Open-label, Observational, Prospective, 9-month Study to Assess the Efficacy of Ofatumumab on Microglia in Patients With Relapsing Forms of Multiple Sclerosis
Phase: PHASE3
Conditions: Relapsing Multiple Sclerosis
Sponsor: Brigham and Women's Hospital

 
1 
 
 
Study Protocol and Statistical Analysis Plan 
 
Title: Open-label, Observational, Prospective, 9-month Study to Assess the Efficacy of 
Ofatumumab on Microglia in Patients with Relapsing Forms of Multiple Sclerosis 
 
NCT number: NCT04510220 
 
Date: December 28, 2023 
 
 
Background and Significance  
 
Historical background 
 
Role of microglia in MS: 
 
Multiple sclerosis (MS) is primarily a demyelinating disease of the central nervous system (CNS), but 
many patients also undergo progressive atrophy, especially in the gray matter (GM). GM atrophy plays 
a particularly prominent role in cognitive and physical decline in MS. White matter (WM) 
demyelination is also correlated with cognitive and physical impairment, albeit GM pathology 
accounts for the most variance in predicting cognitive and physical impairment.  
 
Both neuroinflammation and neurodegeneration occur during all stages of MS.1  Axonal loss 
underlies especially the transition from relapsing to progressive MS, but neurodegeneration with 
axonal transections is known to be present already early in the disease. By studying progressive MS, 
one may thus gain important insights about the degenerative component of MS present in all stages of 
the disease. 
 
Microglia are myeloid cells of the CNS are derived from early embryonic erythromyeloid 
progenitor cells and have migrated into the developing nervous tissue, where they have begun the 
maturation process into brain-resident microglial cells. 2 Microglia constitute approximately 10% of 
the total cell population in a human brain. The key tasks performed by microglia include phagocytosis 
of extracellular debris, antigen presentation to host immune cells, and secretion of inflammatory 
mediators. 3 
 
In the early phase of MS, microglial activation promotes the recruitment of naïve T cells, which 
are activated in the periphery by dendritic cells functioning as antigen-presenting cells. Microglia also 
act as antigen-presenting cells, re-stimulating the auto-reactive memory-T-cells invading through the 
blood–brain barrier (BBB) into the CNS. This is considered a key event in maintaining the chronic 
inflammation in the CNS. 4,5 In active WM lesions, there is a profound infiltration of activated 
microglia and blood-borne macrophages throughout the lesion, whereas in slowly expanding 
(smoldering) or chronic active lesions, the microglia and macrophages are concentrated as a dense rim 

 
2 
 
 
around the lesions. 6 Microglia have also been found diffusely in WM and GM with concomitant 
axonal degeneration and meningeal inflammation. 7-9 Thus, chronic activation of microglia has been 
linked to neurodegeneration in the progressive phase of the disease. 7-9 In progressive MS disease, 
activation of microglia has been detected in areas of the brain which appear normal in conventional 
MRI, the so called normal appearing (NA) WM (NAWM) and NAGM. 10 When microglia are 
activated following an insult, they become mobile and capable of phagocytosis and antigen 
presentation. The phagocytosis of debris by microglia is thought to be essential for enabling the return 
of normal tissue homeostasis after an insult. In chronic neuroinflammation, the equilibrium between 
debris-clearing and inflammation-propagating microglia becomes unbalanced, and the hyper-activated 
microglia cause uncontrolled neuronal damage resulting in a self-propagating vicious cycle and 
progressive neurodegeneration. 11 It is thus possible that prolonged inflammation related to activated 
microglial is one of the driving forces of the chronic progression of MS disease. 
 
Imaging of microglia activation using positron emission tomography: 
 
Positron emission tomography (PET) imaging enables molecular and neuroreceptor imaging in vivo. 
Particularly relevant for the study of MS is PET imaging of activated microglia. Here, several 
radioligands binding to the 18 kDa translocator protein (TSPO)—a protein structure expressed on the 
outer mitochondrial membrane of activated, but not resting microglia—have been developed. 12 
Activated microglia express TSPO, earlier called the peripheral benzodiazepine receptor (PBR) on the 
outer membrane of mitochondria. Several functions have been attributed to TSPO, either directly or 
indirectly, including immunomodulation, regulation of cholesterol transport, synthesis of steroid 
hormones, apoptosis, cell proliferation, porphyrin and anion transport, regulation of mitochondrial 
functions and heme synthesis. 13 Two archetypal ligands binding to TSPO have been identified, namely 
Ro5-4864, the 4-chloro-derivative of diazepam, and PK11195, the isoquinoline carboxamide, 14 the 
latter having been used as a marker for in vivo PET imaging of activated microglia. Numerous second-
generation TSPO radioligands with higher affinity and specificity than their predecessor [11C](R)-
PK11195 have been recently developed.12,15 18F-PBR06 is a second-generation TSPO ligand with 
promising imaging characteristics. 16-19 Recent studies showed increased 18F-PBR06 uptake 
corresponding specifically to activated microglia in animal models.20-22 A robust approach for 
quantitative in vivo assessment of specific 18F-PBR06 binding to TSPO has recently been 
developed.23,24 The Singhal et al. study demonstrated that quantitative TSPO PET with a second-
generation 18F-PBR06 can be used to characterize innate immune responses in MS in vivo and 
provides further evidence supporting an association between the WM TSPO PET signal in lesions and 
disease severity. 23 This approach is practical for extension to studies of the role of the innate immune 
response in MS for differentiation of anti-inflammatory effects of new medicines and their longer-term 
impact on clinical outcome. 
 
Rationale and potential benefits 
 
 There has been an increasing recognition of the role of B-cells in the pathogenesis of MS since the initial 
description of intrathecal immunoglobulin synthesis in 1950. In MS, B-cells are present in parenchymal 

 
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lesions, tertiary follicle like structures in the meninges and cerebrospinal fluid where they are known to 
undergo clonal expansion. B-cell directed therapies using anti-CD20 antibodies have been shown to be 
highly efficacious in preventing relapses in RRMS and slowing progression in primary progressive MS 
although their mechanisms of action are not clear.25,26 In animal models, targeting B-cells using 
antiCD20 antibodies led to reduction in microglial activation and lesion formation.27 In fact, there was 
greater effect of anti-CD20 antibodies on microglial activation and lesion formation in the antibody 
independent model implying that the mechanism of action of these therapies extends beyond antibody 
production and may involve their actions on microglia.27 However, there is no human data to date 
exploring this hypothesis. Ofatumumab is a fully human anti-CD20 antibody that has recently been 
studied for treatment of relapsing MS in multiple phase II and phase III randomized controlled trials 
(ASCLEPIOS I and II). 
 
Prior studies using Ofatumumab have demonstrated the time course of B-cell depletion in the periphery. 
There was 50% reduction by week 2, and >80% reduction by week 4 after administration of Ofatumumab 
at various doses.25 Ofatumumab demonstrated high efficacy for relapse rate reduction, new lesion 
formation and serum neurofilament light chain level reduction in multiple sclerosis patients as compared 
to an active comparator arm of teriflunomide (Aubagio)25,28,29.  Additional secondary endpoints were 
also met, where ofatumumab showed a relative risk reduction in both 3-month confirmed disability 
(CDP) and in 6-month CDP versus teriflunomide in a pre-specified pooled analysis. Ofatumumab was 
found to be safe in MS patients.25,28,29 
 
However, the effects of Ofatumumab on microglia in the brain in MS patients are not known at this time. 
It is important to study the effect of anti-CD20 therapies on the innate immune system to understand 
their role in limiting disease progression in MS. We aim to assess the relationship between peripheral B-
cell depletion and changes in microglial activation, after administration of Ofatumumab. 
 
We aim to assess the effect of Ofatumumab on microglial activation using [F-18]PBR06 PET in MS 
patients in relation to changes in serum markers, MRI abnormalities and clinical impairment 
longitudinally over 9 months. 
 
Specific Aims: 
 
Specific Aim 1: To determine the effect of Ofatumumab on microglial activation in MS over 9 months. 
 
Specific Aim 2: To determine the time course of effect of Ofatumumab on microglial activation and its 
relationship with peripheral B-cell depletion, serum neurofilament light (sNfL) chain and glial-
fibrillary acid protein (GFAP) levels and other serum biomarkers 
 
Specific Aim 3: To determine the relationship of PET changes following Ofatumumab initiation with 
3T MRI changes and clinical parameters. 
  
 
 
 
 

 
4 
 
 
Design:  
 
This is an open-label, observational, prospective, 9-month follow-up study to assess the efficacy of 
ofatumumab on microglia pathology in patients with MS, as measured by changes in microglial 
activation in the lesional and non-lesional, normal appearing white matter, cortical and subcortical grey 
matter, and peri-plaque area of chronic lesions in the brain. 
 
Initial Visit: 
 
During the first visit, subjects will be administered the screening questionnaire (if that has not already 
been done over telephone). Subjects will review and eventually sign the consent form. They will be 
administered a physical examination, clinical assessment and standardized questionnaires for cognitive 
testing and/or other co-morbidities. In addition, blood samples will be drawn for genotype testing, 
infection screening, complete blood count and liver function test. 
 
Demographics, physical examinations and neurologic assessments will be conducted at Brigham MS 
Center, Brigham and Women's Hospital, 60 Fenwood Road, Boston, MA 02115. 
 
Genotype Testing: 
 
Blood sample drawn on the initial screening visit will be used to obtain genomic DNA for genotyping 
for polymorphism within the TSPO gene on chromosome 22q13.2, using a Taqman assay. High 
affinity and medium affinity binders will be included in the study, whereas low affinity binders will be 
excluded from the study. 
 
PET Scanning: 
 
For this study, all subjects will undergo five separate visits for [F-18]PBR06 PET scans, one visit 
before starting Ofatumumab treatment (day 0 baseline) and four more visits at approximately 5, 28, 90 
and 273 days after initiating treatment. During the PET scan visits, all women subjects of child bearing 
age will undergo a stat quantitative serum hCG pregnancy test and only women with a negative test 
will undergo the radiopharmaceutical injection. The radiotracers will be produced using standardized 
procedures. At the time of imaging, the subjects will be positioned in the gantry of a high-resolution 
PET/CT camera. Head alignment will be made, relative to the canthomeatal line, using projected laser 
lines whose positions are known with respect to the slice positions of the scanner. A head support 
apparatus will be used to minimize head motion. Dynamic data over 120 minutes for PET 
quantification will be acquired, according to previously described methods for the tracer. 
 
MRI Scanning: 
 
All subjects will undergo five 3T brain MRIs with and without contrast, one before starting 
Ofatumumab treatment (day 0 baseline) and four more at 5, 28, 90 and 273 days after initiating 
treatment. All women will be queried about their pregnancy status, use of contraception and last 
menstrual period. If a woman is of child bearing potential, she will undergo a urine pregnancy test. 
Subjects will undergo intravenous gadolinium contrast administration, during all visits. Hence, all 
women of child bearing potential will undergo urine pregnancy testing for this study. 
 
Clinical Follow-up Visits: 

 
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Subjects will come in for five clinical visits, one visit before starting Ofatumumab treatment (day 0 
baseline) and four more visits at 5, 28, 90 and 273 days after initiating treatment. However, if the 
patient meets all inclusion criteria at the screening visit, the screening and baseline visits may occur on 
the same day. During these visits, subjects will undergo a physical examination, clinical assessment 
and cognitive assessment. A blood sample will be drawn to determine levels of sNfL, GFAP and other 
serum biomarkers. Visits will be conducted under the supervision of PI and other board-certified 
neurologists. Any adverse event reported by the patient or observed by the investigator will be 
recorded and reviewed at each clinical visit. 
 
Clinical Data: 
 
The following non-imaging, clinical data will be obtained: 
 
Expanded Disability Status Scale (EDSS), Timed 25-feet walk (T25W), 9-Hole Peg Test (9HPT), Four 
component MS Functional Composite (MSFC-4), Symbol-Digit Modality test (SDMT), cognitive and 
symptom questionnaires, vision testing, Levels of serum biomarkers. 
 
Clinical Safety Monitoring: 
 
Safety will be assessed at 0, 3, 6, and 9 months of the study including: 
 
• Pregnancy Testing: All women of childbearing potential will undergo a serum pregnancy test at 
the screening visit and at each of the following scheduled visits to the hospital. The investigator 
will also review the contraception status of subjects at each visit. 
• Electrocardiogram (ECG): An ECG will be performed either at the screening visit or on day 0 
clinical visit and at the end of the study. 
• Routine labs: Blood samples will be collected at the screening visit and at each visit time point. 
Blood samples will be assessed for red blood cell count, hemoglobin, hematocrit, platelets, total 
white blood cell (WBC) count and WBC differential counts (neutrophils, lymphocytes, 
basophils, eosinophils, monocytes) as well as for electrolytes (Na, K, Cl, bicarbonate, Ca, Mg, 
P), random glucose, total protein, blood urea nitrogen, albumin, alkaline phosphatase, ALT, 
AST, GGT, total bilirubin, conjugated bilirubin, creatinine, amylase, C-Reactive protein. 
• Samples for total IgM and IgG levels at baseline, 3, 6 and 9 months 
• Patient diary review 
• Monthly telephone interview 
 
Drug Administration/Dosing: 
 
At the end of the baseline clinic visit, subjects will be instructed by one of the investigators on proper 
drug administration technique. The first injection of ofatumumab will be performed under the guidance 
of an appropriately trained healthcare professional in the clinical space at the Brigham MS Center. 
 
 
We plan to enroll 10 subjects with relapsing MS. All enrolled subjects will receive Ofatumumab 20 mg 
every 4 weeks, subcutaneously for 9 months during the study. Loading doses will be administered 
initially at 1, 7 and 14 days. During the study period, all enrolled subjects will undergo five PET scans 
using [F-18] PBR06 at 0, 5, 28, 90 and 273 days after starting treatment with Ofatumumab. 
 

 
6 
 
 
Ofatumumab (OMB157) is a fully human anti-CD20 monoclonal antibody (mAb) self-administered by 
a once-monthly subcutaneous injection that is in development for MS. Ofatumumab drug product (also 
referred as OMB157) is formulated as 20 mg/0.4 mL (50 mg/mL) solution for injection, provided in 
autoinjectors, for subcutaneous administration. The autoinjectors contain a small overfill to allow for a 
complete withdrawal of the labeled amount (20 mg) of ofatumumab. 
 
Inclusion Criteria: 
• 
Patients diagnosed with active, relapsing MS course (defined by Lublin 2014 criteria)30. 
Active disease is defined by at least 1 relapse during the previous 1 year or 2 relapses 
during the previous 2 years or a positive gadolinium-enhancing MRI scan or MRI scan with 
new or unequivocally enlarging T2 lesions in previous year. 
• 
Age 18 to 60 years 
• 
EDSS 0 to 5.5 
• 
Subjects either untreated or treated with disease modifying therapies other than those listed 
in exclusion criteria 
• 
Agree to start treatment with ofatumumab and comply with study procedures for the 
duration of the study 
• 
No other systemic disease or neurological disorders requiring chronic or acute steroid or 
other immunosuppressive treatment 
• 
No known hypersensitivity reactions to contrast agents 
• 
None of the exclusion criteria 
 
Exclusion Criteria: 
• 
Subjects suspected of not being able or willing to cooperate or comply with study protocol 
requirements in the opinion of the investigator. 
• 
Subjects with primary progressive MS (Polman et al 2011)32 or SPMS without disease 
activity (Lublin et al 2014)30. 
• 
Disease duration of more than 10 years in patients with an EDSS score of 2 or less 
• 
Subjects meeting criteria for neuromyelitis optica (Wingerchuk et al 2015)33. 
• 
Pregnant or nursing (lactating) women, where pregnancy is defined as the state of a female 
after conception and until the termination of gestation, confirmed by a positive hCG 
laboratory test. 
• 
Women of child-bearing potential, defined as all women physiologically capable of 
becoming pregnant, unless they are using highly effective methods of contraception during 
dosing and for at least 6 months after stopping study medication. Highly effective 
contraception methods include: 
• 
Total abstinence (when this is in line with the preferred and usual lifestyle of the subject, if 
accepted by the local regulation). NOTE: Periodic abstinence (e.g., calendar, ovulation, 
symptothermal, post-ovulation methods) and withdrawal ARE NOT acceptable methods of 
contraception 
• 
Female sterilization (have had surgical bilateral oophorectomy with or without 
hysterectomy), total hysterectomy or tubal ligation at least six weeks before taking 
investigational drug. In case of oophorectomy alone, only when the reproductive status of 
the woman has been confirmed by follow up hormone level assessment 
• 
For female subjects on the study, the vasectomized male partner should be the sole partner  
• 
Use of oral, (estrogen and progesterone), injected or implanted hormonal methods of 
contraception or placement of an intrauterine device (IUD) or intrauterine system (IUS) or 
other forms of hormonal contraception that have comparable efficacy (failure rate <1%), for 

 
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example hormone vaginal ring or transdermal hormone contraception. In case of use of oral 
contraception, women should have been stable on the same pill for a minimum of 3 months 
before taking investigational drug. 
• 
In case local regulations deviate from the contraception methods listed above, local 
regulations apply and will be described in the ICF. 
• 
Women are considered post-menopausal and not of child bearing potential if they have had 
12 months of natural (spontaneous) amenorrhea with an appropriate clinical profile (e.g. 
age appropriate, history of vasomotor symptoms) or have had surgical bilateral 
oophorectomy (with or without hysterectomy), total hysterectomy or tubal ligation at least 
six weeks. In the case of oophorectomy alone, only when the reproductive status of the 
woman has been confirmed by follow up hormone level assessment is she considered not of 
child-bearing potential. 
• 
Subjects with active chronic disease (or stable but treated with immune therapy) of the 
immune system other than MS (e.g. rheumatoid arthritis, scleroderma, Sjögren's syndrome, 
Crohn's disease, ulcerative colitis, etc.) or with an immunodeficiency syndrome. 
• 
Subjects with a history of the following: 
o History of any malignancy 
o History of alcohol or drug abuse 
o Primary or secondary immunodeficiency 
o Prior hematopoietic stem cell transplantation 
o History of transplantation or anti-rejection therapy 
• 
Subjects with the following laboratory abnormalities: 
o Abnormal CD19+ B-cell levels at screening (defined as CD19 count <110 cells per 
microliter) 
o Leukopenia (defined as white blood cell (WBC) count <4000 WBCs per microliter) 
o Lymphopenia (defined as lymphocyte count <1000 lymphocytes per microliter) 
o Hypogammaglobulinemia defined as a level of <500mg/dL will be excluded. All 
subjects with low serum immunoglobulins should be evaluated by a hematologic expert 
prior to exclusion in the study.  
o Any abnormality of liver function tests, including ALT/SGPT, AST/SGOT, Alkaline 
phosphatase, total or direct bilirubin or GGT  
• 
Subjects with active systemic bacterial, viral or fungal infections, or known to have 
acquired immunodeficiency syndrome (AIDS). 
• 
Subjects with neurological symptoms consistent with PML or confirmed PML. 
• 
Subjects at risk of developing or having reactivation of syphilis or tuberculosis (eg subjects 
with known exposure to, or history of syphilis, or active or latent tuberculosis, even if 
previously treated).  
• 
Subjects with low affinity binders (LAB) for TSPO radioligand 
• 
Subjects with abnormal serum creatinine levels (defined as >1.3mg/dL) or Subjects with 
estimated glomerular filtration rate (eGFR) <30ml/minute 
• 
Patients with history of significant renal disease (dialysis, kidney transplant, single kidney, 
renal cancer, renal surgery) 
• 
Patient presenting with cardiac disorders defined by at least one of the following 
conditions: 
• 
Patient with recent cardiac history (within 6 months) of:  
o Acute coronary syndrome 
o Acute heart failure (class III or IV of the NYHA classification) 

 
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• 
History of significant ventricular arrhythmia (persistent ventricular tachycardia, ventricular 
fibrillation, resuscitated sudden death) 
• 
Patient with history of cardiac failure class III or IV of the NYHA classification 
• 
Patient with history of severe conduction disorders which are not prevented by permanent 
pacing (atrio-ventricular block 2 and 3, sinoatrial block) 
• 
Syncope without known etiology within 3 months 
• 
Uncontrolled severe hypertension, according to the judgment of the investigator, or 
symptomatic hypertension 
• 
Subjects with any contraindications to PET/CT or MRI procedures (e.g. claustrophobia, 
MRI-incompatible implants or pacemakers, renal failure)   
• 
Subjects with any significant or uncontrolled medical comorbidity   
• 
Subjects with active hepatitis B  
• 
Subjects at risk of hepatitis B reactivation (such as subjects with positive HBsAG or anti-
Hepatitis B core antibodies) should be evaluated by a liver disease expert before inclusion, 
or should be excluded  
• 
Subjects treated with other disease modifying treatments within their respective pre-
specified washout periods will be excluded 
 
Biostatistical Analysis 
 
Study endpoints/variables 
 
The primary endpoint of the study will be the change in PET uptake in the lesional and normal 
appearing white matter, cortical and subcortical grey matter, and peri-plaque area of chronic 
lesions in the brain at early and late time-points (5, 28, 90 and 273 days) as compared to 
baseline. 
 
The secondary endpoints of the study will be the following:  
1) Association of change in PET uptake in the lesional and normal appearing white matter, 
cortical and subcortical grey matter, and peri-plaque area of chronic lesions in the brain with 
changes in the peripheral CD19 counts, serum glial fibrillary acid protein (GFAP) and serum 
neurofilament light chain measurements at 5, 28, 90 and 273 days as compared to baseline. 
2) Association of changes in PET uptake in the lesional and normal appearing white matter, 
cortical and subcortical grey matter, and peri-plaque area of chronic lesions in the brain with 
MRI and clinical changes at 5, 28, 90 and 273 days as compared to baseline.  
 
Sample size and statistical considerations: 
 
We will recruit 10 subjects, and each subject will undergo PET evaluations at day 0 (baseline), 
day 5, day 28, day 90 and day 273. The change with time in PET uptake in the lesional and normal 
appearing white matter, cortical and subcortical grey matter, and peri-plaque area of chronic lesions will 
be estimated using an analysis of response profiles model including a categorical effect of time and an 
unstructured covariance matrix for the residuals.  This approach allows any change with time and places 
no structure on the covariance matrix.  The primary analysis will be the global comparison across the 
five time points.  If a significant difference among the five time points is observed, we will estimate the 

 
9 
 
 
change between day 0 and each of the other measurements, and we will correct for four multiple 
comparisons using a Bonferroni correction.  Since the analysis of response profiles model estimates the 
parameters using maximum likelihood with the mixed command in Stata, this approach will handle 
missing data that is missing at random.  Therefore, subjects who are missing one or more measurement 
will be included in our analysis.  A p-value <0.05 will be considered statistically significant. 
 
In addition to the primary analysis of the change with time, we will also compare the change in 
PET uptake with the change in other measured variables (MRI, clinical, and immunologic measures). 
All of the remaining variables will be measured at the same time points as PET uptake (days 0, 5, 28, 90 
and 273).  First, we will calculate the change from day 0 to each of the other time points for each of the 
measures, and we will estimate the correlation between the changes using Pearson’s correlation 
coefficient.  This analysis will fit several models, but the interpretation of these correlations will be easy 
to interpret.  Second, we will fit a linear mixed model with PET uptake as the outcome and two predictors 
for each of the other measured variables.  In particular, we will include (1) the day 0 measure and (2) the 
change from day 0 so that we can estimate both the cross-sectional and longitudinal association between 
the other measured variables and PET. Our focus will be the longitudinal association estimated using the 
second term. If a subject receives corticosteroids during the study period, we’ll account for that in a 
multivariate analysis by adding it as a covariate.  
 
Paired t-test and repeated measures ANOVA analyses will be performed to assess differences in 
PET measures over time. Correlation analyses will be performed between PET and MRI measures and 
clinical variables. We will also look for correlations between changes in PET uptake and peripheral 
CD19 counts at various time points of the study.  
 
Statistical analyses will be performed using Stata version 16 (StataCorp, College Station, Texas, 
USA). 
 
Sample size calculation: 
 
   
No prior data regarding effect of Ofatumumab on microglial changes is available at this time. 
The sample size of 10 is based on the typical number of patients in published PET-MRI studies with the 
same tracer.  With a sample size of 10 subjects, we will be able to estimate the mean change between 
any pair of time points with a standard error of 0.32 times the standard deviation of the change. Assuming 
a PET effect size of 1.1 noticeable in intracortical and leukocortical lesions11, identified on combination 
of PET and 3T MRI, a sample size of 10 provides 85% power and alpha-error of less than 5% to detect 
a treatment response. This means that we will have a reasonable precision for the design of future studies. 
 
 
 
 
 
 
 
 
 

 
10 
 
 
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