A Novel Targeted Approach to the Treatment of Hemophagocytic Lymphohistiocytosis (HLH)

with NI-0501, an Anti-IFNγ Monoclonal Antibody: First Results from a Pilot Phase 2 Study

in Children with Primary HLH

Michael B. Jordan, F. Locatelli, C. Allen, F. De Benedetti, A. Grom, M. Ballabio, W. Ferlin, and C. De Min,

on behalf of the NI-0501-04 Study Group

HLH is a rare and life-threatening syndrome of extreme immune activation,

often associated with genetic defects of cytotoxic function, and characterized

by distinctive clinical patterns:

Prolonged fever

Splenomegaly/hepatomegaly

Cytopenias

Hyperferritinemia

Hypofibrinogenemia

Hypertriglyceridemia

Hemophagocytosis

Hypercytokinemia

2

Hemophagocytic Lymphohistiocytosis (HLH)

Current treatment of primary HLH

2nd Line HLH treatment No standard of care Alemtuzumab/ATG (no prospective data) - profoundly immunosuppressive

Survival thought to be very poor

1st Line HLH treatment Etoposide / Dexamethasone - myelosuppressive - broadly immune suppressive

Trottestam et al, 2011

Jordan et al, 2004

4

Elevated levels of IFNγ observed in patients with HLH

Henter et al, 1991; Tang et al, 2008, Xu et al, 2011; Bracaglia et al, 2014; Put et al, 2015

These data have led to the development of a neutralizing anti-IFNγ antibody for the treatment of patients with HLH: NI-0501

Pathogenic role of IFNγ in HLH Multiple data point to IFNγ as a rational target

Days after LCMV infection

Perforin-/- mice:

NI-0501 characteristics

Fully human IgG1 binding to human IFNγ with high affinity (KD =1.4 pM)

Recognizes free IFNγ and IFNγ bound to its receptor (R)

5

NI-0501-04 study

International, multicenter, open-label, single-arm, pilot study

Aimed at investigating the neutralization of IFNγ as a targeted innovative therapy for HLH

Study population: children with primary HLH receiving NI-0501 as 1st or 2nd line HLH therapy

Recruitment ongoing

6

Germany

Austria

Spain Italy

Cincinnati

Houston

Phoenix Boston

Wilmington

Sweden

Europe United States

Main inclusion and exclusion criteria Study NI-0501-04

Inclusion criteria

Primary HLH patients, up to and including 18 years of age

Diagnosis of HLH made on the basis of:

a) Genetic findings or family history consistent with primary HLH -OR-

b) Five out of eight clinical criteria fulfilled, as defined by the HLH-2004 study

Exclusion criteria

HLH secondary to rheumatic or neoplastic disease

Treatment with T-cell depleting agents (e.g. ATG) in the 2 weeks prior to enrollment

Active mycobacteria (typical and atypical), Shigella, Salmonella, Campylobacter or Leishmania infections; or evidence of prior or latent tuberculosis

History of malignancy

7

NI-0501-04 study design Concentration controlled study

Study day

8 weeks 4 weeks

NI-0501

0 3 6 9 Week 8

TREATMENT PERIOD FOLLOW-UP PERIOD

8

NI-0501 administered as an i.v. infusion, at the starting dose of 1 mg/kg

Initial interval between infusions: 3 days

Subsequent dose/ interval adjustments based on real-time pharmacokinetic monitoring and clinical assessments

Dexamethasone

(tapered depending on pt conditions)

Demographics and baseline HLH characteristics

Patients enrolled 16

Age range 0.2 - 13 yrs (median 1.2)

Body weight range 4.5 - 53 kg

Gender 8 F, 8 M

Patients with HLH causative mutations 12 (4 FHL2, 2 FHL3, 1 FHL4, 3 GS-2, 1 XLP-1, 1 XLP-2)

No. of patients with CNS involvement 4

No. of patients receiving NI-0501 in 1st line 2

No. of patients receiving NI-0501 in 2nd line 14 Prior HLH treatments: Dexamethasone

Methylprednisone Etoposide ATG CsA Others (IT therapy)

13 2

13 4 6 4

9

NI-0501-04 trial - Summary of outcomes

* Due to subsequent diagnosis of NK/T cell extranodal lymphoma # Received additional HLH therapy (one patient receiving pre-HCT conditioning currently) ^ One patient taken off trial, then NI-0501 was reintroduced on a compassionate use basis 10

16 Patients enrolled

13 Completed therapy

2 Ongoing

4 Insuff. response

9 Favorable response

2 Awaiting HCT

(HLH controlled)

7 Proceeded

to HCT

2# ^

Proceeded to HCT

2 Died

1 Excluded from

analysis*

Follow-up post HSCT

Pt. #1

Pt. #2

Pt. #3

Pt. #5

Pt. #6

Pt. #8

Pt. #12

Pt. #13

HSCT D+30 D+100 6-m 1-y

Post-HSCT follow-up

Pt. died of MOF/GvHD

11

*

* Pt. known to be alive

NI-0501 overall safety and tolerability Study NI-0501-04

12

Total number of patients enrolled 16

Number of patients withdrawn for safety reasons 0

Number of deaths on study 3*

Number of deaths related to NI-0501 0

Number of patients with SAEs 8

Number of SAEs 14

Number of SAEs related to NI-0501 1**

* One patient with post-enrollment diagnosis of NK/T cell extranodal lymphoma (secondary HLH)

** Necrotizing fasciitis following P. Aeruginosa skin infection (resolved), assessed as related to NI-0501 by the investigator, but not by Data Monitoring Committee and Sponsor

NI-0501 was very tolerated (more than 160 NI-0501 infusions performed to date)

No off-target effects have been observed in this ill patient population

The potential effects of IFNγ neutralization on immune defense in humans may be inferred from:

1. Patients with IFNγ receptor deficiency

2. Patients developing neutralizing anti-IFNγ autoantibodies

Atypical mycobacteria, M. Tuberculosis, Salmonella, and Varicella Zoster infections have been reported in these patients

13

Expected effects of IFNγ neutralization on immune defense

Dorman et al, 2004 Hoflich et al, 2004 Koya et al, 2009 Browne et al, 2012

Pre-existing and emerging infections No pathogen favored by IFNγ neutralization found

14

Pathogen Existing infections

at study start Outcome

Emerging Infection during study course

Outcome

Bacteria

Gram-Positive Bacteria

3 1 St. Epidermidis

2 C. Difficile

All resolved 3

St. Epidermidis, C. Difficile, E. Faecium

All resolved

Gram-Negative Bacteria

1 E.Coli

All resolved 3

P. Aeruginosa, K.Pneumoniae, E. Coli

All resolved

Virus

Adenovirus 0 NA 1 Not resolved*

CMV 1 Resolved 2 (reactivations) 1 Resolved 1 Improved

EBV 4 All resolved 1 (reactivation) Not resolved*

Parvovirus 1 Resolved 1 (reactivation) Improved

Parainfluenza T3 0 NA 1 Unknown**

Influenza A 0 NA 1 Not resolved*

Fungus Candida 0 NA 1 Resolved

* All observed in 1 patient with NK/T cell extranodal lymphoma ** Viral status not reassessed prior to patient’s death

Response data for patients enrolled in the NI-0501-04 study having received at least

4 weeks of treatment

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Day 0 EOT

Response to NI-0501 treatment - Neutrophil count A

NC

(n

eu

tro

ph

ils/m

cL)

No. of patients with ANC < 1000/mcl

Baseline (SD0) End of Treatment

10/14 2/14

p < 0.001 (*)

* One-sided single proportion test

16

EOT = end of treatment with NI-0501 Solid lines = pts. with favorable response Dashed lines = pts. with overall insufficient responses

No. of patients with PLT < 100 x103/mcl

Baseline (SD0) End of Treatment

12/14 5/14

p < 0.001 (*)

* One-sided single proportion test

Response to NI-0501 treatment - Platelet count

0

100

200

300

400

Day 0 EOT

Pla

tele

ts (

x10

3/m

cL)

17

-100

-80

-60

-40

-20

0

20

Day 0 EOT

Response to NI-0501 treatment - Ferritin Fe

rrit

in c

han

ge f

rom

bas

elin

e (

%)

18

Median serum levels

Baseline (SD0) End of Treatment

4142 ng/ml 1648 ng/ml

p = 0.0025 (*)

* Paired t-test on log transformed data

19

Bo

dy

tem

pe

ratu

re (°C

) Rapid normalization of fever Hours after first infusion of NI-0501

35

36

37

38

39

0 6 12 18 24 30 36 42 48

Pt. #1

35

36

37

38

39

0 6 12 18 24 30 36 42 48

Pt. #13

Patients with body temperature > 37.5 °C at initiation of NI-0501 treatment

Hours after infusion Hours after infusion

20

Impact of NI-0501 on organomegaly Patients with palpable spleen/liver at start of NI-0501

0

2

4

6

8

10

12

14

16

Day 0 EOT

* Measured by physical examination

Sple

en

(cm

fro

m c

ost

al g

rill)

*

0

2

4

6

8

10

12

14

16

Day 0 EOT

Live

r (c

m f

rom

co

stal

gri

ll)*

21

Response to NI-0501 treatment - CNS involvement

Baseline (SD0) EOT

Pt. #6

Obtundation; Hemiparesis Loss of developmental milestones

Resolved Fully Regained

Elevated protein and neopterin in CSF; Pleocytosis Resolved

Abnormal enhancement on MRI Improved

Pt. #3 Elevated protein in CSF; Pleocytosis Resolved

Pt. #15* Loss of walking ability Regained

Elevated protein and neopterin in CSF; Pleocytosis Resolved

Pt. #4^ Elevated protein in CSF; Pleocytosis Not evaluable

Abnormal infiltrates at MRI Not evaluable

Neurologic status at:

Note: patients received IT therapy, except pt. #4 in whom regular medicated LP was not performed * NI-0501 concentrations were measurable in CSF samples ^ Was not reassessed at EOT

Glucocorticoid tapering during NI-0501 treatment

Median dose

Baseline (SD0) End of Treatment

10.0 mg/m2 4.0 mg/m2

p = 0.023 (*)

No. of pts with tapering 50%:

10/13^

* two-sided Wilcoxon Signed rank test ^ data for 1 patient not yet available

0

2

4

6

8

10

Day 0 EOT22

Dex

amet

has

on

e d

ose

(m

g/m

2) 18

20

IFNγ neutralization during NI-0501 treatment CXCL9, a pharmacodynamic marker of IFNγ bioactivity

CX

CL9

ch

ange

fro

m b

ase

line

(%

)

IFNγ

CXCL9

-100

-80

-60

-40

-20

0

20

40

Day 0 EOT

Conclusions

NI-0501 treatment has shown the potential to improve or resolve relevant clinical and laboratory abnormalities of HLH, including CNS signs and symptoms

Response to NI-0501 is independent of:

the presence and the type of causative mutations

the presence and the type of an infectious trigger

NI-0501 was well tolerated

No safety concerns emerged to date (no myelotoxicity, no broad immunosuppression)

No infections caused by pathogens known to be promoted by IFNγ neutralization have been observed

The neutralization of IFNγ by NI-0501 can offer an innovative and targeted approach to the management of HLH

24

Acknowledgements

Maureen Deehan

Kathy de Graaf

Marie Kosco-Vilbois

Geneviève Lapeyre

Robert Nelson

MarthaL. Reynolds

Philippe Jacqmin

Christian Laveille

Investigators and Co-investigators:

M. Jordan, Cincinnati Children’s Hospital, Cincinnati, Ohio

F. Locatelli, H Pediatrico Bambino Gesu’, Roma, Italy

C. Allen, Texas Children’s Cancer Center, Houston, Texas

S. Cesaro, Policlinico G.B. Rossi, Verona, Italy

F. Fagioli, A.O. Regina Margherita, Torino, Italy

M. Henry, Phoenix Children Hospital, Phoenix, Arizona

C. Rizzari, H San Gerardo, Monza, Italy

C. Rossig, University Children's Hospital, Münster, Germany

J. Sevilla, Hospital Universitario Niño Jesús, Madrid, Spain

A. Filipovich, Cincinnati Children’s Hospital, Ohio, US

Patients and families

Study NI-0501-04

25

Principal Investigator US Michael Jordan E-mail: michael.jordan@cchmc.org

Principal Investigator EU Franco Locatelli E-mail: franco.locatelli@opbg.net

4 months later

Patient 6

Thank you to all patients and families participating in the NI-0501-4 trial

Dendritic cells CD8+ T cells

IFNγ (-) perforin

Macrophages

antigen (+)

Infection

Pathogenesis of Primary HLH Defined in experimental models

Loss of a dominant regulatory feedback loop

Terrell, 2013 Carmo, 2015

Increased activation of CD8+

T cells

Jordan, 2004 Lykens , 2011 Pham, 2012

Increased antigen presentation by dendritic cells

Lykens, 2011 Terrell(2), 2013

Increased IFNγ secretion

Jordan, 2004 Crozat, 2007 Pachlopnik, 2009 Sepulveda, 2013 Kogl, 2013 Jessen, 2013

HLH

IFNγ acts via macrophages to

produce signs of HLH

Zoller, 2011

28

sCD25 course during NI-0501 treatment

100

2000

40000

Day 0 EOT

sCD

25

(p

g/m

l)