Jan Spanholtz Department of Laboratory Medicine Laboratory of Hematology Radboud University Medical Center Nijmegen and Gl...
Adapted from Velardi et al. Curr Opin Immunol 2008, Moretta et al. Immunol Rev 2008 Therapeutic role of alloreative NK cel...
Adoptive transfer of allogeneic NK cells <ul><li>Successful adoptive transfer and in vivo expansion of human haploidentica...
Low cell number T-cell contamination Low cytotoxicity Need extra IL-2 stimulation Low recovery after enrichment Not a stan...
Expansion and differentiation technology for NK cell production expansion differentiation 1 • GBGM + human serum (HS) • SC...
GBGM significantly improved the NK cell generation process  CD56 content  99%  ± 1% using  GBGM Cell expansion  ~50,000 fo...
NK cells cultured in GBGM media display high expression of activating NK cell receptors and mediate a strong cytotoxicity ...
NK cells can be efficiently generated from CB, BM  and mPB derived  CD34+  cells using GBGM medium
Identification of immature stages of  ex vivo -generated NK cells Kaluza™ Analysis Software Gallios™ Flow Cytometer
Acquisition of KIR repertoire of  ex vivo -generated NK cells
GMP-based production of allogeneic NK cell products <ul><li>CliniMACS selection </li></ul><ul><li>CD34 selection </li></ul...
Experimental variables during up-scaling procedure <ul><li>CD34+  selection from frozen UCB </li></ul><ul><li>Container, s...
Efficient CD34+ cell enrichment from cryopreserved UCB units using the CliniMACS system UCB unit Results thawing Results C...
Mean expansion  ~1,300 fold Mean purity 71%  ± 9% Efficient NK cell production using culture bags from  frozen UCB samples...
Efficient NK cell differentiation using the Wave Bioreactor during the differentiation phase CD56 CD34 CD38 CD14 CD117 CD3...
Bag cultures provide sufficient numbers of functional NK cells showing cytotoxicity against various primary AML  NK cells ...
Alloreactive potential of a natural killer-cell subset as a criterion for donor selection C1/C1   Donor cell - + KIR2DL1 N...
KIR-Ligand mismatched NK cell infusion  for elderly AML patients Nijmegen Phase I/II study: 12 patients treated with escal...
<ul><li>Studying of human NK cell development (Poster  Dorit Reiche) </li></ul><ul><li>Submission clinical protocol to eth...
Financial support: Acknowledgements Department of Laboratory Medicine Laboratory of Hematology  & Department of Hematology...
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Freiburg Nk2009 Jan Spanholtz

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  • The experimental setup is as follows: CD34+ expansion for 14 days with expansion medium containing following mixture Followed by NK cell differentiation in a Differentiation medium containing, The NK cell product is characterised at around day 35 of culture by phenotyping and functional analysis.
  • Freiburg Nk2009 Jan Spanholtz

    1. 1. Jan Spanholtz Department of Laboratory Medicine Laboratory of Hematology Radboud University Medical Center Nijmegen and Glycostem Therapeutics Natural Killer (NK) cell immunotherapy in AML using CD34+ derived NK cells
    2. 2. Adapted from Velardi et al. Curr Opin Immunol 2008, Moretta et al. Immunol Rev 2008 Therapeutic role of alloreative NK cells in HLA haploidentical setting D HSC donor NK donor AML patient DC patient T cells patient No GVHD YES GVL
    3. 3. Adoptive transfer of allogeneic NK cells <ul><li>Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Miller et al. Blood, 2005. </li></ul><ul><li>Adoptive transfer of NK-enriched apheresis products after Hi-Cy/Flu conditioning therapy in AML </li></ul><ul><li>Cell dose: up to 2 x 107 cells/kg </li></ul><ul><li>Cell products: 40% ± 2% CD56 + CD3 - NK cells ( range 18%-68% ) </li></ul><ul><li>RESULTS: </li></ul><ul><li>Transient NK engraftment and in vivo expansion </li></ul><ul><li>Hematologic CR in 5 of 19 poor-prognosis AML patients </li></ul><ul><li>BUT: </li></ul><ul><li>T cell administration: 2.1 ± 0.3 (range 0.5-6.5) x 10 5 T cells/kg (risk GVHD) </li></ul><ul><li>B cell contamination: 19% ± 2% (1 patient developed EBV-lymphoma) </li></ul>
    4. 4. Low cell number T-cell contamination Low cytotoxicity Need extra IL-2 stimulation Low recovery after enrichment Not a standardized protocol Need for expansion system Need extra aphaeresis for multiple infusions Bottlenecks to develop sufficient products for NK cell immunotherapy isolated from peripheral blood Low cell number
    5. 5. Expansion and differentiation technology for NK cell production expansion differentiation 1 • GBGM + human serum (HS) • SCF, Flt3L, IL-7, TPO • Clinical grade Heparin CD34+ cells Day 0-9 Day 9-14 Expansion medium: Differentiation medium 2: • SCF, IL-7, IL-2, IL-15 NK progenitors Mature NK cells STEP 1 STEP 2 differentiation 2 Day 14-35 Differentiation medium 1: • SCF, IL-7, Flt3L, IL-15 • Clinical grade Heparin Low-dose cytokine cocktail • Low-dose cytokine cocktail • Low-dose cytokine cocktail • • GBGM + HS • GBGM + HS Formulation of a novel GMP-grade medium designated GBGM (Glycostem Basal Growth Medium) www.glycostem.nl
    6. 6. GBGM significantly improved the NK cell generation process CD56 content 99% ± 1% using GBGM Cell expansion ~50,000 fold using GBGM
    7. 7. NK cells cultured in GBGM media display high expression of activating NK cell receptors and mediate a strong cytotoxicity E:T ratio 2:1 E:T ratio 2:1 E:T ratio 2:1
    8. 8. NK cells can be efficiently generated from CB, BM and mPB derived CD34+ cells using GBGM medium
    9. 9. Identification of immature stages of ex vivo -generated NK cells Kaluza™ Analysis Software Gallios™ Flow Cytometer
    10. 10. Acquisition of KIR repertoire of ex vivo -generated NK cells
    11. 11. GMP-based production of allogeneic NK cell products <ul><li>CliniMACS selection </li></ul><ul><li>CD34 selection </li></ul>Washing & volume reduction NK cell generation + Umbilical cord blood Product release Fresh vs. frozen material Titer plates vs. bags
    12. 12. Experimental variables during up-scaling procedure <ul><li>CD34+ selection from frozen UCB </li></ul><ul><li>Container, surfaces </li></ul><ul><li>Medium </li></ul><ul><li>Cytokines </li></ul><ul><li>Cell inoculation </li></ul><ul><li>Medium refreshment </li></ul><ul><li>Cell density </li></ul>
    13. 13. Efficient CD34+ cell enrichment from cryopreserved UCB units using the CliniMACS system UCB unit Results thawing Results CD34 CliniMACS UCB Volume (ml) NC (x10 6 ) CD34 (%) Recovery (%) CD34 (%) CD34 (x10 6 ) Recovery (%) 1 80 368 0.88 76 52 1.47 50 2 69 469 0.92 69 77 1.99 53 3 75 653 0.47 62 70 2.36 73 4 70 819 1.04 78 92 6.34 73 5 53 583 0.36 56 54 1.74 76 6 74 829 0.30 68 65 1.70 79 7 71 440 0.45 88 64 1.70 82 8 53 403 0.49 68 73 1.42 69 9 80 248 1.04 69 88 1.32 72 range 53-80 248-819 0.3-1.04 56-88 52-92 1.32-6.34 50-82
    14. 14. Mean expansion ~1,300 fold Mean purity 71% ± 9% Efficient NK cell production using culture bags from frozen UCB samples Mean purity 97% ± 2% Mean expansion ~3,500 fold
    15. 15. Efficient NK cell differentiation using the Wave Bioreactor during the differentiation phase CD56 CD34 CD38 CD14 CD117 CD3 CD19 CD15 SSC CD45 NK cell products are devoid of T- and B- cells
    16. 16. Bag cultures provide sufficient numbers of functional NK cells showing cytotoxicity against various primary AML NK cells per experiment fold expansion CD34+ cells CD56+ (%) NK cells CB0109 1770 1.7x10 6 63 1.9x10 9 CB0209 759 1.4x10 6 80 8.6x10 8 CB0309 1291 1.3x10 6 70 1.2x10 9 CB0709 2861 0,81x10 6 95 2.2x10 9
    17. 17. Alloreactive potential of a natural killer-cell subset as a criterion for donor selection C1/C1 Donor cell - + KIR2DL1 NK cell from C1/C1 donor Tolerance Killing C2/C2 Recipient tumor cell HLA-C1 + - +
    18. 18. KIR-Ligand mismatched NK cell infusion for elderly AML patients Nijmegen Phase I/II study: 12 patients treated with escalating NK cell doses AML patient C1/C1 C2/C2 High Cy/Flu conditioning NK cell infusion UCB unit C2/C2 C1/C2 C1/C1 GVL reaction
    19. 19. <ul><li>Studying of human NK cell development (Poster Dorit Reiche) </li></ul><ul><li>Submission clinical protocol to ethical review board (CCMO) </li></ul><ul><li>Up-scaling of UCB-derived NK cells in closed system (Wave Bioreactor) </li></ul><ul><li>CD133+ selection vs. CD34+ selection </li></ul><ul><li>Develop clinical-grade in vivo monitoring method ( 19 F MRI) </li></ul><ul><li>Adoptive transfer studies in Rag2 -/-  c -/- mice bearing human AML </li></ul><ul><li>Monitoring and follow up of NK cell study </li></ul><ul><ul><ul><ul><ul><li>PostDoc position available!! </li></ul></ul></ul></ul></ul>Work in progress and Future perspectives
    20. 20. Financial support: Acknowledgements Department of Laboratory Medicine Laboratory of Hematology & Department of Hematology Radboud University Nijmegen Medical Centre Marleen Tordoir Carel Trilsbeek Jos Paardekoper Bijan Moshaver Frank Preijers Michel Schaap Theo de Witte Harry Dolstra Department of Laboratory Medicine Laboratory of Medical Immunology Radboud University Nijmegen Medical Centre Diana Eissens Arnold van der Meer Irma Joosten Glycostem Therapeutics Dirk Groenewegen
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