Engineering Artificial Antigen Presenting Cells for Efficient Expansion of T Cell Subsets
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1 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
2 Magnetic Bead-Based Expansion of Effector T Cells wo/feeder Cells Artificial APC: Bead Anti-CD3 Anti-CD28 TcR/CD4 Signal 1 CD28 CTLA4 + Growth 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
3 Total Cells Polyclonal Replicative Potential of Adult CD4 T Cells In Vitro Figure 3 1E+20 1E+19 1E+18 1E+17 1E+16 1E+15 1E+14 1E+13 1E+12 1E+11 1E+10 1E+09 1E+08 1E+07 1E+06 IL-2 Added Day 49 No IL-2 Day 50 Exponential growth (days) Mean log10 growth (fold) 9-12 Mean Pop Doubling Day
4 Interaction of LV-aAPC with CD8+ T Cell 2 microns CD8 T LV-aAPC Suhoski, M., Molecular Therapy 2007 May;15(5):981-8
5 1 st and 2 nd 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
6 1 st and 2 nd Generation aapc for T Cell 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
7 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)
8 Two Tiered Cell Banking System
9 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
10 Challenges of Treg Isolation, Expansion, and Testing 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
11 CD25 Adult Peripheral Blood Cord Blood Adult CD25+ Selected Cord CD25 Selected Godfrey, W. R. et al. Blood 105: , 2005 CD4
12 Treg Expansion with aapc CD (Miltenyi) Golovina et al. J Immunol (4):
13 Foxp3 Expression on In Vitro Expanded Tregs Day 14 Golovina et al. J Immunol (4):
14 Expanded Treg In Vitro Potency Assay PBMC control Tregs: PBMC 1:8 1:4 1:2 1:1
15 Cell-Based aapcs are Superior to CD3/28 Bead Expanded Cord Blood T REGS in Suppression of CD8+ T Cell Proliferation CD3/CD28 Beads KT64 86 aapc No beads control + CD3 beads control + Tregs 1/2 + Tregs 1/4 + Tregs 1/8
16 Population Doubling Level K562 aapc Effector T Cell Expansion Expansion of CD8+ T cells by KT.A2.41BBL KTA2.41BBL.64 KTA2.41BBL.64 (fresh/cryo) CD3/CD28 beads restimulation Day of Culture
17 K562 aapc s Cell Bank Status Research grade aapc s KT64:86 Treg aapc cgmp MCB KTA2.41BBL.64 CTL aapc cgmp MCB aapc Line #3 GMP MCB cgmp WCB BMF 4/09 IND #1 IND #2 Clinical Trials Clinical Trials cgmp WCB BMF (future) GLP WCB s -validations -to collaborators IND #1 IND #2 Clinical Trials Clinical Trials GLP/GMP WCB s prn -validations -to collaborators -BMF s -IND s -Clinical Trials
18 PENN - TRP Tatiana Golovina Samik Basu Xiaochuan Shan Rong Liu Tatiana Mikheeva Carl June Jim Riley NCI R01-CA NCI Intramural B2B JDRF Becton Dickinson PENN - CVPF Andrea Brennan Anne Chew Julio Cotte Dawn Maier Dan Powell Tamara Tripic Ashley Vogel Zoe Zheng NCI, DTM Crystal Mackall David Stroncek Naomi Taylor Marianna Sabatino Mark Dudley 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
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