Bioassays for Quality Control of Cell & Gene Therapy Products
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1 Bioassays for Quality Control of Cell & Gene Therapy Products Erik Rutjens, Cell & Gene Therapy, Novartis Pharma AG CASSS Bioassays, Silver Spring, March2015
2 CTL019 Introduction CARTs = Chimeric Antigen Receptor transduced T cells CART19 = T cells engineered to express CD19 specific CAR Chimeric protein consists of recognition domain of anti- CD19 antibody (scfv) and intracellular CD3ζ and 4-1BB signaling domains MHC-independent, every surface molecule is a target Autologous product 2 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
3 CTL019 Introduction The chimeric antigen receptor consists of T- cell activation domains coupled to anti-cd19 single-chain variable fragments The intracellular T-cell receptor CD3-zeta chain signaling domain induces T-cell activation The CD137 (4-1BB) co-stimulatory domain enhances the cytolytic function of T cells and T-cell mediated responses The CD137 (4-1BB) domain impacts in vivo persistence by preferentially expanding memory cytotoxic lymphocytes and facilitating survival of memory cells Anti-CD19 antibody fragments bind to CD19- expressing cancer cells. The activated T cells are thus able to kill tumor cells in an antigen-dependent manner 3 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
4 CTL019 Overview of CTL019 Manufacturing Leukapheresis: The patient s own T cells are harvested T cells are activated and genetically transduced ex vivo with a lentiviral vector encoding the anti-cd19 chimeric antigen receptor (CAR) Chemotherapy: patient may receive a preparative lymphodepleting regimen before T cell infusion Slide adapted from K Walker CTL019 cells are re-infused into the patient where they expand and target CD19+ cells for destruction 4 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
5 CTL019 T cells Mode of Action Recognition of a common protein (CD19) by chimeric antigen receptor (CAR) Signaling through CD3 intracellular pathway Activation of Th and CTL responses - Expansion of the cells - High Cytotoxic Granule content - Strong expression of cytotoxic agents (FasL, IFN-g) - High expression potential of necessary cytokines / chemokines Cartellieri M, J Biomed Biotech CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
6 T cell activation a multifaceted process Cytokine production initiated, e.g. IFNγ, TNFα, IL-2, IL-6 Upregulation of surface marker expression, e.g. CD25, Fas, CD137 Shed surface receptors (e.g. CD25, Fas, CD137) and ligands (e.g. Fas ligand) T cells may Proliferate Kill targets Some/all of the above in sequence/ simultaneously 6
7 Functional bioassay for CTL019 Requirements Potency assays should be Simple reflecting potential Mode of Action (MoA) accurate, precise, robust and transferable be validated acc. to GMP 7 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
8 Functional potency bioassay for CART19 in vitro model of mode of action T cell (patient or reference) Cytokines / chemokines CART19 CD19 CD19+ cell cytotoxic granules Incubate Potential readouts 8 Cytotoxicity, apoptosis Degranulation markers by FACS (CD107a) Cytotoxic granule release (Perforin, Granzyme B,...) Cytokine release T cell activation (CD25, FasL)
9 Screening for readout Luminex CTL019 product was incubated with CD19 expressing cells (target) or control cells A 1:1 ratio of effector cells to target cells were incubated for 0, 6, and 20 hours. Culture supernatants were analyzed using a 17-plex Luminex kit. Conclusion Cytokine expression was specific to the co-incubation of CTL019 product and target cells. Cytokine (pg) Time Hrs 9 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
10 Potency bioassay for CART19 Cytokine release by ELISA Coculture with target cells Representative cytokine for T cell activation Cytokine readout (ELISA) Readout by commercial kit, relatively easy to use Reproducible, robust, and antigen-specific production linked to mechanism of action Shows early potential to distinguish responding from non-responding products 10 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
11 Final Assay Layout Cytokine release by ELISA Potency test: Coculture Product cells (50.000) CD19 expressing cells (stabily transfected) Controls (system suitability) Pos control: - Product cells (50.000) - PMA/Ionomycin Negative control: - Product cells (50.000) - Target cells expressing irrelevant protein Readout: Elisa for cytokine Acceptance criteria: Product exposed to CD19+ cells must show >5* negative control read 11 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
12 Validation: Intermediate Precision Intermediate precision improvement Three samples, four operators. Each operator worked independently Cell count and viability with Multisizer 4 and microscopy CD19 stimulated 4 Samples, 4 operators Each operator worked independently NC200 used for cell counts and viability CD19 stimulated pg/ml Sample 1 Op.1 Op.2 Op.3 CV 39.3% Sample 2 Sample 3 Op.1 Op.2 Op.3 Op.1 Op.2 Op.3 CV 34.2% CV 50.8% pg/ml Sample 1 Sample 2 Op.1 Op.2 Op.3 Op.4 Op.1 Op.2 Op.3 Op.4 CV 10.4% CV 6.9% Conclusion Changing from conventional viability and cell count (Trypan Blue) to automated fluorescent cell count improved int. precision from 50.8% (inacceptable) to 10.4% (Acceptable) 12 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
13 Linearity: number of target cells 50,000 CTL019 cells plated in co-culture with different numbers of target cells (12, ,000). Conclusion Linear correlation between effector and target cells 13 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
14 Linear response to stimulation by increasing numbers of CTL019 cells Transduced and non-transduced cells from the same donors were plated in different ratio with the total number of 50,000 cells per well. Conclusion Linear correlation between effector and target cells 14 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
15 Robustness, Cell Hold at 37C Pre-plating of cells Cells were thawed and held at 37C for various times Conclusion CTL019 cells can be held for upto 6 hours before co-culture Target cells can be held for upto 24 hours before co-culture 15 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
16 Robustness, Supernatant Hold Supernatant hold before freezing and after thaw Supernatant from CD19 stimulated co-cultures was harvested and held in room temperature before and after freezing. Conclusion Supernant can be held for upto 3 hours before freeze Supernant can be held for upto 24 hours after thaw 16 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
17 Characterization is key Future developments for Potency and Characterization Optimization of functional assay: Replace target cells with standardized CD19 source to reduce biological variation - (immobilized Rec. CD19 / activating antibody) Additional assay: Killing of CD19 expressing target cells Proliferation of CTL019 cells after stimulation with CD19 Extensive Phenotypic characterization of the T-cell populations 17 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
18 Thank you! Acknowledgements: Carlos Romero Therese Vallerskog Tamika Mayse-Webb Dirk Haubert Margit Jeschke Stefan Wildt Novartis Campus, Basel 18 CASS Bioassay 2015 Erik Rutjens 23MAR15 CTL019 Bioassay Business Use Only
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