Natural Killer Cells: Angels and Devils for Immunotherapy

In e na ional Jou nal o
Molecula Sciences
Re iew
Na u al Kille Cells: Angels and De ils
o Immuno he apy
Bea iz Ma ín-An onio 1,2,*ID , Guille mo Suñe 1,2, Lo ena Pe ez-Amill 1, Ma ia Cas ella 1,2
and Al a o U bano-Ispizua 1,2
1Depa men o Hema ology, Hospi al Clinic, Ins i u d’In es igacions Biomèdiques Augus Pi i
Sunye (IDIBAPS), 08036 Ba celona, Spain; gsune@ca e as esea ch.o g (G.S.); [email p o ec ed] (L.P.-A.);
[email p o ec ed] (M.C.); [email p o ec ed] (A.U.-I.)
2Josep Ca e as Leukaemia Resea ch Ins i u e, 08036 Ba celona, Spain
*Co espondence: [email p o ec ed]; Tel.: +34-93-227-5400 (ex . 4528)
Recei ed: 1 Augus 2017; Accep ed: 19 Augus 2017; Published: 29 Augus 2017
Abs ac :
In ecen yea s, he ele ance o he immune sys em o igh cance has led o he
de elopmen o immuno he apy, including he adop i e cell ans e o immune cells, such as
na u al kille (NK) cells and chime ic an igen ecep o s (CAR)-modi ied T cells. The disco e y o
dono NK cells’ an i- umo ac i i y in acu e myeloid leukemia pa ien s ecei ing allogeneic s em
cell ansplan a ion (allo-SCT) was he igge o conduc many clinical ials in using NK cells.
Su p isingly, many o hese s udies did no ob ain op imal esul s, sugges ing ha many di e en NK
cell pa ame e s combined wi h he bes clinical p o ocol need o be op imized. Va ious pa ame e s
including he high a ay o ac i a ing ecep o s ha NK cells ha e, he sou ce o NK cells selec ed o
ea pa ien s, di e en cy o oxic mechanisms ha NK cells ac i a e depending on he a ge cell and
umo cell su i al mechanisms need o be conside ed be o e choosing he bes immuno he apeu ic
s a egy using NK cells. In his e iew, we will discuss hese pa ame e s o help imp o e cu en
s a egies using NK cells in cance he apy. Mo eo e , he chime ic an igen ecep o (CAR)
modi ica ion, which has e olu ionized he concep o immuno he apy, will be discussed in he
con ex o NK cells. Las ly, he da k side o NK cells and hei in ol emen in in lamma ion will also
be discussed.
Keywo ds: na u al kille (NK); immuno he apy; umo cell su i al mechanisms; in lamma ion
1. Na u al Kille (NK) Cell Modula ion Ac i i y
Na u al kille (NK) cells a e cells o he inna e immune sys em wi h high an i- umo , an i i al
and an imic obial ac i i y. In heal hy indi iduals, 90% o NK cells in pe iphe al blood (PB) a e ma u e
and cy o oxic, and cha ac e ized by he exp ession o CD16
b igh
and CD56
dim
. The emaining 10% o
NK cells ep esen he imma u e subse o NK cells ha a e cy okine p oduce s, and exp ess CD16
dim
o CD16
−
, CD56
b igh
and CD25
+
[
1
]. The ac i i y o NK cells is modula ed by an a ay o inhibi o y
and ac i a ing ecep o s, which a e undamen al in con olling NK cell ac i i y. Whe eas inhibi o y
Kille -cell immunoglobulin-like (KIR) ecep o s inhibi NK cell cy o oxici y by in e ac ing wi h he
Human Leukocy e An igen (HLA)-I in human cells, ac i a ing ecep o s ac i a e NK cells by in e ac ing
wi h hei ligands in a ge cells induced upon umo ans o ma ion, i al in ec ion and cell s ess.
In physiological condi ions, inhibi o y KIR-HLA-I in e ac ion holds NK cell cy o oxici y agains no mal
cells in he body. Howe e , when a KIR-HLA-I misma ch o HLA-I down- egula ion occu s a e
i al in ec ion o in some umo cells, inhibi o y KIRs canno in e ac wi h hei ligands ac i a ing
NK cell cy o oxic unc ion [
2
]. This inding obse ed by Rugge i e al. in 2002 in acu e myeloblas ic
leukemia (AML) pa ien s a e allogeneic s em cell ansplan a ion (allo-SCT), allowed using he
In . J. Mol. Sci. 2017,18, 1868; doi:10.3390/ijms18091868 www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2017,18, 1868 2 o 20
inhibi o y KIR (dono )-HLA-I (pa ien ) misma ch as a p ognos ic ac o in AML pa ien s ecei ing
an allo-SCT [
3
]. This “missing sel ” ecogni ion leads o allo- eac i i y a e allo-SCT, and consequen ly,
NK cells lyse leukemia blas s, ecipien dend i ic cells (DCs) and ecipien T cells, which ansla es
in o a educ ion o elapse, p e en ion o G a s. Hos Disease (GVHD), and p e en ion o g a
ejec ion, espec i ely. The “missing sel ” ecogni ion led o he p oposal o he “missing ligand
model” as a powe ul algo i hm o p edic a po en an i-leukemia e ec and, consequen ly, a a o able
ou come a e allo-SCT [
3
,
4
]. Howe e , in non-myeloid malignancies, such as acu e lymphoblas ic
leukemia (ALL), he GVHD educ ion and p e en ion o g a ejec ion was no always accompanied
by an inc ease o he g a s. leukemia e ec obse ed in AML [
5
]. These con lic ing esul s led o
applying he “missing ligand model” mos ly in AML [
6
], and in non-myeloid malignancies o use
his model jus o NK-media ed killing o DCs and T cells o p edic a educed GVHD and educed
g a ejec ion, espec i ely. The g a s. leukemia e ec occu ing mos ly in AML could be media ed
by HLA down- egula ion, which does no occu in o he malignancies such as ALL. To suppo his,
clinical s udies in ALL con i med ha low HLA-I exp ession le els in blas s con e ed a bene icial
e ec media ed by allo- eac i e NK cells [7].
Howe e , NK cells ha e a high a ie y o ac i a ing ecep o s, which also modula e hei cy o oxic
ac i i y. These ecep o s will play a undamen al ole in he ecogni ion o o he ypes o umo cells
which do no down- egula e HLA-I. Some o hese ecep o s a e exp essed only a gi en s ages o
di e en ia ion o by speci ic NK cell subse s [
8
]. Su p isingly, his high numbe o ac i a ing ecep o s
in NK cells is esponsible o he exis ence o 6000–30,000 di e en pheno ypic NK popula ions
in each heal hy indi idual, which p o ide lexibili y o espond o pa hogens and umo cells [
9
].
Ac i a ing ecep o s include di e en amilies such as he: (i) ac i a ing KIRs which in e ac wi h
TYRO p o ein y osine kinase binding p o ein (DAP-12); (ii) C- ype lec in-like ecep o s, including he
ac i a ing CD94/NKG2C and NKG2D. NKP80, included in his amily, exe s an au onomous con ol
o NK cells agains excessi e in lamma o y esponse causing sel -NK cells media ed cy olysis [
10
–
12
];
(iii) na u al cy o oxici y ecep o s (NCR) which include NKP30, NKP44 and NKP46 and in e ac wi h
ligands o e exp essed on umo cells and i al in ec ed cells; and (i ) signaling lymphocy e ac i a ing
molecule (SLAM) amily o ecep o s which include SLAMF1, 2B4, NTB-A, CD48, CD84, Ly9, and
CRACC. They ansmi ac i a ing signals o media e NK cy o oxici y. Mo eo e , NK cells exp ess CD16,
which is he Fc ecep o ha binds Ig-G media ing an ibody dependen cellula cy o oxici y (ADCC).
2. NK Cell Classic Cy o oxici y Mechanisms
Upon ecogni ion, NK cells elimina e a ge cells apidly (wi hin 30–60 min) by di e en
mechanisms. The wo classic NK cell cy o oxic mechanisms include he dea h ecep o pa hway
and he g anule dependen pa hway. The dea h ecep o pa hway is ac i a ed by he umo nec osis
ac o (TNF)- ela ed apop osis inducing ligand amily (TRAIL) and by Fas-Ligand (FASL) (CD95L)
which a e exp essed on NK cells [
13
], and in e ac wi h hei ligands in a ge cells. In e ac ion o
FASL wi h FAS and TRAIL wi h TRAIL ecep o s allows he o ma ion o a dea h-inducing signaling
complex ha includes Fas-associa ed dea h domain p o ein (FADD), caspase-8, and caspase-10 [
14
].
Ac i a ion o caspase-8 esul s ei he in di ec ac i a ion o he o he caspases o in he p o eolysis o
Bid, wi h elease o cy och ome C and subsequen caspase ac i a ion. E en i i we e belie ed ha
hese ecep o s only ac i a e apop osis, new s udies ha e e ealed ha o he ypes o non-apop o ic
in lamma o y ypes o cell dea h can be ac i a ed [
15
,
16
]. The g anule dependen pa hway is ini ia ed
a e NK cells adhe e o he a ge cell, wi h subsequen deli e y o cy o oxic g anules con aining
pe o in and p o eases called G anzymes owa d he bound a ge cell [
17
]. In humans, he e a e
i e di e en ypes o G anzymes (A, B, H, K and M) and each one will ac i a e di e en cell dea h
pa hways, ei he apop o ic o non-apop o ic. G anzymes A and B a e he mos s udied ones. Whe eas
G anzyme B ac i a es apop osis h ough ac i a ion o caspases o elease o cy och ome C om he
mi ochond ia, appa en ly, G anzyme A ac i a es non-apop o ic cell dea h [
18
]; howe e , he e a e
con lic ing esul s ega ding he cy o oxic ole o G anzyme A [
19
]. The o he G anzymes ha e been
In . J. Mol. Sci. 2017,18, 1868 3 o 20
less s udied, al hough i has been shown ha G anzyme K is eleased by CD56b igh cells media ing
non-apop o ic umo cell dea h [
20
]. G anzyme M shows an i- umo p ope ies a e adop i e NK
cell ans e [
21
]. G anzyme H helps G anzyme B kill adeno i us-in ec ed cells [
22
]; and G anulysin,
which is ano he NK cy o oxic molecule eleased in he cy o oxic g anules, ac i a es Endoplasma ic
Re iculum s ess leading o cell dea h [
23
]. All hese di e en cy o oxici y mechanisms ha NK cells
show, enable hem o elimina e di e en ypes o umo cells wi h hei in insic cha ac e is ics.
3. Immuno he apy S a egies Using NK Cells
As p e iously men ioned, he an i- umo NK cell ac i i y desc ibed a e allo-SCT added o he
inding ha unlike dono T cells, NK cells do no induce GVHD, led o he de elopmen o many
clinical ials in using NK cells in pa ien s ecei ing an SCT. We ha e e iewed published s udies
in using NK cells as an immuno he apy op ion. Resul s a e no op imal; hey a e summa ized in
Table 1. In mos o hese s udies, NK cells we e ac i a ed
in i o
and/o
in i o
wi h In e leukin (IL)2,
and adminis e ed a e immunosupp essi e ea men based on luda abine and cyclophosphamide.
Pa ien disease, disease s a us, numbe o NK cells in used and numbe o NK cell in usions di e ed
om one s udy o ano he . All published s udies ag ee ha NK cell in usion is a sa e and well- ole a ed
p ocedu e and does no associa e o GVHD. Whe eas, o myeloid malignancies, mos s udies used NK
cells om haploiden ical dono s conside ing he KIR-HLA-I misma ch, in non-myeloid malignancies,
NK cells we e ei he au ologous o allogeneic and expanded in i o.
In myeloid malignancies, haploiden ical NK cells wi h KIR-HLA-I misma ch ha e been used ei he
as a consolida ion he apy [
24
,
25
] o in high isk and e ac o y pa ien s [
26
,
27
]. As a consolida ion
he apy, in bo h child en wi h AML and elde ly AML pa ien s, NK cells showed a clea bene i .
A e a median ollow up o ou yea s, pedia ic pa ien s emain in emission, and elde ly pa ien s
wi h high isk AML showed p olonged disease ee su i al (DFS) a e NK cell in usion [
24
,
25
,
28
].
These s udies concluded ha NK cells could be a p omising consolida ion he apy s a egy in pa ien s
who a e no candida es o ecei ing an allo-SCT. Ano he no el consolida ion he apy s a egy in
AML consis ed on he in usion o NK cells de i ed om hema opoie ic p ogeni o cells (HPC) ob ained
om a co d blood (CB) uni . This echnique allowed ob aining enough numbe o NK cells which
we e well ole a ed. Unlike o he p ocedu es, his s udy did no adminis a e IL2 in o pa ien s, and
esponses we e de ec ed, sugges ing ha his echnique allows ob aining e ec i e NK cells which
migh be used o - he-shel when equi ed [29].
In e ac o y pa ien s, esul s a e no good. Howe e , i was no iced ha in usion o haploiden ical
NK cells combined wi h IL2 diph he ia oxin usion p o ein (IL2DT) o deple e hos T- eg cells led
o highe NK cell expansion, imp o ed comple e emission a e and disease- ee su i al wi h no
inc eased oxici y in AML pa ien s [
26
]. Lee e al. also showed du able esponses associa ed wi h CD56+
cells deli e ed in myeloid malignancies [
27
]. Ano he s udy in AML and MDS ound esolu ion o
dysplas ic ea u es in 50% o myelodisplas ic sind ome (MDS) pa ien s, and 16% o comple e emission
(CR) in AML; howe e , hey could no ind NK cells in pe iphe al blood (PB) [
30
]. In pedia ic
e ac o y AML pa ien s who ecei ed o did no ecei e an allo-SCT p e iously, esponses highe
han 50% we e de ec ed a e NK in usion, and combined wi h allo-SCT a e wa ds, 27% and 36% o
DFS we e achie ed, espec i ely, a six yea s [
31
]. Ano he app oach es ed in high isk AML pa ien s,
consis ed in he in usion o haploiden ical NK cells p e iously p imed wi h umo cell lysa es. IL2 was
no adminis a ed o pa ien s, and s ill NK cells seemed o exe an i-leukemia e ec in 57% o he
pa ien s. Howe e , a 2 yea s 85.7% pa ien s died [
32
]. S udies in myeloid malignancies indica ed ha
be e esponses we e achie ed when in using NK cells in pa ien s in emission sugges ing ha NK
cells canno o e come la ge umo bu dens. The e o e, mo e ecen app oaches aim a expanding NK
cells in i o o ob ain a high numbe o NK o la ge umo mass.
In . J. Mol. Sci. 2017,18, 1868 4 o 20
Table 1. Clinical s udies pe o med wi h NK (na u al kille ) cells as immuno he apy ea men .
nPa ien s Disease NK Sou ce T ea men be o e
NK In usion NK Ac i a ion De ec ion o NK
in PB
Median Numbe o
In used NK (×106/Kg)
G a s.
Hos Disease Ou come. Clinical T ial Numbe (Re e ence)
10. AML in CR (pedia ic) Haplo Flu/Cy IL2 pos -in usion Yes 29 No All in emission a 964 days [24]
13. AML: 38.4% in AD, 15.3%
MR, 46% CR Haplo Flu/Cy IL2 pos -in usion Yes 2.74 No
AD: 20% achie ed ansien CR
MR: 100% achie ed CR
CR: 50% DFS a e 34, 32, 18 mon hs.
NCT00799799 [25]
16. AML in CR Haplo Flu/Cy IL2 pos -in usion Yes F om 1.29 o 5.53 No
A 22.5 mon hs: 56% DFS, 44% elapse. Highe NK
cell numbe associa ed o highe DFS.
NCT00799799 [28]
10. AML in CR
Allo NK de i ed
om CD34+ HSPC
om CB
Flu/Cy IL15 and IL2 Yes (in 21%) F om 3 o 30 No 20% became MRD nega i e o 6 mon hs [29]
57. Re ac o y AML (15
ecei ed IL2DT) Haplo Flu/Cy IL2 pos -in usion
Yes: in 10% o
pa ien s, and in 27%
o pa ien s ecei ing
IL2DT)
26 No
CR: 53% (IL2DT) s. 21% (no IL2DT)
DFS: 33% (IL2DT) s. 5% (no IL2DT)
NCT00274846, NCT01106950 [26]
21. AML, MDS, CML Haplo Flu, Bu IL2 p e and
pos -in usion NA F om 0.22 o 8.32 No associa ed o NK
Su i al associa ed wi h CD56+ cells deli e ed;
24% du able CR (no associa ion o KIR-HLA
misma ch). NCT00402558. NCT01390402 [27]
Re ac o y 6: AML, 2: MDS Haplo Flu/Cy IL2 pos -in usion No 10.6 No 16% CR; 83% Disease p og ession. NCT00871689
[30]
29. Pedia ic e ac o y AML
Coho 1: no p io allo-SCT (14)
Coho 2: elapsed a e
allo-SCT (15)
Haplo Clo/E o/Cy IL2 pos -in usion Yes F om 3.5 o 103 No
Coho 1: 71% esponse; 86% unde wen allo-SCT;
36% DFS a 6 yea s.
Coho 2: 66.6% esponse and unde wen allo-SCT;
27% DFS a 6 yea s.
NCT00697671 and NCT00187096 [31]
7. High isk AML Haplo Flu/TBI
Tumo -p imed NK
cells wi h umo
lysa e
Yes 3 doses: 1, 5, 10 No
A 6 mon hs: 42.8% in CR emained in emission,
14% in PR achie ed CR, 28% elapse, 14% died.
A 1 yea : 14% emained in CR.
A 2 yea s: 85.7% died. Median OS: 400 days [32]
10. Relapsed MM Haplo Flu/Mel/Dx IL2 p e and pos
in usion Yes (un il day 14) 1.7 No 50% CR o nea CR, 20% PR, 10% SD and 20% PD
[33]
17. Lymphoma (2), ad anced
solid umo s (15) Allo
Non
immunosupp essi e
egimen
IL2 (MG4101
me hod) Yes
F om 1 o 30 (1 and 3 doses)
No
Lymphoma: 50% SD, 50% PD
Solid umo s: 47% SD, 53% PD. PFS in SD: 4
mon hs. NCT01212341 [34]
5. Relapsed MM Au o Len, Bo
IL2,
K562-mb15-41BBL
cells
Yes (7.5)x2 No
80% disease s abiliza ion; 40–50% educ ion in BM.
NCT02481934 [35]
In . J. Mol. Sci. 2017,18, 1868 5 o 20
Table 1. Con .
nPa ien s Disease NK Sou ce T ea men be o e
NK In usion NK Ac i a ion De ec ion o NK
in PB
Median Numbe o
In used NK (×106/Kg)
G a s.
Hos Disease Ou come. Clinical T ial Numbe (Re e ence)
8. Relapsed MM Au o/Haplo Bo /Cy/Dx/Flu
K562-mb15-41BBL
cells
IL2 pos -in usion
Yes (in 62%) 100 No 28% pa ial esponse [36]
12. Relapsed MM CB Len/Mel K562-mb21-41BBL
cells Yes (in 50%) 4 doses: 5 , 10, 50 and 100 No
83% VGPR, 66% NCR; 33% elapse (a 21 mon hs);
16% dead (a 21 mon hs) [37]
6. Pedia ic e ac o y solid
umo s Haplo Flu/Bu/Thio/Mp IL15 Yes F om 3 o 27 No
66% clinical esponse: 16% VGPR, 33% PR, 16%
SD. A 310 days all pa ien s died. NCT01337544
[38]
14. O a ian
6. B eas Haplo Flu/Cy/TBI (in 7 p ) IL2 p e and
pos -in usion
In 1 pa ien (no
de ec ion associa ed
o T- eg p esence)
21.6 No Toxici y associa ed o TLS. NCT01105650 [39]
61.Hepa ocellula ca cinomoa
C yosu ge y (26)
C yosu ge y+NK (35)
Allo C yosu ge y K562-based sys em NA NA No
Inc eased PFS: 9.1 s. 7.6 mon hs
Inc eased Response a e: 60% s. 46.1%
Inc eased disease con ol a e: 85.7% s. 69.2% [
40
]
7. Me as a ic melanoma
1. Renal cell ca cinoma Au o Flu/Cy IL2 Yes 4.7 No 0% esponse. NCT00328861 [41]
5. CRC (1), .HC (1), RCC (2),
CLL (1) Allo
Ta/Mp (in 2 pa ien s)
IL2 p e and pos Yes F om 1 o 50 No 20% PR [42]
Haplo: haploiden ical; Allo: allogeneic; Allo-SCT: allogeneic s em cell ansplan a ion; Flu: Fluda abine; Bu: Busul an, ATG: An i-Thymocy e Globulin; Ta: Tac olimus, Mx: Me ho exa e;
Cy: Cyclophosphamide; Cs: Cyclospo ine; Len: Lenalidomide; Bo : Bo ezomib; Dex: Dexame hasone; Mel: Melphalan; Clo: Clo a abine, E o: E oposide; Thio: hio epa; Mp:
me hylp ednisolone; TBI: o al body i adia ion; TLS: umo lysis synd ome; BM: bone ma ow; AML: acu e myeloid leukemia; MDS: myelodisplas ic sind ome; CML: ch onic Myeloid
Leukemia; CLL: Ch onic Lymphocy ic Leukemia; NHL: Non-Hodgkin Lymphoma; MM: mul iple myeloma; HC: Hepa ocellula ca cinoma; CRC: colo ec al ca cinoma; RCC: Renal cell
ca cinoma; CB: co d blood; HSPC: hema opoie ic s em p ogeni o cells; VGPR: e y good pa ial esponse; NCR: nea comple e esponse; PR: pa ial esponse; CR: comple e emission; AD:
ac i e disease; MR: molecula elapse; SD: s able disease; PB: pe iphe al blood; PD: p og essi e disease; PFS: p og ession ee su i al; DFS: disease ee su i al; OS: o e all su i al; NA:
in o ma ion no p o ided in he s udy; IL2DT: IL2 diph he ia oxin usion p o ein; NA: in o ma ion no speci ied.

In . J. Mol. Sci. 2017,18, 1868 6 o 20
In non-myeloid malignancies, bo h haploiden ical NK cells and NK cells expanded
in i o
om o he sou ces ha e been used. In usion o haploiden ical NK cells in mul iple myeloma
(MM) elapsed pa ien s, be o e an au ologous-SCT (au o-SCT), ob ained 50% o comple e o nea
comple e esponses [
33
]. Yang e al. expanded allogeneic NK cells
in i o
allowing in usion
o epe i i e adminis a ions o NK cells in ad anced lymphoma and ad anced solid umo s.
They ound ha ac i a ed and expanded NK cells a e also sa e ob aining 47.1% o s able disease.
In e es ingly, hey obse ed ha T- eg cells and myeloid-de i ed supp esso cells we e educed
a e NK adminis a ion [
34
]. In elapsed MM, di e en p o ocols in using
in i o
expanded NK
cells wi h K562 a i icial An igen P esen ing Cells (aAPCs) ha e been es ed. Fo ins ance, Lei as
e al. combining au ologous expanded NK cells wi h an i-MM d ugs (Lenalidomide/Bo ezomib)
showed 80% o disease s abiliza ion and ha he combina ion o NK cells wi h Lenalidomide was
he bes one. They did no adminis a e IL2 in o he pa ien s [
35
]. Szmania e al. in used ei he
au ologous o haploiden ical expanded NK cells combined wi h Bo ezomib-Dexame hasone based
an i-MM ea men , and IL2
in i o
adminis a ion, ob aining much lowe esponses (28% PR) [
36
].
Shah e al. [
37
] in used expanded NK cells de i ed om a CB uni (CB-NK) and combined hem wi h
Lenalidomide be o e au o-SCT. Eigh y- h ee pe cen o pa ien s showed e y good pa ial esponses;
a 21 mon hs, 33% o pa ien s elapsed. These h ee s udies showed ha NK cell
in i o
expansion
using aAPC K562-based sys em is an e icien echnique o ob ain a high numbe o ac i a ed NK cells,
which a e sa e o pa ien s. The NK cells
in i o
expansion echnique wi h aAPC K562-based sys em
can be isualized in Figu e 1.
In . J. Mol. Sci. 2017, 18, 1868 6 o 20
In non-myeloid malignancies, bo h haploiden ical NK cells and NK cells expanded in i o
om o he sou ces ha e been used. In usion o haploiden ical NK cells in mul iple myeloma (MM)
elapsed pa ien s, be o e an au ologous-SCT (au o-SCT), ob ained 50% o comple e o nea comple e
esponses [33]. Yang e al. expanded allogeneic NK cells in i o allowing in usion o epe i i e
adminis a ions o NK cells in ad anced lymphoma and ad anced solid umo s. They ound ha
ac i a ed and expanded NK cells a e also sa e ob aining 47.1% o s able disease. In e es ingly, hey
obse ed ha T- eg cells and myeloid-de i ed supp esso cells we e educed a e NK
adminis a ion [34]. In elapsed MM, di e en p o ocols in using in i o expanded NK cells wi h
K562 a i icial An igen P esen ing Cells (aAPCs) ha e been es ed. Fo ins ance, Lei as e al.
combining au ologous expanded NK cells wi h an i-MM d ugs (Lenalidomide/Bo ezomib) showed
80% o disease s abiliza ion and ha he combina ion o NK cells wi h Lenalidomide was he bes
one. They did no adminis a e IL2 in o he pa ien s [35]. Szmania e al. in used ei he au ologous o
haploiden ical expanded NK cells combined wi h Bo ezomib-Dexame hasone based an i-MM
ea men , and IL2 in i o adminis a ion, ob aining much lowe esponses (28% PR) [36]. Shah e
al. [37] in used expanded NK cells de i ed om a CB uni (CB-NK) and combined hem wi h
Lenalidomide be o e au o-SCT. Eigh y- h ee pe cen o pa ien s showed e y good pa ial
esponses; a 21 mon hs, 33% o pa ien s elapsed. These h ee s udies showed ha NK cell in i o
expansion using aAPC K562-based sys em is an e icien echnique o ob ain a high numbe o
ac i a ed NK cells, which a e sa e o pa ien s. The NK cells in i o expansion echnique wi h
aAPC K562-based sys em can be isualized in Figu e 1.
Figu e 1. Clinical expansion o Na u al Kille (NK) cells om aphe esis p oduc s o co d blood (CB)
uni s. Ac i a ed NK cells can be gene a ed s a ing ei he om mononuclea cells (MNC) o wi h
magne ically selec ed NK cells. Fi s , ei he CB o aphe esis p oduc s a e icolled o ge he MNC,
and hen hey a e ei he added di ec ly o a bio- eac o o subjec ed o magne ic CD56+ selec ion.
These CD56+ cells will be added o he bio- eac o . Then, hey a e expanded in i o o se en days
wi h a i icial an igen p esen ing cells (aAPCs). IL2 is added exogenously e e y o he day. aAPCs
a e K562-based aAPCs exp essing 41BB ligand, CD64, CD86 and ei he memb ane bound IL21 o
IL15. They a e co-cul u ed in a 2:1 aAPC:MNC o NK cells a io. On Day 7, esh aAPCs a e added
again in he same a io and co-cul u ed in he same condi ions o an addi ional se en days. On Day
7 and Day 14, cells a e CD3-deple ed, only in case he expansion was s a ed wi h MNC. Expansion
can be con inued o a o al o 4 weeks epea ing he same p ocedu e. PB: pe iphe al blood.
In he con ex o non-hema ological malignancies, haploiden ical NK cells ha e been used wi h
no op imal esul s. In pedia ic pa ien s wi h e ac o y solid umo s, haploiden ical NK cells we e
in used a e haplo-SCT. No oxic e ec s we e obse ed, and 66% o he pa ien s showed a clinical
Figu e 1.
Clinical expansion o Na u al Kille (NK) cells om aphe esis p oduc s o co d blood (CB)
uni s. Ac i a ed NK cells can be gene a ed s a ing ei he om mononuclea cells (MNC) o wi h
magne ically selec ed NK cells. Fi s , ei he CB o aphe esis p oduc s a e icolled o ge he MNC,
and hen hey a e ei he added di ec ly o a bio- eac o o subjec ed o magne ic CD56+ selec ion.
These CD56+ cells will be added o he bio- eac o . Then, hey a e expanded
in i o
o se en days
wi h a i icial an igen p esen ing cells (aAPCs). IL2 is added exogenously e e y o he day. aAPCs a e
K562-based aAPCs exp essing 41BB ligand, CD64, CD86 and ei he memb ane bound IL21 o IL15.
They a e co-cul u ed in a 2:1 aAPC:MNC o NK cells a io. On Day 7, esh aAPCs a e added again
in he same a io and co-cul u ed in he same condi ions o an addi ional se en days. On Day 7 and
Day 14, cells a e CD3-deple ed, only in case he expansion was s a ed wi h MNC. Expansion can be
con inued o a o al o 4 weeks epea ing he same p ocedu e. PB: pe iphe al blood.
In . J. Mol. Sci. 2017,18, 1868 7 o 20
In he con ex o non-hema ological malignancies, haploiden ical NK cells ha e been used wi h
no op imal esul s. In pedia ic pa ien s wi h e ac o y solid umo s, haploiden ical NK cells we e
in used a e haplo-SCT. No oxic e ec s we e obse ed, and 66% o he pa ien s showed a clinical
esponse. Howe e , all pa ien s had died a e 310 days [
38
]. In adul s wi h ecu en o a ian and
b eas cance , he bene icial e ec o NK cells could no be di e en ia ed om he chemo he apy
egimen [
39
]. Ano he app oach in hepa ocellula ca cinoma combined allogeneic
in i o
expanded
NK cells showing KIR-HLA-I misma ch wi h c yosu ge y. Bene icial e ec s we e de ec ed in e ms
o enhanced immune unc ion, inc eased p og ession ee su i al (PFS) and imp o ed o pa ien s’
quali y o li e [
40
]. In me as a ic melanoma and enal cell ca cinoma, au ologous NK cells did no
show any an i- umo ac i i y. Al hough NK cells pe sis ed in PB hey had los NKG2D exp ession
and needed o be e-ac i a ed wi h IL2 [
41
]. In 2008, Alici e al. also de eloped a echnique o expand
NK cells wi hou he need o eede cells. They managed o ob ain a high numbe o ac i a ed NK
cells s a ing om Pe iphe al Blood Mononuclea Cells (PBMC), by adding an i-CD3 an ibody o
he i s i e days and IL2 [
43
]. A e wa ds, hey compa ed di e en expansion sys ems by using
lasks, cell cul u e bags and bio eac o s and showed ha bio eac o s wi hou he need o eede
cells ob ained he bes esul s [
44
]. A e wa ds, hey used his echnique in a clinical ial wi h i e
e ac o y cance pa ien s who ecei ed dono -de i ed expanded NK cells a e allo-SCT. In one pa ien
(20%) wi h hepa ocellula ca cinoma pa ial esponses we e obse ed wi h ma kedly dec eased se um
alpha- e op o ein le els [
42
]. O in e es , o he ype o eede cells (Eps ein-Ba i us immo alized
lymphoblas oid B-cell lines: EBV-LCL) ha e also been es ed o expand NK cells, also ob aining a high
numbe o ac i a ed NK cells wi h e icien an i- umo ac i i y in mouse melanoma models [
45
].
Mo e in o ma ion abou clinical s udies in using ac i a ed NK cells in o pa ien s has been add essed
in o he e iews [46].
All hese s udies demons a ed ha : (1) he e is a lack o expansion and pe sis ence o NK
cells in PB a long e m, which is due o allo- eac i e T cells elimina ing NK cells [
47
,
48
]; (2) he
nega i e immunosupp essi e e ec o T- eg cells migh be imp o ed wi h he use o IL2DT [
26
];
(3) NK cells migh be a be e choice o consolida ion he apy a he han o e ac o y pa ien s; (4) in
non-myeloid e ac o y malignancies, NK cells do no achie e du able esponses and he KIR-HLA-I
ligand misma ch migh no be always e icien . Mo eo e , a e NK
in i o
expansion, he KIR-HLA-I
misma ch e ec in some occasions can be bypassed, and he exp ession o NK ecep o s become
homogenous because o he expansion. The e o e, in non-myeloid malignancies, ac i a ing ecep o s
could be mo e ele an [
49
,
50
]; (5) di e en expansion echniques such as he aAPC K562-based sys em,
NK cells de i ed om HPC and NK-92 cell line ha e been de eloped o o e come he limi a ion in
ob aining la ge numbe o NK cells o he ea men o la ge umo masses. These echniques allow
ob aining a high numbe o NK cells eady o in use o - he-shel [29].
4. Chime ic An igen Recep o s (CAR) Modi ied NK Cells
Whe eas CAR-T cell he apy has appea ed in he las yea s as a e olu iona y immuno he apy
op ion o he ea men o hema ological malignancies, CAR-modi ied NK cells is a ield s ill unde
de elopmen . A CAR is a chime ic molecule composed o h ee egions: (1) an ex acellula domain
de i ed om he single chain a iable agmen (scFV) o a monoclonal an ibody (mAb), which
edi ec s he speci ici y o T cells owa ds a speci ic a ge exp essed in umo cells wi hou he need
o an igen p esen a ion; (2) a ansmemb ane domain; and (3) an in acellula domain de i ed om
he
ζ
chain o he T cell ecep o (TCR)/CD3 complex which ac i a es he ly ic pa hway o T cells.
Mo eo e , co-s imula o y signaling endodomains (CD28, 4-1BB o OX40) ac i a e T cell p oli e a ion
a e encoun e o he a ge cell. The numbe o co-s imula o y domains can di e be ween he
di e en CAR [
51
]. Fi s clinical s udies in using CAR-T cells showed he e icacy o hese cells in
e ac o y pa ien s [
52
]; consequen ly, an inc easing numbe o clinical s udies in using CAR-T cells
a e cu en ly being execu ed.
In . J. Mol. Sci. 2017,18, 1868 8 o 20
The in insic an i- umo ac i i y o NK cells added o he high numbe o ac i a ing ecep o s
ha ini ia e hei cy o oxic ac i i y would lead us o hypo hesize ha NK cells do no need a CAR.
Howe e , he nega i e clinical esul s in using NK cells, especially in e ac o y pa ien s, indica e ha
o he op ions a e needed. The addi ion o a CAR in o NK cells migh add an addi ional mechanism o
umo cell ecogni ion, speci ically use ul in pa ien s wi h down- egula ion o he ligands equi ed o
ac i a ion o NK ecep o s. Fu he mo e, a e ecognizing he umo cell, he CAR will induce NK cell
p oli e a ion inc easing NK cell pe sis ence in pa ien s.
Howe e , up o da e, only p eclinical s udies using CAR-NK cells ha e been published.
These include CAR-NK a ge ing CD19 and CD20 o B cell malignancies [
53
–
55
], CD5 o T cell
malignancies [
56
], and CD138 and CS1 o MM [
57
,
58
]. In solid umo s, many p eclinical s udies
ha e also been published a ge ing among o he s He 2, GD2 and EGFR o b eas cance , enal cell
ca cinoma, o a ian cance , melanoma, neu oblas oma and glioblas oma [
59
–
62
]. Mos o hese s udies
ha e used NK-92 cells. Howe e , o he NK cell sou ces es ed include NK cells p e iously ob ained
om HPCs [
63
], and NK cells om CB and expanded
in i o
wi h aAPC K562-based sys ems [
53
].
Mo e de ailed in o ma ion abou cu en p e-clinical s udies on-going wi h CAR-NK cells can be ound
in o he e iews [64].
Some ad ances made in hese s udies indica e ha he addi ion o IL15 in o he CAR cons uc
inc eases NK cell pe sis ence
in i o
[
53
]. Mo eo e , he inabili y o NK cells o a ic o umo
si es has been elimina ed by he addi ion o C-X-X mo i chemokine ecep o 4 (CXCR4) in he CAR
cons uc [
65
]. Clinical s udies in using CAR-NK a e e y sca ce up o da e and hey a e s ill ec ui ing
pa ien s. These s udies a ge CD19 o B cell malignancies, CD33 o CD33+ AML and CD7 o
leukemias and lymphomas. Mos o hese s udies use ei he NK-92 cells o NK cells expanded
in i o
wi h aAPC K562-based sys ems. They a e summa ized in Table 2.
Table 2. Clinical s udies on-going in using CAR-NK cells in cance pa ien s.
NCT Numbe Ins i u ion Type o NK/CAR-Co-
S imula o y Domains Disease T ea men /Doses
NCT02892695
Pe sonGen BioThe apeu ics
NK-92
An i-CD19-CD28, 4-1BB
Relapsed/ e ac o y
ALL, CLL, FL, BCL,
DLBCL
NK be o e SCT
NCT02944162
Pe sonGen BioThe apeu ics
NK-92
An i-CD33-CD28, CD137
Relapsed/ e ac o y
CD33+ AML NK on Days 0, 3 and 5
NCT02742727
Pe sonGen BioThe apeu ics
NK92
An i-CD7- CD28, 4-1BB
Relapsed/ e ac o y CD7
posi i e leukemias and
lymphomas
NK
NCT03056339
M.D.Ande son Cance Cen e
CB-NK expanded wi h K562-mb21
An i-CD19, 4-1BB, CD28, iCasp9,
IL15
B-cell malignancies: ALL,
CLL, NHL
Day-5 o -3: Flu, Cy, Mesna
Day 0: NK
AP1903 in case o CRS
o GVHD
NCT02839954
Pe sonGen BioThe apeu ics NA
Relapsed/ e ac o y
Muc1 posi i e
solid umo s
NA
NCT01974479
Na ional Uni e si y Heal h
sys em, Singapo e
Haploiden ical NK expanded wi h
K562-mb15-41BBL
An i-CD19, 4-1BB
Re ac o y ALL NK
NCT00995137
S . Jude Child en‘s Resea ch
Hospi al
Haploiden ical NK expanded wi h
K562-mb15-41BBL
An i-CD19, 4-1BB
Re ac o y ALL NK
ALL: acu e lympoblas ic leukemia; CLL: ch onic lymphocy ic leukemia; FL: ollicula lymphoma; BCL: B cell
lymphoma; MCL: man le cell lymphoma; DLBCL: di use la ge cell lymphoma; AML: acu e myeloid leukemia;
NHL: non Hodgkin Lymphoma; Flu: luda abine; Cy: cyclophosphamide; NA: in o ma ion no speci ied
5. CB De i ed NK Cells (CB-NK): A Sou ce o Highly Ac i a ed NK Cells Which Ini ia e a
T ansmissible Cy o oxici y
The aAPC K562-based sys em used o expand NK cells appea s as one o he p e e ed echniques
o expand NK cells in all published s udies. These cells can ha e ei he memb ane-bound IL15 o IL21,
In . J. Mol. Sci. 2017,18, 1868 9 o 20
which a e equi ed o NK cell di e en ia ion [
66
]. The e o e, his sys em, s a ing om mononuclea
cells, allows ob aining a la ge numbe o di e en ia ed NK cells. We ha e used his echnique o
expand NK cells om a CB uni . These NK cells a e e med CB de i ed NK cells (CB-NK). A he end o
he
in i o
expansion, a la ge numbe o highly ac i a ed NK cells a e ob ained. The e o e, ully es ed
and HLA- yped NK cells a e a ailable o - he-shel [
50
]. In e es ingly, in physiological condi ions, he
CD56 b igh NK cells a e he imma u e popula ion, wi h longe elome es han CD56dim NK cells [
67
].
Howe e , a e
in i o
expansion, CB-NK become a CD56b igh cell popula ion wi h a homogenous
pheno ype in e ms o inhibi o y KIRs and ac i a ing ecep o s, such as NKG2D and he NCR amily o
ecep o s [
50
]. Mo eo e , CB-NK show longe elome es han eshly ob ained NK cells om CB [
68
],
and a e highly ac i a ed in e ms o bo h cy okine p oduc ion and cy o oxici y [43,64].
We ha e s udied he speci ic cy o oxici y o CB-NK agains MM cells, which exp ess HLA-I, and
compa ed i agains ha o umo cells wi h HLA-I down- egula ion (K562 cells) [
69
]. We obse ed
ha CB-NK cy o oxici y di e s o each ype o umo cell, in e ms o NK cell ecep o s, cy o oxic
molecules and ypes o cell dea h ac i a ed. Whe eas NKG2D and NKP30 ac i a ing NK cell ecep o s
do no ha e any impac in CB-NK cy o oxici y agains K562, o MM cells, hese ecep o s as well
as NKG2D ligands ha e a signi ican ole. Mo eo e , whe eas G anzyme B is in ol ed in CB-NK
cy o oxici y agains K562 cells media ing a Caspase-3 dependen cy o oxici y; in MM cells, G anzyme B
does no impac he CB-NK cy o oxici y, which, in addi ion, is Caspase-3 independen . Mo eo e , his
cy o oxici y agains MM cells in ol es ca hepsin elease om lysosomes, which media e a ype o cell
dea h e med “lysosomal cell dea h”. This dependence on ca hepsins occu s only in MM cells and no
in K562 cells [
69
], indica ing he a ie y o cy o oxic mechanisms ha NK cells can ac i a e depending
on each ype o umo cell.
In e es ingly, when CB-NK and MM cells become in con ac , bo h NKG2D and NKP30 ecep o s
a e ans e ed o MM cells. This ans e o NK cell ecep o s co-localizes wi h lipid s uc u es.
When Filipin-III—a lipid a inhibi o —is added, his NK cell ecep o ans e o MM cells dec eases,
sugges ing a ole o lipid me abolism in con olling he s abili y o hese ecep o s in NK cells.
NK cell ac i a ing ecep o s a e con inuously being ecycled and deg aded by endocy ic pa hway [
70
].
Mo e speci ically, he ans e o NKG2D and NKP30 co-localizing wi h p o eins o he endocy ic
pa hway (Rab1, Rab7 and Rab11) was con i med [
69
]. Impo an ly, he ans e and deg ada ion
o hese ecep o s in MM cells, migh explain why cance pa ien s ha e NK cells ha show wi h
down- egula ion o ac i a ing ecep o s [71,72].
Mo eo e , a e CB-NK con ac , MM cells become s essed and inc ease cell–cell communica ion
be ween hem. This inc eased cell–cell con ac enables a seconda y ans e o NKG2D and NKP30
NK cell ecep o s om MM cells exposed o CB-NK o neighbo ing MM cells non-exposed o
CB-NK. In e es ingly, his seconda y ans e be ween MM cells o CB-NK p o eins ansla es in o
a ansmissible cy o oxici y media ed by MM cells, as ini ial MM cells exposed o CB-NK a e able o
ans e lipid-p o ein esicles o neighbo ing MM cells non-exposed o CB-NK, causing cy o oxici y
in o a p opo ion o hese neighbo ing MM cells (Figu e 2) [69].
In . J. Mol. Sci. 2017,18, 1868 16 o 20
38.
Pe ez-Ma inez, A.; Fe nandez, L.; Valen in, J.; Ma inez-Rome a, I.; Co al, M.D.; Rami ez, M.; Abad, L.;
San ama ia, S.; Gonzalez-Vicen , M.; Si en , S.; e al. A phase I/II ial o in e leukin-15–s imula ed na u al
kille cell in usion a e haplo-iden ical s em cell ansplan a ion o pedia ic e ac o y solid umo s.
Cy o he apy 2015,17, 1594–1603. [C ossRe ] [PubMed]
39.
Gelle , M.A.; Cooley, S.; Judson, P.L.; Gheb e, R.; Ca son, L.F.; A gen a, P.A.; Jonson, A.L.;
Panoskal sis-Mo a i, A.; Cu singe , J.; McKenna, D.; e al. A phase II s udy o allogeneic na u al kille cell
he apy o ea pa ien s wi h ecu en o a ian and b eas cance . Cy o he apy
2011
,13, 98–107. [C ossRe ]
[PubMed]
40.
Lin, M.; Liang, S.; Wang, X.; Liang, Y.; Zhang, M.; Chen, J.; Niu, L.; Xu, K. C yoabla ion combined
wi h allogenic na u al kille cell immuno he apy imp o es he cu a i e e ec in pa ien s wi h ad anced
hepa ocellula cance . Onco a ge 2017. [C ossRe ]
41.
Pa khu s , M.R.; Riley, J.P.; Dudley, M.E.; Rosenbe g, S.A. Adop i e ans e o au ologous na u al kille cells
leads o high le els o ci cula ing na u al kille cells bu does no media e umo eg ession. Clin. Cance Res.
2011,17, 6287–6297. [C ossRe ] [PubMed]
42.
Ba khol , L.; Alici, E.; Con ad, R.; Su lu, T.; Gilljam, M.; S ellan, B.; Ch is ensson, B.; Gu en, H.;
Bjo ks om, N.K.; Sode dahl, G.; e al. Sa e y analysis o ex i o-expanded NK and NK-like T cells
adminis e ed o cance pa ien s: A phase I clinical s udy. Immuno he apy
2009
,1, 753–764. [C ossRe ]
[PubMed]
43.
Alici, E.; Su lu, T.; Bjo ks and, B.; Gilljam, M.; S ellan, B.; Nahi, H.; Quezada, H.C.; Gah on, G.;
Ljungg en, H.G.; Dilbe , M.S. Au ologous an i umo ac i i y by NK cells expanded om myeloma pa ien s
using GMP-complian componen s. Blood 2008,111, 3155–3162. [C ossRe ] [PubMed]
44.
Su lu, T.; S ellan, B.; Gilljam, M.; Quezada, H.C.; Nahi, H.; Gah on, G.; Alici, E. Clinical-g ade, la ge-scale,
eede - ee expansion o highly ac i e human na u al kille cells o adop i e immuno he apy using an
au oma ed bio eac o . Cy o he apy 2010,12, 1044–1055. [C ossRe ] [PubMed]
45.
G anzin, M.; S ojano ic, A.; Mille , M.; Childs, R.; Huppe , V.; Ce wenka, A. Highly e icien IL-21 and
eede cell-d i en ex i o expansion o human NK cells wi h he apeu ic ac i i y in a xenog a mouse
model o melanoma. Oncoimmunology 2016,5, e1219007. [C ossRe ] [PubMed]
46.
Gao, X.; Mi, Y.; Guo, N.; Xu, H.; Xu, L.; Gou, X.; Jin, W. Cy okine-Induced Kille Cells As Pha macological
Tools o Cance Immuno he apy. F on . Immunol. 2017,8, 774. [C ossRe ] [PubMed]
47.
Bisha a, A.; de San is, D.; Wi , C.C.; B au ba , C.; Ch is iansen, F.T.; O , R.; Nagle , A.; Sla in, S. The bene icial
ole o inhibi o y KIR genes o HLA class I NK epi opes in haploiden ically misma ched s em cell allog a s
may be masked by esidual dono -allo eac i e T cells causing GVHD. Tissue An igens
2004
,63, 204–211.
[C ossRe ] [PubMed]
48.
Lowe, E.J.; Tu ne , V.; Handg e inge , R.; Ho wi z, E.M.; Benaim, E.; Hale, G.A.; Wooda d, P.; Leung, W. T-cell
allo eac i i y domina es na u al kille cell allo eac i i y in minimally T-cell-deple ed HLA-non-iden ical
paedia ic bone ma ow ansplan a ion. B . J. Haema ol. 2003,123, 323–326. [C ossRe ] [PubMed]
49.
Kim, S.; Pou sine-Lau en , J.; T usco , S.M.; Lyba ge , L.; Song, Y.J.; Yang, L.; F ench, A.R.; Sunwoo, J.B.;
Lemieux, S.; Hansen, T.H.; e al. Licensing o na u al kille cells by hos majo his ocompa ibili y complex
class I molecules. Na u e 2005,436, 709–713. [C ossRe ] [PubMed]
50.
Shah, N.; Ma in-An onio, B.; Yang, H.; Ku, S.; Lee, D.A.; Coope , L.J.N.; Decke , W.K.; Li, S.; Robinson, S.N.;
Sekine, T.; e al. An igen p esen ing cell-media ed expansion o human umbilical co d blood yields log-scale
expansion o na u al kille cells wi h an i-myeloma ac i i y. PLoS ONE
2013
,8, e76781. [C ossRe ] [PubMed]
51.
Do i, G.; Sa oldo, B.; B enne , M. Fi een yea s o gene he apy based on chime ic an igen ecep o s: “A e
we nea ly he e ye ?”. Human Gene The . 2009,20, 1229–1239. [C ossRe ] [PubMed]
52.
Maude, S.L.; F ey, N.; Shaw, P.A.; Aplenc, R.; Ba e , D.M.; Bunin, N.J.; Chew, A.; Gonzalez, V.E.; Zheng, Z.;
Lacey, S.F.; e al. Chime ic an igen ecep o T cells o sus ained emissions in leukemia. N. Engl. J. Med.
2014,371, 1507–1517. [C ossRe ] [PubMed]
53.
Liu, E.; Tong, Y.; Do i, G.; Shaim, H.; Sa oldo, B.; Mukhe jee, M.; O ange, J.; Wan, X.; Lu, X.;
Reynolds, A.; e al. Co d blood NK cells enginee ed o exp ess IL-15 and a CD19- a ge ed CAR show
long- e m pe sis ence and po en an i- umo ac i i y. Leukemia 2017. [C ossRe ] [PubMed]
54.
Shimasaki, N.; Fujisaki, H.; Cho, D.; Masselli, M.; Lockey, T.; Eld idge, P.; Leung, W.; Campana, D. A clinically
adap able me hod o enhance he cy o oxici y o na u al kille cells agains B-cell malignancies. Cy o he apy
2012,14, 830–840. [C ossRe ] [PubMed]

In . J. Mol. Sci. 2017,18, 1868 17 o 20
55.
Chu, Y.; Hochbe g, J.; Yah , A.; Ayello, J.; an de Ven, C.; Ba h, M.; Czuczman, M.; Cai o, M.S. Ta ge ing
CD20
+
Agg essi e B-cell Non-Hodgkin Lymphoma by An i-CD20 CAR mRNA-Modi ied Expanded Na u al
Kille Cells In Vi o and in NSG Mice. Cance Immunol. Res. 2015,3, 333–344. [C ossRe ] [PubMed]
56.
Chen, K.H.; Wada, M.; Pinz, K.G.; Liu, H.; Lin, K.W.; Ja es, A.; Fi o , A.E.; Shuai, X.; Salman, H.;
Goligh ly, M.; e al. P eclinical a ge ing o agg essi e T-cell malignancies using an i-CD5 chime ic an igen
ecep o . Leukemia 2017. [C ossRe ] [PubMed]
57.
Jiang, H.; Zhang, W.; Shang, P.; Zhang, H.; Fu, W.; Ye, F.; Zeng, T.; Huang, H.; Zhang, X.; Sun, W.; e al.
T ans ec ion o chime ic an i-CD138 gene enhances na u al kille cell ac i a ion and killing o mul iple
myeloma cells. Mol. Oncol. 2014,8, 297–310. [C ossRe ] [PubMed]
58.
Chu, J.; Deng, Y.; Benson, D.M.; He, S.; Hughes, T.; Zhang, J.; Peng, Y.; Mao, H.; Yi, L.; Ghoshal, K.; e al.
CS1-speci ic chime ic an igen ecep o (CAR)-enginee ed na u al kille cells enhance
in i o
and
in i o
an i umo ac i i y agains human mul iple myeloma. Leukemia 2014,28, 917–927. [C ossRe ] [PubMed]
59.
Schon eld, K.; Sahm, C.; Zhang, C.; Naundo , S.; B endel, C.; Odendahl, M.; Nowakowska, P.;
Bonig, H.; Kohl, U.; Kloess, S.; e al. Selec i e inhibi ion o umo g ow h by clonal NK cells exp essing
an E bB2/HER2-speci ic chime ic an igen ecep o . Mol. The . 2015,23, 330–338. [C ossRe ] [PubMed]
60.
Zhang, C.; Bu ge , M.C.; Jennewein, L.; Genssle , S.; Schon eld, K.; Zeine , P.; Ha ingen, E.; Ha e , P.N.;
Mi elb onn, M.; Tonn, T.; e al. E bB2/HER2-Speci ic NK Cells o Ta ge ed The apy o Glioblas oma. J. Na l.
Cance Ins . 2016,108. [C ossRe ] [PubMed]
61.
Seidel, D.; Shibina, A.; Siebe , N.; Wels, W.S.; Reynolds, C.P.; Huebene , N.; Lode, H.N.
Disialoganglioside-speci ic human na u al kille cells a e e ec i e agains d ug- esis an neu oblas oma.
Cance Immunol. Immuno he . 2015,64, 621–634. [C ossRe ] [PubMed]
62.
Genssle , S.; Bu ge , M.C.; Zhang, C.; Oelsne , S.; Mildenbe ge , I.; Wagne , M.; S einbach, J.P.; Wels, W.S.
Dual a ge ing o glioblas oma wi h chime ic an igen ecep o -enginee ed na u al kille cells o e comes
he e ogenei y o a ge an igen exp ession and enhances an i umo ac i i y and su i al. Oncoimmunology
2016,5, e1119354. [C ossRe ] [PubMed]
63.
Lowe, E.; T usco , L.C.; de Oli ei a, S.N. In Vi o Gene a ion o Human NK Cells Exp essing Chime ic
An igen Recep o Th ough Di e en ia ion o Gene-Modi ied Hema opoie ic S em Cells. Me hods Mol. Biol.
2016,1441, 241–251. [PubMed]
64.
Rez ani, K.; Rouce, R.; Liu, E.; Shpall, E. Enginee ing Na u al Kille Cells o Cance Immuno he apy.
Mol. The . 2017,25, 1769–1781. [C ossRe ] [PubMed]
65.
Mulle , N.; Michen, S.; Tie ze, S.; Top e , K.; Schul e, A.; Lamszus, K.; Schmi z, M.; Schacke , G.; Pas an, I.;
Temme, A. Enginee ing NK Cells Modi ied Wi h an EGFR III-speci ic Chime ic An igen Recep o o
O e exp ess CXCR4 Imp o es Immuno he apy o CXCL12/SDF-1
α
-sec e ing Glioblas oma. J. Immuno he .
2015,38, 197–210. [C ossRe ] [PubMed]
66.
Si o i, S.; Can oni, C.; Pa olini, S.; Ma cena o, E.; Con e, R.; Mo e a, L.; Mo e a, A. IL-21 induces bo h
apid ma u a ion o human CD34
+
cell p ecu so s owa ds NK cells and acquisi ion o su ace kille Ig-like
ecep o s. Eu . J. Immunol. 2003,33, 3439–3447. [C ossRe ] [PubMed]
67.
Romagnani, C.; Juelke, K.; Falco, M.; Mo andi, B.; D’Agos ino, A.; Cos a, R.; Ra o, G.; Fo e, G.; Ca ega, P.;
Lui, G.; e al. CD56
b igh
CD16
−
kille Ig-like ecep o
−
NK cells display longe elome es and acqui e
ea u es o CD56dim NK cells upon ac i a ion. J. Immunol. 2007,178, 4947–4955. [C ossRe ] [PubMed]
68.
Denman, C.J.; Senyuko , V.V.; Somanchi, S.S.; Pha a peka , P.V.; Kopp, L.M.; Johnson, J.L.; Singh, H.;
Hu on, L.; Mai i, S.N.; Huls, M.H.; e al. Memb ane-bound IL-21 p omo es sus ained ex i o p oli e a ion
o human na u al kille cells. PLoS ONE 2012,7, e30264. [C ossRe ] [PubMed]
69.
Ma in-An onio, B.; Najja , A.; Robinson, S.N.; Chew, C.; Li, S.; Y on, E.; Thomas, M.W.; Mc Niece, I.;
O lowski, R.; U bano-Ispizua, A.; e al. T ansmissible cy o oxici y o Mul iple Myeloma cells by NK cells
media ed by esicle a icking. Cell Dea h Di e . 2015,22, 96–107. [C ossRe ] [PubMed]
70.
Masilamani, M.; Pe uzzi, G.; Bo ego, F.; Coligan, J.E. Endocy osis and in acellula a icking o human
na u al kille cell ecep o s. T a ic 2009,10, 1735–1744. [C ossRe ] [PubMed]
71.
Hue go-Zapico, L.; Acebes-Hue a, A.; Gonzalez-Rod iguez, A.P.; Con es i, J.; Gonzalez-Ga cia, E.;
Paye , A.R.; Villa-Al a ez, M.; Fe nandez-Guizan, A.; Lopez-So o, A.; Gonzalez, S. Expansion o NK cells
and educ ion o NKG2D exp ession in ch onic lymphocy ic leukemia. Co ela ion wi h p og essi e disease.
PLoS ONE 2014,9, e108326. [C ossRe ] [PubMed]
In . J. Mol. Sci. 2017,18, 1868 18 o 20
72.
Pesce, S.; Tabellini, G.; Can oni, C.; Pa izi, O.; Col ini, D.; Rampinelli, F.; Ma a, J.; Vi ie , E.; Mo e a, A.;
Pa olini, S.; e al. B7-H6-media ed down egula ion o NKp30 in NK cells con ibu es o o a ian ca cinoma
immune escape. Oncoimmunology 2015,4, e1001224. [C ossRe ] [PubMed]
73.
Roge s, R.S.; Bha acha ya, J. When cells become o ganelle dono s. Physiology
2013
,28, 414–422. [C ossRe ]
[PubMed]
74.
Islam, M.N.; Das, S.R.; Emin, M.T.; Wei, M.; Sun, L.; Wes phalen, K.; Rowlands, D.J.; Quad i, S.K.;
Bha acha ya, S.; Bha acha ya, J. Mi ochond ial ans e om bone-ma ow-de i ed s omal cells o
pulmona y al eoli p o ec s agains acu e lung inju y. Na . Med. 2012,18, 759–765. [C ossRe ] [PubMed]
75.
Yasuda, K.; Khanda e, A.; Bu iano skyy, L.; Ma uyama, S.; Zhang, F.; Nasjle i, A.; Goligo sky, M.S. Tunneling
nano ubes media e escue o p ema u ely senescen endo helial cells by endo helial p ogeni o s: Exchange
o lysosomal pool. Aging 2011,3, 597–608. [C ossRe ] [PubMed]
76.
Xie, J.; Liu, M.; Li, Y.; Nie, Y.; Mi, Q.; Zhao, S. O a ian umo -associa ed mic oRNA-20a dec eases na u al
kille cell cy o oxici y by down egula ing MICA/B exp ession. Cell. Mol. Immunol.
2014
,11, 495–502.
[C ossRe ] [PubMed]
77.
Zhang, X.; Rao, A.; Se e, P.; Deibe , C.; Pome an z, A.; Kim, W.J.; Kohanbash, G.; Chang, Y.; Pa k, Y.;
Engh, J.; e al. IDH mu an gliomas escape na u al kille cell immune su eillance by down egula ion o
NKG2D ligand exp ession. Neu o Oncol. 2016,18, 1402–1412. [C ossRe ] [PubMed]
78.
Song, H.; Kim, J.; Cosman, D.; Choi, I. Soluble ULBP supp esses na u al kille cell ac i i y ia
down- egula ing NKG2D exp ession. Cell. Immunol. 2006,239, 22–30. [C ossRe ] [PubMed]
79.
Seme a o, M.; Rusakiewicz, S.; Mina d-Colin, V.; Delahaye, N.F.; Eno , D.; Vely, F.; Ma abelle, A.; Papoula , B.;
Pipe oglou, C.; Ponzoni, M.; e al. Clinical impac o he NKp30/B7-H6 axis in high- isk neu oblas oma
pa ien s. Sci. T ansl. Med. 2015,7. [C ossRe ] [PubMed]
80.
Weil, S.; Memme , S.; Lechne , A.; Huppe , V.; Gianna asio, A.; Becke , T.; Mulle -Run e, A.; Lampe, K.;
Beu ne , D.; Quaas, A.; e al. Na u al Kille G oup 2D Ligand Deple ion Recons i u es Na u al Kille Cell
Immunosu eillance o Head and Neck Squamous Cell Ca cinoma. F on . Immunol.
2017
,8, 387. [C ossRe ]
[PubMed]
81.
Lue, H.; Dewo , M.; Leng, L.; Bucala, R.; Be nhagen, J. Ac i a ion o he JNK signalling pa hway by
mac ophage mig a ion inhibi o y ac o (MIF) and dependence on CXCR4 and CD74. Cell Signal.
2011
,23,
135–144. [C ossRe ] [PubMed]
82.
Shi, X.; Leng, L.; Wang, T.; Wang, W.; Du, X.; Li, J.; McDonald, C.; Chen, Z.; Mu phy, J.W.; Lolis, E.; e al.
CD44 is he signaling componen o he mac ophage mig a ion inhibi o y ac o -CD74 ecep o complex.
Immuni y 2006,25, 595–606. [C ossRe ] [PubMed]
83.
K ockenbe ge , M.; Domb owski, Y.; Weidle , C.; Ossadnik, M.; Honig, A.; Hausle , S.; Voig , H.; Becke , J.C.;
Leng, L.; S einle, A.; e al. Mac ophage mig a ion inhibi o y ac o con ibu es o he immune escape o
o a ian cance by down- egula ing NKG2D. J. Immunol. 2008,180, 7338–7348. [C ossRe ] [PubMed]
84.
Ali, S.A.; Shi, V.; Ma ic, I.; Wang, M.; S oncek, D.F.; Rose, J.J.; B udno, J.N.; S e le -S e enson, M.;
Feldman, S.A.; Hansen, B.G.; e al. T cells exp essing an an i-B-cell ma u a ion an igen chime ic an igen
ecep o cause emissions o mul iple myeloma. Blood 2016,128, 1688–1700. [C ossRe ] [PubMed]
85.
G upp, S.A.; Kalos, M.; Ba e , D.; Aplenc, R.; Po e , D.L.; Rheingold, S.R.; Teachey, D.T.; Chew, A.;
Hauck, B.; W igh , J.F.; e al. Chime ic an igen ecep o -modi ied T cells o acu e lymphoid leukemia.
N. Engl. J. Med. 2013,368, 1509–1518. [C ossRe ] [PubMed]
86.
Ruella, M.; Ba e , D.M.; Kende ian, S.S.; Shes o a, O.; Ho mann, T.J.; Pe azzelli, J.; Klichinsky, M.;
Aikawa, V.; Nazimuddin, F.; Kozlowski, M.; e al. Dual CD19 and CD123 a ge ing p e en s an igen-loss
elapses a e CD19-di ec ed immuno he apies. J. Clin. In es ig.
2016
,126, 3814–3826. [C ossRe ] [PubMed]
87.
Kailayangi i, S.; Al a e , B.; Spu ny, C.; Jami zky, S.; Schelhaas, S.; Jacobs, A.H.; Wiek, C.; Roellecke, K.;
Hanenbe g, H.; Ha mann, W.; e al. Ta ge ing Ewing sa coma wi h ac i a ed and GD2-speci ic
chime ic an igen ecep o -enginee ed human NK cells induces up egula ion o immune-inhibi o y HLA-G.
Oncoimmunology 2017,6, e1250050. [C ossRe ] [PubMed]
88.
Ma ín-An onio, B.; Suñe, G.; Najja , A.; Pe ez-Amill, L.; Velasco-de And és, M.; Lozano, F.; Lozano, E.;
Bueno, C.; Es anyol, J.-M.; Muñoz-Pinedo, C.; e al. Na u al kille cells ans e an imic obial and an i umo al
His one H2AZ o kill mul iple myeloma cells con ibu ing o ansmissible cy o oxici y. Blood
2016
,128, 2115.
In . J. Mol. Sci. 2017,18, 1868 19 o 20
89.
Benson, D.M., J .; Bakan, C.E.; Mish a, A.; Ho meis e , C.C.; E ebe a, Y.; Becknell, B.; Baiocchi, R.A.; Zhang, J.;
Yu, J.; Smi h, M.K.; e al. The PD-1/PD-L1 axis modula es he na u al kille cell e sus mul iple myeloma
e ec : A he apeu ic a ge o CT-011, a no el monoclonal an i-PD-1 an ibody. Blood
2010
,116, 2286–2294.
[C ossRe ] [PubMed]
90.
Ray, A.; Das, D.S.; Song, Y.; Richa dson, P.; Munshi, N.C.; Chauhan, D.; Ande son, K.C. Ta ge ing PD1-PDL1
immune checkpoin in plasmacy oid dend i ic cell in e ac ions wi h T cells, na u al kille cells and mul iple
myeloma cells. Leukemia 2015, 10. [C ossRe ] [PubMed]
91.
Lee, Y.H.; Ma in-O ozco, N.; Zheng, P.; Li, J.; Zhang, P.; Tan, H.; Pa k, H.J.; Jeong, M.; Chang, S.H.; Kim, B.S.;
e al. Inhibi ion o he B7-H3 immune checkpoin limi s umo g ow h by enhancing cy o oxic lymphocy e
unc ion. Cell Res. 2017,27, 1034–1045. [C ossRe ] [PubMed]
92.
Liu, Y.; Cheng, Y.; Xu, Y.; Wang, Z.; Du, X.; Li, C.; Peng, J.; Gao, L.; Liang, X.; Ma, C. Inc eased exp ession o
p og ammed cell dea h p o ein 1 on NK cells inhibi s NK-cell-media ed an i- umo unc ion and indica es
poo p ognosis in diges i e cance s. Oncogene 2017. [C ossRe ] [PubMed]
93.
Talle ico, R.; C is iani, C.M.; S aa , E.; Ga o alo, C.; So ile, R.; Capone, M.; Pico de Coana, Y.; Madonna, G.;
Palella, E.; Woloda ski, M.; e al. IL-15, TIM-3 and NK cells subse s p edic esponsi eness o an i-CTLA-4
ea men in melanoma pa ien s. Oncoimmunology 2017,6, e1261242. [C ossRe ] [PubMed]
94.
Schmid , S.; T amsen, L.; Hanisch, M.; La ge, J.P.; Huenecke, S.; Koehl, U.; Leh nbeche , T. Human na u al
kille cells exhibi di ec ac i i y agains Aspe gillus umiga us hyphae, bu no agains es ing conidia.
J. In ec . Dis. 2011,203, 430–435. [C ossRe ] [PubMed]
95.
Spo i, R.; Jolle , N.; Albe s, U.; Hilbi, H.; Oxenius, A. MyD88-dependen IFN-
γ
p oduc ion by NK cells is
key o con ol o Legionella pneumophila in ec ion. J. Immunol. 2006,176, 6162–6171. [C ossRe ] [PubMed]
96.
Ga cia-Lao den, M.I.; S oo, I.; Te ps a, S.; Flo quin, S.; Medema, J.P.; de Vos, A.F.; an de Poll, T. Exp ession
and Func ion o G anzymes A and B in Esche ichia coli Pe i oni is and Sepsis. Media . In lamm.
2017
,2017,
4137563. [C ossRe ] [PubMed]
97.
Ga cia-Lao den, M.I.; S oo, I.; Blok, D.C.; Flo quin, S.; Medema, J.P.; de Vos, A.F.; an de Poll, T.
G anzymes A and B Regula e he Local In lamma o y Response du ing Klebsiella pneumoniae Pneumonia.
J. Inna e Immun. 2016,8, 258–268. [C ossRe ] [PubMed]
98.
Baschuk, N.; Wang, N.; Wa , S.V.; Halse, H.; House, C.; Bi d, P.I.; S ugnell, R.; T apani, J.A.; Smy h, M.J.;
And ews, D.M. NK cell in insic egula ion o MIP-1
α
by g anzyme M. Cell Dea h Dis.
2014
,5, e1115.
[C ossRe ] [PubMed]
99.
Wei, H.M.; Lin, L.C.; Wang, C.F.; Lee, Y.J.; Chen, Y.T.; Liao, Y.D. An imic obial P ope ies o
an Immunomodula o —15 kDa Human G anulysin. PLoS ONE 2016,11, e0156321. [C ossRe ] [PubMed]
100.
S enge , S.; Hanson, D.A.; Tei elbaum, R.; Dewan, P.; Niazi, K.R.; F oelich, C.J.; Ganz, T.; Thoma-Uszynski, S.;
Melian, A.; Bogdan, C.; e al. An an imic obial ac i i y o cy oly ic T cells media ed by g anulysin. Science
1998,282, 121–125. [C ossRe ] [PubMed]
101.
Lu, C.C.; Wu, T.S.; Hsu, Y.J.; Chang, C.J.; Lin, C.S.; Chia, J.H.; Wu, T.L.; Huang, T.T.; Ma el, J.;
Ojcius, D.M.; e al. NK cells kill mycobac e ia di ec ly by eleasing pe o in and g anulysin. J. Leukoc. Biol.
2014,96, 1119–1129. [C ossRe ] [PubMed]
102.
Dalbe h, N.; Callan, M.F. A subse o na u al kille cells is g ea ly expanded wi hin in lamed join s.
A h i is Rheuma ol. 2002,46, 1763–1772. [C ossRe ] [PubMed]
103.
An hony, D.A.; And ews, D.M.; Chow, M.; Wa , S.V.; House, C.; Aki a, S.; Bi d, P.I.; T apani, J.A.; Smy h, M.J.
A ole o g anzyme M in TLR4-d i en in lamma ion and endo oxicosis. J. Immunol.
2010
,185, 1794–1803.
[C ossRe ] [PubMed]
104.
Sha ma, M.; Me kulo a, Y.; Rai ha ha, S.; Pa kinson, L.G.; Shen, Y.; Coope , D.; G an ille, D.J. Ex acellula
g anzyme K media es endo helial ac i a ion h ough he clea age o p o ease-ac i a ed ecep o -1. FEBS J.
2016,283, 1734–1747. [C ossRe ] [PubMed]
105.
Wensink, A.C.; Kemp, V.; Fe mie, J.; Ga cia Lao den, M.I.; an de Poll, T.; Hack, C.E.; Bo enschen, N.
G anzyme K syne gis ically po en ia es LPS-induced cy okine esponses in human monocy es. P oc. Na l.
Acad. Sci. USA 2014,111, 5974–5979. [C ossRe ] [PubMed]
In . J. Mol. Sci. 2017,18, 1868 20 o 20
106.
Jiang, W.; Chai, N.R.; Ma ic, D.; Bieleko a, B. Unexpec ed ole o g anzyme K in CD56b igh NK
cell-media ed immuno egula ion o mul iple scle osis. J. Immunol.
2011
,187, 781–790. [C ossRe ] [PubMed]
107.
San iago, L.; Menaa, C.; A ias, M.; Ma in, P.; Jaime-Sanchez, P.; Me ka , S.; Comas, L.; E ill, N.;
Gonzalez-Rumayo , V.; Esse , E.; e al. G anzyme A Con ibu es o In lamma o y A h i is in Mice Th ough
S imula ion o Os eoclas ogenesis. A h i is Rheuma ol. 2017,69, 320–334. [C ossRe ] [PubMed]
108.
Deng, A.; Chen, S.; Li, Q.; Lyu, S.C.; Claybe ge , C.; K ensky, A.M. G anulysin, a cy oly ic molecule, is also
a chemoa ac an and p oin lamma o y ac i a o . J. Immunol. 2005,174, 5243–5248. [C ossRe ] [PubMed]
109.
Falschlehne , C.; Emme ich, C.H.; Ge lach, B.; Walczak, H. TRAIL signalling: Decisions be ween li e and
dea h. In . J. Biochem. Cell Biol. 2007,39, 1462–1475. [C ossRe ] [PubMed]
110.
Ba nha , B.C.; Legemb e, P.; Pie as, E.; Bubici, C.; F anzoso, G.; Pe e , M.E. CD95 ligand induces mo ili y
and in asi eness o apop osis- esis an umo cells. EMBO J. 2004,23, 3175–3185. [C ossRe ] [PubMed]
111.
Qadi , A.S.; Ceppi, P.; B ockway, S.; Law, C.; Mu, L.; Khoda e , N.N.; Kim, J.; Zhao, J.C.; Pu zbach, W.;
Mu mann, A.E.; e al. CD95/Fas Inc eases S emness in Cance Cells by Inducing a STAT1-Dependen Type I
In e e on Response. Cell Rep. 2017,18, 2373–2386. [C ossRe ] [PubMed]
112.
Ha wig, T.; Mon ina o, A.; on Ka s ed , S.; Se ko, A.; Su ino a, S.; Chak a a hy, A.; Ta abo elli, L.;
D abe , P.; La on , E.; A ce Va gas, F.; e al. The TRAIL-Induced Cance Sec e ome P omo es
a Tumo -Suppo i e Immune Mic oen i onmen ia CCR2. Mol. Cell.
2017
,65, 730–742. [C ossRe ]
[PubMed]
113.
Su, Z.; Yang, Z.; Xie, L.; DeWi , J.P.; Chen, Y. Cance he apy in he nec op osis e a. Cell Dea h Di e .
2016
,
23, 748–756. [C ossRe ] [PubMed]
114.
Del Fabb o, E.; Sko o, N.; B ian, C. The p e alence o cachexia among pa ien s wi h solid and hema ologic
malignancies a a Na ional Cance Ins i u e (NCI)-designa ed cance cen e in he 12 mon hs p io o dea h.
J. Clin. Oncol. 2014,32. [C ossRe ]
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