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Cancer Immunotherapy as the Breakthrough of the Year 2013

December 2013, Science

Human NK Cells; Basic Biology and Therapeutic Applications

Rizwan Romee, MD

• No relevant disclosures

FSC

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CD45SS

CFSC

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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)

CD56bright 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)

CD56dim 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

CD56right and CD56dim NK Cell Subsets

Norell H et al. J Leukoc Biol 2013;94:1123-1139

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-E5. 2B4 binds CD486. 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

LeukemiaRemission

Long TermDisease 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

Modified from Romee et al, Blood 2012

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;2013:247-253

©2013 by American Society of Hematology

Acknowledgments

Todd Fehniger, MD, PhD

Fehniger Lab members:

Stephanie SchneiderJeff LeongRyan SullivanMax 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)

http://news.sciencemag.org/breakthrough-of-the-year-2013

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:AMLN=10Median 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 -7Flu dose: 25mg/m^2/day for 5 doses (-6 to -2)

Donor apheresis on day -1Apheresis 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 ( 0.097 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 performedon days 2, 7, 14, 21, and 28 after transplantation

-Chimerism studies of immunologically sorted NK cells were performed bystandard 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

-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 weeksOne 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 SchneiderJeff LeongRyan SullivanMax 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