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Cancer Immunotherapy as the Breakthrough of the
Year 2013
http://news.sciencemag.org/breakthrough-of-the-year-2013
December 2013, Science
Human NK Cells; Basic Biology
and Therapeutic Applications
Rizwan Romee, MD
• No relevant disclosures
FSC
FSC
SSC
FTOF
NK cells in the peripheral blood
CD45
FSC
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)
CD56right and CD56dim NK Cell Subsets
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
NK cell development and maturation
Caliguiri et al, Trends in
Immunol, 2013
Norell H et al. J Leukoc Biol 2013;94:1123-1139
NK cells express a wide variety of receptors
Vivier, Science, 2012
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
Dynamic regulation of NK Cell Function
Vivier, Science, 2012
Killer immunoglobulin-like receptors (KIRs)
Romagnane 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
Romagne et al, 2013
KIR haplotype A vs. haplotype B
KIR B haplotypes are further classified into:
Cen B, Tel B
Cen B, Tel A
Cen A, Tel B
Romagne et al, Curr Opin Immuno, 2012
Why are KIRs important?
• 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)
Role of KIRs in mediating NK cell allo-reactivity in
hematopoietic stem cell transplantation
KIR ligand mismatch
Handgretinger, ped res, 2012
KIR ligand mismatch is important for mediating NK cell GvL effect and also
potentially increases engraftment and decreases GvHD
Donor B haplotype leads to enhanced GvL effect after HSCT
Cooley et at Blood, 2009 and 2010
Hsu et al, NEJM, 2012
Adoptive transfer of NK cells by Minnesota group
(Miller et al)
Haploidentical Donor:
leukapheresis
Patient s/p lymphodepleting chemo
(Cy/Flu)
Purify/Enrich
NK cells
Leukemia
Remission
IL-2
IL-2
Infuse
NK cells
Long Term
Disease Free Survival
• 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
Miller et al, Blood 2005
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 memorylike 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
CIML NK first in human trial status
• FDA approved / IND granted
• Expect IRB approval this week
• First patient expected to be enrolled next month!
Funding sources
-
SCC R&D award
BJHF award
BJHF/ICTS Team Science Award
ICTS translational award
Two major breakthroughs in the T cell immunotherapy world
BiTE
CAR
Rosenberg et al Nat. Rev. Clin. Oncol., 2013
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
John F. DiPersio, MD, PhD
Fehniger Lab members:
Daniel C. Link, MD
Stephanie Schneider
Jeff Leong
Ryan Sullivan
Max Rosario
Timothy J. Ley, MD
GMP Facility at Siteman Cancer
Center
Meagan Cooper MD, PhD
William Swaney PhD
Jeffrey Miller MD (my mentor at
UMN)
Cancer Immunotherapy as the Breakthrough of the
Year 2013
http://news.sciencemag.org/breakthrough-of-the-year-2013
December 2013, Science
Cancer Immunotherapy as the Breakthrough of the Year
2013
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
December 2013, Science
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 ( 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
Post NK cell infusion monitoring
-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
-Bone marrow minimal residual disease (MRD) was determined by flow
cytometry at enrollment and at 1, 2, and 4 months after the NK infusion
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)
Clinical outcomes
 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% !
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
John F. DiPersio, MD, PhD
Fehniger Lab members:
Daniel C. Link, MD
Stephanie Schneider
Jeff Leong
Ryan Sullivan
Max Rosario
Timothy J. Ley, MD
GMP Facility at Siteman Cancer
Center
Meagan Cooper MD, PhD
William Swaney PhD
Jeffrey Miller MD (my mentor at
UMN)
Complexity of the KIR locus in mammals
Parham et al, 2012