Post on 07-Jul-2018
Erik Rutjens, Cell & Gene Therapy, Novartis Pharma AGCASSS Bioassays, Silver Spring, March2015
Bioassays for Quality Control of Cell & Gene Therapy Products
CTL019
2 | CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
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
Introduction
CTL019
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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
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
Introduction
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CTL019Overview 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
CTL019 cells are re-infused into the patient where they expand and target CD19+ cells for destructionSlide adapted from K Walker
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
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CTL019 T cellsMode of Action
Cartellieri M, J Biomed Biotech 2010
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
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
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/ simultaneously6
Potency assays should be• Simple• reflecting potential Mode of Action (MoA)• accurate, precise, robust and transferable• be validated acc. to GMP
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Functional bioassay for CTL019Requirements
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
Functional potency bioassay for CART19in vitro model of mode of action
Incubate
T cell (patient or reference)
CD19+ cell
CD19
CART19cytotoxic granules
Cytokines / chemokines
Potential readouts• Cytotoxicity, apoptosis• Degranulation markers by FACS (CD107a) • Cytotoxic granule release (Perforin, Granzyme B,...)• Cytokine release• T cell activation (CD25, FasL)
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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.
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LuminexScreening for readout
0
1000
2000
3000
4000
5000
6000
0 6 20
Cyto
kine
(pg)
Time Hrs
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
Representative cytokine for T cell activation
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
Potency bioassay for CART19Cytokine release by ELISA
Cytokine readout (ELISA)Coculture with target cells
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
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
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Final Assay LayoutCytokine release by ELISA
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
Validation: Intermediate Precision
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Intermediate precision improvement
0
20000
40000
60000
CD19 stimulated
pg/m
L
Sample 1Op.1 Op.2 Op.3
CV 39.3%
Three samples, four operators. Each operator worked independentlyCell count and viability with Multisizer 4 and microscopy.
Sample 2Op.1 Op.2 Op.3
Sample 3Op.1 Op.2 Op.3
CV 34.2% CV 50.8%
0
5000
10000
15000
20000
25000
CD19 stimulated
pg/ m
LSample 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%
4 Samples, 4 operators Each operator worked independentlyNC200 used for cell counts and viability.
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
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)
Linearity: number of target cells
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50,000 CTL019 cells plated in co-culture with different numbers of target cells (12,500 – 100,000).
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
Conclusion
Linear correlation between effector and target cells
Linear response to stimulation by increasing numbers of CTL019 cells
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Transduced and non-transduced cells from the same donors were plated in different ratio with the total number of 50,000 cells per well.
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
Conclusion
Linear correlation between effector and target cells
Robustness, Cell Hold at 37C
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Pre-plating of cells
Cells were thawed and held at 37C for various times
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
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
Robustness, Supernatant Hold
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Supernatant from CD19 stimulated co-cultures was harvested and held in room temperature before and after freezing.
Supernatant hold before freezing and after thaw
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only
Conclusion
Supernant can be held for upto 3 hours before freeze
Supernant can be held for upto 24 hours after thaw
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
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Characterization is keyFuture developments for Potency and Characterization
| CASS Bioassay 2015| Erik Rutjens | 23MAR15 | CTL019 Bioassay | Business Use Only