SINGLE CELL ANALYSIS OF VIRAL TRANSDUCTION AS A NOVEL TOOLBOX FOR AN IMPROVED CHARACTERISATION OF CELL THERAPY PRODUCTS

Dr. Nicole NicholasSenior analytical development scientist Cell and Gene Therapy Catapult

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About us

Bridge the gap between businesses and academic research

Provide access to unique technical facilitiesand expertise to help adopt, develop and exploit innovations

Established by Innovate UK as a not-for profit, independent centreto transform UK’s capability for innovation in CGT

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Accelerate the commercialisation of innovations from research

Collaboration with academia and industry to create world leading solutions to meet the needs of the industry.

Cell and Gene Therapy Catapult

Large scale manufacturing

development centre

• 7,200m2 manufacturing centre designed specifically for cell and gene therapies

• Located at Stevenage Bioscience Catalyst• 12 unique supported modules

Development facility

• 1200m2 custom designed cell and gene therapy development facility

• Heart of the London clinical research cluster

• 170 permanent staff

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Cell and Gene Therapy Catapult – Manufacturing Centre

Modules are architecturally segregated (1000 sq ft)

Independent modules can be operated at your choice of grade B or grade C

The centre’s architecturally segregated modules allowing companies to develop their process while retaining intellectual property (IP) and know how.

Walk in infrastructure for large scale GMP manufacture

Access established ATMP supply chain

Operate under our MHRA licences for clinical and commercial supply

Collaborate with industry specialists

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SINGLE CELL ANALYSIS OF VIRAL TRANSDUCTION

Generation of gene-modified cell therapy products

Leukapheresis from the patient

Cell enrichment & T cell activation

Viral transduction

Expansion

Formulation and freeze

Re-infusion in the patient

Lot release

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A high number of clinical trials in the UK make use of integrating vectors

Risks associated with use of gene therapy products:

• Initiate insertional mutagenesis

• Contribute to the generation of replication-

competent viruses

Viral Copy Number (VCN) as a Critical Quality

Attribute

Kymriah (first FDA-approved CAR T cell therapy):

→ VCN is measured on final product: average number

transgene/cell in the population

→ VCN for Post-Marketing Requirements Study

Cell and Gene Therapy Catapult UK Clinical Trial Database 2017

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Quantification of VCN

Common method to detect VCN - qPCR

Relative quantification:

• Reliant on standard curve (both viral and copy number reference

genes)

Challenges:

• Highly dependent on primer efficiency

• Stability of the standard material over time

• Independent qPCR assays imply a harder standardization across

labs

Standard curve

Cqx

log[x]

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The evolution of PCR: droplet digital PCR

Droplet generation QX200 – Droplet reading

Poisson distributionAbsolute No. of copies

End point PCR

Advantages:

• The quantification does not depend on primer efficiency

• There is no need for a standard curve → reduction of costs

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VCN estimation in a cell population using the ddPCR technology

Experimental setup:

• Primary T-cell from healthy donors transduced with a lentiviral vector encoding for GFP.

• Custom design and validation of three TaqMan assays to target different regions in the viral genome.

• Measure the absolute copy number for viral and human genes using droplet digital PCR (BioRad).

Reference Gene 1

Reference Gene 2

Commercially available assays ashuman copy number reference gene

Viral gene 1

Viral gene 2

Viral gene 3

6 combinations of duplex TaqMan reactions

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VG1 + RG1 VG2 + RG1 VG3 + RG1

VG1 + RG2 VG2 + RG2 VG3 + RG2

Mean

Use Poisson distribution toderive the absolute number ofcopies for each TaqMan assay.

VCN/cell = 2 ∗viral gene copies

reference gene copies

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VG = viral geneRG = reference gene

VCN estimation in a cell population using the ddPCR technology

Is population heterogeneity a safety concern for immunotherapy?

population average

VCN estimation on cell population

VCN estimationon single cells

Why we need a single cell method?

• Measure cell-to-cell variability within a cell therapy product.

• High variability may underlie higher risk of therapy-induced secondary diseases.

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VCN

C1TM Single-Cell Auto Prep System

Single Cell Viral Copy Number Assay

Single cell capture

DNA extraction

Targeted pre-amplification

ddPCR assays

Two copy number

reference genesThree LV-

specific genes

Fluidigm C1 system + OpenApp IFC

High Throughput cell imagerInCell

QX200 droplet digital PCR

Bayesian statistical

frameworkFACS sorting

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Single Cell Viral Copy Number Assay

The average of the six combo matches the

gDNA (non-preamplified) value

LV1 /R

G1

LV2 /R

G1

LV3 /R

G1

LV1 /R

G2

LV2 /R

G2

LV3 /R

G2

gDN

A

0

2

4

6

8

1 0

S t u d y 2 5

VC

N

N=83

RG1RG2

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Bayesian statistical framework was used to assign an integer VCN to each single cell

Each point represents the average of six combinations with both Ref Genes

Predicted mean VCN = 1.47Bulk-VCN = 1.43 ± 0.04

e.g. Cell x has a y% probability to have z viral copies

3818

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6 VCN 1

VCN 2

VCN 3

VCN 4

VCN ND

Predicted mean VCN = 2.44Bulk-VCN = 2.19 ± 0.36

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18

943

1

26

VCN 1

VCN 2

VCN 3

VCN 4

VCN 5

VCN 6

ND

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Higher bulk-VCN are accurately represented by the sc-VCN assay

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Conclusion

Take home message

• The sc-VCN assay overall appears to be robust,

and accurate at different VCN

• The assay can be customized to meet the product

requirements

Future plans

• Increase accessibility - increased automation.

• Provide an assay that can be easily implemented as

off line monitoring of the manufacturing

processes.

Viral gene therapy

Non-viral gene therapy

Transgene quantification

Non-integrated viruses

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• Monitor the safety of cell-

based immunotherapy

products to reduce the risk of

off-target toxicity in patients

How can single cell VCN impact gene therapies?

Leukapheresis from the patient

Cell enrichment & T cell activation

Viral transduction

Expansion

Formulation and freeze

Re-infusion in the patient

Lot release

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• Process development – improve

consistency and cost of

manufacturing.

Post-marketing requirements

study

• Post-marketing requirements study

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Vincenzo Di CerboIlaria SanteramoEnas HassanRhys MacownBeata Surmacz-CordleDamian Marshall

Acknowledgements

Frank GesellchenXin LiuElena Shvets

John S. Bridgeman

Thank you for your attention

Questions?

Access established supply chain

Raw materials

Fill and finish

Product release

Cryostorage

Clinical sites

Transport and logistics

including international

airports

Link into UK supply chain and NHS

Unique software solution to collate, track, and document process information

• Enhanced visibility of the entire cell therapy process

Fisher BioServices’ European CryoHub located at CGT Catapult manufacturing centre

• GMP controlled temperature storage

• Ultra-cold chain logistics