Novel pathway to control interferons and inflammation

Jean-Philippe Herbeuval, DR1 CNRSChemistry & Biology, Modeling & Immunology for Therapy (CBMIT)

CNRS UMR8601, Université de Paris

Novel pathway to control interferons and inflammation

ISG: up to 300 genes!

ü Type I IFN are powerful molecules: induce activation of 300-600 genes (called ISG)ü IFNAR are expressed by virtually all the human cells

Interferon-Inflammation: friends or foes?

Immune Surveillance

Viral infection

Bacterial infection

Type I IFN: the good, the bad, and the ugly

Tumor

Chronic viral and bacterial infections

Depression

Autoimmune diseases

Interferono-pathies

SLE

ü Immune cells discovered in 1997, role during innate and adaptive immunity

ü Professional of IFNa production (up to 1000x than other cell type)

ü Powerful but very rare cells: less than 0,5% of PBMC

ü Express innate immune sensors of the Toll-like receptors family (TLR-7 and -9)

ü Express high levels of IRF-7 (regulating IFN genes) and the chemokine receptor CXCR4

ü pDC represent promising cellular target for interferonopathies (SLE, sclerodermia..)

The IFN producers: the plasmacytoid DCs

Sascha Rodes and Nikaïa Smith, Ulm University-CBMIT

Interferons and interferon producing cells

NCI, NIH USA (2001-2006)

pDC

IFN-aTRAIL

(Killer pDC)

This is the first demonstration that natural molecules could block pDCs

TLR7/9

Natural amines block IFN in pDC

Gene Shearer Lab’s, Experimental Immunology Branch, NCI, NIH

R848

RNA/DNA

Virus

HistamineDopamineSerotonin

• Herbeuval et al, Blood 2005• Herbeuval et al, Blood 2005• Herbeuval et al, PNAS 2005• Herbeuval et al, PNAS 2006• Hardy et al, PNAS 2007 • Herbeuval et al, Clin Immu, 2007• Herbeuval et al, AIDS 2009

GlucocorticoidsAnti-BDCA2Antii-TLR7

PGE2

Modulation of interferon production

CNRS, Necker Hospital (2006-2012)

Clobenpropit (CB)

§ Naturel Ligand of HR (GPCR)§ Very unstable (Histaminase)§ Endogenous monoamine § Km (H1R et H2R) = 1000nM§ Km (H3R et H4R) = 5-10 nM

Histamine

§ Pharmacologic analogue§ Very stable§ Agoniste H4R – Km = 1nM§ Antagoniste H3R – IC50 = 10nM

ü Histamine overproduction in atopic children increases viral infection

Modulation of pDC viral activation by histamineEffect of Histamine/CB on pDC

CNRS UMR8147, Hôpital Necker, Michel DY

Flu

Flu+ H

A

Flu+ C

B

50

100

150

200

0

IFN

-apr

oduc

tion

(ng/

mL)

-EL

ISA

Mock

******

**

ü IFN-α production by HIV-stimulated pDC

Non Stimulated 5 10 20 40

BDC

A-4

Flu + CB (µM)

BDC

A-4

BDC

A-4

BDC

A-4

BDC

A-4

BDC

A-4

Flu

IFNa

Histamine

Clobenpropit (CB)

Flu

IFN-IELISAFACS

pDC

Or

Effect of Histamine and CB on pDC activation

pDC

Influenza A virusX31strain (H3N2)

- IFNa, IFNb in BAL- Cytokines - High activation of pDC,

Effect of Histamine/CB in vivo

X31 very pathogenic Flu:

IFN-a IFN-b

S129S8 12 week mice

CB or Histamine450µg/30µL/mouse18h before infection

Influenza A virusX31 (H3N2)

Broncho-alveolar Wash 3 days post-infection

Elisa : IFNa, IFNb

A. Wack (F. Crick Institute)

Effect of Histamine/CB in vivo

Flu Flu Flu Flu Smith et al Nat Comm 2017

Identification of the receptor controlling interferon production

CBMIT, Faculté des Saints-Pères(since 2012)

Fundamental research:Production and regulation of IFN-I

Translational research:Evaluation of therapeutic molecules

Cellular model: innate immunepDC, Monocytes

Integrated model:Human tonsils

In silico modeling

Organic chemistry

Molecular/cellular screening Selection of best molecules

IFN screeningPatients

Clinical network

In silico dockingFunctional screening

Organic chemistryOptimization

In vitro Ex vivo In vivo

Auto-immune pathologies

Interferonopathies

Patients

Auto-immunityInterferonopathies

Viral infection

Chemistry & Biology, Modeling & Immunology for Therapy (CBMIT)Understanding the regulation of type I interferons and inflammation

The chemokine receptor CXCR4 hypothesis

CBMIT, CNRS UMR8601, Université de Paris

HypothesisAmine’s activity is due to:

Their receptor(HR,SR..)

Bind to Toll-like R A common receptor

No No

Could CXCR4 be the common receptor?

ü Amino compounds bind to CXCR4 expressed by neurons

ü CXCR4 is highly expressed by human pDC

ü CXCR4 is a member of the GPCR family (as amine natural receptors)

Nicolas PietrancostaCR1, CNRS

Nikaïa SmithPhD-Posdoc

The chemokine receptor CXCR4: pleiotropic activity

Cell migration HIV coreceptor Tumor migration

Expressed by Hematopoietic Immune cells

NeuronsTumor cells

Chimiotaxism

GPCR family member 7 transmembrane

Natural ligand: CXCL12

Could CXCR4 be the common receptor?

Physiological Pathological

Extracellular domain

0

20

40

60

80

100

120

140

160

Rel

ativ

e m

RN

Aex

pres

sion

leve

ls

siCTR - - + -- ++siCXCR4 - + - ++ --

- + -++ - +-

IFNα Histamine CB Spermine

/

Mock

Silencing of CXCR4 in primary cells

0

20

40

60

80

100

120

140

160

Rel

ativ

e m

RN

Aex

pres

sion

leve

ls

siCTR - - + -- ++siCXCR4 - + - ++ --

- + -++ - +-

IFNα Histamine CB Spermine

Flu

/

Mock

CXCR4

siCTL / siCXCR4

Primary human pDC

CXCR4 siRNAHistamine or CB

+Flu

Flu

§ Synthetic ligand of CXCR4 § Very stable§ Developed by Novartis§ IC50 CXCR4 = 5-10 nM§ EC50 antagonist: 10 nM

IT1t

§ Other name Plerixafor§ Stable/clinically used§ IC50 CXCR4 = 5-10 nM§ EC50 antagonist: 10 nM

AMD3100Plerixafor

CB / IT1t

Structural similarties

CXCR4 specific ligands effect on type I IFN

CXCL12

§ Natural Ligand§ Protein§ Metastasis migration

CB is not a CXCR4 specific ligand, so what about CXCR4 ligands?

Inhibition of IFNa production by CXCR4 ligands

Immunomodulation of IT1t versus AMD3100

Purified pDC

R848

NS 0 0.5 1 5 10 50 µM AMD20

SSC

-A

IFN!

81.6 81.9 82.4 84.3 84.1 82.1 81.51.29

NS 0 0.5 1 5 10 20 500

50

100

150

IFNα

pro

duct

ion

(% o

f R84

8 st

imul

ated

pDC

)

R848 + Compound (µM)

**,xx

**** *,xxxx

***,xx

SSC

-A

R848

NS 0 0.5 1 5 10 50 µM IT1t2081.6 78.8 76.7 49.2 20 3.27 0.921.29

IFN!

Targeting CXCR4 ex vivo with IT1t:SLE model

• Systemic lupus erythematous (SLE) is defined by a complex clinical syndrome (e.g., arthritis,skin rashes, serositis, glomerulonephritis, nervous system involvement) and the production ofantinuclear antibodies (ANAs).

• Some individuals develop a type of skin disease, called cutaneous lupus erythematosus.

Systemic lupus erythematous: symptoms

Kaul et al., (2016) Nat Rev Diseases Primer

pDC & IFN: central players in lupus pathology

Role of IFN-I in systemic lupus erythematous (SLE)

Rodero et al., (2017) J Exp Med

Detecting the Undetectable: Ultrasensitive SiMoA Immunoassay. Digital IFN-a ELISA Complex interferonopathies

v Increased serum levels of IFN-α observed in many SLE patients correlate with both disease activity and key diseasemarkers

Guiducci et al. Nature 2010

v Inhibition of IFN-α/β induced TRAIL expression may reduce symptoms in SLE by stopping pathogenesis of autoantibodyproduction and autoimmune tissue injury

Zahn et al. British j. derm 2011

v Over-production of Type I interferon (IFN-I) is strongly associated with SLE and is involved in disease pathogenesis.Rodero et al. JEM 2017

Lupus is an interferonopathy

Brigitte Badder-MeunierPierre Quartier

HCP9P9 + IT1t

Gated on CD3+ cells

pSTAT1

1

2

3

4

pSTA

T1 (M

FI x

103 )

HD NS IT1t

**

Gated on CD3+ cells

pSTAT3

HCP9P9 + IT1t

2

4

6

8

pSTA

T3 (M

FI x

102 )

HD NS IT1t

**

Mathieu Rodero

+/- IT1t

overnightØ STAT activationØ IFNa productionØ Simoa

PBMC

SpI JuvenilLupus Patients

Ø pSTAT1 (= IFN-I, II)

Ø pSTAT3 (= IL-6)

0.1

1

10

100P6

P4

P5

P3IFNα(

fg/m

l)

IT1t - +

Ø Simoa: IFNa

Ex vivo effect of IT1ton SLE patient

Simoa

HDsP7P8P9

JSLEP4P5 JSLE

HDsP7

P9JSLEP8

P3

P6

Darragh Duffy

Lupus patients

+ R848+/- IT1t

overnightØ IFNa production

Anne-Sophie Korganow

PBMC

0.1

1

10

100

1000

10000

IFNα

(fg/

ml)

Unst 0 5 20R848 + IT1t (µM)

HC3HC4HC5HC6

P3P4P5P6

SLE

*****

ns

SSC

-A

BDCA-4

SSC

-A

IFNa

NS R848 R848 + IT1tHC2

0

1

10

20

1

1

P2

0.34

0.29

Ø Ultrasensitive digital IFNa ELISA (Simoa) Ø Intracellular staining of IFN-a

Ex vivo effect of IT1ton SLE patient

N. Bekadour N. Smith

Targeting CXCR4 with IT1t in vivo:Pristane-induced lupus in mice

10 Male DBA1/J

Pristane (IP) daily+/- Prednisolone+/- IT1t 3 doses

Cytokine in serumAnti-DNA antibodies

at Week 4,8,10

Pristane-induced SLE in mouse model

10 weeks

IT1t

Extracellular

Intracellular

CXCR4

CXCR4

Binding pocket

SLE mice model set upPristane SLE mouse model

0

20

40

60

IL-1β

(pg/

mL)

Week 4 Week 10

******

***

0

10

20

30

40

IL-1

7 (pg

/mL)

Week 4 Week 10

********

*******

0

2

4

6

8

101520

TRAI

L (p

g/m

L)

Week 10

****

*

• SLE is characterized by over-production of cytokines: IL-1b, IL-6, IL-17 and TRAIL• IL-17 Accurate Biomarkers for Systemic Lupus Erythematosus Disease Activity

IL-1b IL-17

TRAIL

0.0

0.5

1.0

1.5

2.0

dsDN

A (1

/50, O

.D.)

VehiclePrednisoloneIT1t - 3mpkIT1t - 10mpkIT1t - 30mpk

Week 4 Week 8 Week 10

* **

*****

****

IT1t effect on cytokine production in SLE in mice

• Systemic lupus erythematous (SLE) is defined by a complex clinical syndromeand the production of antinuclear antibodies (ANAs).

0.0

0.5

1.0

1.5

2.0

anti-

dsDN

A (1

/50,

O.D

.) VehiclePrednisoloneIT1t - 3mpkIT1t - 10mpkIT1t - 30mpk

Week 4 Week 8 Week 10

* **

*****

****

Vehic

lePr

edni

solo

ne3m

pk10

mpk

30m

pk

0

2

4

6

8

Mice

(num

ber)

GPT+GPT-

IT1t

0.0

0.5

1.0

1.5

2.0

dsD

NA

(1/5

0, O

.D.)

VehiclePrednisoloneIT1t - 3mpkIT1t - 10mpkIT1t - 30mpk

Week 4 Week 8 Week 10

* **

*****

****

IT1t effect on anti-dsDNA production SLE mice

Smith et al, 2019

Auto-immune diseases

Biased agonistAntagonist

Anti-tumoral

Inhibition cellular migration Inhibition of immune cells

The CXCR4 drug target

Novel activity

The chemokine receptor CXCR4 (GPCR)

Well-known activity

Cell migration Immune modulation

IFN-driven auto-immune diseases Inflammation-driven auto-immune diseases

Developpment of new anti-inflammatory drugs :Creation of Ermium therapeutics

Pascal NeuvilleCEO-Founder

Thierry LaugelPresident

Hadrien BouchezAssocitae

Nathalie LennePorfolio Director

Joel CrouzetCEO

JP HerbeuvalScientific Founder

Drug design and optimizationHeat/leads development

Preclinical studiesClinical studies

Simon HayekResearcher

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Interface Immunology VirologyNeurology Oncology Chemistry

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