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Webinar SeriesWebinar SeriesScienceScienceCulturing Patient Cells for Cancer Immunotherapy:Culturing Patient Cells for Cancer Immunotherapy:

Challenges and OpportunitiesChallenges and Opportunities18 November, 201318 November, 2013

Sponsored by:

Participating Experts:

Brought to you by the Science/AAAS Custom Publishing Office

Stephen Minger, Ph.D.GE Healthcare Life SciencesCardiff, Wales, UK

Bruce Levine, Ph.D.University of PennsylvaniaPhiladelphia, PA



Culturing Patient Cells for Cancer Immunotherapy:

Challenges and Opportunities

Bruce Levine, Ph.D.

Department of Pathology and Laboratory MedicineUniversity of Pennsylvania

• Declaration of financial interest due to intellectual property and patents in the field of cell and gene therapy.

• Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight

• Funding support for trial and data to August, 2012: Alliance for Cancer Gene Therapy, Leukemia and Lymphoma Society of America

Conflict of Interest Statement

• Following chemotherapy or stem cell transplants, T cell counts and immune function is depressed for months to years

• Tumors have evolved sophisticated means to avoid immune detection.

• Hypothesis: Select, re-educate and expand T cells ex vivo outside of tumor milieu and re-infuse to prevent relapse and/or infections

• Following chemotherapy or stem cell transplants, T cell counts and immune function is depressed for months to years

• Tumors have evolved sophisticated means to avoid immune detection.

• Hypothesis: Select, re-educate and expand T cells ex vivo outside of tumor milieu and re-infuse to prevent relapse and/or infections

Rationale for T Cell Immunotherapy

• Be Potent

• Be Numerous (Effector to Target Ratio)

• Have Substantial Proliferative Capacity

• Persistent (Memory)

If we could design T cells for the treatment of disease, they would:

Ex Vivo Approaches for Adoptive T Cell Therapy

*

Redirected by gene transfer

Ex Vivo Approaches for Adoptive T Cell Therapy

*

Activated T Cell Therapy for Infection or Malignancy9-10 Day Ex Vivo Process

2. Enrich, deplete, or isolate cells

4. Clinical scale cell expansion

7. Reinfuse cells

5. Wash and concentrate cells

3. Stimulate cells with artificial Antigen Presenting Cells

(insert genes)

6. Quality Control

1. Source = patient or directed donor by leukapheresis or blood draw

TerumoBCT Elutra

Counterflow Centrifugal Separation of Lymphocytes and Monocytes

Incr

easi

ng ra

te o

f Buf

fer F

lowCaridianBCT Elutra

Counter Flow Centrifuge

Positive or Negative Immunomagnetic Selection of Cell Subsets

J Immunol 1997; 159: 5921 Science 1997; 276: 273Immunol. Rev. 1997; 160: 43Mol. Ther. 2004; 9; 902Exp. Opin. Biol. Ther. 2008; 8: 475

J Immunol 1997; 159: 5921 Science 1997; 276: 273Immunol. Rev. 1997; 160: 43Mol. Ther. 2004; 9; 902Exp. Opin. Biol. Ther. 2008; 8: 475

Anti-CD3Anti-CD3 Anti-CD28Anti-CD28

Artificial APC: Bead Artificial APC: Bead

Signal 1Signal 1

GrowthGrowth

CD28 CTLA4CD28 CTLA4TcR/CD3TcR/CD3

++

Activation and Expansion of T cells Via Bead-Immobilized Antibodies

CD3/CD28 Co-Stimulation Increases Cytokine Production 1-2 Log10 Over anti-CD3 mAb + IL-2

J. Immunol. 159:5921-5930, 1997

Ex Vivo Exponential Growth For 3 Months Polyclonal CD4+ T Cells

Nc020795.xls

Exponential growth (days) 60-100Mean log10 growth (fold) 9-12Mean Pop Doubling 30-40

Baxter Lifecell - Gas permeable bags •Closed system with minimal risk of contamination•Ease of large volume fluid transferWave Bioreactor

WAVE Feed and Harvest via Perfusion

Intraoperative Blood Transfusion Equipment

loadreservoir

waste

productwash

Source: Apheresis or blood draw, may be cryopreserved

Final product:If gene modified,

always cryopreserved

Transforming the Myth of the Chimera Into Potent and Durable Cancer Immunotherapy

Two Basic Approaches to Overcome Tumor SuppressionCreation of Re-directed T cells

TCR heterodimer approach “CAR” approach- off the shelf- MHC independent

Extracellular

Intracellular

Chimeric Protein

Tumor binding domain

Autologous T Cells Transduced w/ Anti-CD19 mAb spliced to TCR- and 4-1BB Signaling Domains

• Lentiviral vector to deliver construct• CD3- and 4-1BB signaling domains

• Anti-CD3/anti-CD28 mAb coated bead stimulation

4-1BB 4-1BB

Chimeric Antigen Receptor Modified T Cell Immunotherapy in Chemotherapy Resistant or Refractory CD19+ CLL

• Eligibility criteria– no available curative treatment options (such

as autologous or allogeneic SCT)– limited prognosis (several months to < 2 year

survival) with currently available therapies

• Dose– max 5 x 109 CAR T Cells

Blood Marrow

CART-19 Cells in UPN03 Blood and Marrow:Engraftment, Expansion, Persistence to

6 Months (now 36)

LLOQ = 2 copies per ug 1,600 copies per ug = ~1% marking

Day -21UPN 01

Day 177

R/B R/B B

CARs

Days from Infusion-80 -40 120 160 200

Cel

ls(x

10-3/m

m3 )

0

10

20

30

40

50

60

70

80WBCALC

Corticosteroidsstarted

80400

UPN 03

UPN 02

Pre-Therapy 3 Months

Clinical Response After CART-19 Infusion: 1st 3 Pts

Sci Transl Med. 2011 Aug 10;3(95):95ra73 and NEJM 2011 Aug 25;365(8):725-33

Chimeric Antigen Receptor Modified T Cells in Chemotherapy Resistant or Refractory

CD19+ Leukemia (Pediatric)

• Eligibility criteria– ALL without curative options for therapy,

including those not eligible for allogeneic SCT because of age, comorbid disease, lack of suitable donor or prior SCT

• Dose– 1 x 108 CAR T cells/kg up to max 5 x 109

Pediatric CART19 ALL StudyPI: Stephan Grupp, MD PhD

• Subject #1: 7yoF pre-B ALL. Karyotype: high risk• Dx May 2010: standard COG ALL induction• Relapse #1: 10/2011• Relapse #2: 2/2012• 3/2012: high dose cytoxan/clofaribine: persistent ALL• Marrow 4/16/2012: 60% blasts w/kidney, liver, spleen

lesions• Autologous CART19 4/17/2012

Total dose CART19: 1.2 x10^7 CAR cells/Kg• CAR T cells infused with no additional chemotherapy

Rapid Induction of Remission in Pediatric ALL Marrow

T Cells

B Cells(tumor)

day +6 day +23

Rapid Induction of Remission in Pediatric ALL Marrow

T Cells

B Cells(tumor)

day +6 day +23

• Deep remission induced in 23 days

• Status: CR (2+)

• MRD <0.01% cells

Molecular remissions in pediatric ALL

Stephan Grupp, et al. NEJM 2013 Apr 18;368(16):1509-18

Patient Tissue Timepoint (day)

Number of input genomes (cell equivalents)

Total TCRb reads

Total IGH reads

Dominant clone reads

Tumor clone

frequency (%)

CHP959-100 Blood -1 111,340 525,717 189 185 97.88

Marrow -1 317,460 348,687 59,791 59,774 99.97

23 362,819 1,712,507 37 33 89.19

87 645,333 425,128 10 10 100.00

180 952,381 800,670 45 0 0.00

• ~ 5 log tumor reduction in CHP959-100• No chemotherapy was given to CHP959-100

Chimeric Antigen Receptor Longevity in vivo

15 year monitoring for delayed adverse events required by FDA (2006 Guidance).

Patients from 3 studies in HIV gene therapy utilizing a retroviral vector expressing the CD4- CAR in CD4/8 T cells were evaluated annually for persistence of genetically modified T cells.

Longitudinal Analysis of CD4CAR

Combined Data of all 3 Studies Shows Decade Long Persistence of Gene Modified T Cells

AnnualFollow ups Half-life

Yrs 1-9 13.9 yrs

Scholler et al.Science Trans. Med.2012 May 2;4(132):132ra53

Platform Cancer Immunotherapy Technology: Allows design and targeting against other tumors

Target Cancer

CD19 B cell leukemiasand lymphomas

Mesothelin Mesothelioma, pancreatic, ovarian

New Targets

Other cancers

Adoptive Transfer of Engineered T-cells:

Easily accessed, large numbers can be rapidly produced ex vivoPlatform technology to Enhance and Redirectimmunity.Persist for months to years in vivoOpportunities/ApplicationsImmune reconstitution, vaccine augmentation, engineering HIV resistance, retargeting with gene transduced chimeric antigen receptors

Challenges in Cell Manufacturing

• Science, Translation, Pre-clinical Models– Process development from small scale to clinical

scale• Reagents and Supplies

– Good Manufacturing Practices or clinical grade– Secondary suppliers

• Equipment– Scaleable Bioreactors– Cell washing and concentration

The First Cell Therapy Assembly Lines

Modern Cell Therapy Assembly Lines

Clinical Cell and Vaccine Production Facility

Department of Pathology and Laboratory MedicineDivision of Transfusion Medicine and Therapeutic PathologyDirector, Don Siegel

Department of Pathology and Laboratory MedicineDivision of Transfusion Medicine and Therapeutic PathologyDirector, Don Siegel

Study Participants

Department of Medicine, Division of Hematology/OncologyNoelle FreyDavid PorterChildren’s Hospital of PhiladelphiaStephan Grupp

Alliance for Cancer Gene Therapy, NIH/NCI, Jeffrey J. Weinberg Memorial Foundation, Leukemia and Lymphoma Society

Abramson Cancer CenterTranslational Research ProgramCarl June, Director

Sponsored by:



Stephen Minger, Ph.D.GE Healthcare Life SciencesCardiff, Wales, UK




Culturing Patient Cells for Cancer Immunotherapy: Challenges and Opportunities

Stephen Minger Chief ScientistLife SciencesGE Healthcare

45

Cellular Immunotherapy

Cell Separation

Cell Collection

Cell Selectionand ActivationCell Harvest &

Concentration

Cell Infusion into Patient

Cell Expansion

46

Typical cell dose = 1x108/kg

20 kg patient= 2 x 109 cells

100 kg patient= 1 x 1010 cells

47

Scale Up

Typical cell dose = 1x108/kg

20 kg patient = 10 x T225 flasks100mls @ 2x106/ml

48

Scale Up

100 kg patient

= 50 x T225 flasks100mls @ 2x106/ml

Typical cell dose = 1x108/kgTypical cell dose = 1x108/kg

49GE Title or job number

12/2/2013

Key Requirements of Cell Therapy Manufacturing ProcessesScalable.Sample contained in 1 vesselEasy to scale out to make most efficient use of manufacturing space

Automated to minimize the chance of human error

Single Use to eliminate cross contamination with other patient cells

Closed system to eliminate chance of contamination with adventitious agents due to handling

Robust and Compliant. To ensure consistency of product and satisfaction of regulatory requirements

50

Creating the right environment to maximize cell densityEfficient gas transfer

• Motion creates large turbulent surface for oxygen transfer

• Wave action sweeps up cells and prevents settling.

• Oxygen transfer to cells without shear force or bubble formation.


12/2/2013

Optimization Studies: Rocking angle & rateObjective: Maximize the expansion of viable T cells in a 10 day period

Rocking rate (per minute)

2 10 18

Roc

king

Ang

le

(deg

ress

) 2

6

9

52

0 1 2 3 4 55 6 7 8 9 10Day of culture

Experimental Design

Perfuse 500mls

Perfuse 1LPerfuse 750mls

Daily monitoring of: • Cell proliferation/viability• Glucose/Lactate/Ammonia


12/2/2013

Results

Important impact of the rocking speed on fold expansion (steep slope)

Minor impact of the rocking angle on fold expansion (low slope)

Speed residuals (rpm)

Fold

exp

ansi

on s

um re

sidu

als

Fold

exp

ansi

on s

um re

sidu

als

Angle residuals (°)


12/2/2013

Optimization Fo

ld e

xpan

sion

sum

Optimized speed and angle: 15.02 rpm, 5.625 º


12/2/2013

OptimizationAt least 20% more growth after 5 days of culture at 15rpm compared to 10rpm.

0

2

4

6

8

10

12

14

16

5 6 7 8 9 10

10 rpm, 6° 15 rpm, 6°Cel

l con

cent

ratio

n (1

06/ m

L)

Day of Culture

56

Creating the right environment to optimize cell densityAutomated media exchange

• The addition of fresh media keeps nutrient levels high

• Media perfusion enables cells to be cultured at high densities without loss of viability (i.e. >2x106 cells/ml)

• The removal of spent media keeps metabolite levels low

57

Creating the right environment to minimize contaminationA disposable System

• Disposable system mitigates cross contamination risks

• Allows high cell numbers to be grown within a single contained unit

58

Create the right environment for scalable immunotherapy

1.0E+07

1.0E+08

1.0E+09

1.0E+10

1.0E+11

0 2 4 6 8 10 12 14

Generation of 20 billion cells

Days of culture

Viab

le C

ells

100 x

1 x

or

Based upon 2x1010 cells grown in T225 flasks at 2x106cell/ml in 100mls per flask

59

Measuring outcomes in clinical trials

Complete RemissionPartial ResponseNon-responder

60

?

What is the effect of expansion on T-cell phenotype and function?

61

0 1 2 3 4 55 6 7 8 9 10QC analysis

Experimental Design

Phenotype monitoring of: • CD4/CD8 ratio• CD27/CD28 expression to assess differentiation state• CD57 expression to assess the presence of senescent cells• CD62L expression to assess migratory ability

1211 13 14

62

0

20

40

60

80

100

0 5 10 14

CD4/CD8 ratio after expansion

Day of Culture

Pro

porti

on o

f T c

ells

CD4+CD8+

T cells activation with CD3/CD28 expander beads and culture in the bioreactor promotes the

preferential expansion of CD8+ T cells.

63

Phases of T-cell differentiation.

CD27

CD

28early/naiveintermediate

Late/Senescent

64

Post-expansion differentiation status.

0

20

40

60

80

100

0 5 10 140

20

40

60

80

100

0 5 10 14

Day of culture

Per

cent

age

of p

opul

atio

n

CD4+ CD8+

Cultured cells remain in an early/intermediate differentiation state

Naïve/EarlyIntermediateLate/Senescent

65

CD57 – a marker of replicative senescence.

CD57

CD

3

0

10

20

30

40

50

0 2 4 6 8 10 12 14

CD

57+

(%)

Day of Culture

T cells expansion in the bioreactor does not result in the accumulation of senescent (CD57+)

cells

66

CD62L – a measure of migration capability.

CD

62L+

(%)

Day of Culture

Cultured T cells are primed to migrate to secondary tissues upon transfer

CD62L

CD

3

0

20

40

60

80

100

0 10

CD4+CD8+

67

TH1 TH2

Sec

rete

d C

ytok

ine

(pg/

ml)

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

Expanded T-cells have a dominant TH1 phenotype.

Culture day 10

68

The challenge of scaling-out

69

• Advanced control of cell expansion through setup, process monitoring and remote operation

• FDA Compliant with GAMP 5 and 21 CFR part 11 software

• Remote monitoring & notifications

Progressing into commercialization

Xuri™ Cell Expansion System W25

70

Cellular Immunotherapy: The GE approach

Scale Upand

Scale Outto produce

Healthy Cells


12/2/2013

Cell Bioprocessingwww.gelifesciences.com/xuri

GE, Imagination At Work and GE Monogram are trademarks of General Electric Company. Wave Bioreactor, Cellbag and Xuri are trademarks of General Electric Companies.

© 2013 General Electric Company – All rights reserved. First published October 2013GE Healthcare Life Sciences, a General Electric company.

www.gelifesciences.com, GE Healthcare Life Sciences. Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK

WAVE Bioreactor, Xuri Cell Expansion System W25 and W5, and Ficoll-Paque products are not medical devices nor CE marked and are not for use in diagnostic processes.Drug manufacturers and clinicians are responsible for obtaining the appropriate IND/BLA/NDA approvals.

All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information.

Sponsored by:



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and click .Stephen Minger, Ph.D.GE Healthcare Life SciencesCardiff, Wales, UK




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Science Webinar Series slides_GE4... · download slides and more info LinkedIn login shows slide window ... Cardiff, Wales, UK Bruce Levine, Ph.D. University of Pennsylvania Philadelphia,