Stephen Marley | Mesenchymal stem cell production

Mesenchymal Stromal Cell production How can we make MSC therapies efficacious: the next steps

Stephen Marley

1. Replace the lost tissue

“Stem Cell Therapies” 3. Paracrine effect 2. Transdifferentiation

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

Tissue damage

Inflammation

Cell death

3

Tissue repair promoting activity

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

‘Immunosuppressive activity’

Stephen Marley | Mesenchymal stem cell production

Mesenchymal stromal cells CD45CD31CD73+ CD150+ PDGFR+Sca-1+ CD146+/Nestin+ Progenitors

Bone

Chondrocytes

Adipocyte

Muscle

MSC have ‘tissue repair’ activity MSC have ‘anti-inflammatory’ activity

• Renal ischaemia

• Arthritis

• Lung fibrosis

• Multiple sclerosis

• Toxic liver injury

• IBD

• Stroke

• Sepsis

• Myocardial infarction

• Asthma

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

MSC exhibit immunosuppressive activities

Krampera et al, Blood 2003

Proliferative response to HY (cpm)

The inhibitory effect is not dependent on MHC class I expression

Control

MSC

3T3-F442A Krampera et al, Blood 2003

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

The anti-proliferative effect is a property of stroma 100

100

MSC

100

SF

50

50

50

0

0

0

LF

100

CH

100

50

DF

50

0

0

1:100

1:10

1:5

1:100

Neurons

1:10

1:5

Endothelial cells 200

100

150 50

100 100 0

0

1:100

1:10

1:5

1:100

1:10

1:5

MSC induced anergy is ultimately mediated by soluble factors

- IDO - NO - PGE2 - Galectins - HO-1 - TGFβ - IL6 - IL10 - HLA-G

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Mediators AND inducers of immunosuppressive populations

Stephen Marley | Mesenchymal stem cell production

The anti-proliferative property needs ‘licensing’

110 100 90 80 70 60 50 40 30 20 10 0 SC only

SC +T

+PHA

+aCD3

Source of supernatant Jones et al. J Immunol 2007

‘Licensing’ is modulated by inflammatory cytokines

Wang et al, 2012

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

Inhibition of NFkΒ activation prevents ‘licensing’

sh-iKKb

Weng et al, 2012

Prophylactic regimen: single dose of MSC

days

0

7

14

21

28

49

56

20x106

PBMC + 3x106 MSC

% huCD45

Weight (gr))

100

30

80 60 15 40 20 0

0 3

4

5

1

6

Weeks

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

2

3

4

5

6

7

Weeks

Stephen Marley | Mesenchymal stem cell production

Preventive treatment: multiple doses of MSC

days

0

7

14

21

28

49

56

20x106

PBMC + 3x106 MSC

3x106 MSC

% huCD45

Weight (gr))

100 30 80 60 15 40 20 0

0 3

4

5

1

6

2

3

4

5

Weeks

6

7

Weeks

The efficacy of MSC on GVHD depends on time of infusion Day 0

Day 2

Day 20

Day 30

Polchert et al, EJI 2008

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

Incidence and severity of GVHD after prophylactic administration of MSC

BM

PBSC

MSC + BM/PBSC

aGVHD > II

25%

29%

28%

cGVHD lim

42%

22%

40%

cGVHD ext

21%

21%

19% Lazarus et al, BBMT 2005

MSC increase survival of aGVHD patients

Le Blanc et al, Lancet 2008

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

Clinical response to MSC: the Hammersmith experience (34 cases) Early aGvHD (53%) Late aGvHD (15%) cGvHD (2%)

The inflammatory microenvironment determines clinical responses to multipotentmesenchymal stromal cells for graft-versus-host disease. Phase II clinical trial within the British Society of Blood and Marrow Transplantation (BSBMT) network •

open to all UK adult and paediatric allograft centres.

•

40 patients with newly diagnosed severe steroid-resistant acute GvHD to receive MSC in addition to the standard of care selected at the Transplant Centre.

•

Blood sample and biopsy of the involved site (generally gut, skin and liver if clinically indicated) to be processed fresh (unfixed) for transcriptomics, immunohistochemistry and cytokine production.

•

Patients will then receive 1 dose of 3x106 MSC/kg intravenously the day after the diagnosis of steroid-refractory GvHD will have been made. 20

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

The inflammatory microenvironment determines clinical responses to multipotentmesenchymal stromal cells for graft-versus-host disease. MSC manufacturing: •

MSC will be evaluated and selected according to a potency assay. We will only select high-potency MSC as defined by the ability to: a) Acquire immunosuppressive function when incubated at ≤20U/ml of IFN-γ and b) suppress, once ‘licensed’, T-cell proliferation (50%) at least at 1:20 MSC:T cell ratio.

21

Developing a potency assay for clinical grade Mesenchymal Stromal Cells. •

To develop an assay to quantitate the functional properties of the immunosuppressive activity of MSC ('potency assay') and accordingly determine the variability of clinical grade MSC preparations from different donors and sources

•

To correlate the parameters of the 'potency assay' with the in vivo activity in a pre-clinical model of GvHD

•

To correlate clinical results with the results of the potency assays 22

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

Potency assay – human MSC are not licensed by activated murine splenocytes, but do suppress them. Fixed IFN/variabl MSC:Spl ratio = potency Variable IFN/fixed MSC:Spl ratio = sensitivity to licensing huIFN-γ

Human MSC

Suppression

X

Murine splenocyte CD3/CD28

Licensing Murine factors do not license human MSC

MSC batches exhibit different IS activity

24

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

Summary • MSC immunosuppressive activity depends on the inflammatory environment to which MSC are exposed • Need for stratifying patients according to their inflammatory stage/status • MSC sensitivity and potency may be 25 significant

Clinical Scale Mesenchymal Stromal Cell production

26

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

MSC at the Hammersmith •

System streamlined and adapted for ATMP production from 2009

•

Specials License obtained in 2011 – permits use of MSC in a broad spectrum of applications by prescription only on an individual basis

•

Clinical trial in Multiple Sclerosis (STREAMS) 2012 – in collaboration with Dr Paolo Muraro as part of a broader international study

27

1. Hardware – seeding sets

28

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

2. Hardware – Incubators Remote monitored, filtered CO2 supply, modified insulation and rear filtration to reduce particle shedding, sterilisation cycle.

29

3. Hardware – Incubator capacity 22 levels, comprising 4x 5-stack and 2x 1-stack cell factories

30

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

MSC Culture – process overview Marrow 10-50 million TNC/stack in

Seed

Seed P1 culture

11-22 stacks

MSC 0.1-1 million/stack In 22 stacks

Day 3 media change

Day 14 media change

D14 media change

harvest

harvest

Up to 20 million MSC/stack

Up to 50 million MSC/stack (up to 1,100 million MSC) Approx. 4 adult doses

Infusion

infusion 31

Platelet lysate pool preparation GMP-compliant (NBS sourced, TSE regulations) Faster cell growth than serum-free + cytokines No requirement to pre-coat culture surfaces Utilises a ‘wasted’ resource Batch testing

Expired platelets

Pooled Platelets

COBE 3000rpm, 10 minutes

25mL lots

-80C storage

32

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

Harvesting layout

33

Harvest – Draining the medium Waste medium is saved to block trypsin activity at end of detachment period

34

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

Harvest – washing off residual protein

35

Harvest – trypsinisation

36

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

Harvest – checking detachment

37

Harvest – harvesting Trypsin activity is blocked with waste medium and cells are collected for washing

38

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

Harvest – cell washing An 11-level harvest typically takes three COBE spins to compact all the cells plus one wash in CliniMACS buffer Wash settings – 1500rpm for 5 minutes

39

Harvest– clean room burden High consumables useage High time burden – harvesting time 2-3hours High risk of environmental contamination

40

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

Process algorithms – Matched donor/recipients Aspirate

Primary MSC culture (11-22 levels)

Primary cells ready in 3 weeks Passage cells ready in 5 weeks

Passage 1 MSC (22 levels)

Total harvest per recipient should exceed 2x adult dose at 4million cells/kg

Bulk of cells cryopreserved (infusion or further culture)

Infusion

Disadvantages – donor aspirates can be low volume and poor quality, especially from adults. Culture performance is at best a guide and can fail to yield therapeutic doses.

41

Process algorithms – 3rd party donors Primary cells ready in 3 weeks First cells for infusion ready in 5 weeks Typical yield per marrow aspirate is 12 seed lots

Aspirate Primary MSC culture (11-22 levels)

Passage 1 (22 levels)

Passage 1 (22 levels)

Cells cryopreserved in multiple identical lots

Passage 1 (22 levels)

Passage 1 (22 levels)

Typical harvest per seed lot is 440 million cells = 1 adult dose Can be up to 1,100 million cells = 4 adult dose Disadvantages – first cells for infusion not ready for 5 weeks+ Advantages – total yield per donor in excess of 12 adult doses Culture performance is uniform and predictable

42

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stephen Marley | Mesenchymal stem cell production

Ling Weng Ilaria Marigo Andrea Guerra Andrew Innes Lucien Lopes Jeong Hun Ko Cristina Trento Antonio Galleu Alice Wang N Horwood J Dyson Guido Franzoso F Marelli-Berg

EBMT Autoimmune Diseases and Immunobiology Working Parties | 16-17 Nov 2012 | Paris, France

Stem cell biology