Engineering Artificial Antigen Presenting Cells for Efficient … · • Julio Cotte • Dawn Maier
Transcript of Engineering Artificial Antigen Presenting Cells for Efficient … · • Julio Cotte • Dawn Maier
Engineering Artificial Antigen Presenting Cells for
Efficient Expansion of T Cell Subsets
Bruce Levine, Ph.D.
Division of Transfusion Medicine
Department of Pathology and Laboratory Medicine
Abramson Cancer Center Translational Research Program
University of Pennsylvania
J Immunol 1997; 159: 5921
Science 1997; 276: 273
Immunol. Rev. 1997; 160: 43
Mol. Ther. 2004; 9; 902
Exp. Opin. Biol. Ther. 2008; 8: 475
Anti-CD3 Anti-CD28
Artificial APC: Bead
Signal 1
Growth
CD28 CTLA4TcR/CD4
+
Magnetic Bead-Based Expansion of
Effector T Cells wo/Feeder Cells
Polyclonal Replicative Potential of
Adult CD4 T Cells In VitroFigure 3
1E+06
1E+07
1E+08
1E+09
1E+10
1E+11
1E+12
1E+13
1E+14
1E+15
1E+16
1E+17
1E+18
1E+19
1E+20
0 10 20 30 40 50 60 70 80 90
Day
To
tal
Cell
s
IL-2 Added Day 49
No IL-2 Day 50
Exponential growth (days) 60-100
Mean log10 growth (fold) 9-12
Mean Pop Doubling 30-40
Interaction of LV-aAPC with CD8+ T Cell
2 microns
CD8 T LV-aAPC
Suhoski, M., Molecular Therapy 2007 May;15(5):981-8
1st and 2nd Generation aAPC for
T Cell Expansion
CD3/28 coated beads
•GMP reagents available
•Safety record in humans, ~800 infusions
Cell Based aAPC
•Ability to use natural ligands
•Multiple costimulatory signals
•Secretion of cytokines
•Antigen specific expansion
CD3/28 coated beads Cell Based aAPC
Preparation
-purchase Dynal/Invitrogen ClinExVivo
or
-coat “naked” beads w/mAbs, QC
Preparation
-expand in culture
-clear non-specific Ab from CD64
-irradiate
-load anti-CD3 (CD28) mAb
1st and 2nd Generation aAPC for
T Cell Expansion
Master Cell Bank Qualification- Quality control
Sterility Testing- to test for absence of bacteria, fungi, mycoplasmas, viruses other than lentiviral vector (CMV, HIV-1 & 2, HTLV1 &2, EBV, HBV, HCV, etc).
Identity Testing- to establish that all significant properties of the cells are stable and maintained throughout the entire manufacturing process. Includes lentiviral transgene expression and RCL testing.
Purity Testing- to detemine identity and quantity of other cells- determine % of target aAPCs in MCB
Functional Testing- to determine potency of aAPCs in stimulating Tregs (as measured by CD4+, CD25+, and FoxP3 expression, functional assay)
Closed System Production of MCB and WCB
Source Cells
Expand in Wave
Harvest and
Cryopreserve MCB
Thaw and
Expand in Wave
Antibody Clearance
Irradiation
Anti-CD3 mAb Loading
Cryopreserve WCB
Tregs are uncommon: rigorous in vitro selection
process is required.
Tregs are hypoproliferative: ex vivo culture can
select for effector T cells
Establishing the best aAPC to expand human PB
and UCB Tregs: Cell based aAPCs are superior
to beads in expanding functional T reg cells
Challenges of Treg Isolation,
Expansion, and Testing
Adult Peripheral Blood Cord Blood
Adult CD25+ Selected Cord CD25 Selected
CD4
CD
25
Godfrey, W. R. et al. Blood 105:750-758, 2005
Cell-Based aAPCs are Superior to CD3/28 Bead Expanded
Cord Blood TREGS in Suppression of CD8+ T Cell Proliferation
No beads control
+ CD3 beads control
+ Tregs 1/2
+ Tregs 1/8
+ Tregs 1/4
CD3/CD28 Beads KT64 86 aAPC
K562 aAPC Effector T Cell Expansion
Expansion of CD8+ T cells by KT.A2.41BBL.64
0
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0 5 10 15 20 25
Po
pu
lati
on
Do
ub
lin
g Le
vel
Day of Culture
KTA2.41BBL.64
KTA2.41BBL.64 (fresh/cryo)
CD3/CD28 beads
restimulation
K562 aAPC’s Cell Bank StatusResearch grade aAPC’s
cGMP WCB
BMF 4/09
KT64:86
“Treg” aAPC
cGMP MCB
KTA2.41BBL.64
“CTL” aAPC
cGMP MCB
IND #1 IND #2
Clinical
Trials
Clinical
Trials
GLP WCB’s
-validations
-to collaborators
cGMP WCB
BMF
(future)
IND #1 IND #2
Clinical
Trials
Clinical
Trials
-validations
-to collaborators
-BMF’s
-IND’s
-Clinical Trials
aAPC Line #3
GMP MCB
GLP/GMP
WCB’s prn
PENN - TRP• Tatiana Golovina
• Samik Basu
• Xiaochuan Shan
• Rong Liu
• Tatiana Mikheeva
• Carl June
• Jim Riley
PENN - CVPF• Andrea Brennan
• Anne Chew
• Julio Cotte
• Dawn Maier
• Dan Powell
• Tamara Tripic
• Ashley Vogel
• Zoe Zheng
Univ. Minnesota• Stephen Porter
• Keli Hippen
• Paul Harker-Murray
• Aryel Londer
• Megan Bina
• Bruce Blazar
• Claudio Brunstein
• Margaret Macmillan
• David McKenna
• Diane Kadidlo
UCSF, BSRI• Jeff Bluestone
• Greg Szot
• Amy Putnam
• EJ Read
Univ. Florida• Mark Atkinson
NCI, DTM• Crystal Mackall
• David Stroncek
• Naomi Taylor
• Marianna Sabatino
• Mark Dudley
NCI R01-CA-105216
NCI Intramural B2B
JDRF
Becton Dickinson