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Webinar Series
Culturing Human Cells
Optimizing Growth Conditions for Immunotherapy
October 1, 2014
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Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts
Laurence Cooper, M.D., Ph.D.
MD Anderson Cancer Center
Houston, TX
Michelle Janas, Ph.D.
GE Healthcare
Cardiff, Wales
Webinar Series
Sponsored by:
Culturing Human Cells
Optimizing Growth Conditions for Immunotherapy
October 1, 2014
Culturing Human T Cells: Optimizing Growth Conditions for
Immunotherapy of Cancer
Laurence J.N. Cooper
October 1, 2014
T-cell therapy for oncology
• Premise:
– Ex vivo culture can genetically modify, activate, and propagate T cells to improve their in vivo potency
• Promise:
– Investigator-initiated trials demonstrate that infused genetically modified and unmodified T cells can treat hematologic malignancies and solid tumors
Migrating from the proof-of-principle to the standard-of-practice
• Currently, medical centers both produce and infuse T cells at disparate points-of-care
• This limits the number of: – Patients infused – Number of T cells administered to the maximally-
manufactured-dose (MMD) • Not necessarily the maximally-tolerated dose
• This introduces heterogeneity in the manufacturing processes
• Bioprocessing is asynchronous with manufacturing
Translational pipeline
Regulatory affairs
Trials implementation
Correlative studies
Research Development
Manufacture
Regulatory pathway for genetically modified T
cells for phase I/II trials
(at MDACC)
cGLP to cGMP
Application
to IBC
Application
to NIH-OBA
(RAC)
Infusion of
genetically
modified T
cells protocol
and consent
Responses
to Appendix
M
Submitted via ORERM
CMC and
IND
Application
to CRC
Application
to IRB
IND
Sponsor
Genetic modification and propagation
of clinical-grade genetically modified T cells
Open trial
Gene therapy
long term follow
up clinical
protocol and
consent
Submitted via ORERM
Application
to FDAMedical Monitoring
Vector(s)
(Non-viral, viral)
Approval by
Institutional Compliance
Resources regarding federal regulatory affairs
• http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/ucm072587.htm
• http://www.immunotherapyofcancer.org/content/1/1/5
• http://www.fda.gov/BiologicsBloodVaccines/guidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/default.htm
• http://osp.od.nih.gov/office-biotechnology-activities/rdna/nih_guidelines_oba.html
• http://www.asgct.org/archived_course_materials/training_course/docs/2011/Reiser.pdf
Good T cells to target bad
B cells
Manufacture of T cells expressing CD19-specific
chimeric antigen receptor (CAR)
11
In process testing
and release testing
Source of T cells
• Steady-state apheresis
• Mobilized apheresis
• Venipuncture
• Umbilical cord blood
In process testing
and release testing
Ex vivo activation
• None
• Beads
• Artificial antigen presenting cells
• Co-stimulation
• Cytokines
– Soluble
– Tethered
In process testing
and release testing
Gene transfer
• Viral – Retrovirus
– Lentivirus
• Non-viral – DNA
• Mini-plasmids
• Episomal
– Sleeping Beauty or other transposition
– In vitro-transcribed mRNA
In process testing
and release testing
Propagation
• None
• Beads
• aAPC
• Co-stimulation
• Cytokines
• Time in culture
In process testing
and release testing
Product formulation
• Fresh
• Thawed
• Storage conditions
• Vialing
– Thawing at bedside
• Shipping
In-process testing undertaken on CD19-specific CAR+ T cells (at MDACC)
Parameter Test Method Notes
Phenotype
Cell surface expression of CAR* Flow Cytometry Typically >80% T cells express CAR
Total CAR expression Western Blotting A 73 kDa chimeric CD3 band is seen
for 2nd generation CAR
Potency** Specific lysis Chromium Release Assay CAR-dependent lysis of CD19+ target
cells
Potential for persistence
Memory/Naïve Phenotype Flow Cytometry Includes expression of CD28, CD62L,
CCR7, CD45R0
Markers of exhaustion Flow Cytometry Includes expression of PD1 and, CD57
Telomere Length Flow-FISH Not degraded compared to
unmanipulated control T cells
Safety
CAR copy number Q-PCR Typically one to two integrants per T-
cell genome
Stable integration of DNA
plasmid coding for SB11 by PCR
PCR Absence of PCR band by agarose gel
electrophoresis
TCR Repertoire
Flow Cytometry for TCR V
and/or Direct TCR expression
assay for TCR V & V
Absence of skewing (emergence of
oligoclonality or monoclonality in
pattern of TCR abundance)
Karyotype G-banding Normal *This assay is also undertaken as part of release testing **An assay is being developed to be compliant with guidance document from U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/UCM243392.pdf
Immunol Rev. 2014 Jan;257(1):181-90.
Release testing undertaken on CD19-specific CAR+ T cells (at MDACC)
Tests** performed to generate the certificate
of analysis Specification
Sterility
Visual inspection
No growth at 14 days (BD BACTEC)
PCR negative for Mycoplasma
Endotoxin LAL < 5 EU/kg
Viability (Trypan Blue Exclusion) ≥ 70%
Immunophenotyping (ungated)*** CD32+7-AADneg < 5%
CD19+7-AADneg < 5%
Immunophenotyping
(gated on lymphocytes)
CD3+ ≥ 80%
CAR+ ≥ 10%
Autonomous Growth 10% cells viable at day 18 after withdrawal of
cytokines and aAPC
*K562-derived aAPC (e.g., clone #4 that endogenously expresses CD32 and genetically modified to co-express CD19, CD64, CD86, CD137L, mIL15) **To establish chain-of-custody low resolution HLA class I may be confirmed with donor of T cells ***To assess presence of aAPC Immunol Rev. 2014 Jan;257(1):181-90.
Approaches to manufacture and distribution of bio-engineered T cells
CONFIDENTIAL 2014 25
Challenges to bioprocessing
• The investment in current clinical trial and resources to undertake trial may limit innovation
• Overcome “One technician one product”
• Too many variables to evaluate and lack of pre-clinical in vitro and in vivo models to guide “optimization” of the manufacturing process
• Correlative studies that inform on the therapeutic potential need to be linked to changes in bioprocessing
Points to consider
• Avoid single-vendor suppliers – Beads to activate T cells via cross-linking CD3
• Closed (unbreached) system – Lack of GMP space
• Automation – Lack of GMP technicians
• Serum-free conditions • Time in culture
– Quantity versus quality
• Reagents suitable for Phase I/II trials may not be suitable for Phase III • Potency assay
– http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/UCM243392.pdf
Applied Cellular Therapy (ACT)
Conclusions
• Distribution is a rate-limiting step to the broad application of T-cell therapy for oncology
• Multiple variables need to be addressed with imperfect knowledge base
• The human experience is the only valued experience
• There is an ongoing need to refine the T-cell culturing process based on clinical data to meet the needs of future recipients
• (713) 563-5393
• http://www.mdanderson.org/children/
• https://www.facebook.com/pages/Laurence-Cooper-Cell-Therapy/195075373908115
• http://www.researchgate.net/profile/Laurence_Cooper/
Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts
Laurence Cooper, M.D., Ph.D.
MD Anderson Cancer Center
Houston, TX
Michelle Janas, Ph.D.
GE Healthcare
Cardiff, Wales
Webinar Series
Sponsored by:
Culturing Human Cells
Optimizing Growth Conditions for Immunotherapy
October 1, 2014
Imagination at work
The use of perfusion in high density T-cell cultures Michelle Janas PhD Oct 01, 2014
See tutorial regarding confidentiality disclosures. Delete if not needed.
T-cell therapy The next generation in cancer treatment
Cell Separation
Cell Collection
T-cell Selection, Activation and
Expansion Cell Harvest & Concentration
Cell Infusion into Patient
Genetic modification
See tutorial regarding confidentiality disclosures. Delete if not needed.
The logistical challenge of delivering autologous T-cell therapies
100 kg patient Typical cell dose = 1x108/kg
= 50 x T225 flasks
100mls @ 2x106/ml
See tutorial regarding confidentiality disclosures. Delete if not needed.
Key Requirements of Cell Therapy Manufacturing Processes Scalable. Sample contained in 1 vessel Easy to scale out to make most efficient use of manufacturing space
Automatable 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
See tutorial regarding confidentiality disclosures. Delete if not needed.
XuriTM Cell Expansion Systems
• Rocking motion: keeps cells in suspension and ensures efficient oxygen transfer
• Media perfusion: enables cells to be cultured at high densities without loss of viability
• Disposable Cellbag™ bioreactor: mitigates cross contamination risks and allows T-cells to be grown in a single vessel
See tutorial regarding confidentiality disclosures. Delete if not needed.
What is Media Perfusion?
Fresh Media in Spent Media out
See tutorial regarding confidentiality disclosures. Delete if not needed.
Experimental Protocol
0 5 14
Day of Culture
perfusion
Cell Density
(x106/ml)
Perfusion rate
(per 24hrs)
< 2 0
2-10 50%
10-15 75%
> 15 100%
Scalable to 1 and 5L culture volumes
See tutorial regarding confidentiality disclosures. Delete if not needed.
2.5E+07
2.0E+07
1.5E+07
1.0E+07
5.0E+06
0.0E+00
Ce
ll Co
nc
en
tratio
n (/m
l) Media Perfusion enables T-cells to be grown at high cell densities
Viable Cells
0.0E+00
5.0E+09
1.0E+10
1.5E+10
2.0E+10
2.5E+10
0 2 4 6 8 10 12 14
Day of Culture
To
tal V
iab
le C
ells
Inoculate
Cellbag
50%
75%
100% With perfusion
1L culture
See tutorial regarding confidentiality disclosures. Delete if not needed.
2.5E+07
2.0E+07
1.5E+07
1.0E+07
5.0E+06
0.0E+00
Ce
ll Co
nc
en
tratio
n (/m
l) Without media perfusion growth arrest occurs
0.0E+00
5.0E+09
1.0E+10
1.5E+10
2.0E+10
2.5E+10
0 2 4 6 8 10 12 14
Day of Culture
Via
ble
Ce
lls
With perfusion
Without perfusion
Inoculate
Cellbag
50%
75%
100%
1L culture
See tutorial regarding confidentiality disclosures. Delete if not needed.
Without media perfusion T-cell viability declines
50
60
70
80
90
100
5 6 7 8 9 10 11 12 13 14
Day of Culture
Via
bili
ty (
%)
With perfusion
Without perfusion
50% 75% 100%
Inoculate
Cellbag
See tutorial regarding confidentiality disclosures. Delete if not needed.
100 75% 50%
Media perfusion removes unwanted metabolites from the culture
Lactate Ammonia
0
5
10
15
20
25
30
35
6 7 8 9 10 11 12 13 14
Day of Culture
Lac
tate
(mm
ol/
L)
0.0
1.0
2.0
3.0
4.0
5.0
6 7 8 9 10 11 12 13 14
Day of Culture
Am
mo
nia
(mm
ol/
L)
With perfusion
Without perfusion
100 75% 50%
See tutorial regarding confidentiality disclosures. Delete if not needed.
Lactate is detrimental to both T-cell growth and viability
50%
60%
70%
80%
90%
100%
0.0
0.5
1.0
1.5
2.0
0mM 10mM 20mM 30mM 40mM
Via
bili
ty (
)
Fo
ld E
xpa
nsi
on
()
Lactic Acid Addition
See tutorial regarding confidentiality disclosures. Delete if not needed.
Media perfusion provides crucial growth factors to the culture
0
1
2
3
4
5
6
7
8
5 6 7 8 9 10 11 12 13 14
Day of Culture
IL-2
(ng
/ml)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
5 6 7 8 9 10 11 12 13 14
Glu
co
se (g
/L)
Day of Culture
With perfusion
Without perfusion
Glucose IL-2
100 75% 50% 100 75% 50%
See tutorial regarding confidentiality disclosures. Delete if not needed.
Continual IL-2 supply is critical for T-cell viability
Initiate perfusion or IL-2 addition
0.0E+00
5.0E+09
1.0E+10
1.5E+10
2.0E+10
2.5E+10
5 6 7 8 9 10 11 12 13 14
Tota
l via
ble
ce
lls
Day of Culture
50
60
70
80
90
100
5 6 7 8 9 10 11 12 13 14
Day of Culture
% v
iab
le c
ells
With perfusion
IL-2 addition without perfusion
T-cell growth T-cell viability
100 75 50 100 75 50
Determining cell concentration
without sampling
See tutorial regarding confidentiality disclosures. Delete if not needed.
Key Requirements of Cell Therapy Manufacturing Processes Closed system to eliminate chance of contamination with adventitious agents
due to handling
Sampling port
See tutorial regarding confidentiality disclosures. Delete if not needed.
The Xuri™ W25 allows continuous monitoring of dissolved oxygen in the Cellbag bioreactor
The optical sensor port in embedded in the bottom of the Cellbag and connected externally to an optical fiber cable
See tutorial regarding confidentiality disclosures. Delete if not needed.
Continuous monitoring of dissolved oxygen
• Dissolved Oxygen (DO) is a relative measure of the amount of oxygen that is dissolved in the culture medium
• Freshly prepared media has a DO concentration of 100% (saturation)
• Media incubated in the presence of 5% CO2 has a DO concentration of 95%
See tutorial regarding confidentiality disclosures. Delete if not needed.
The relationship between dissolved oxygen and cell density in T-cell cultures is linear
0
10
20
30
40
50
60
70
80
90
100
0.E+00 1.E+07 2.E+07 3.E+07 4.E+07 5.E+07
DO
(%
)
Cell Concentration (per ml)
R2 = 0.94
See tutorial regarding confidentiality disclosures. Delete if not needed.
DO readings can be used to extrapolate T-cell concentrations
0
10
20
30
40
50
60
70
80
90
100
0.E+00 1.E+07 2.E+07 3.E+07 4.E+07 5.E+07
DO
(%
)
Cell Concentration (per ml)
See tutorial regarding confidentiality disclosures. Delete if not needed.
DO readings can be used to automate perfusion rates
50
55
60
65
70
75
80
85
90
95
100
0.0E+00 5.0E+06 1.0E+07 1.5E+07 2.0E+07
DO
(%
)
Cell Concentration (per ml)
50%
75%
100%
50% 75% 100%
Cell Density
(x106/ml)
Perfusion rate
(per 24hrs)
< 2 0
2-10 50%
10-15 75%
> 15 100%
See tutorial regarding confidentiality disclosures. Delete if not needed.
Summary
• Media perfusion enables T-cell to be grown at high densities
Unwanted Metabolites
Critical Growth Factors
• DO is a predictor of T-cell density
• DO readings can be used to set perfusion rates and monitor the growth kinetics of T-cell cultures without having to sample
Imagination at work
GE Healthcare, Cardiff UK Paul Bowles Andrew Hall Ray Ismail Michelle Janas Angela Marenghi Ou Li Claudia Nunes Vincent Sauvage Sarah Stone Bill Shingleton
GE Global Research, NY USA Anshika Bajaj Andrew Burns Brian Davies
GE and GE monogram are trademarks of General Electric Company. Cellbag, UNICORN, Xuri, WAVE, and WAVE bioreactor are trademarks of General Electric
Company or one of its subsidiaries.
©2014 General Electric Company—All rights reserved. First published Apr. 2014.
Xuri Cell Expansion Systems W5 and W25 are not a medical devices nor CE marked and should not be used in diagnostic processes. Drug manufacturers &
clinicians are responsible for obtaining the appropriate IND/BLA/NDA approvals for clinical applications.
Cellbag bioreactors with integrated optical sensors are sold under a sublicense from Sartorius Stedim Biotech under US patent numbers 6,673,532, 7,041,493
and/or its foreign equivalents.
GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK
Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts
Laurence Cooper, M.D., Ph.D.
MD Anderson Cancer Center
Houston, TX
Michelle Janas, Ph.D.
GE Healthcare
Cardiff, Wales
Webinar Series
Sponsored by:
Culturing Human Cells
Optimizing Growth Conditions for Immunotherapy
October 1, 2014
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Brought to you by the Science/AAAS Custom Publishing Office
Culturing Human Cells
Optimizing Growth Conditions for Immunotherapy
October 1, 2014