Bioassays for Quality Control of Cell & Gene Therapy Products · Erik Rutjens, Cell & Gene Therapy, Novartis Pharma AG. CASSS Bioassays, Silver Spring, March2015. Bioassays for Quality

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

3

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

4

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


5

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


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

7

Functional bioassay for CTL019Requirements


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)

8

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.

9

LuminexScreening for readout

0

1000

2000

3000

4000

5000

6000

0 6 20

Cyto

kine

(pg)

Time Hrs


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


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

Final Assay LayoutCytokine release by ELISA


Validation: Intermediate Precision

12

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.



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.


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

13

50,000 CTL019 cells plated in co-culture with different numbers of target cells (12,500 – 100,000).


Conclusion

Linear correlation between effector and target cells

Linear response to stimulation by increasing numbers of CTL019 cells

14

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

Robustness, Cell Hold at 37C

15

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

Robustness, Supernatant Hold

16

Supernatant from CD19 stimulated co-cultures was harvested and held in room temperature before and after freezing.

Supernatant hold before freezing and after thaw


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

17

Characterization is keyFuture developments for Potency and Characterization


18

Thank you!

Acknowledgements:

Carlos RomeroTherese VallerskogTamika Mayse-WebbDirk HaubertMargit JeschkeStefan Wildt

Novartis Campus, Basel


Documents

Bioassays for Quality Control of Cell & Gene Therapy Products · Erik Rutjens, Cell & Gene Therapy, Novartis Pharma AG. CASSS Bioassays, Silver Spring, March2015. Bioassays for Quality