Cancer Immunotherapy as the Breakthrough of the Year 2013 December 2013, Science

Human NK Cells; Basic Biology and Therapeutic Applications Rizwan Romee, MD

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FSC FTOF CD45 SSC FSC NK cells in the peripheral blood

Major NK cell functions  Secrete immunoregulatory cytokines (e.g. IFN-γ)  Kill virus-infected cells  Key contribution to human reproduction  Exhibit anti-tumor responses  Longitudinal study correlated low NK cell activity with increased risk of developing cancer Imai et al, Lancet, 2000  Cancer patients have defective NK cell number and/or function  Kill cancer target cells (e.g., leukemic blasts) without prior sensitization  Key mediators of ADCC (Rituximab, Cetuximab etc)

CD56 bright NK cells: Immature, constitute only 5-15% of the peripheral blood NK cells (dominant population in lymph nodes) Low expression of FcγR3a (CD16) Exhibit weak cytokine and cytotoxic responses to tumor target cells (in the resting state) CD56 dim NK cells: Mature, dominant population in peripheral blood High CD16 expression (and other maturity markers) Exhibit potent cytokine and cytotoxic responses to tumor target cells CD56 right and CD56 dim NK Cell Subsets

Norell H et al. J Leukoc Biol 2013;94: NK cell development and maturation Caliguiri et al, Trends in Immunol, 2013

Vivier, Science, 2012 NK cells express a wide variety of receptors

Key inhibitory NK cell receptors and their ligands 1.KIRs (killer cell Ig-like receptors) 2.NKG2A binds HLA-E Key activating NK cell receptors and their ligands 1.CD16 (FcγRIIIA),, binds Fc part of IgG to mediate ADCC 2.NCRs : NKp30 binds B7-H6 (expressed on certain tumor cells) NKp44 binds MLL5 NKp46 binds ? 3.NKG2D binds MIC A/B and ULBP proteins 4.NKG2C binds HLA-E 5.2B4 binds CD48 6.DNAM-1 binds PVR and Nectin-2

Vivier, Science, 2012 Dynamic regulation of NK Cell Function

Killer immunoglobulin-like receptors (KIRs) Romagnane et al, 2013

Romagne et al, 2013 C1 vs. C2 HLA-C alleles classified based on amino acid polymorphisms at positions 80 (asp vs. lysine) of alpha-1 chain

KIR haplotype A vs. haplotype B Romagne et al, Curr Opin Immuno, 2012 KIR B haplotypes are further classified into: Cen B, Tel B Cen B, Tel A Cen A, Tel B

Presence of KIR2DS2 associated with decreased B cell ALL in children (Ahmad et al, Blood 2012) Increased ALL incidence in children with Haplo A/A ( Wiemels et al Blood 2014) KIR3DS1 in presence of its cognate HLA-Bw4 leads to slower HIV progression (Carrington et al, Nat genetics, 2002) KIR2DS1 in presence of its cognate ligand HLA-C2 promotes placentation (Moffet et al, JCI 2013) Why are KIRs important?

Role of KIRs in mediating NK cell allo-reactivity in hematopoietic stem cell transplantation Handgretinger, ped res, 2012 KIR ligand mismatch

KIR ligand mismatch is important for mediating NK cell GvL effect and also potentially increases engraftment and decreases GvHD

Cooley et at Blood, 2009 and 2010 Hsu et al, NEJM, 2012 Donor B haplotype leads to enhanced GvL effect after HSCT

Purify/Enrich NK cells IL-2 Leukemia Remission Long Term Disease Free Survival Haploidentical Donor: leukapheresis Infuse NK cells Patient s/p lympho- depleting chemo (Cy/Flu) IL-2 Adoptive transfer of NK cells by Minnesota group (Miller et al)

Miller et al, Blood 2005 Only some success with AML patients Limited expansion and persistence of the transferred cells But some dramatic responses Responses correlated with expansion of the NK cells Preferential expn of Tregs

Other adoptive NK cell trials Autologous NK cells ( Childs group at NIH, McKenna at UMN) KIR-Ligand mismatched NK cells (Roberto M. Lemoli, Italian group) Post transplant NK cell infusions (Rizzeri et al Duke) Post induction NK cell consolidation (Leung et al St Jude’s) Mixed results Limited persistence and expansion of the adoptively transferred NK cells IL-2 side effects and preferential Treg expansion

NK cells exhibit memory! Hapten specific memory by von Andrian group (Nat Immunol, 2006) Hapten hypersensitivity seen in Rag2 -/- mice (Lack T or B cells but have NK cells) This hapten specific memory was transferred upon adoptive transfer Only liver NK cells expressing CXCR6 MCMV specific memory by Lanier group (Nature, 2009) In B6 mice MCMV infection caused preferential expansion of Ly49H+ cells Upon re-challenge with MCMV more robust response Able to transfer this enhanced response upon adoptive transfer of Ly49H+ cells from mice previously injected with MCMV

CMV infection in humans induces memory- like NK cells  Preferential expansion of NKG2C + CD57 + cells after solid and Stem cell transplantation (Lanier et al, PNAS 2010, Miller et al Blood 2010)  These NKG2C + CD57 + cells exhibit enhanced function  Similar population of cells also expands in patients with Hanta virus (Ljunggren JEM, 2011) and Chikungunya virus ( Vincent Vieillard Plos Path, 2011)

Cytokine activation induced memory-like NK cells in mice Pre-Activation (or control) Rest (return to ‘basal’ state) Restimulation Enhanced IFN-γ Response Cooper et al. … Yokoyama PNAS, 2009

Modified from Romee et al, Blood 2012

Romee et al, ASH Abstract, 2013

CIML NK cells express high affinity IL-2 receptor Leong et al, BBMT, 2014

Pre-clinical xenogenic mouse model

Mouse model of CIML NK cell immunotherapy Cerwenka et al, JEM, 2012

So can we use the concept of cytokine induced memory-like NK cells in clinic?

Correlative science planned (Fehniger Lab) -Persistence -Look for expansion -Activation -Functional analyses -KIR ligand mismatch impact on outcomes

FDA approved / IND granted Expect IRB approval this week First patient expected to be enrolled next month! CIML NK first in human trial status Funding sources -SCC R&D award -BJHF award -BJHF/ICTS Team Science Award -ICTS translational award

Rosenberg et al Nat. Rev. Clin. Oncol., 2013 CAR Two major breakthroughs in the T cell immunotherapy world BiTE

BiKE- and TriKE-mediated NK cell targeting to tumor-associated antigens. Miller J S Hematology 2013;2013: ©2013 by American Society of Hematology

Acknowledgments Todd Fehniger, MD, PhD Fehniger Lab members: Stephanie Schneider Jeff Leong Ryan Sullivan Max Rosario Meagan Cooper MD, PhD John F. DiPersio, MD, PhD Daniel C. Link, MD Timothy J. Ley, MD GMP Facility at Siteman Cancer Center William Swaney PhD Jeffrey Miller MD (my mentor at UMN)

Cancer Immunotherapy as the Breakthrough of the Year 2013 December 2013, Science

Cancer Immunotherapy as the Breakthrough of the Year 2013 December 2013, Science The Runner-ups -CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) -CLARITY (Makes brain imaging super clear) -Human Stem Cells From Cloning (By SCNT) -Mini-organs (mini-organoids) -Cosmic Particle Accelerators identified -Perovskite Solar Cells (Graphene based high effn solar cells) -Why We Sleep (Studies with mice showed that the brain cleans itself — by expanding channels between neurons and allowing more cerebrospinal fluid to flow through — much more efficiently during sleep. The finding suggests that restoration and repair are among the primary purposes of sleep) -Our Microbes, Our Health -In Vaccine Design, Looks do Matter

Acute Myeloid Leukemia (AML) is a bad disease  Treatment includes Induction chemotherapy with 7+3 and once in complete remission, consolidation with 3-4 cycles of Ara-C  Prognosis determined by cytogenetics, mutational status and prior chemo/rxt (secondary and therapy related)  Up to 50% of the AML patients relapse (most relapses in first two years)  Chemotherapy alone:30% of the favorable risk and 54% of the intm risk  Allo-HSCT is a curative option but TRM, GVHD and relapse still a problem (20 % TRM and 40-50% relapse rate)  Urgent need to develop novel therapies

Pilot study with the intention to determine feasibility and safety of using NK cell adoptive transfer in patients with AML Patient characteristics: AML N=10 Median age of the patients was 2.5 years (range: 8 months to 21 years) Favorable and intm risk ( because unfavorable risk patients underwent transplant) 4 CBF+ AML, 6 had intermediate risk All MRD negative at the time of enrollment (s/p consolidation chemotherapy)

Cy dose: 60 mg/kg bw on day -7 Flu dose: 25mg/m^2/day for 5 doses (-6 to -2) Donor apheresis on day -1 Apheresis product processed on day 0 with 2 step process using Miltenyi’s CliniMACS NK cell product infused on day 0 with no prior IL-2 stimulation IL-2 (1million U/m^2) given subcutaneously for 6 doses on alternate days starting on day -1

NK cell product characteristics Median NK cell dose of 29 x 10^6/kg bw (range : 5 to 81 x 10^6/kg bw) Minimal B cell contamination ( x 10^6/kg bw) T cells detected in only one product but very low (1 x 10^3/kg bw, log fold lower than the threshold for inducing GVHD

9/10 patients with KIR-ligand mismatch

- NK cell chimerism, phenotyping, and functional assays were performed on days 2, 7, 14, 21, and 28 after transplantation -Chimerism studies of immunologically sorted NK cells were performed by standard variable number of tandem repeats techniques -NK cell phenotyping was determined by direct measurement of surface expression of KIRs by flow cytometry -Cytotoxicity of peripheral blood NK cells was measured in vitro by europium release assays -Bone marrow minimal residual disease (MRD) was determined by flow cytometry at enrollment and at 1, 2, and 4 months after the NK infusion Post NK cell infusion monitoring

Toxicity Well tolerated both chemo, IL-2 and NK cell infusions One patient had swelling and erythema at the IL-2 injection site One patient had a viral URI One needed to be hospitalized for 2 days with neutropenic fevers Median time for neutrophil recovery (ANC > 500) was 12 days (9-56 days) All patients had neutrophil and platelet recovery by day 21, except one patient whose ANC recovered after 56 days and platelets after 127 days None of the patients developed GVHD

NK cell numbers post adoptive transfer

NK cell numbers post adoptive transfer in individual patients

Donor NK cell engraftment / chimerism All patients with transient NK cell engraftment (Median 10 days, range : 2 to 189 days) Median peak NK chimerism of 7% ( range : 1 to 30%) 3 patients had stable donor NK cells at 4 weeks One patient had prolonged NK engraftment (2% at on day +189)

 Median follow up of 964 days (range: 569 to 1,162 days)  None of these patients relapsed  All remained MRD negative (at 1, 2, and 4 months)  2-year even free survival of 100% ! Clinical outcomes

Summary  Nicely demonstrates the safety and feasibility of this approach  Potentially could replace HSCT and its complications  Major critique -Small patient numbers (10) -Only favorable and intm risk patients -Included only pediatric patients -Only transient NK cell engraftment demonstrated -No information on NK cell homing -No information on Treg expansion

Acknowledgments Todd Fehniger, MD, PhD Fehniger Lab members: Stephanie Schneider Jeff Leong Ryan Sullivan Max Rosario Meagan Cooper MD, PhD John F. DiPersio, MD, PhD Daniel C. Link, MD Timothy J. Ley, MD GMP Facility at Siteman Cancer Center William Swaney PhD Jeffrey Miller MD (my mentor at UMN)

Parham et al, 2012 Complexity of the KIR locus in mammals