Natural Killer cells and Hematopoietic Stem Cell Transplantation Jeffrey S. Miller, M.D.

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1 Natural Killer cells and Hematopoietic Stem Cell Transplantation Jeffrey S. Miller, M.D. University of Minnesota Cancer Center Associate Director of Experimental Therapeutics Division of Heme/Onc/Transplant Minneapolis, MN

2 NK cells are important Cancer treatment and tumor surveillance Infection disease control Autoimmunity Pregnancy (placental angiogenesis) NK cell functions Killing targets Produce cytokines Interferon-γ Tumor necrosis factor Many others

3 NK cells after transplant are increased = Normal DONOR = RECIPIENT Percentage Donor and Recipient NK cells (TCD vs. UBM) NK % NK Cells TCD UBM Cooley et al Blood 106:4370, 2005

4 Chr. 19 determines the personality of NK cells: Killer-immunoglobulin receptor (KIR) gene locus Ligand: HLA-C N80 HLA-C N80 HLA-C K80 HLA-G HLA-Bw4 HLA-C K80 HLA-A KIR: 3DL3 2DL3 3DL1 2DS2 2DL2 2DL5B 2DS3 2DP1 2DL1 3DP1 2DL4 2DL5A 2DS4 2DS5 2DS1 3DL2 3DS1 No. of Alleles: NKG2 family recognizes HLA-E From Peter Parham

5 NK cells are very different after URD HCT Donor Recipient % CD56 % CD % KIR % CD56 60% % % KIR % KIR Recipients have < KIR > CD56 +bright > NKG2A < Function Cooley et al Blood 110:578, %CD % %CD % % NKG2A % NKG2A % NKG2A

6 NKG2A/KIR expression distinguishes populations of CD56 +dim NK cells A B C ADULT BLOOD.fcs ADULT BLOOD.fcs BRIGHT 1% 5% ADULT BLOOD.fcs DIM 42% 12% 8% 86% 16% 30% CD3 PerCP-Cy5-5-H NK2Ga PE-H NK2Ga PE-H KIR - /NKG2A - subset: 19.4 ± 2.8% of CD56 +dim NK cells healthy donors (n=26) These cells do not kill targets or make IFN, thus are hyporesponsive (immature) Cooley et al Blood 110:578, 2007.

7 Hypothesized NK cell development schema NK precursors left shifted into the blood after transplant? HSC=CD34 + /Lin - /CD38 - Marrow Lymphoid committed progenitor CD34 + /CD7 - CD34 + /CD7 + CD56 - CD56 - NK cell precursor NKp CD34-/CD7 + CD56 - NK cell commitment NK CD56 + KIR - LN Stage 4 IFN-γ producing NK NK CD56 +bright KIR - Blood Stage 5 & 6 MatureFc + cytotoxic NK NK CD56 +dim KIR + KIR CD94

8 Absolute Count per µl Outpatient subcutaneous IL-2 promotes in vivo NK cell expansion but NK cells are not maximally activated NK T 100 Pre-IL Days On IL2 Miller et al, Biol Blood Marrow Transplant 3:34, 1997

9 Autologous NK Administration in Cancer Patients Recovery from autologous HCT IL-2 PB IL-2 NK IL-2 NK cells more activated using this approach

10 NK cell-based autologous Immunotherapy to prevent relapse (HD, NHL, BC) Burns et al, Bone Marrow Transplant, 32: , 2003 Conclusions Enhanced activation of NK cells. A matched paired analysis with our data and data from the IBMTR showed no apparent efficacy (survival or time to disease progression).

11 Hypothesis: Autologous NK cell therapy failed due to inhibitory receptors that recognize MHC Auto Allo NK NK apoptosis KIR - MHC class I match-> No Killing To Kill or not to kill KIR - MHC mismatch-> Lysis occurs

12 AML Transplant trials based on promoting NK cell alloreactivity Transplant Graft Outcome Ruggeri et al Science 3/2002 Haploidentical KIR-L Mismatch TCD Benefit in AML Davies et al URD UBM No Benefit Blood 11/2002 KIR-L Mismatch Giebel et al URD In Vivo TCD Benefit Blood 8/2003 KIR-L Mismatch

13 How can we best exploit NK cells? Adoptive Transfer? Transplant Pros and cons Safer Transient Can expand in vivo (IL-2) More TRM Permanent Too risky 2 o GVHD risk

14 Related Donor Haploidentical NK Infusions After High Dose Chemotherapy HD Rx Cy 60 mg/kg x 2 Flu 25 mg/m 2 x 5 PB NK 2-8 x 10 7 MNC/kg TCD IL-2 IL-2 10 MU QOD x 6

15 Patients and eligibility Poor prognosis AML (n=19) Primary refractory disease Relapsed disease not in CR after 1 or more cycles of standard re-induction therapy Secondary AML from MDS Relapsed AML > 3 months after HCT. No active infections

16 KIR Ligand mismatched donor correlates with achieving AML CR (5 of 19=26%) P= KIR L Match n=15 KIR L MM n=4 % Achieving CR KIR L MM KIR L Match

17 CR patients had higher numbers of functional NK cells after haplo NK cells % NK cells at Day p=0.027 % NK cells NK cells did not expand with lower dose preparative regimens CR non-cr Miller et al, Blood 105:3051, 2005

18 In vivo expansion of haploidentical NK cells in AML After cell infusions No Donor D1 D2 D7 D14 D28 H2O HLA-B7 B-act 100% 10% 1% 0.1% 0.01% 0.001% No Donor PB CD56 + PB CD3 + PB CD19 + BM CD56 + BM CD3 + H2 0 Donor Specific HLA-A31 ß-actin

19 Circulating donor cells were functional NK cells 14 days after Haplo NK cell infusions 92% % Specific Lysis K562 Raji Effector:Target Ratio Verified by VNTR and G-banding

20 Hi-Cy/Flu induces in vivo expansion of donor cells (all patients by prep) 30 Renal - Flu AML Hi-Cy/Flu 20 % Donor Day after NK cell infusion

21 Hi-Cy/Flu induces endogenous IL-15 which correlates with in vivo NK cell expansion IL-15 Concentration (pg/ml) ** Cy/Flu Flu 20 0 Pre Day after cell infusion

22 Interpretation of cytokine data Every time we give lymphodepleting chemotherapy (±TBI), we see a sustained surge in endogenous IL-7 and IL-15 May explain high fevers when adding exogenous IL-2 in this setting.

23 Questions Why NK cells don t expand in everyone? Would other cell sources be superior to adult blood NK cells?

24 Hypothesis The best strategy may be to combine adoptive transfer and in vivo expansion followed by HCT Adoptive Transfer + Transplant The best of both worlds?

25 Umbilical Cord Blood ml cord blood Usually discarded High concentration of hematopoietic and NK cell progenitors Stem cell source for related donor transplant

26 Cord blood is rich in NK cell precursors FRESH UCB Testing this population clinically CD3 PerCP FRESH UCB 20.0% 2.3% 8.4% CD7 PE **Percents shown are based on the total CD3 depleted product as the denominator

27 Full Prep Triple UCB strategy: UCB NK + double UCB Transplant for patients with refractory AML Pablo Rubinstein New York Blood Center KIR-L MM if possible UCB 1 HLA < 2 ag mm HLA < 2 ag mm TNC >1.5 x 10 7 /kg UCB 2 HLA < 2 ag mm TNC >1.5 x 10 7 /kg THAW THAW CD3 deplete IL-2 Overnight (1000 U/ml) UCB 3 HLA < 2 ag mm TNC >1.5 x 10 7 /kg Patient Eligibility Age 2-45 years Refractory AML BMBx FLU CY FLU CY FLU TBI BMBx BMBx BMBx IL-2 x 6 doses for In vivo NK expansion TIME CsA (day -1 to 6 months) MMF (day -1 to day 35 unless GVHD) 28

28 Conclusions NK cells are important in cancer therapy and transplant. Better methods to optimally activate NK cells are still needed for refractory AML patients. KIR genotyping may be of value in selecting donors in addition to HLA-typing.

29 Acknowledgements Miller Lab Purvi Gada Veronika Bachenova Valarie McCullar (Research) Gong Yun Karen Peterson Michelle Pitt Todd Lenvik Becky Haack Feng Xiao Sue Fautsch (Translational) Sarah McNearney Rosanna Warden Kirsten Malvey Liz Narten Michelle Gleason Ginny Kohl HLA typing lab - Harriet Noreen NMDP/CIBMTR (Confer, Klein, Wang, Spellman, Maiers) U of MN Faculty Phil McGlave Arne Slungaard Linda Burns John Wagner Claudio Brunstein Bruce Blazar Dave McKenna (GMP Facility) Chap Le/Tracy Bergemann (Biostat) Dan Weisdorf, Sarah Cooley, MD Stanford Peter Parham Univ of Washington Dan Geraghty Children s Hospital Oakland Beth Trachtenberg London Steve Marsh NK PPG working group