Pr. Daniel Olive, INSERM UMR 1068, Institut Paoli

Calmettes, Aix Marseille Université

Targeting of Novel

Immunomodulatory Pathways in

Lymphomas

Regulating costimulation

Inhibiting inhibitory pathways including Checkpoint

blockade inhibitors and others

Regulating different immune effector populations

Innate effectors and B cells

Classes of Cosignaling Molecules

Presentation

Escape mechanisms in lymphomas: role of BTLA-

HVEM pathway involving innate effectors

Treg and NHL: regulation by the ICOS-ICOSL

pathway

HVEM LTR

LT

LIGHT PD1 CTLA-4 BTLA

PDL-1 PDL-2 B7-1 B7-2

CD160

TNFR-TNF family CD28-B7 family

W/O viral receptors UL144 and

HSV gD

T Cells & BTLA HVEM

BTLA = B and T lymphocyte attenuator

KO BTLA increased:

- T proliferation & cytokine production (Liang & Sha,Curr Opin Immunol,2002)

- severity and duration of EAE (Watanabe et al.,Nat Immunol,2003)

- hypergammagobulinemia and autoantibodies to nuclear antigens, increased numbers

of activated T cells (Oya et al., Arthritis and Rheumatism, 2008)

- duration of infl. in the lung after airway challenge (Deppong et al.,J.Immunol,2006)

- reject of mismatched cardiac allografts (Tao et al., J.Immunol,2005)

- T cell survival (Deppong et al.,J.Immunol,2008)

- survival of mice infected by MHV3 due to decreased apoptosis of macrophages

through TRAIL (Yang et al., Gut, 2013)

BTLA mainly negatively regulates the T immune response in

these settings

BTLA-HVEM Serves as Negative Regulator

of Immune Responses in Cancer

• Melanoma and leukemic specific T cell responses

BTLA highly expressed on melanoma antigen-specific effector CD8 T cells

BTLA activation suppresses cancer-specific CD8 T cells

(melanomas, AML)

– Derre et al., JCI (2010) Fourcade et al., Cancer Res (2012), Hobo et al., J. Immunol., 2012

• γδ T-mediated anti-lymphoma responses

BTLA strongly expressed on resting γδ T cells

BTLA-HVEM ligation suppresses γδ T responses

Lymphoma cells suppress γδ T cells proliferation via BTLA-HVEM ligation

Suppression blocked by BTLA-HVEM mAbs

– Gertner-Dardenne et al., Blood (2013)

• B cell responses

CpG upregulates BTLA in vitro & in vivo in melanoma

BTLA-HVEM ligation suppresses B cell responses

Suppression blocked by BTLA-HVEM mAbs

– Thibult et al., J Mol Med (2013)

HVEM

HVEM Expression in Melanoma

BTLA Clusters with Vg9Vd2 T CR after

Activation

TC

RV

d2

B

TLA

M

ER

GE

D

API

Resting 1h BrHPP

Escape Mechanism Involving BTLA and HVEM in NHL

TNFSFR14/HVEM in Lymphomas

HVEM expressed on human B cell malignancies

HVEM is a possible tumor suppressor gene since HVEM cross linking

induces lymphoma cell death and sensitizes to Fas killing (Costello, Leukemia

2003; Pasero, Eur.J. Immunol., 2009)

TNFRSF14 gene alterations might be correlated to prognosis in FL

(Cheung et al., Cancer Research, 2010; Launay et al., Leukemia 2011)

BTLA is highly expressed in lymphoma microenvironment (Mhidi et al.,

Am. J. Clin. Pathol., 2009, Gertner-Dardenne et al., Blood, 2013).

HV INF FL0

20

40

60

80

100

p = 0.0312

p = 0.0583

%gd

BT

LA

positiv

e c

ells

BTLA Expression on gd T cells in NHL

Microenvironment

BTLA

FL

62 3.4

NK

0.8%

gd

0.3%

CD3

CD3

VIV

ID

CD

56

CD

4

Vg9

Count

BTLA

Characterization of HVEM Antibodies for the Ability

to Modulate BTLA –Ig Binding to Cell Surface HVEM

00.

010.

03 0.1

0.3 1 3 10 30

0

100

200

300

PD1-3.1HVEM-11.8

HVEM18.10

mAb concentration (Log10 µg/ml)

No

rmaliz

ed m

Abs M

FI

BTLA Blockade Restores Autologous gd T Cell

Proliferation in Coculture with its FL NHL

IgG1 Antag. BTLA Fab Antag.BTLA Agon. HVEM Antag.HVEM

49 86 70 19.5 87

CellTrace

CONCLUSIONS

• BTLA is expressed on Vg9Vd2 T cells

• BTLA engagement inhibits selectively Vg9Vd2 T cells proliferation/activation

(via partial S phase arrest) and accelerates their differentiation

• BTLA belongs to the class of molecules regulating cell cycle progression like

CTLA-4 and PD-1

• BTLA regulates homeostatic proliferation of Vg9Vd2 T cell

• BTLA blockade restores Vg9Vd2 T cell proliferation in co-culture with

HVEM+ lymphoma cells

Escape mechanisms in lymphomas: role of

BTLA-HVEM pathway involving innate effectors

Treg and NHL: regulation by the ICOS-ICOSL

pathway

Objectives

V

V

V

C

ICO

S

ICOS-

L

CM

H TC

R

Activated T

cells, TFH,

Tregs DC, B cells,

Monocytes

Differentiation

Cytokine production

IL-10 , IL-4, IL-21, IFN-γ, TNF-α,…

B TF

H

Cooperation

(germinal center formation

and antibody response)

Th1

Th17

Th0

Th2

Survival and proliferation

TFH

Inducible T Cells Costimulator (ICOS)

ICOSL downregulation

Tregs: Prognosis Marker in Tumor

Tregs

Tregs Tregs

Tregs

CCR4 CXCR

4

CCR5 Tregs

Tumor

cell

MDSC

TAM

Migration

CXCL

17

CXCL

22

CCL5

Tumor site

APC

Tumor

cell MDSCs TAM

sTGF-

β

Tregs Tregs TCR

MHC

II

Tregs ICOS

MHC

II

TCR ICOS

ICOS

L MV

IL-2

Expansion

IDO

pDC

ICOS

L

↓ Anti tumoral

responses

• Adverse Prognosis marker on solid

tumors(breast), positive prognosis

marker (H&N, FL) Mougiakakos et al, 2010

Yang et al, 2007, 2009

Martin-Orozco et al, 2010

Faget et al, 2012

Conrad et al, 2012

TCD4

TCD8

Tγδ

NK

Tcon

v Tcon

v

IL-10

APC TAM

MDSC Tumor

cell

CD70

CD27

Tcon

v

TCR TCR

Conversi

on

sTGF-

β PGE2

COX-

2

MHC

II MHC

II

CD28

CD80

CD86

Accumulation of Tregs and ICOS+ Tregs in FL

CXCR5 PD-1 GITR CTLA-4 Ki67

67.5 7.57

15.9 8.98

78.3 5.23

11.5 5

73.5 9.95

8.62 7.89

31.2 54.4

1.99 12.4

8.56 77

1.08 13.3

ICO

S

Gated on Tregs

CD

127

CD25

CD

8

CD4

Tregs

Tconv TCD8

TCD4

ICOSL is absent from B lymphoma cells, pDC, mDC from FL but present on DLBCL

ICOS / ICOSL Interaction

B cells mDC pDC

ICOSL ICOS

FL DLBCL

TCD4

B

cells

0.7 2 1.5

53.0 90.6

22.0

CD86 ICOSL

MFI

331

MFI

69

MFI

35

MFI

280

ICOS /ICOSL interaction induces downregulation of ICOSL on follicular lymphoma B cells

ICOS / ICOSL Interaction

ICOSL

CD

19

B alone B + TCD4 B + TCD4

Ctrl IGG1

B + TCD4

Anti-ICOS

59.4 13.5 14.1 57.3

23.5 25.9 25.1 28

CD

19

CD86

ICOS / ICOSL Interaction

ICOS /ICOSL interaction induces enrichment of Treg in Follicular lymphoma

Foxp3

CD

25

TCD4 + B TCD4

Cell trace

CD25+ Foxp3+ CD25high Foxp3high

ICOS

ICOS / ICOSL Interaction

ICOS /ICOSL induced enrichment of Treg in Follicular lymphoma is prevented by

antagonist ICOS mAbs

Role of Tregs in the Development of FL B Cells

Lymphoma infiltrating- Tregs suppress lymphoma B cells responses

CD80

B alone B / Treg

39.4

%

14.1

%

Role of Tregs in Development

of Lymphoma B Cells

Lymphoma infiltrating- Tregs suppress T conv induced

lymphoma B cells help

B alone B / Tconv /

Treg B / Tconv

39.4

%

49.6

% 31.7

%

CD80

Conclusions

Treg

ICOS ICOSL

Lymphoma

B cells

Lymphoma

B cells

PD-1

Ki67

HLA-DR

CD39

Treg

Treg

Treg

Agonist anti-ICOS mAb would enhance Tregs proliferation and function

Whereas antagonist anti-ICOS mAb would prevent Treg survival and function

Agonist and Antagonist BTN3A mAbs Regulate

Vg9Vd2 T Cell Functions Against Lymphoma

γδ T cells alone

γδ T cells + Control Isotype

+DAUDI

γδ T cells + anti BTN3 20.1

+DAUDI

γδ T cells + anti BTN3 103.2

+DAUDI

1. Cancer : Transcriptome, IHC,

Deep sequencing, RNA Seq

TAA and TSA typing

heterogeneity

HLA typing

Preexisting immune response

4. Combo personalized medicine

Immunosuppressive mechanisms

Cosignaling, galectins, enzymes,

Tregs, MDSC…

2. Exogenous Immunisation

Peptides

Proteins

Viral vectors

DNA

anti CD mAb fused to

TSA or TAA + adjuvant

3. Endogenous Immunisation

Chemo

Rx

Oncolytic viruses

Therapeutic Mabs

5. Monitoring

PFS

Immune parameters (FCM,

transcriptome, RNAseq)

Cytométrie

Transcriptome

ARNseq Olive D. , Cancer Vaccines, 2014

Collaborative approach

– Marseilles

• INSERM UMR 1068

• Aix-Marseilles University

• Marseilles Cancer Research Center

• Paoli Calmettes Institute

• Marseilles University Hospital

– Rennes: INSERM U917, University

Hospital

– Lyon : Lyon Cancer Research Center

Industrial valorization

– Creation of Imcheck

Therapeutics

– Patents licensed to GSK and

Janssen

– Other IP available

– Research partnerships

Multi-source financial support

– French National Agency for

Research

– French National Cancer Intitute

– SIRIC

– Fondation pour la Recherche

Médicale

– Institut Universitaire de France

– ARC Foundation

– GSK

Novel Immunomodulatory Pathways in

Lymphomas Discovered by CALYM

50 29 25

+ PDL1-Fc + PDL2-Fc + IgG1 + HVEM-Fc

27

CFSE

0

20

40

60

80

100

none HVEM PDL1 PDL2

% d

ivis

ion

BTLA-HVEM Interaction Inhibits Vg9Vd2 T

Cell Proliferation

* * *

ITIM(V/IxYxxL/V)

ITSM (TxYxxV/I)

PD-1

Y

Y

P

d γ 2

ZAP70

ERK P

P

P

PKC P

AKT

BTLA

Y

Y

Y

Y

ITIM

ITSM

YxN

YxN

GRB2

Inhibitory function of PD-1 and BTLA

?

CD28

P

Bcl-XL

IIL-2

TCR signals

NF-B

P

P

PI3K p110

P

p85 PI3K p110 p85

P

TNFSFR14 HVEM in Lymphomas

Mutations and deletions of TNFRSF14 gene have been identified in FL

patients (Cheung et al., Cancer Research 2010, Launay et al., Leukemia 2011)

73 non synonymous mutations out of 387 patients

12 were missense mutations affecting CRD1 of which 7 affect BTLA

binding