the course of two years 12

13 T Cell Redirection with BiTEs Concept for KILR TCR assay with Blinatumomab T Cell BiTE-mediated T cell recruitment, activation and target cell killing Engineered Target Cell No antibody Very low signal 13 BiTE: Bi-Specific T cell Engager

14 Potent and Rapid Killing Kinetics for TCR with KILR CD16 Effectors Faster results relative to pan T Cells Effector Cells: Pan T Cells Target Cells: KILR Raji Cell Pool (E:T= 10:1); 24 h incubation Effector Cells: KILR CD16 cells Target Cells: KILR Raji Cell Pool (E:T= 5:1); Incubation times: Varies % C ytotoxicity E:T=0:1 E:T=10:1 24 h Blinatumomab [ng/ml] % C ytotoxicity no effector cells 4 h 5 h 6 h 6 h 7 h Blinatumomab (ng/ml) Sample Max Cytotoxicity, % EC 50, pg/ml Time Point Max Cytotoxicity, % EC 50, pg/ml 14 No effectors (E:T= 0:1) E:T= 10: Reported EC 50 of Blinatumomab= pg/ml 4 h 20% h 33% h 58% h 31% 13.6

15 KILR CD16 Effector Cells Driving robust and reproducible effector cells performance for cytotoxicity assays Feature Single-donor derived primary human effector cells Effector cells engineered to stably express CD16 Benefit Eliminate donor variability from your ADCC assays and get results with high reproducibility Biologically-relevant true measure of ADCC High lot-to-lot reproducibility Drive and measure cell death of target cells Cryopreserved ready-to-use (24 hours post-thaw) primary cells Higher signal to background ratios Enable long-term support of QC lot release bioassays Reflective of true mechanism of action with target cell death driven by human effectors QC-friendly protocol for rapid adoption in any lab Improved performance compared to PBMCs enabling use in lot release assays 15

16 KILR CD16 Effector Cells Effector cells and supporting products Product Name Product configuration Catalog Number KILR CD16 Effector Cells 1 Vial KILR CD16 Effector Cells 5 Vials AssayComplete Cell Culture Kit ml Bottle G Recombinant Human IL-2, Cell Culture Grade 1 Vial (10 µg) Product Configuration 1 vial or 5 vials 20 million cells/vial (± 15%) Certificate of analysis User manual Shipping and Storage Shipped on dry ice Transfer to vapor phase of liquid nitrogen immediately upon receipt 16

17 Want More Information? Product Manager: Tech Support Team 17