Engineering Artificial Antigen Presenting Cells for Efficient ...

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Engineering Artificial Antigen Presenting Cells for Efficient ...

Engineering Artificial Antigen Presenting Cells forEfficient Expansion of Regulatory T CellsBruce Levine, Ph.D.Division of Transfusion MedicineDepartment of Pathology and Laboratory MedicineAbramson Cancer Center Translational Research ProgramUniversity of PennsylvaniaMagnetic Bead-Based Expansion ofEffector T Cells wo/Feeder Cells +J Immunol 1997; 159: 5921Science 1997; 276: 273Immunol. Rev. 1997; 160: 43Mol. Ther. 2004; 9; 902Exp. Opin. Biol. Ther. 2008; 8: 4751


Polyclonal Replicative Potential ofAdult CD4 T Cells In VitroTotal Cells1E+201E+191E+181E+171E+161E+151E+141E+131E+121E+111E+101E+091E+081E+071E+06IL-2 Added Day 49No IL-2 Day 50Exponential growth (days) 60-100Mean log10 growth (fold) 9-12Mean Pop Doubling 30-400 10 20 30 40 50 60 70 80 90DayInteraction of LV-aAPC with CD8+ T Cell2 micronsCD8 TLV-aAPCSuhoski, M., Molecular Therapy 2007 May;15(5):981-8 2


Challenges of Treg Isolation,Expansion, and TestingTregs are uncommon: rigorous in vitro selection processis required.- What is the right balance between yield and purity?Tregs are hypoproliferative: ex vivo culture can select foreffector T cells-Rapamycin: friend to TregsPurity and potency-What % of FoxP3 expressing cells will be consideredenough for adoptive T cell therapy?Establishing the best aAPC to expand human PB and UCBTregs: Cell based aAPCs are superior to beads in expandingfunctional T reg cellsAdult Peripheral Blood Cord BloodCD25Adult CD25+ SelectedCord CD25 SelectedGodfrey, W. R. et al.Blood 105:750-758, 2005CD43


Potential advantages for umbilical cordblood vs. adult blood as a source of Tregs• Less complexity in the T cell compartment• T cells have longer telomeres• Readily accessible source• Can mediate efficient suppression of thirdpartyalloresponses.• Possible autologous cord blood sourcesChoosing the right aAPC for Treg ExpansionCD3/28 coated beadsGMP reagents availableSafety record in humansCell Based aAPCAbility to natural ligandsMultiple costimulatory signalsStable expressionSecretion of cytokinesAntigen specific expansion4


Treg Expansion with aAPCCD4+25+127-(Miltenyi)Golovina et al. J Immunol. 2008 81(4):2855-68.Foxp3 Expression on In VitroExpanded TregsDay 14Golovina et al. J Immunol. 2008 81(4):2855-68.5


aAPC for Treg ExpansionCD3/28 coated beadsCell Based aAPCPreparation-purchase Dynal/Invitrogen ClinExVivoor-coat “naked” beads w/mAbs, QCPreparation-expand in culture-clear non-specific Ab from CD64-irradiate-load anti-CD3 (CD28) mAbTwo Tiered Cell Banking System6


Closed System Production of MCB and WCBSource CellsExpand in WaveHarvest andCryopreserve MCBThaw andExpand in WaveAntibody ClearanceIrradiationAnti-CD3 mAb LoadingCryopreserve WCBLarge-Scale Validations:Non-Specific Ab ClearanceMouse IgG2a PEMouse IgG1 PELoading media, 24HLoading media, 18HLoading media, 6HLoading media, 4HLoading media, 2HLoading media, 0HK562M, no loading mediaSpherotech beads7


MCB and WCB QC TestingTest Name Test Result Test Name Test ResultSterility Test-Immersion (USP/21CFR 610.12; Sterility including1 Bacteriostasis+Fungistasis Negative 14 HCV Negative2 Mycoplasma (PTC) Negative 15 CMV NegativeIdentification of Cell Line Species3 (Isoenzyme electrophoresis) Human 16 EBV Negative4 In Vitro Adventitious Virus Negative 17 HHV-6 A and HHV-6B Negative5 In Vivo Adventitious Virus Negative 18 HHV-7 NegativePolymerase Chain Reaction-BasedReverse Transcription (PBRT)6 Assay No RT activity 19 HHV-8 NegativeDetection of Bovine Adventitious7 Agents None 20 Parvo B19 NegativeSubcellular Components andDetection of Viral Particles (ThinSections)Negative for viralparticles; subcellularcomponents 21Endotoxin (ChromogenicEndpoint LAL, KineticChromogenic LAL or Gel Clot)8NegativeAnchorageindependent,Replication Competent LentivirusDetermination of Cellularlymphoblastoid,9 (RCL) Negative 22 Morphology and Growth logarithmic growth10 HIV-1 Negative 23 Viability (Trypan blue) ≥90% viable11 HIV-2 Negative 2412 HTLV I & II Negative 2513 HBV NegativeSurface protein stability: CD64,CD86 (Flow cytometry)Proliferation of CD4+ CD25+Tcells following stimulationExpresses proteinsTreg expansionExpanded Treg In Vitro Potency AssayTregs: PBMC1:81:4PBMC control1:21:18


In Vitro Suppression Assay:Day 17 K562 Expanded TregsWCBWCB+RAPACD3 stimulated Effector Cells OnlyUnstimulated Effector Cells Only1: 2 Tregs: Effectors1: 4 Tregs: Effectors1: 8 Tregs: Effectors1:16 Tregs: Effectors1:32 Tregs: Effectors10 0 10 1 10 2 10 3 10 10 0410 1 10 2 10 3 10 4FL1-H: CFSEFL1-H: CFSECell-Based aAPCs are Superior to CD3/28 Bead ExpandedCord Blood T REGS in Suppression of CD8+ T Cell ProliferationCD3/CD28 BeadsKT64 86 aAPCNo beads control+ CD3 beads control+ Tregs 1/2+ Tregs 1/4+ Tregs 1/89


Small Contamination with Effectors:Loss of Suppressive Activity95% CD25brights 100 % CD25 brightsCD3 beadsNo beads1/21/41/81/16FL1-H: CFSEDay 21FL1-H: CFSEK562 aAPC Effector T Cell ExpansionExpansion of CD8+ T cells by KT.A2.41BBL.64Population Doubling Level18161412108642KTA2.41BBL.64KTA2.41BBL.64 (fresh/cryo)CD3/CD28 beadsrestimulation00 5 10 15 20 25Day of Culture10


PENN - TRP• Tatiana Golovina• Samik Basu• Xiaochuan Shan• Rong Liu• Tatiana Mikheeva• Chanelle Case• Gwenn Danet• Carl June• Jim RileyNCI R01-CA-105216NCI Intramural B2BJDRFBecton DickinsonPENN - CVPF• Andrea Brennan• Linda Cheung• Anne Chew• Julio Cotte• Hong Huynh• Lori Landgrebe• Dawn Maier• Dan Powell• Ashley Vogel• Zoe ZhengNCI, DTM• Crystal Mackall• David Stroncek• Naomi Taylor• Marianna Sabatino• Mark DudleyUniv. Minnesota• Stephen Porter• Keli Hippen• Paul Harker-Murray• Aryel Londer• Megan Bina• Bruce Blazar• Claudio Brunstein• Margaret Macmillan• David McKenna• Diane KadidloUCSF, BSRI• Jeff Bluestone• Greg Szot• Amy Putnam• EJ ReadUniv. Florida• Mark Atkinson11

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