Tumor Microenvironment Hijacking the Immune System [Read-Only]

James H Finke Ph.D.

Department ImmunologyLerner ResearchCleveland Clinic

Tumor Microenvironment; Hijacking The Immune System

Disclosures

Research Support: Pfizer, GSK, Immatics and ChemDiv

Cellular Composition of the Tumor

• The tumor microenvironment is not only composed of malignant cells it contains:

• neovasculature, fibroblast and myeloid cells

• Immune cells critical for mounting an immune response to tumor.-T lymphocytes, CD4 and CD8 subsets- Dendritic cell subsets- NK/NKT cells -B cells/plasma cells

• Many of the tumor infiltrating immune cells are dysregulated, functionally impaired and contribute to tumor progression.

-T cells (anergic)-T-regulatory cells-Macrophages-Myeloid derived suppressor cells

Cellular Constituents of Immune Escape within the T umor MicroenvironmentKerkar SP, Restifo NP.Cancer Res. 2012 Jul 1;72(13):3125-30. Epub 2012 Jun 21. Review

• In metastatic RCC patients peripheral blood CD4+

and CD8+ T cells showed a skewing toward a TH-2

response against EphA2 and MAGE-6-derived

peptides except NED patients

Diminished Type 1 T cell Response in mRCC is Linked to Active Disease.

Tatsumi T et al. Cancer Res 2003;63:4481-4489

• CD8+ EphA2 specific T cells detected in

patients who are long term survivors

with residual disease and NED.

Features of some solid tumors that may mediate poor immune recognition or reduce immune destruction of tumor .

T cell Priming-Reduced recruitment of Dendritic subsets for antigen presentation-Inadequate expression of co-stimulatory molecules on tumor cells or infiltrating DC

Effector Phase-Inadequate recruitment of activated effector cells• chemokines

Presence of dominant immune inhibitor mechanisms th at suppress T cell effectorfunction• Suppressive cells (Treg, MDSC, Macrophages, neutrophils)• Inhibitory cytokines (IL-10, TGFβ)• Inhibitory receptors (CTLA4, PD1/PDL-1)

(n=22) (n=56) (n=40)

Tregs in Blood and Tumor of RCC patie nts

Finke J et al , unpublished and Clin Cancer Res 2008,

Treg Foxp3+ cells

A. B. T-regulatory cell facts

• Foxp3+ Treg cells suppress T effectors via different mechanisms .

• Difficult to selectively reduce Treg cell numbers.

• For adoptive T cell therapy the use of cytoablativestrategies to deplete Treq enhances clinical responses. (Dudley EM et al. J Clin Oncology 2008)

• Within different tumor types there is variable correlationbetween the degree of Treg infiltration and overall survival.

• In colon cancer Treg numbers have not correlated withreduced survival (Loddenkemper C. et al J Trans Med 2006).

Myeloid-derived Suppressor Cells

• Heterogeneous population of immunosuppressive myelo id cells• Normally present in very small amounts but systemic ally accumulate under pathologic conditions – tumor-bearing• Accumulation associated with:

VEGF, SCF, GM-CSF, G-CSF, S100A9, and M-CSF

• Murine MDSC: Granulocytic (CD11b+Gr1hi+),

Monocytic (CD11bGr1low)• Human MDSC multiple subsets:

Granulocytic (CD33lowHLADR-CD15+CD14-)Monocytic (CD33lowHLADR-CD15-CD14+)

Linage Negative (CD33lowHLADR-CD15-CD14-)

Plasticity of MDSCGranulocytic MDSC differentiate into CD31 Endothelial cells Monocytic MDSC differentiate into Tumor Associated MacrophageMonocytic MDSC differentiate into Granulocytic MDSC

Ko J et al Can Res 2010 and unpublished data

MDSC in Cancer Patients

MDSC subset levels in blood and tumor of RCC pts

Baseline levels of Monocytic and Granulocytic MDSC negatively correlate with overallsurvival

MDSC subset morphology .A.

B.

C.

G-MDSC M-MDSCLinage

Negative

G-MDSC Linage

Negative

M-MDSC

Walter S, et al Nat Med. 2012 Aug

Patient Granulocytic MDSC and Neutrophils: Suppress ive and Angiogenic Activities

% T

ce

ll

sup

pre

ssio

n

PatientG-MDSC Normal4:1 8:1 16:12:1 4:1 8:1 16:12:1 4:1 8:1 16:12:1

Neutrophil

T cell suppression

MDSC promote angiogenesis of RCC implanted in SCID mice

Ko J et al Manuscript in preparation

MDSC

ArginaseiNOS

InduceTreg

Macraphage M2 Cell IL-12

Th2Cell

IL-10

Tumor Progression

ArginineReductionCysteine Reduction

CD3ζ ζ ζ ζ CD4

T cell

CD3ζ ζ ζ ζ CD8

T cell

Cell Cycle Arrest

ROS NO (H2O2, peroxynitrate)

CD4T cell

CD8T cell

Apoptosis Nitrate TCR

MDSC –Mechanisms of Suppression

Granulocytic subsetROS, Arginase

Monocytic subset NO, Arginase

Neutrophils

• Elevated blood neutrophils and elevated neutrophil/lymphocytes ratio is associated with poor clinicaloutcome in several human cancers (RCC, melanoma colorectal, lung , ovarian etc) ((Donskov F. Seminar in Cancer Biology 2013)

• In non-metastatic localized clear cell RCC the presence of intratumoral CD66+ neutrophils was associated with short recurrence-free survival and overall survival (Jensen HK et al J Clin Oncology 2009)

• Neutrophils from cancer patients but not healthy donors can suppress T cell function and produce elevated levels of proangiogenic proteins (Schmielau J. and Finn O., Can Res 2001 , Rodriguez PC et al. Can Res 2009)

• The relationship between granulocytic -MDSC and patients neutrophils is being assessed (functional andgene array studies).

• Tumor microenvironment promotes pro-tumor neutrophils (TAN )Claudia A. Dumitru , et al Seminars in Cancer Biology Volume 23, Issue 3 2013 141 - 148

IL-1ββββTNFIL-6

IL-23IL-12

LPS IFNγγγγ/TNF

MHCII

M1 M2

IL-4, IL-10& IL-13

ImmuneComplexes+ IL-1/LPS

TGFββββIL-10

CCL22

Arg

MHCII

Functions

Stimuli Stimuli

Tumor Associated Macrophages

Type 1 Inflammation

Th1 ResponsesTumoricidal

Type II Inflammation

Th2 Responses

Tumor Promotion

Functions

Tissue Macrophages

PDL-1

Angiogenesis

Impair T Cell Activation

Macrophages

Gabrilovich et al Nat Rev Immunol. 2012 Mar 22;12(4):253-68

Multiple Subsets of TAM are Induced by the Tumor M icroenvironment

TAMs in RCC

RCC associated TAM can mediate immune suppression and tumor escape via the activation of the 15-Lipoxygenase-2 pathway.

Daukrin I. et al. Can Res ,2011

15-LOX

15-(S) HETEBioactive lipid

CCL2 MonocyteRecruitment

IL-10

T cell

FOXP3CTLA4Up-regulation

TAM

Tumor Hypoxia (Hif1-αααα)

Treg

CCL28

Angiogenesis

MDSC

Tumor Hypoxia (Hif1-αααα)

ArginaseNO Production

TAM

TAM

DCCD40CD80CD86MHC Class IICCR7

TNFαIL1βCCR5

SuppressiveFunction

PDL-1 (B7-H1) Expression by RCC and Its Suppression of T Cell Function

PDL-1

PDL-1

PDL-1

PDL-1 Expression in RCC-Poor Outcome

Thompson H et. al. Clin Can Res 13:1757-1761, 2007

Can be expressed on- Tumor- Macrophages-Dendritic Cells

Secreted form (sB7-H1)Immunosuppressive

Frigola X et al Clin Can Res 17:1915-1923,2011)

Gaurisankar Sa et al (Unpublished)

PD-L1

Bright Field

PDL-1 in tumor supernatant binds T cells which is blocked by anti-PDL-1 Ab

(Cancer J 2013;19: 353Y364)

Pre-Tx Cycle 1 Cycle 2 Cycle 4

G-MDSC ( ), CD3+ ( )

One Example of Modulating The Tumor Microenvironment : Sunitinib Treatment

Blood Tumor

Ko et a Clin Cancer Res. 15:2148-57, 2009 and unpublished data

5 10 15 20 25 300

500

1000

1500

2000

2500

3000UntreatedSUT OnlyVAC OnlySUT/VAC

1000

1500

MO

5 T

umor

Vol

ume

(mm

3 )

Days Post Tumor Inoculation

VAC

SUT

(0/30)

(0/30)

(0/30)

(12/50)

****** **

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5 10 15 20 25 300

500

1000

1500

2000

2500

3000UntreatedSUT OnlyVAC OnlySUT/VAC

1000

1500

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5 T

umor

Vol

ume

(mm

3 )

Days Post Tumor Inoculation

VAC

SUT

(0/30)

(0/30)

(0/30)

(12/50)

****** **

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Bose et al. Int. J. Cancer 2011; 129: 2158 - 2170.

Superior Anti-Tumor Efficacy of Vaccine + TKI Co-Therapy

MO5 (B16.OVA)

injected s.c .

(2 x 10 5)

+/- oral Sunitinib(0.1 mg/day, d10

+/- s.c . DC/OVA 257 -262

vaccines d10, d17

Monitor tumor size

TME analysis (d34)

Immune monitoring (d34)

MO5 (B16.OVA)

injected s.c .

(2 x 10 5)

+/- oral Sunitinib(0.1 mg/day, d10-16)

+/- s.c . DC1/OVA 257- 262

vaccines d10, d17

Monitor tumor size

TME analysis (d34)

Immune monitoring (d34)

Bose et al. Int. J. Cancer 2011; 129: 2158 - 2170.

Vaccine/TKI Co-Therapy Promotes the Inhibition of Regulatory Cells and the Activation/Recruitment of

Protective CD8+ T cells

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(CD11b+Gr1+)

Treg (CD4+/ Foxp3 +)

DC (CD11c+/CD86+)

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ell

Num

ber

in T

umor

(x 1

06 )

Days Post-Inoculation

UntreatedSUT OnlyVAC OnlySUT/VAC

UntreatedSUT OnlyVAC OnlySUT/VAC

CD8-T CD4-TMDSC

(CD11b+Gr1+)

Treg (CD4+/ Foxp3 +)

DC (CD11c+/CD86+)

VEGF

HIF-1ααααHIF-2αααα

PDGFRββββ

Un

tre

ate

d

SU

T

VA

C

SU

T/V

AC

ββββ−−−−Actin

CXCL9

CXCL10

CXCL11

CXCR3IFN-γγγγ

Combination Therapy Results in a

Type-1 Biased Immune Profile in

the Tumor

Combination Treatment with Vaccine

and Sunitinib Improved T cell Response

Clinical Trials with Sunitinib Plus Vaccine therapy

Argos Therapeutics

DC loaded with autologous total tumor RNA + sunitinib

Immatics Biotechnology

Multipeptide vaccine + sunitinib

UPMC/Cleveland Clinic

DC pulsed with tumor blood vessel-associated antigens (TBVA) + sunitinib

Conclusions

• There is heterogeneity in patient response to immune-based Immunotherapy.

• A significant component of that heterogeneity comes from differences at the level ofthe tumor microenvironment.

• Key determining factors in the tumor (RCC) environment include recruitment of T effector cells, local production of chemokines and the presence of localimmunosuppressive mechanisms.

• Further Identifying the different mechanisms mediating immune suppression and angiogenesis by tumor stromal cells has already yielded new strategies forimproving cancer therapy (CTLA4 and PD1 antibodies) and is likely to

yield additional targets.

• The fact that different histological types of tumors all share in common the infiltrationof stromal cells should provide unique universal targets for therapy of solidtumors including RCC.