(Schaum )Ira Levine-Solucionario Fisicoquimica Levine 5 Edicion
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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
Webinar SeriesWebinar SeriesScienceScienceCulturing Patient Cells for Cancer Immunotherapy:Culturing Patient Cells for Cancer Immunotherapy:
Challenges and OpportunitiesChallenges and Opportunities18 November, 201318 November, 2013
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:
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
Webinar SeriesWebinar SeriesScienceScienceCulturing Patient Cells for Cancer Immunotherapy:Culturing Patient Cells for Cancer Immunotherapy:
Challenges and OpportunitiesChallenges and Opportunities18 November, 201318 November, 2013
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.
51GE Title or job number
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
53GE Title or job number
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 (°)
54GE Title or job number
12/2/2013
Optimization Fo
ld e
xpan
sion
sum
Optimized speed and angle: 15.02 rpm, 5.625 º
55GE Title or job number
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
71GE Title or job number
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:
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
To submit your questions, type them into the text box
and click .Stephen Minger, Ph.D.GE Healthcare Life SciencesCardiff, Wales, UK
Bruce Levine, Ph.D.University of PennsylvaniaPhiladelphia, PA
Webinar SeriesWebinar SeriesScienceScienceCulturing Patient Cells for Cancer Immunotherapy:Culturing Patient Cells for Cancer Immunotherapy:
Challenges and OpportunitiesChallenges and Opportunities18 November, 201318 November, 2013
Look out for more webinars in the series at:webinar.sciencemag.org
For information related to this webinar, go to:www.gelifesciences.com/xuri
To provide feedback on this webinar, please e‐mailyour comments to [email protected]
Sponsored by:
Brought to you by the Science/AAAS Custom Publishing Office
Webinar SeriesWebinar SeriesScienceScienceCulturing Patient Cells for Cancer Immunotherapy:Culturing Patient Cells for Cancer Immunotherapy:
Challenges and OpportunitiesChallenges and Opportunities18 November, 201318 November, 2013