An Observational, Multicenter Study to Evaluate Interferon Gamma (IFNγ) and Other Inflammatory Mediators in Patients With Malignancy-Associated Hemophagocytic Lymphohistiocytosis (M-HLH)
Conditions: Hemophagocytic Lymphohistiocytosis
Sponsor: Swedish Orphan Biovitrum

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Protocol NI-0501-07 version 1.0, dated 12 Feb 2016 
 
 
 
An observational, multicenter study to evaluate interferon 
gamma (IFN) and other inflammatory mediators in patients 
with malignancy-associated hemophagocytic 
lymphohistiocytosis (M-HLH) 
 
 
 
Protocol number: 
NI-0501-07 
Version: 
Version 1.0 
Date: 
12Feb 2016 
Sponsor: 
NovImmune S.A. 
14 Chemin des Aulx 
1228 Plan-les-Ouates 
Switzerland 
Confidentiality Agreement 
This document is a confidential communication of NovImmune S.A. Acceptance of this document 
constitutes the agreement by the recipient that no unpublished information contained within will be 
published or disclosed without prior written approval, except as required to permit review by responsible 
Ethics Committees and Health Authorities or to obtain informed consent from potential subjects. 
 
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Contact list 
 
Investigator: 
Naval Daver, MD 
Assistant Professor 
Leukemia Department 
MD Anderson Cancer Center 
1515 Holcombe Blvd 
Houston, TX 77030  
Phone: +01 713 794 4392 
Sponsor: 
NovImmune S.A. 
14 Chemin des Aulx 
1228 Plan les Ouates 
Switzerland 
NovImmune staff: 
 
Chief Medical Officer 
 
 
 
Clinical Science Leader (primary contact): 
Cristina de Min, MD 
Phone: +41 22 593 5116 
Email: cdemin@novimmune.com 
 
Emmanuel Monnet, PhD 
Phone: +41 22 593 8233 
Email: emonnet@novimmune.com 
 
Exploratory Science and Translational 
Medicine Head: 
 
 
Walter Ferlin, PhD 
Phone: +41 22 593 5120 
Email: wferlin@novimmune.com 
 
Data Management: 
Sébastian Meier Piantanida 
Phone: +41 22 593 5124 
Email: smeierpi@novimmune.com 
 
 
Clinical Operations: 
MarthaLee Reynolds 
Phone : +41 79 916 65 15 or +1 804 683 2541 
 
Email : mreynolds@novimmune.com  
 
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Protocol signature page 
 
 
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Contents 
Contact list .................................................................................................................................... 2 
Protocol signature page ................................................................................................................. 3 
Contents ........................................................................................................................................ 4 
Background and Rationale ............................................................................................................ 5 
Study Objectives: ........................................................................................................................ 10 
Sample Size: ................................................................................................................................ 10 
Study Population: ........................................................................................................................ 10 
Inclusion Criteria: ....................................................................................................................... 10 
Study Design: .............................................................................................................................. 10 
Study Duration: ........................................................................................................................... 11 
Exploratory Parameters: ............................................................................................................. 11 
Statistical Analysis: ..................................................................................................................... 13 
Medications: ................................................................................................................................ 13 
Informed consent: ....................................................................................................................... 13 
Data collection and patient confidentiality: ................................................................................. 14 
Study administration: .................................................................................................................. 14 
 
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Background and Rationale 
Interferon gamma (IFN) in secondary HLH 
HLH is a syndrome of severe immune activation and deregulation characterized by hyperactive 
macrophages and lymphocytes, pro-inflammatory cytokine hypersecretion, hemophagocytosis, 
and tissue and organs damage. HLH occurs as either a primary (pHLH, familial) or secondary 
(sHLH, sporadic) disorder (Henter et al., 1998; Janka, 1983). pHLH is an autosomal recessive 
disease with an incidence of 1/50,000 live-born children (Henter et al. 1991; Arico et al. 1996). 
Patients often have a clear familial inheritance or genetic mutation. pHLH is caused by 
homozygous mutations in the genes coding for perforin, Munc13-4, syntaxin 11, Munc18-2 and 
other proteins involved in cytotoxic granule activation, polarization, priming, fusion, or function  
(Jordan et al., 2011; Zhang et al.,  2014). These mutations result in defective immune effector cell 
function, uncontrolled activation of histiocytes, and pathognomic clinical manifestations of HLH. 
Secondary HLH often occurs as a result of pathological immune activation in response to a 
trigger. Frequently noted triggers include malignancies (especially hematological malignancies 
including acute leukemia’s and lymphomas), infections (especially EBV) and rheumatologic 
disorders (sJIA, SLE). The list of triggers associated with secondary HLH is extensive (Henter et 
al., 1997). Primary HLH is reported to be more frequent in childhood. 
The pathogenesis of secondary HLH remains a matter of debate. Reports have recently described 
patients developing Macrophage Activation Syndrome (MAS)  in the context of systemic 
juvenile idiopathic arthritis (sJIA). These patients were identified to be heterozygous for rare 
variants in the primary HLH-associated genes (Zhang et al., 2014) highlighting potential 
etiological, clinical, and pathological similarities between familial and acquired forms of HLH. A 
link between mutations in the perforin gene and predisposition to hematological malignancies has 
been described (Clementi et al., 2005; Zhang et al., 2014), further supporting a possible 
contribution of hypomorphic mutations in HLH-related genes to the development of M-HLH 
(Lehmberg 2015).  
Immune-activation and immune mediated pathology likely play a central role in the evolution of 
HLH. Secondary HLH is characterized by acute clinical signs and symptoms of immune 
activation including hepatomegaly, jaundice, adenopathy, rash, seizures, and focal neurologic 
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Protocol NI-0501-07 version 1.0, dated 12 Feb 2016 
 
deficits as well as laboratory measures such as strikingly high serum levels of numerous 
cytokines including interferon gamma (IFNγ), tumor necrosis factor α (TNF-α), interleukin 6 (IL-
6), IL-10, and macrophage–colony-stimulating factor (M-CSF) (Akashi et al., 1994; Ohga et al., 
1993). Furthermore, biopsies of lymphoid tissues or histological examination of liver tissue from 
HLH patients reveal highly activated macrophages and lymphocytes, further supporting striking 
activation of the immune system. Secondary HLH carries a significant mortality rate with 
approximately 10 to 30% of the diagnosed patients succumbing from the disease (Celkan et al., 
2009; Minoia et al., 2014; Grom et al., submitted). Categorizing patients into primary or 
secondary HLH based on clinical manifestations is often difficult and may be of limited value. 
Treatment should be initiated expediently if the diagnosis of HLH is suspected, regardless of the 
classification/trigger. 
Secondary HLH that develops in the setting of malignancy is also known as malignancy-
associated hemophagocytic lymphohistiocytosis (M-HLH). The majority of malignancies 
triggering secondary HLH in children and adults are lymphomas and leukemia. While primary 
forms of HLH are more common in younger patients, M-HLH is more frequent in adults (Ramos-
Casals et al., 2014). Machaczka et al. (2011)  estimated that HLH affects 0.9% of adults with 
hematological cancer, but the prevalence may be as high as 20% in patients with specific types of 
B-cell lymphoma’s (intravascular B-cell lymphoma or B-cell lymphoma without peripheral 
adenopathies) and T-cell lymphoma (nasal natural-killer-cell or panniculitis-like subtypes) 
(Ramos-Casals et al., 2014). 
M-HLH may manifest during the treatment (iatrogenic) of a known malignancy or as the 
presenting feature of a yet undiagnosed malignancy (non-iatrogenic). It is postulated that the 
hyperinflammation is triggered by the neoplasm or its treatment, due to an excessive secretion of 
pro-inflammatory cytokines by the malignant cells or by T-cell activating immunotherapies for 
the treatment of leukemia/lymphoma and emerging for treatment of solid tumors (e.g. bispecific 
monoclonal antibody blinatumomab, CAR T-cell therapies) (Hijiya et al., 2005; Teachey et al., 
2013; Lee et al., 2014).  
The malignancy induced immunodeficiency combined with therapy likely predisposes to 
infections and defective T-/NK-cell function reducing the threshold for triggering HLH in these 
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patients (Celkan et al., 2009). Indeed, similarly to primary HLH, infections are often identified as 
co-triggers in iatrogenic M-HLH.  
The clinical course of M-HLH is often aggressive, and characterized by poor outcome, which are 
further impacted by the nature and status of the underlying malignancy. A single center 
retrospective study in adults reported only 1 of 8 patients survived despite identification of HLH 
and implementation of HLH directed therapies (Machaczka et al., 2011). A recent retrospective 
study in children and adolescents (n=27) reported a 6-month survival rate of 67% in non-
iatrogenic M-HLH and 63% in iatrogenic M-HLH (Lehmberg et al., 2015). The group at MD 
Anderson Cancer Center retrospectively identified pathologic hemophagocytosis and/or 
lymphohistiocytosis in 61 adult patients with underling malignancies between 1991-2001. Thrity 
five patients had additional systemic variables conferring a high suspicion for M-HLH. The 
median survival among the 35 patients was a dismal 1.5 months. The median survival was 1.2 
months among patients treated with HLH directed therapy as compared to 1.8 months among 
those not treated with HLH directed therapies. 31% of patients were alive at 6-months 
(Tamamyam, Daver et al.; manuscript submitted). 
An effective therapeutic approach to M-HLH (iatrogenic and non-iatrogenic) remains unknown. 
Of note is that agents that are currently considered as standard therapies for HLH (corticosteroids, 
etoposide) are often part of the treatment of malignancies known to be able to cause M-HLH.  
Furthermore, the addition of etoposide, alemtuzumab, dexamethasone may induce worsening 
myelosuppression and immunodeficiency in patients who have an underlying malignancy and/or 
recent systemic therapy. A non-myelosuppressive and less immunosuppressive therapy would be 
an ideal approach to evaluate in M-HLH patients. A prompt initiation of an HLH-directed 
regimen (before or in combination with the malignancy-directed therapy) is of critical importance 
to decrease the high mortality rate in these patients. Furthermore, studies have described the use 
of tocilizumab, an anti-IL-6R antibody, to manage cytokine release syndrome occurring after 
treatment with T-cell engaging therapies (Barrett et al., 2014). 
NI-0501 is a fully human anti-interferon gamma (IFNγ) monoclonal antibody (mAb) that binds to 
and neutralizes IFNγ. NovImmune is developing NI-0501 as the first targeted monoclonal 
therapy for HLH. Several lines of evidence support the investigation and use of therapeutic 
agents targeting IFNγ in HLH: 
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1. Two animal models of pHLH and two animal models of sHLH have been investigated in the 
context of the NI-0501: 
 Perforin knockout mice and Rab27-deficient mice develop all the diagnostic and many of 
the clinical and laboratory characteristics of human primary HLH when infected with 
lymphocytic choriomeningitis virus (LCMV) as a trigger. Neutralization of the high 
circulating IFNγ levels in both animal models led to the reversal of HLH clinical and 
laboratory abnormalities, and dramatically improved survival in the animal model 
characterized by high mortality (pfp -/-  mice) (Pachalopnik Schmid et al., 2009, Jordan et 
al., 2004).  
 Repeated administration of CpG, causing TLR9 stimulation, has been used to mimic a 
chronic severe hyperstimulation in healthy mice as a model of HLH secondary to 
infection. Although these mice do not necessarily die, they develop typical clinical and 
laboratory features of HLH (Behrens et al., 2011). Administration of an anti-IFNγ 
antibody led to reversal of clinical and laboratory features of HLH. Importantly, in this 
model it was demonstrated that administration of the anti-IFNγ antibody also led to full 
neutralization of IFNγ effects in relevant target tissues, such as liver and spleen (Buatois 
et al, submitted). 
 To study the physiopathology of sHLH in the context of rheumatic diseases, an animal 
model has been generated using IL-6 transgenic mice expressing high levels of IL-6, to 
mimic what occurs in patients with sJIA. When triggered with TLR ligands, these mice 
die with many of the features of the human HLH (Stippoli et al. 2012). In these mice, 
when IFNγ was neutralized by administration of an anti-IFNγ antibody, survival was 
markedly improved and HLH laboratory parameters reverted. 
In conclusion, in all presented animal experiments, IFNγ was demonstrated to be a key 
pathological effector in disease onset and progression. 
2. Primary and secondary HLH patients have hypercytokinemia with elevated levels of several 
pro-inflammatory cytokines including IFNγ (Henter et al., 1991; Janka et al., 1998; Xu et al. 
2012). More recently, high levels of IFNγ were demonstrated both in patients with HLH 
secondary to infections and in sJIA patient developing MAS (Buatois et al., manuscript 
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submitted; Bracaglia et al. manuscript submitted). The levels of CXCL9, CXCL10 and 
CXCL11, three chemokines known to be induced by IFNγ, were also significantly elevated. 
Noteworthy, levels of IFNγ and of the three IFNγ-inducible chemokines were found to 
significantly correlate with laboratory parameters of disease severity, such as ferritin, platelet 
count and transaminases. 
3. A Phase 2 pilot study is presently ongoing to evaluate the efficacy and safety of treatment 
with NI-0501, an anti-IFNγ monoclonal antibody, in pHLH. Based on data gathered so far, 
NI-0501 treatment has been well tolerated and shown a favorable impact on all relevant 
clinical and laboratory features of HLH such as fever, splenomegaly, cytopenia, 
hyperferritinemia, hypofibrinogenemia, and also CNS signs/symptoms (Jordan et al, 2015). 
No animal models specific to M-HLH have been described (Brisse et al., 2015). The levels of 
IFNγ and other pro-inflammatory cytokines at diagnosis and their correlation with clinical 
evolution and severity of the HLH manifestations during and after therapy have been poorly 
characterized for M-HLH. The fact that primary HLH and HLH secondary to 
inflammatory/autoimmune diseases or infections have many pathophysiological and clinical 
features in common with M-HLH, suggests that IFNγ might also play a key role in M-HLH. The 
present observational study is designed to provide a better understanding of the potential use of 
IFNγ and IFNγ-inducible chemokines as markers of M-HLH disease activity and potential 
prognosis and of the possible role of IFNγ as a therapeutic target in M-HLH, and to explore the 
genetic profiles in patients with M-HLH with regard to HLH-related genes. 
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Study Objectives: 
 To determine the levels of inflammatory markers including, but 
not limited to interferon gamma (IFN), interleukin 1 beta (IL-
1), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 17 
(IL-17), soluble IL-2 Receptor α (sCD25), C-X-C chemokine 
ligand 9 (CXCL9), C-X-C chemokine ligand 10 (CXCL10), C-X-
C chemokine ligand 11 (CXCL11), soluble CD163 (sCD163), 
neopterin and Tumor Necrosis Factor alpha (TNFα) in patients 
diagnosed with M-HLH and whenever possible, to monitor the 
evolution over time of these markers. 
 To assess the relationship between the above mentioned 
inflammatory cytokines and disease activity. 
 To assess the relationship between genetic variants of the genes 
typically causing primary HLH and M-HLH and associated 
inflammatory markers. 
Sample Size: 
A minimum of 14 (maximum of 50) M-HLH patients will be 
studied, including a minimum of 7 non iatrogenic M-HLH and 7 
iatrogenic M-HLH. A control group of 10 patients with a diagnosis 
of hematological malignancy in absence of HLH will also be 
evaluated.  
Study Population: 
Male and female (adult and children) patients who are diagnosed 
with M-HLH and that meet the inclusion criteria listed below.  
In addition, patients with a diagnosis of hematological malignancy in 
the absence of HLH will be included as a control group. 
Inclusion Criteria: 
 
 
 
 
 
 
1. Patients diagnosed with HLH in the context of a malignancy. 
The diagnosis of M-HLH will be established by the treating 
physician.  
2. The patient or patient’s legal representative (in case the patient is 
< 18 years old) must have consented to the use of their clinical 
data for research purposes at the site. 
 
3. For the control group, patients with a diagnosis of hematological 
malignancy and never diagnosed with HLH or no suspicion of 
HLH (at any time, including time after sample collection in case 
of use of retrospective sample). 
 
Study Design: 
This is a non-interventional study designed to determine the levels of 
pro-inflammatory markers (as listed above) in patients diagnosed 
with M-HLH and to assess the relationship between the biomarkers 
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and disease activity and prognosis during the M-HLH course. 
Inflammatory markers will also be measured in a group of control 
patients diagnosed with hematological malignancy in absence of 
HLH (as described above). 
Data collection may be performed retrospectively if sufficient 
clinical information is available to allow for a meaningful 
interpretation of the biomarker results. 
An approximate volume of 500 µl – 1 ml of serum or plasma is 
required per time point. Whenever possible, serum (and not plasma) 
should be collected, as this is the preferred matrix for the biomarker 
analysis. 
In addition to the serum samples for the biomarker analysis, relevant 
information gathered by the treating physician will be collected in a 
data collection form. This will include information on the clinical 
presentation of M-HLH, the type of underlying malignancy, the date 
of onset of malignancy in relation to HLH manifestations, treatment 
regimen prior to and/or ongoing at the time of M-HLH onset, 
laboratory parameters, M-HLH disease activity (e.g. newly 
diagnosed, reactivation), specific HLH therapy with best response to 
the therapy, other concomitant medications, patient follow-up and 
disposition, stem cell transplant status, duration of response, and 
survival. Whenever possible, collection of serum samples for 
biomarker analysis and relevant information should occur at M-HLH 
diagnosis, at regular time intervals during the treatment course, at 
resolution and/or reactivation of the disease. 
In addition to the above a minimum of 1 ml of whole blood will be 
collected 
(EDTA 
tubes) 
for 
genetic 
characterization 
and 
identification of the presence of genetic variants of the genes 
typically causing primary HLH. A specific consent for genetic 
testing must be given by the patient. Genetic samples may also be 
collected from existing bank of samples (frozen whole blood in 
EDTA or frozen extracted DNA), if proper consent has been 
obtained. 
Study Duration: 
This study collects information on M-HLH patients at diagnosis and 
during treatment of the disease, and will last until a minimum of 14 
(maximum of 50), including at least 7 iatrogenic and 7 non-
iatrogenic M-HLH patients and up to 10 hematological malignancy 
patients in absence of HLH have been recruited. 
Exploratory 
Parameters: 
1. Laboratory parameters, at the time of sample collection, 
assessed at the local laboratory, according to local clinical 
practice, such as: 
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 Hemoglobin, Hematocrit, Red blood cells, white blood cells 
and differential count, platelets 
 Fibrinogen 
 Ferritin  
 Fasting triglycerides 
 AST, ALT, Alkaline Phosphatase 
 BUN, serum creatinine  
 Albumin (if available) 
 Sodium (if available) 
 Beta2 microglobulin (if available) 
 Serum IgG levels (if available) 
 Lactate dehydrogenase 
 Total and conjugated Bilirubin  
 D-dimers 
 CRP 
 NK-cell activity (if available) 
2. Inflammatory markers of disease activity. The following 
assessments will be performed by the NovImmune Bioanalytical 
Assay Laboratory: 
 IFN 
 sCD25 
 IL-1β, IL-6, IL-10, IL-17 
 TNFα 
 CXCL9, CXCL10, CXCL11 
 sCD163 and neopterin 
3. Additional parameters recorded at the time of diagnosis will 
be collected when available for a posteriori diagnosis:  
 Temperature 
 Splenomegaly 
 Hb concentration 
 Platelet and neutrophils count 
 Triglycerides 
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 Fibrinogen 
 Hemophacocytosis 
 NK cell activity 
 Ferritin 
 sCD25 
Statistical Analysis: 
All analyses are exploratory. Statistical analyses of the data collected 
in this study will be focused on providing tabular and graphical 
summaries of the levels of inflammatory markers and of the clinical 
features of the disease both at diagnosis and during the course of 
treatment. Levels of inflammatory markers will be compared 
between patients with active M-HLH and the control group (i.e. 
hematological malignancy in absence of HLH). In addition, the 
degree of correlation between inflammatory markers, and between 
inflammatory markers and the severity of clinical manifestations, 
response to therapy, and survival of the patients will be assessed. 
Analyses may also be performed to study these relationships in 
various subgroups of patients, depending on the demographic, 
genetic and clinical characteristics observed, including a posteriori 
diagnosis based on HLH-2004 diagnostic criteria (Henter et al., 
2007).  
Criteria for diagnosis are: 
i) 
Fever 
ii) Splenomegaly 
iii) Cytopenia (affecting ≥ 2 of 3 lineages in peripheral blood):  
 Hb <90 g/L (in infants < 4weeks: Hb<100 g/L) 
 Platelets<100 x 109/L 
 Neutrophils <1.0 x 109/L 
iv) Hypertriglyceridemia 
and/or 
hypofibrinogenemia: 
Fasting 
triglycerides ≥3.0 mmol/L; Fibrinogen ≤1.5 g/L 
v) Hemophagocytosis in bone marrow or other specimens 
vi) Low or absence NK-cell activity (according to local laboratory 
reference) 
vii) Ferritin ≥ 500 µg/L 
viii) sCD25 ≥ 2400U/ml 
Medications: 
Any medications that are considered necessary for the patient’s 
welfare are given based on the decision of the treating physician. 
Informed consent: 
 
It is the responsibility of the investigator to obtain written informed 
consent from each patient participating in this study, after adequate 
explanation of the aims, methods and potential hazards of the study. 
An informed consent form will be signed. An additional signature 
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will be requested specifically for genetic analyses. 
Patients may withdraw their consent at any time; it is the 
responsibility of the investigator to contact the sponsor for specific 
patient withdrawal of consent while maintaining the confidentiality 
by communicating only patient codes. The sponsor will proceed with 
the destruction of all left over patient material within 30 days. The 
sponsor will also inform and request confirmation from potential 
collaborators to destroy aliquots that may have been transferred 
under collaboration agreements for the purpose of the research 
described in this protocol. The data generated prior withdrawal of 
consent will be maintained. 
Data collection and 
patient 
confidentiality: 
Relevant clinical information including date of diagnosis, medical 
history and previous and current treatments will be gathered by the 
treating physician. 
The investigator must assure that patients’ anonymity will be 
maintained and that their identities are protected from unauthorized 
parties. On Data Collection Forms or other documents or material 
submitted to the sponsor, patients should not be identified by their 
names, but by an identification code. The investigator should keep a 
patient log showing codes, names and addresses. The investigator 
should maintain documents not for submission to NovImmune, e.g., 
patients’ written consent forms, in strict confidence. 
Study 
administration: 
 
Record Retention 
The investigator will ensure that essential records are kept in a secure 
archiving facility for the retention period stipulated in the study 
contract. Essential documents include, but are not limited to, the 
following: 
 Signed informed consent documents for all patients 
 Subject identification codes 
 Record of all communications between the investigator and 
the IEC 
 Composition of the IEC 
 Record of communications between the investigator, 
NovImmune and their authorised representative 
These records will be held in the Investigator’s archives. If the 
Investigator is unable to meet this obligation, the Investigator must 
ask NovImmune for permission to make alternative arrangements. 
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Details of these arrangements should be documented in the clinical 
study centre TMF. 
Financial Disclosure 
Investigator is required to provide financial disclosure information to 
allow NovImmune to submit complete and accurate certification or 
disclosure statements in accordance with applicable national and 
local regulations upon request. In addition, investigator must provide 
NovImmune with a commitment to promptly update this information 
if any relevant changes occur during the course of the investigation 
and for 1 year following the completion of the study. 
Disclosure of Protocol and Study Results and Publication Policy 
Publication or communication of results will be decided upon 
completion of the study or earlier if data are considered sufficiently 
novel and robust. NovImmune will comply with the requirements for 
publication of results involving human research.  
Authorship will be determined by mutual agreement and in line with 
ICMJE authorship requirements. Any formal publication of the study 
in which contribution of NovImmune personnel exceeded that of 
conventional monitoring will be considered as a joint publication by 
the investigator and the appropriate NovImmune personnel. 
So-called ‘ghost writing’ is not permitted. All contributors who do 
not meet the criteria for authorship should be listed in an 
acknowledgments section. Examples of those who might be 
acknowledged include a person who provided purely technical help, 
writing assistance, or a department chairperson who provided only 
general support. 
Any inventions and resulting patents, improvements, and/or know-
how originating from the use of data from this study will become and 
remain the exclusive and unburdened property of NovImmune, 
except where agreed otherwise. 
Monitoring and Auditing 
As the aim of this study is mainly to test blood for research purpose 
no specific monitoring at site is foreseen by NovImmune. However 
for compliance with applicable government regulations with respect 
to current GCP and standard operating procedures, direct access to 
the on-site study documentation and medical records must be 
ensured to any audit or inspections. 
 
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