Cancer Immunotherapy Targeting

Cancer and Innate Immune Cells

Hun Sik Kim

University of Ulsan

College of Medicine

2011 춘계면역학회

Problems of traditional cancer therapies

Many therapies are aiming at stopping or slowing the growth of tumor, but not eradicating it. This may lead to long-term remission with developing a resistance to the drug.

Moreover, chemotherapy is nearly of no use to control metastasis and involves a series of side effects including disruption of the body’s immune system.

Contribution of the immune system to the

success of anti-tumor therapy

The importance of immune system for the efficacy of anti-tumor radiotherapy in mice is shown.

More frequent tumor (both spontaneous and carcinogen-induced) development in immunodeficient mice.

Harnessing the immune system to treat cancer

Developing approaches to manipulate conventional cancer

therapies to prime immunity is now an exciting area of

investigation.

Boosting the immune system to battle cancer

In order to completely eradicate cancer cells, the immune system must be activated at a stage at which the tumor has been reduced to a minimum using conventional therapy.

Consideration of immunomodulatory effects of

dying cells associated with traditional therapies

Chemotherapy and radiotherapy can target tumor cells

for apoptotic destruction.

Apoptotic cells lead to immunosuppression after uptake

by macrophages, while necrotic cells induce

inflammatory responses.

Therefore, chemotherapeutic agents able to induce

immunogenic cancer cell death have a great potential to

optimise anticancer immunotherapy.

Modulation of the immune system by dying cells

What receptor is responsible for sensing DAMPs to induce pro-inflammatory response?

DAMPs: damage-associated

molecular patterns

Nat Rev Immunol, 2008

Toll-like receptor 2

senses -cell death

and contributes to

the initiation of

autoimmune diabetes

Kim et al., Immunity 27,

321-333 August 2007

Cover Paper

Essential role of TLR2 but not TLR4 in pro-

inflammatory response after primary or secondary

necrotic cell encounter

0

1000

2000

3000

4000TLR2+/+

TLR2-/-

UV STZ Eto FT PN PGN LPS

SN

TN

F-

(p

g/m

l)

0

200

400

600

800

1000TLR2+/+

TLR2-/-

_SN PNApop

Primary MEF

TN

F-

(p

g/m

l)

Does antigen presenting cells from TLR KO mouse

affect antigen-specific T cell proliferation in vivo?

SN cells trigger TLR2-dependent stimulation of

APC and priming of antigen-specific T cells in PLN

TLR2

HMGB1 mediates endogenous TLR2 activation and brain tumor regression,

Curtin et al., PLOS Med, 2009, 6, e1000010.

Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate

metastasis, Kim et al., Nature, 2009, 457, 102.

Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-

dependent immunosuppressive function of mouse and human myeloid-

derived suppressor cells, Chalmin et al., J Clin Invest, 2010, 120, 457.

High-mobility group box 1 is involved in the initial events of early loss of

transplanted islets in mice, Matsuoka et al., J Clin Invest, 2010, JCI41360.

TLR4

Toll-like receptor 4-dependent contribution of the immune system to

anticancer chemotherapy and radiotherapy, Apetoh et al., Nature Med, 2007,

13, 1050.

CD36 ligands promote sterile inflammation through assembly of a Toll-like

receptor 4 and 6 heterodimer, Stewart et al., Nature Immunol, 2010, 11, 155.

Immunogenic cell death associated with

stimulation of Toll-like receptors

Induction of immunogenic cell death and its use in

cancer immunotherapy-focusing on TLR signaling

Cancer therapy Autoimmune diseases

Alzheimer’s disease

Allograft rejection

Ischemia injury

Development of innate

and adaptive immunity

Boosting

immunity

by targeting

TLR and its

associated

signaling

Gasser, S. et al. 2006 Cancer Res., 66, 3959

Tumor cells responding to chemotherapy or radiotherapy

express ‘danger’ signals to stimulate innate killer cells

NK cell functions

Natural killer (NK) cells are lymphocytes of the innate immune system that are able to kill tumor cells and infected cells without prior sensitization.

NK cells regulate adaptive immune responses by activating DCs, macrophages and T cells.

Eric Vivier et al. 2008 Nature Immunol., 9, 503

NK cell effector functions

NK cells as a promising therapeutic

target in human cancer

Cancer stem cell – Drug resistant and radioresistant

NK cells recognize and kill human glioblastoma cells with stem cell-like

properties, Castriconi et al., J Immunol, 2009, 182, 3530.

Primitive quiescent CD34+ cells in chronic myeloid leukemia are targeted

by in vitro expanded NK cells, which are functionally enhanced by

bortezomib, Yong et al., Blood, 2009, 113, 875.

NK cells kill human melanoma cells with characteristics of cancer stem

cells, Pietra et al., Int Immunol, 2009, 21, 793.

Regulatory T cells – Immunosuppression

NK cells lyse T regulatory cells that expand in response to an

intracellular pathogen, Roy et al., J Immunol., 2008, 180, 1729.

Myeloid derived suppressor cells – Immunosuppression

Mononuclear myeloid-derived "suppressor" cells express RAE-1 and

activate NK cells, Nausch et al., Blood, 2008, 112, 4080.

Antagonistic effect of NK cells on alternatively activated monocytes: a

contribution of NK cells to CTL generation, Geldhof et al., Blood, 2002,

100, 4049.

Missing-self hypothesis

- NK

No Kill

Normal

NK

Kill

Cancer

MHC Class I

Karre et al. Nature 1986

Integration of inhibitory and activating NK cell

receptor signals regulates NK decision to kill

The dynamic regulation of NK cell effector function

E Vivier et al. Science 2011;331:44-49

The NK cell-target cell ‘zipper’

Eric Vivier et al. 2008 Nature Immunol., 9, 503

How signals from discrete receptors are integrated to determine functional outcome?

Inhibitory signaling via SHP-1 recruitment to ITIMs

I T I

M

KIR

Src kinase P

P

SHP

-1

P

P

SHP

-1

Activation

Slide by Carsten Watzl

ITIM consensus:

V/IxYxxL

Burshtyn et al., 1996 Immunity

MHC I

Activation receptors

Adapters Receptors

ITAMs: FcR , TCR CD16 (FcRIII)

FcR , TCR , DAP12 NCRs (NKp46, NKp44, NKp30)

DAP12 Activating KIR, Ly49, CD94/NKG2C

DAP10: DAP10 NKG2D

No adapter: 2B4

DNAM-1 (CD226)

CD2

NKp80

CD27 (TNFR family)

etc.

Integrins: L2 (LFA-1)

41 (VLA-4)

ITAM and NKG2D signaling pathway

ITAM-coupled receptor

Degranulation

Syk

ZA

P70

Y

Y

- P

P

+ LA

T

3BP2

SLP76 Vav PI3K

PLC2

PIP3 PIP2 DAG

IP3

Ca2+ Rac, Cdc42

ERK

Actin reorganization

PKC

Ras

SOS

Grb2

PI3K

PIP3

Vav1 Grb2

ERK Adhesion,

Polarization

NKG2D/

DAP10

PIP2

CrkL

Rac, Cdc42 Rap1

The multiple receptor-ligand interactions between NK cells and

target cells make it difficult to dissect the function of individual

NK receptors.

By employing insect cells as surrogate target cells, the specific

ligands of NK cell receptors can be expressed to test NK cell

activation.

Drosophila cell line as a customized target

for human NK cells

CD48

Apart from CD16 (FcRIIIA), which is sufficient for activation of

resting NK cells, natural cytotoxicity by resting NK cells is only

induced by mutual costimulation of activating receptors, such as

NKG2D and 2B4.

Bryceson YT et al.

2006 Immunol. Rev.,

214, 73

Synergy-dependent activation of natural cytotoxicity in

human resting NK cells

NKG2D and 2B4

synergy as a

platform model for

NK cell activation

- NKG2D plays a

critical role in

immunosurveillance

of certain tumor and

infected cells.

NKp46

2B4

CD2

NKG2D

DNAM-1

CD16

ADCC Natural Cytotoxicity

Degranulation

Cytokine release

Degranulation

Cytokine release

Synergy

Degranulation

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1.30

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13.4

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0.41

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19.2CD

56

CD107a

rNK alone S2-ULBP1 S2-ULBP1+CD48

S2-CD48 S2+IgGS2

100 101 102 103 104

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%ofM

ax

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fM

ax

%fM

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oa

x

S2-ULBP1+CD48S2-CD48

S2 S2-ULBP1

cIgGULBP1CD48

S2 Target cells

Synergistic activation of resting NK cells induced

by NKG2D and 2B4 ligands

Imaging live NK cells on lipid bilayers by

total internal reflection fluorescence (TIRF) microscopy

Imaging live NK cells on lipid bilayers by

total internal reflection fluorescence (TIRF) microscopy

ICAM-1+ULBP1+CD48

pY759-PLC 2

pY1217-PLC 2

PLC 2

0 22 5 15 0 22 5 15 0 22 5 15 min

NKG2D 2B4 NKG2D+2B4

pS473-AKT

AKT

p-ERK1

ERK1ERK2

p-ERK2

Synergistic activation of PLC-2 and ERK but not

PI3K/Akt in human resting NK cells

Critical role of Vav1 but not Vav2 or Vav3 in

NKG2D and 2B4 synergy

Vav1 is required for synergistic calcium mobilization, cytotoxicity,

and cytokine production

0 100 200 3000

50

100

150

200siControl

siVav1

siVav2

siVav3

[Ca ]

i2

+

Time (sec)

NKG2D+2B4

0

20

40

60

80

1.25:1 5:1 10:12.5:1

E:T ratio

Specific

lysis

(%)

siControl

siVav1

siVav2

siVav3

NKG2D+2B4

0

100

200

300

400siControlsiVav1

NKG2D 2B4 NKG2D

+2B4

cIgG1

IFN

-pro

duction

(pg

/ml)

0

500

1000

1500

2000siControlsiVav1

cIgG1 NKG2D 2B4 NKG2D

+2B4

MIP

-1

pro

duction

(pg/m

l)

Vav1

siCont

rol

siVav1

siVav2

siVav

3

Vav2

Vav3

Actin

c-Cbl but not Cbl-b limits NK cell activation and imposes

a requirement for synergy

Knockdown of c-Cbl but not Cbl-b potentiates calcium

mobilization, degranulation, and IFN- production by NKG2D

and 2B4 synergy

s iC

o n tro l

s ic -

Cb l

s iC

b l -b

c -C b l

C b l -b

A c tin

NKG2D 2B4 NKG2D+2B4

[Ca

]

i2+

Time (sec)

0 100 200 3000

50

100

150

200siControlsic-CblsiCbl-b

0 100 200 3000

50

100

150

200

[Ca

]

i2+

Time (sec)

0 100 200 3000

50

100

150

200

[Ca

]

i2+

Time (sec)

0

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1600

2400

3200

S2 S2ULBP1

S2CD48

S2ULBP1CD48

siControlsic-Cbl

G

ran

zym

e B

(p

g/m

l)

0

200

400

600

800siControlsic-Cbl

cIgG1 NKG2D 2B4 NKG2D+2B4

IFN

- p

rod

uctio

n (

pg

/ml)

Increased Vav1 expression overcomes inhibition by c-Cbl

NKG2D or 2B4 alone becomes sufficient for NK cell activation by

overexpression of Vav1 or Vav1 CAAX

YFP control YFP-Vav1YFP-Vav1 CAAX

0

10

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30

40

50

60

cIgG1 NKG2D 2B4 NKG2D+2B4

YFP controlYFP-Vav1YFP-Vav1 CAAX

Sp

ecific

lysis

(%

)

0

400

800

1200

1600

2000

S2 S2ULBP1

S2CD48

S2ULBP1CD48

YFP controlYFP-Vav1YFP-Vav1 CAAX

G

ran

zym

e B

(p

g/m

l)

Cytotoxicity Degranulation

Cellular localization

NK cell activation by synergy is regulated at the level

of Vav1 by a hierarchy of inhibitory mechanism

Synergistic activation

Activating Receptor

Activating Ligand

Vav1 Rac1

P P P

RAFT

KIR

MHC I

SHP

-1

Target Cell

Actin c-Cbl

Kim et al.,

Immunity (2010)

Dissecting the contribution of individual receptors to NK cell activation using Drosophila S2 cells expressing ligands of NK cell receptors

Screening potential drug candidates that enhance NK cell function by targeting specific NK cell receptor or key signaling molecules

Activation signal Inhibitory signal

Insect cell

CD16 CD2

LFA3

2B4

CD48 ICAM1

LFA1 KIR CD94/NKG2A

HLA-C HLA-E

Anti S2 cell

Rabbit serum

ITI M

ULBP

NKG2D

DAP10

NK cell

ITI M

ITA M

Analysis of NK cell activation with insect target cell system

Developing systems approach for complete

eradication of tumor cells

Chemotherapy

Radiotherapy

Immunotherapy

Vaccines

Tumor cell

Induction of

stress (DNA

damage)

response NKG2D-L

Stressed Cell

Killer cell

response

NK cell

CTL

response

TH1 cell

response

Bystander

activation

Boosting

immune

responses

Cancer targeting Immune cell targeting

Acknowledgements

SMC, SKKU University

Myung-Shik Lee

Eunsil Kim

Myoung Sook Han

Namjoo Cho

Sunshin Kim

Hyun Ah Lee

Moon-Kyu Lee

Kwang-Won Kim

Osaka University

Shizuo Akira

Kiyoshi Takeda

NIAID/NIH

Eric Long

Sumi Rajagopalan

Asmita Das

Catharina Gross

Dongfang Liu

Mike March

Mary Peterson

Susina Vielkind

Harvard University

Diane Mathis

Christopher Benoist