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
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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)
Two Tiered Cell Banking System
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
Treg Expansion with aAPC
CD4+25+127-
(Miltenyi)
Golovina et al. J Immunol. 2008 81(4):2855-68.
Foxp3 Expression on In Vitro
Expanded Tregs
Golovina et al. J Immunol. 2008 81(4):2855-68.
Day 14
Expanded Treg In Vitro Potency Assay
1:1
1:2
1:4
1:8
Tregs: PBMC
PBMC control
Cell-Based aAPCs are Superior to CD3/28 Bead Expanded
Cord Blood TREGS in Suppression of CD8+ T Cell Proliferation