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Cord Blood Natural Killer Cells for Immunotherapy
- 1. Cord Blood Natural Killer Cells for Immunotherapy Nina Shah, M.D. Assistant Professor Department of Stem Cell Transplantation and Cellular Therapy M.D. Anderson Cancer Center Houston, TX
- 2. OBJECTIVES • NK cell tumor immunity • Generation of CB NK cells • Modulating the NK response • Clinical implications
- 3. BACKGROUND: NATURAL KILLER (NK) CELLS • Non T/B cytotoxic lymphocytes • CD56+/CD3- • Mechanisms of action • ADCC • Killer Immunoglobulin-like Receptor (KIR)-MHC Class I mismatch
- 4. NCRs + ± + KIRs, CD94 2B4, NTBA NKG2D ? HLA I CD48? MICA, MICB, ULBPs KIR-HLA I MISMATCH: “MISSING SELF” HYPOTHESIS1 1. Moretta et al, Nature Immunology, 2002 NK Cell Target Cell
- 5. Donor KIR Type (HLA type) Recipient HLA Type 2DL2/3 (C1) C2 homozygous 2DL1 (C2) C1 homozygous 2DL2/3 + 2DL1 (C1 + C2) C1 or C2 homozygous 3DL1 (Bw4) Bw4 negative ALLOREACTIVE DONOR KIR-RECIPIENT HLA I COMBINATIONS
- 6. NK CELLS AND ALLOREACTIVITY • Allo-reactive NK cells predict better outcome in haplo-SCT’s for AML1 • Lower relapse rate in patients transplanted in CR • Better EFS in patients transplanted in relapse or remission • Reduced risk of relapse or death • KIR-ligand incompatibility associated with improved outcome after UCBT for acute leukemia • Improved LFS, OS • Decreased relapse rate • Results more evident for AML 1. Ruggeri et al, Blood 2007 2. Willemze et all, Leukemia, 2009
- 7. WHY DON’T NK CELLS PROMOTE GVHD? Ruggeri et al, Blood, Cells, Mol and Dis, 2004 GVL GVHD Ruggeri et al, Blood, Cells, Mol and Dis, 2004
- 8. ARE ALLOGENEIC NK CELLS SAFE? • Haplo-identical NK cell infusion well-tolerated without evidence of GVHD and some transient CR’s1 • MDACC protocol 2005-0508 and 2010-0099: • AML/MDS, CML pts (15 +5 so far) • Up to 8.24 x 106 CD56+ cells/kg have been infused • No infusional toxicities, grade 3 GVH or delayed engraftment thus far • U of AK experience2: Relapsed/refractory myeloma • N=10 • Haplo-identical KIR-mismatched NK cells with auto-HCT • No GVH • No autograft rejection • 50% nCR +CR; PFS 0-533 days 1. Miller, Blood 2005 2. Shi, Br J Haematol, 2008
- 9. WHY DON’T AUTOLOGOUS NK CELLS WORK? • Altered balance of inhibitory and activating receptors on autologous NK cells1 • Altered ligands on tumor cells - requiring more active NK cells than at baseline2 • Change in distribution of NK cell subpopulations (LN, PB) 3 • Direct immunosuppression by tumor cell –produced soluble factors (cytokines, ligands) 1, 4 • NK cells from MM patients express PD-15 • Increased Class I on MM cells in advanced disease6 1. Lion, Leukemia, 2012 2. Veuillen, JCI, 2012 3. Gibson, Hum Pathol, 2011 4. Reiners, Blood, 2013 5. Benson, Blood, 2010 6. Carbone, Blood, 2005
- 10. CORD BLOOD AS A SOURCE OF NK CELLS • Peripheral blood (PB) • Requires collection • (Mis)-Matching • Cord blood (CB) • Immediate availability • More flexibility in matching • More naïve T cell repertoire
- 11. CB NK CELLS: CHALLENGES • Ability to expand CB NK cells • Expanded CB NK cells have appropriate phenotype • CB NK cells are as active as PB NK cells • Must use frozen cord blood units
- 12. TECHNIQUES OF CB NK CELL EXPANSION
- 13. CB NK CELL EXPANSION: TECHNIQUES • CD56 selection + IL-2 • Feeder cells: K562 antigen-presenting cells expressing Fc-IL-21 or IL-15 +IL-2 • Gas permeable culture system
- 14. IL-21 CD86 4-1BBL CD19 FcγRI IL-15 4-1BBL K562-cl9-mIL21 K562-mb-IL15- 41BBL ARTIFICIAL APC’S Courtesy of Dr. L Cooper Courtesy of Dr. D. Campana
- 15. GAS PERMEABLE EXPANSION FLASKS
- 16. GAS PERMEABLE EXPANSION FLASKS Gas permeable membrane allows for more efficient gas exchange for cells Media automatically feeds cells by convection and diffusion
- 17. Frozen cord Blood unit Ficoll MNC Culture condition: 2(γ-irradiated)APC : 1 cord MNC IL-2 100u/ml GP500 bioreactor Day 7 CD3 depletion (CliniMACS) CD3-depleted NK cells Culture condition: 2(γ-irradiated)APC : 1 CD3 - cell IL-2 100u/ml For another 7 days Day 14 CD3 depletion (CliniMACS) CD3-depleted NK cells CD3+ cells CD3+ cells Flow cytometry on day7 & 14 CD56 CD16 CD3 CD19 CD14 CD45 Clinical NK Expansion From Cryopreserved Umbilical Cord Blood Thaw Day 0
- 18. 1 10 100 1000 10000 Fresh + IL-2 Fresh + APC Frozen + APC FoldExpansionofNKCells A 0.1 1 10 100 1000 10000 0 7 14 NKCellExpansion,x106 Days Fresh + IL-2 Fresh + APC B 0.1 1 10 100 1000 10000 Fold NK increase # NK Cells Produced (x 10e6) # CD3+ Cells (x 10e6) IL-15 IL-21 C * * * n=3 n=10 n=7 P <0.01 P <0.01 * P <0.01 P <0.01 APC CB-NK expansion from fresh or cryopreserved CB units yields significantly greater fold expansion of NK cells than expansion of CD56+ cells with IL-2 alone Fold NK increase Absolute NK (x 10e6) Absolute CD3 (x 10e6) IL-15 2659.5 883.1333333 0.872 IL-21 4093.66 6667 1471 4.495666667
- 19. 10 0 101 102 103 10 4 FL2 H CD(16 56) PE 10 0 10 1 102 10 3 10 4 FL4-H:CD3APC 39.3 0.22 7.5253 10 0 10 1 10 2 10 3 10 4 FL2 H CD(16 56) PE 10 0 101 10 2 103 10 4 FL4-H:CD3APC 0.22 0.39 77.521.9 10 0 10 1 10 2 10 3 10 4 FL2 H CD(16 56) PE 10 0 101 102 10 3 104 FL4-H:CD3APC 0.032 0.15 96.43.43 0 200 400 600 800 1000 0 200 400 600 800 1000 SSC-H:SideScatter 93.6 0 200 400 600 800 1000 0 200 400 600 800 1000 SSC-H:SideScatter 78.6 0 200 400 600 800 1000 0 200 400 600 800 1000 SSC-H:SideScatter 74.8 Day 0 Day 14Day 7 CD3SSC FSC A CD56 aAPC Eomes T-bet B C CD16 NKp30 NKp46 NKp44 KIR2DL1/DS1 KIR2DL2/DL3 KIR3DL 1 NKG2A CD94NKG2C IL-2 alone
- 20. AFTER EXPANSION, CB NK CELLS ARE AS ACTIVE AS PB NK CELLS Pre-Expansion Post-Expansion Xing et al, J of Immunotherapy, 2010
- 21. K562 RPMI 8226 Primary CD138+ cells U266ARP-1 A CD138+ CD138- B APC-expanded NK cells synapse with tumor targets. 0 10 20 30 40 50 60 %NK:TargetSynapseFormation
- 22. A C -10 0 10 20 30 40 50 60 70 80 10:1 1:1 0.1:1 %Cytotoxicity Effecto:Target Ratio K562 RPMI 8226 ARP-1 U266 Autologous 0 5 10 15 20 25 10:1 1:1 0.1:1 %Cytotoxicity Effector:Target Ratio CD138+ B 0 10 20 30 40 50 60 70 80 10:1 1:1 0.1:1 %Cytotoxicity Effector: Target Ratio IL-2 APC APC-expanded NK cells are cytotoxic to various myeloma targets
- 23. 0 5000 10000 15000 20000 25000 30000 35000 40000 8 15 22 ARP-1 ARP-1+ NK Days after ARP-1 injection LuminescenceinROI,p/s 0 500000000 1E+09 1.5E+09 2E+09 2.5E+09 3E+09 3.5E+09 4E+09 4 8 11 15 18 22 ARP-1 ARP-1 + NK P <0.05 at each time point Days after ARP-1 injection Serumkappa,ng/mL d.4 d.8 d.11 d.15 d.18 d.22 ARP-1 ARP-1 +NK P <0.01 at each time point CB-NK cells delay development of myeloma in a NSG murine model
- 24. -8 -7 -5 0 Lenalidomide 10 mg daily High dose melphalan, 200 mg/m2 CB NK cells Autologous graft PHASE I/II STUDY OF UMBILICAL CORD BLOOD-DERIVED NATURAL KILLER CELLS IN CONJUNCTION WITH HIGH DOSE CHEMOTHERAPY AND AUTOLOGOUS STEM CELL TRANSPLANT FOR PATIENTS WITH MULTIPLE MYELOMA -2
- 25. EXPANDED CB NK CELLS ARE ACTIVE AGAINST CLL EX CB NK CLL-B EX CB NK CLL-B EX CB NK CLL-B EX CB NK CLL-B
- 26. Day -8 to -2: lenalidomide 10 mg PO daily Day -7 to -4: Fludarabine 40 mg/m2 IV daily Day -4: Melphalan 140 mg/m2 IV x1 Day -16 to -2 Identify 2 CB units Use 20% fraction of one unit to generate NK cells -16 -2 0 Ex vivo NK generation from 20% fraction Day 0 Infuse unmanipulated CB units 100% CB# 1 80% CB# 2 Day -2 Infuse expanded NK cells Protocol 2011-0493: Pilot study of double cord blood transplantation with ex- vivo expanded cord blood derived natural killer cells to enhance the graft-versus- leukemia effect in patients with relapsed/refractory lymphoid malignancies. NK cell dose: 3- 7 x108 (can be obtained from the 20% fraction of the CB unit) Principal Investigator: Chitra Hosing
- 27. ENHANCING CB NK ACTIVITY: LENALIDOMIDE
- 28. LENALIDOMIDE • Immunomodulatory agent • First-line novel agent in myeloma • Direct anti-tumor effects via apopotosis • Disrupts tumor microenvironment (anti-angiogenic and anti-osteoclastic effects) • Immunomodulatory effects, possibly on NK cells
- 29. Chromium Cytotoxicity Assay Treatment of target myeloma cells with lenalidomide enhances CB NK cytotoxicity
- 30. TREATMENT OF EFFECTOR AND TARGET WITH LENALIDOMIDE ENHANCES CELL KILLING OF PRIMARY CLL 0 10 20 30 40 50 60 70 10:1 1:1 0.1:1 No Revlimid Plus Revlimid p < 0.001 at 1:1 and 10:1 E:T Ratio PercentSpecificLysis
- 31. CAN LENALIDOMIDE REVERSE NK DYSFUNCTION? …Can lenalidomide enhance normal NK function? Ramsay, Blood, 2012
- 32. CB NK CELL -PROJECTS IN DEVELOPMENT
- 33. FUCOSYLATION OF NK CELLS TO IMPROVE HOMING TO BM √ P-, L- & E- Selectin? BM endothelial cell A * * * * * * * * * * * * * * CD34+ CB NK
- 34. NK MM FcR Elotuzumab NK CAR Potential Targets for MM • CS1 • Kappa/ lambda light chain • CD38
- 35. CONCLUSIONS • NK cells are a potential therapy for hematologic malignancies • Autologous NK cells do not provide sufficient anto- tumor activity • CB NK cells may be a safe and effective allogeneic option to shift balance towards GVL and away from GVH • CB NK cells can be expanded to clinical scale • CB NK cells have activity against myeloma and CLL (and other hematologic malignancies)
- 36. ACKNOWLEDGEMENTS Cell Therapy Laboratory Elizabeth J. Shpall Katy Rezvani Ian McNeice Beatriz Martin Antonio Simon Robinson Hong Yang Eric Yvon Amer Najjar Simrit Parmar Michael Thomas Xiaoying Liu Sufang Li Junjun Lu Van Nguyen Indreshpal Kaur Baylor College of Medicine- Center for Cell and Gene Therapy Catherine Bollard Stephanie Ku, Benjamin Tzou Department of Stem Cell Transplantation and Cellular Therapy Richard Champlin Chitra Hosing Muzaffar Qazilbash Department of Pediatrics Laurence Cooper Dean Lee Department of Lymphoma/Myeloma Robert Orlowski Michael Wang Qing Yi Celgene Corporation
- 37. THANK YOU!