Novel ‘elements’ of immune suppression within the tumor ... · Novel ‘elements’ of immune suppression within the tumor microenvironment Nicholas P. Restifo . National Cancer

Novel ‘elements’ of immune suppression within the tumor microenvironment

Nicholas P. Restifo

National Cancer Institute NIH, Bethesda, Maryland USA NCAB meeting Sept 7, 2016

At the center of the galaxy of increasingly successful cancer immunotherapies

Cancer Vaccines T cell:

Tumor cell

CAR/TCR/TIL-based treatments

Checkpoint blockade anti-PD-(L)1, anti-CTLA-4

Hypoxia O 0

Understanding the tumor microenvironment during initiation and growth of tumor

Metastasis is the cause of >90% of all cancer deaths

• Successful metastasis requires evasion of immunity at the secondary site

• The lung is a common site of metastasis for many cancers

• Vascular architecture has historically explained cancer’s predisposition to disseminate to the lung

Hypothesis Site-specific environmental factors – such as Oxygen – help establish immunologically permissive sites for metastasis

How do anti-tumor T cells ‘sense’ Oxygen, and does this affect their function?

T cells use prolyl hydroxylase domain (PHD) containing proteins

These dioxygenase (O2) sensors containing non-heme–binding iron (Fe) that catalyzes the

hydroxylation of proline residues

_5 ___ ____,i>llaaa __ ,....6 ___ _____. ~-

~---4 ..,;;;-,:::;;:-,c...~::::..:::.::=:13:= 7 His-548 8 Ar -55

- --o .. -······ O~- CH2. ~-·

02 ==-==~ ~~ . }- c~2 CO2 C-terminal region

H ..... -< .. o 2

R'HNOC ....... 0 Asp-489His-487

1 <---------r-i

N-terminal I

COR .

region

PHD proteins hydroxylate proline residues

Proline residue on PHD target protein

·.N ·.

I JV\

praline

0 . 0

o· 0

2.-oxog I uta r.ate

0 1=0 CO2

~ J .... PHD2

OH /

"'

+ -o ... · 0

4-hyd roxyp ro Ii n e succinate

The PHD enzyme splits dioxygen into hydroxylated proline and succinate

NORMOXIA

HYPOXIA

transcription

nucleus

PHD enzymes degrade hypoxia inducible factor (HIF) – and possibly other proteins –

in the presence of oxygen

1 aa J:=F::i===== ::::,::::=;;;-:;;;:::=========:::;;:::==========:::::::;;:;:::==~=:;::::::===1426aa / -----···l ---------1 _/ ·--------r> ..

iaa 239aa ..=- ·u~·------... ____ ..... ,~-_------··r ·_·············----··--, _-··········_··· .-·····_····· '·--l /-' ·---~

EGLN genes encoding PHD oxygen sensors are located at three different sites in human genome

PHD1 (EGLN2): 19q13.2

PHD2 (EGLN1): 1q42.1

PHD3 (EGLN3): 14q13.1

l

l

Studying T cell-intrinsic oxygen sensing required a triple KO mouse

X Does oxygen affect anti-tumor immunity?

Egln1fl/fl Egln2fl/fl

Can oxygen sensing be F2 manipulated to improve X

generation: cancer immunotherapy?

F2 generation:

F2 generation:

Egln1fl/fl Egln3fl/fl

Egln2fl/fl

X

Egln1fl/fl Egln2fl/fl

Egln3fl/fl

PHD-tKO Egln1fl/fl Egln2fl/fl

Egln3fl/fl CD4Cre+/-

CD4Cre+/+

D Clever, Cell, August 25, 2016

WT Egln1fl/fl Egln2fl/fl

Egln3fl/fl CD4Cre-/-

Immune homeostasis

Suppressed pulmonary effector

response

Tumor colonization

Tumor growth

Tumor clearance

Anti-tumor immunity

Oxygen Sensing by T Cells Establishes an Immunologically Tolerant Metastatic Niche

D Clever, R Roychoudhuri . . . A Goldrath, Y Belkaid and NP Restifo, Cell, August 25, 2016

T-cell intrinsic PHD proteins suppress spontaneous pulmonary inflammation

WT PHD-tKO

WT PHD-tKO

AST ALT Amylase

60 NS 35 NS

3 NS

• • •• ....-. C't')

+ • 30 C)

• • ~

:::? 40 • >< 2 • • T ....-. _J •• _J -- -- -- ¼ :::) :::)

-f :::) • - -25 - • • •• I- I- Q) + •• Cf) _J en

<( 20 <( ••• cu 1 • • • 20 ~

~

• E <(

• •• 0 15 0

WTPHD WT PHO WTPHD tKO tKO tKO

T-cell intrinsic PHD proteins do not trigger spontaneous inflammation in the gut

WT

PHO tKO

Spleen LP meLN Lung Lung T RM

23.8 19.6 17.8

~ o 33.1 30.9 (.) ........_..,,...............,...............,..............,.., '-r-~~~.............-- ·.........___,~~~~ '-r-..............-...........----............... .......,.....,. ..........._,~ ~~ .............

IFN-y

CD4+ T cells lacking PHD proteins are prone to produce IFN-γ after stimulation

D Clever, Cell, August 25, 2016

1.6x 1.2x 1.4x 2.1x 3.1x 80

** ** NS **** **

.-.

* •

60 ~ • .... ~ • 0 .. ..._. + •

* ?- -f- • • I

:z 40 • + LL ~ • •

-l-+ • • co 0 20 • (_) • -I-

• 0

Spleen LP meLN Lung Lung

• VVT • PHD-tKO TRM

CD8+ T cells lacking PHD proteins are prone to produce IFN-γ after stimulation

/ ~

50 **

~ 40 • 0

~ 30 I- • g> 20 -t-:::J

...J 10 •• • • ••1••

0

T cell-intrinsic expression of PHD proteins licenses tumor colonization in the lung but not SQ tissue

IV AND SubQ NS

B16 WT or PHD-tKO P < 0.01 melanoma

WT PHD tKO WT

PHD tKO

WT PHD tKO

...- 300 N ...... E E ...... -co 200 (l) '-co '-0 E 100 :::J I-

0 0 7 14 21

Day Post Implantation

T cell-intrinsic expression of PHD proteins licenses tumor colonization in the lung but not SQ tissue

IV AND SubQ NS

P < 0.01B16 WT or PHD-tKO melanoma

Subcutaneous WT PHD-tKO

WT

PHO tKO

Naive

IL4,5, 13

0.4

0.1

. . 1.9

0.4

WT

PHO tKO

PHD proteins suppress type I responses against innocuous house dust mite (HDM) Ag

BAL

Summary

1. T-cell intrinsic PHD proteins suppress spontaneous pulmonary inflammation

2. CD8+ and CD4+ T cells lacking PHD proteins are prone to produce IFN-γ after stimulation

3. T cell-intrinsic expression of PHD proteins licenses tumor colonization in the lung but not SQ tissue

4. PHD proteins suppress type I responses against innocuous house dust mite (HDM) Ag

Tumor C II

The Problem

Normal Hyper-responsiveness to homeostasis innocuous Ag

Tumor Tumor colonization clearance

A Solution

Knockout or drug PHD proteins only in T cells specific for tumor antigens while

leaving all other T cells intact

proline 2-oxogluta rate

o-o CO2

\........1.., PHD2

OH / -c;½,o +

\/\rt .N'-

4-h yd roxyproli ne

0

Jl ./--.._. ..o­-o~ . ....._,, n 0

succinate

DMOG blocks the oxygen sensing PHD proteins

Dimethyloxalylglycine (DMOG)

Q) L...

0 (.)

~ 0.6 C:

~ 0.4 ..c: -~ 0.2 C:

w 0.0

(.) ·c ......, ~ 15 n

"'O

PHD-tKO/WT UP

NES: 4.18 P-value < 0.0001

Q) ~ 0 1.-----========~ ;;;;;;~ C:

I !5

cu 0:: -10 .._ _____________ _

0 1500 3000 Rank in Ordered Dataset

Gene set enrichment analysis (GSEA) shows that DMOG/vehicle induces similar gene expression

changes as PHD-tKO/WT

600

::::. E 400 -... C)

a ~ ~ 200 -

****

Inhibition of PHD proteins with DMOG before adoptive cell transfer immunotherapy

SubcutaneousTransferred Cell Function OR Pulmonary

Tumors Veh

TRP-1+

Splenocytes

Ex vivo expansion

+ DMOG

0.11

3.16

0.11

.. ·, .

I 20.1] .... _______ .... 0.0

Foxp3~---- ,-

Inhibition of PHD proteins with DMOG before adoptive cell transfer immunotherapy

SubcutaneousTransferred Cell Phenotype OR Pulmonary

Tumors Veh

TRP-1+

Splenocytes

Ex vivo expansion

+ DMOG

Inhibition of PHD proteins with DMOG improves adoptive cell transfer immunotherapy

No Cells Trp-1 VEH Trp-1 DMOG

D Clever, Cell, 2016

400 ...-...

N

~ 300 co ~ 200 co s.... 0 E 100 ::, I-

, f' ** ' ' ' ' ' ' ' ' ' ' '

.i i ' ' ' ' '

**

o ............... ~~-~--o 11 23 35 47

Day Post Transfer

co

100- - - - ....... No Cells ,_______. ....... Th0 VEH

....... Th0 DMOG

~ 50 ** ::,

Cf)

** 0-------~-~

0 20 40 60 Day Post Transfer

Improved efficacy of DMOG-cultured cells for established subcutaneous tumors

D Clever, Cell, 2016

40 NS

* 1i

30 • • 20 -r • •

• 10

0------

Foxp3+ iTreg fate specification of human CD4+ T cells cultured with DMOG

Foxp

3+ (

%) Donor A

Donor B

Donor C

[O2] 2% 20% 20%

DMOG - - +

Summary

1. DMOG blocks the oxygen sensing PHD proteins as evidenced by RNA seq and gene set enrichment analysis (GSEA)

2. Inhibition of PHD proteins with DMOG changes the function and phenotype of T cells . . .

3. . . . and improves adoptive cell transfer immunotherapy

4. Finally, similar maneuvers can be done with human CD4+ T cells

How do tumor immune suppressive mechanisms change with progressive growth?

PHD function [02]

0

Hypoxia 0 0

o K+ o \.___o

0

0 Cellular

. necrosis

j Release of K+

Increased hypoxia accompanies progressive tumor growth

Restifo, In Preparation, 2016

The tumor microenvironment is characterized by a high tissue density of necrosis

Pt 4007-1 Pt 4007-2

Tumor

Necrosis

Stroma

Necrosis

4x 4x

.... 11.__ ...

11 I . , .-.I I 1 I ~ I ,1 . ~

ii 11 • ,. ' I I ~ • •• I

I I_ • I • I • Il l

• I

I ,. .. . , --, -._,. ._, __ I • • --------~---·····

Severe tumor necrosis is associated with a poor prognosis

Cum

ulat

ive

surv

ival

rat

e

100 -

None (n=15)

50 -

Moderate (n=69)

Severe (n=14) 0 -

2000

4000 Time from resection (days) Komori et al. Anticancer Res. 2013

n- ir- -- -- --------------=--=-= --~----=---=--=--=--=--=-===~

lo-_,) '--======~) ~

Healthy Tissue

- - - - - - -

Necrosis releases intracellular ions into the extracellular space

Healthy Tissue

[K+]i ≈ 145 [Na+]i ≈ 5

++++++ Interstitial

(extracellular) space [K+]e ≈ 5 [Na+]e ≈ 145

[K+]

Tumor + necrosis

15 15 • • E 10 • E 10 • ::::J

•• ::::J

-I-L.. L.. Q) Q)

(f)

I (f) . . •• LL LL - I- 5 I- 5

• ~~~

0 ~

0 ~ ~ Na K Cl Ca Mg Na K Cl Ca Mg

Tumor interstitial fluid (TIF) has an elevated concentration of extracellular potassium ([K+])

Mouse Human

+ + - 2+ 2+ + + - 2+ 2+

120

100 • 80 .

• •• • 60 • . •"

... ...... i . . . . .. 40

. 20 -·

0 0 20 40 60 80 100

Cell death correlates with levels of K+ in the extracellular space

Anne

xin

V+ ce

lls x

106 /g

of t

umor

R2 = 0.32 P=0.017

TIF [K+] mmol

R Eil, Nature (In Press), Fall, 2016

Background and Experimental Question

1. Human tumors persist and progress despite infiltration by tumor-specific effector T cells

2. Mouse and human tumors contain dense areas of cell necrosis

3. Cell necrosis leads to the release of an intracellular ion, potassium, into the extracellular space

4. Do elevated concentrations of extracellular potassium ([K+]) have any effect on T cell function?

i

Elevated [K+] acutely inhibits T cell effector function

Vehicle [K+] 1.8 0.65.3 20.9

90.1 7.439.9 33.9 IFNγ

R Eil, Nature (In Press), Fall, 2016

IL-2

60 **** 50 **** **** **** ...-... ...-...

'#. 45 f!. 40 .._... .._.. -+ -+ co co C C 30 u 30 u ~ ~

0 0 20 -+ + > >

I

15 I z z

10 LL LL - -0 0

CTLA-4 - - + + PD-L 1 + -- -t [K ]e - + - + t, [K]e - + - -

Hyperkalemia augments checkpoint inhibition of T cells that may already be in place

R Eil, Nature (In Press), Fall, 2016

Tumor Interstitial Fluid (TIF) contains ~ 40 mm of K+

1. Elevated [K+] produces profound suppression of human and mouse T cell TCR induced effector function

2. Hyperkalemia produces profound suppression of T cell receptor-induced transcripts including IL-2 and IFN-γ

3. Tumor associated hyperkalemia augments checkpoint inhibition of T cells that may already be in place

K K \ [K ]i

Kv 1.3

Ctrl Kcna3

. - - - -

- .

·­

.-

J3-actin I ____ ____J

Naturally-occurring T cells express low levels of the potassium ion channel

Kcna3 encoding Kv1.3

R Eil, Nature (In Press), Fall, 2016

PHD function [02]

0

Hypoxia 0 0

o K+ o \.___o

0

0 Cellular

. necrosis

j Release of K+

Increased hypoxia accompanies progressive tumor growth

Restifo, In Preparation, 2016

K K \ [K ]i

Kv 1.3

Ctrl Kcna3

. - - - -

- .

·­

.-

J3-actin I ____ ____J

Genetically engineering anti-tumor T cells to over-express the potassium ion

channel Kcna3

R Eil, Nature (In Press), Fall, 2016

> I z

LL -

Ctrl Pmel

6.D · -.·

T L in vivo

Kcna3 Pmel •

.. · .. . · ... .

... . .. A . -~

I _. ~

, .... .. ·-•• I

•",j

I

SSC-A --------- ---

Kcna3 gene-engineered T cells make more IFN-γ in vivo

X

-0

-

Anti-tumor T cells over-expressing Kcna3 have enhanced therapeutic efficacy

0

100

200

300

Tum

our

area

(m

m2 )

*

PBS

Ctrl

Kcna3_PD Kcna3

0 10 20 30 40

Days after cell transfer R Eil, Nature (In Press), Fall, 2016

Overall summary

1. Tumor cell death creates elevated [K+] in the tumor microenvironment.

2. This local hyperkalemia produces profound suppression of human and mouse T cells

3. T cells can be gene-engineered for resistance to hyperkalemia by over-expressing the [K+] ion transporter Kcna3

4. Anti-tumor T cells over-expressing Kcna3 have enhanced therapeutic efficacy

CD4+Treg

CD8+ T cell

IL-10

~ CD8•Treg / 1D0

CD8+ / Tcell y

• T cell

~ B7-DC Ar!¥-lase I

<

ICOS-L

CD4+Treg

\

L-10 GF,6

Regulatory DC

TAM (lv12)

CD8+ Tcell

--1 • B7-Hl TGF,6 Ar!¥-lase I

NK cell

CD8+ Tcell Immature DC

Progenitor VEGF

MUC 1 rn ucins COX-2/PGE2

VEGF CCL2 CXCLl CXCL5

CD4+ T cell

Immature DC

VEGF ~ Progenitor

IL-13 GM-CSF COX-2/PGE2 ROS

MUC I rn ucins ___ c'---o~ _X-2/PGE2

IL- 6 VEGF IL-10 TGF,6

CCL2

Lactic acid COX -2/PGE2

Arl;;;ej IDO CCL3 CCL4 CCL5

CD4+ Treg

T cell

t IL-~ Macro . phage

TO~~ -y ~ · CD8+ ~ · Tcell

B7-Hl

\ ~t:sel

\-- CD4• T cell

Mature, ~ Activated • • CD8+

T cell DC CD8+ T cell

Tumor-induced immunosuppression is complicated

Hargadon, Front. Immunol., 2013

Element Symbol Percentage in Body

Oxygen 0 65.0

Carbon C 18.5

Hydrogen H 9.5

Nitrogen N 3.2

Calcium Ca 1.5

Phosphorus p 1.0

Potassium K 0.4

Sulfur s 0.3

Sodium Na 0.2

Chlorine Cl 0.2

Magnesium Mg 0.1

Trace elements include boron (B), chromium (Cr), cobalt (Co), copper (Cu), fluorine (F), iodine (I), iron (Fe), manganese (Mn), molybdenum (Mo), less than 1.0 selenium (Se), silicon (Si), tin (Sn), vanadium (V), and zinc (Zn).

Composition of a human being

1

H Hydrogen

.,..

*

I 57 I 58

:e

105

Db 106

Sg 107

Bh Dubnlum Seaborglum Bohrlum

59

Pr 60 Nd

61

Pm

108

Hs

27

Co Cobalt

45

Rh hodlum

77

Ir :rtdlum

109

Mt 110

Ds 111

Rg 112

Cn Hasslum Meltnerlum Darmst.adtlum Roentgenlum Copemiclum

62 Sm

63

Eu 64 Gd

65

Tb 66

Dy 67 Ho

?rium Praseodymium Neodymium Promethium Samarium Europium Gadolinium Terbium Dysprosium Holmlum

I A~ Actinium

I 30 Th

Thorium

91

Pa Protact inium

92

u Uranium

93

Np Neptunium

94 Pu

Plutonlum

95

Am Americ ium

96

Cm Curium

97

Bk Berkelium

98 Cf

Californium

99

Es Einsteinium

114

Fl Flerovlum

68

Er Erbium

100

Fm Fermium

7

N

69 I 70 I T

2

He Helium

What is the immunology of the elements and how can it be used to destroy cancer?

David Clever Robert Eil

Acknowledgements

Restifo Lab: Past and present

Robert Eil David Clever Madhu Sukumar Douglas C Palmer Suman Vodnala Shashank Patel Christopher Klebanoff Raul Vizcardo Zhiya Yu Ping-Hsien Lee Devikala Gurusamy Christine Kariya Rigel Kishton Anthony Leonardi Marta Bosch-Marce Arash Eidizadeh Amanda Henning

[email protected]

Luca Gattinoni Yun Ji Rahul Roychoudhuri Enzo Bronte Willem Overwijk Christian Hinrichs Nick Acquavella Joe Crompton Nick Klemen Tori Yamamoto Naritaka Tamaoki Rafiqul Islam

Rosenberg Lab: Eric Tran Alena Gross

David Stroncek Franco Marincola Ena Wang

Collaborators:

John O’Shea Jon Yewdell Yasmine Belkaid Ananda Goldrath Rafi Ahmed Carl June Francis Collins

Clinical Team: James Yang Udai Kammula Rick Sherry Stephanie Goff Paul Robbins Steve Feldman Robert Somerville Steve Rosenberg

PHD function [02]

! Function of Kv1 .3

0

Hypoxia 0 0

o K+ o \.___o

0

0 Cellular

. necrosis

j Release of K+

Increased hypoxia accompanies progressive tumor growth

Restifo, In Preparation, 2016