Clinical trial NCT03853746
Conditions: Multiple Sclerosis
Sponsor: Johns Hopkins University
Start date: 2019-04-01; Completion date: 2024-07-01
Enrollment: 10 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Several disease-modifying therapies (DMTs) have been shown to be effective in reducing the disease activity in patients with relapsing forms of multiple sclerosis (MS) but these treatments, often need to be used continuously for an unknown duration, rendering the long-term use extremely expensive. In addition, chronic administration of DMTs is often associated with undesirable side effects. Among these medications, B-cell depleting monoclonal antibodies might have the properties of an ideal group of medications: i) B-cell depleting antibodies have proven to be extremely potent in reducing or stopping the disease activity in relapsing MS, ii) B-cell depleting antibodies are very safe if used for a short period and use for a short duration may stop the inflammatory disease activity over long term, although current clinical practice and protocols are based on continuing B-cell depletion for an unknown period of time. Indeed, early phase clinical trials of rituximab and ocrelizumab suggested that a short course treatment with B-cell depleting antibodies can have long term effects and disease activity will not return even long after B-cell repopulation in the blood.

This long-term effect might be related to the specific pattern of B-cell tolerance defect in patients with MS and the potential of its normalization with B-cell depleting antibodies. By analyzing the reactivity of recombinant antibodies expressed from single B-cells, the investigators' collaborators have demonstrated that the pattern of B-cell tolerance defect is different in people with MS who only display an impaired removal of developing autoreactive B-cells in the periphery while central B-cell tolerance in the bone marrow is functional in most patients. In contrast, patients with rheumatoid arthritis (RA), type-1 diabetes (T1D) or Sjögren's syndrome (SS) show defective central and peripheral B-cell tolerance checkpoints. As a consequence, while anti-B-cell therapy does not correct defective early B-cell tolerance checkpoints in T1D and only temporarily slows down autoimmune processes before newly generated autoreactive B-cells likely induce patient relapse, the investigators postulate that the efficacy of B-cell depleting antibodies in MS may be linked to the B-cell depleting antibodies' normal central B-cell tolerance and the production of a normal B-cell and T-cell compartment after anti-B-cell therapy.

The investigators' goal is to provide proof-of-concept that a short duration of treatment with B-cell depleting antibodies can correct B-cell tolerance defects in MS and allow for medication-free prolonged freedom from disease activity, at least in a proportion of subjects with relapsing MS.

In an open label study, 10 patients with active relapsing MS will be treated with two courses of ocrelizumab and will be followed clinically and radiologically for at least two and a half years. Time to the return of disease activity (defined as clinical relapses or new or enhancing lesions on the MRI) will be the primary outcome of the study. The investigators will harvest B-cells before starting the treatment and after B-cell repopulation and assess the central and peripheral tolerance defects. The investigators hypothesize that in most participants, the disease activity will not come back, and this prolonged response to anti cluster of differentiation 20 (CD-20) therapy is associated with normalization of B-cell tolerance defect in these patients. Considering the safety of this approach, it can be adopted widely among people with MS. Hence, the proposed B-cell analyses before and after B-cell depletion in people with MS will provide novel insights regarding the mechanisms underlying the beneficial effect of B-cell depleting antibodies and the potential long-term suppression of disease activity. This strategy can therefore improve the approach to treatment of many people with relapsing MS.

Detailed description:

Arms: arm 1: All participants will receive Ocrelizumab
Interventions: intervention 1: Ocrelizumab
Intervention descriptions: intervention 1: Patient will be treated with two courses of ocrelizumab (Ocrevus) for one year and then will stop the medication and will be monitored for the return of the disease activity. Those who experience return of the disease activity can go back on the medication.

Protocol documents:
Prot_SAP_001.pdf: 
1 
Protocol Title:  Evaluating the effects of short-term B-cell depletion on long-term disease 
activity and immune tolerance in relapsing multiple sclerosis.  
Date:  March 28, 2020 
NCT03853746


Version: 2.0 
Date: 3/8/2019 
2 
 
Contents 
Synopsis: ....................................................................................................................................................... 4 
Introduction .................................................................................................................................................. 6 
Background and rational for performing this study ................................................................................. 6 
Disease modifying treatments and relapsing multiple sclerosis .......................................................... 6 
B-cell depleting antibodies and relapsing MS ....................................................................................... 6 
Anecdotal and early phase clinical trials evidence of long-term efficacy of short-term B-cell 
depletion ............................................................................................................................................... 7 
B-cell tolerance defects in MS and other autoimmune diseases ......................................................... 7 
Specific aims and study overview ................................................................................................................. 8 
Risk/benefit assessment ............................................................................................................................... 8 
Known potential benefits .......................................................................................................................... 8 
Known Potential Risks ............................................................................................................................... 9 
Study objectives ............................................................................................................................................ 9 
Primary objective ...................................................................................................................................... 9 
Secondary objective .................................................................................................................................. 9 
Investigational plan ....................................................................................................................................... 9 
Study design .............................................................................................................................................. 9 
Rationale of study design ........................................................................................................................ 10 
End of Study Definition ........................................................................................................................... 10 
Study Population ......................................................................................................................................... 10 
Inclusion criteria: .................................................................................................................................... 11 
Exclusion criteria: .................................................................................................................................... 11 
SCREEN FAILURES .................................................................................................................................... 11 
Intervention ................................................................................................................................................ 11 
Investigational treatments ...................................................................................................................... 11 
Treatment arms ...................................................................................................................................... 11 
Treatment assignment ............................................................................................................................ 11 
Study intervention discontinuation and participant discontinuation/withdrawal ..................................... 12 
Discontinuation of study intervention .................................................................................................... 12 
Lost to follow-up ..................................................................................................................................... 12 
Schedule of events ...................................................................................................................................... 12 
Screening visit: ........................................................................................................................................ 14 


Version: 2.0 
Date: 3/8/2019 
3 
 
MRI procedures:...................................................................................................................................... 14 
Outcomes: ................................................................................................................................................... 14 
Primary outcome: ................................................................................................................................... 14 
Secondary outcome: ............................................................................................................................... 15 
Disability: six-month confirmed worsening or improvement of disability: ........................................ 15 
Quality of life measures: ..................................................................................................................... 15 
Assessment of T and B-cell phenotypes and function at baseline and 18-24 months post-B-cell 
depletion ............................................................................................................................................. 15 
Analysis of the central and peripheral B-cell tolerance checkpoints .................................................. 16 
Safety and adverse effect monitoring: ................................................................................................... 16 
Data management ...................................................................................................................................... 17 
Sample size .................................................................................................................................................. 18 
Statistical analysis ....................................................................................................................................... 18 
Ethical considerations ................................................................................................................................. 19 
Regulatory and ethical compliance ......................................................................................................... 19 
Informed consent procedures ................................................................................................................ 19 
Publication of the study protocol and results ......................................................................................... 20 
Protocol adherence ................................................................................................................................. 20 
 
 
 
 
 
 
 
 
 
 
 
 
 


Version: 2.0 
Date: 3/8/2019 
4 
 
Synopsis: 
Protocol title: Evaluating the effects of short-term B-cell depletion on long-term disease activity and 
immune tolerance in relapsing multiple sclerosis. 
Indication: Relapsing multiple sclerosis (MS). 
Objectives 
- 
To determine whether two courses of treatment with ocrelizumab is associated with long-term 
suppression of disease activity in people with highly active relapsing MS.  
- 
To test if two courses of treatment with ocrelizumab corrects the B-cell tolerance defects in 
patients with relapsing MS. 
Study design: Open-label intervention.  
Number of patients: 10 
Target population: Adult patients with relapsing MS  
Inclusion criteria: 
- Age 18 years and older.  
- Diagnosis of RRMS based on the latest revision of McDonald criteria 
- At least two Gd-enhancing lesions on the brain or spinal cord MRI done in the prior three months 
- Naïve to disease modifying therapies or using an injectable therapy (interferons or glatiramer 
acetate); or, if history of receiving natalizumab, fingolimod, dimethyl fumarate and teriflunomide, 
no exposure for past three months   
- Expanded Disability Status Scale (EDSS) score at the time of screening =<3 
Exclusion criteria: 
- 
Contraindication to treatment with anti-CD20 antibodies, including  being seropositive for 
HBsAg or HIV antibody or T-spot (or Quantiferon-Gold) positive 
- 
Ever received B-cell depleting antibodies (rituximab, ocrelizumab, ofatumumab), alemtuzumab, 
daclizumab, mitoxantrone or hematopoietic stem-cell transplant 
- 
Female who are pregnant, nursing or unwilling to use contraception up to six months after the 
second course of the infusion (or 12 months after the first infusion) 
-     Treatment with steroids in the past 30 days 
-    Clinically unstable medical or psychiatric disorders at the time of screening that require acute 
treatment as determined by the PI 
  
Study duration:  2.5 years after the last dose of B-cell depleting antibody, ocrelizumab.  
Description of study intervention: Participants will receive two courses of treatment with ocrelizumab, 
an FDA-approved medication for treatment of relapsing MS. The ocrelizumab infusions will not be 


Version: 2.0 
Date: 3/8/2019 
5 
 
repeated after these two courses. Patient will be monitored for the return of clinical or MRI disease 
activity for at least 2.5 years after the last dose ocrelizumab. Immune cell phenotype and B-cell tolerance 
characteristics will be assessed before start of the treatment with ocrelizumab and 12-18 months after 
the second course of ocrelizumab infusion.  
Assessments: 
The primary endpoint (the failure event) of this study is the time to return of disease activity after the 
third month post-first-infusion, objectively demonstrated by development of new T2 hyperintense lesions 
or Gd-enhancing lesions on the MRI or a clinical relapse that is confirmed with an objective change in the 
neurological examination or a corresponding new or enhancing lesion on the MRI that is obtained after 
the onset of symptoms. 
Mechanistic studies will be performed on the peripheral blood mononuclear cells obtained before the 
infusions and periodically after the infusions. 
Study locations: This study will be conducted at Johns Hopkins University (JHU).  
Sample size and statistical analysis 
This is a pilot proof of concept study and no formal sample size calculations are performed.  
We will recruit 10 patients in this study.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Version: 2.0 
Date: 3/8/2019 
6 
 
Introduction 
Background and rational for performing this study  
Disease modifying treatments and relapsing multiple sclerosis 
Controlling and even stopping the inflammatory disease activity (i.e. clinical relapses and gadolinium 
enhancing or new T2 lesions on the MRI) is nowadays possible in most patients with relapsing forms of 
multiple sclerosis (MS). However, in most instances, it requires continuous and long-term treatment with 
a disease modifying treatment (DMT), which is costly, disruptive to the life of patients, associated with a 
sense of having a chronic disease and potentially adverse effects that are commonly seen with prolonged 
immunosuppressive therapies (opportunistic infections and cancers). To control the disease activity, 
almost all the available DMTs need to be used continuously. Stopping them is associated with return of 
the disease activity and in the case of two medications (fingolimod and natalizumab) with the possibility 
of more severe return of the disease activity compared to the pre-DMT baseline (rebound phenomenon). 
There are currently two strategies that can result in medication-free prolonged freedom of disease activity 
in a proportion of patients: hematopoietic stem cell transplantation and treatment with alemtuzumab. 
Intense immunosuppression, followed by autologous bone marrow transplantation stopped the disease 
activity in more than 80% of patients with highly active relapsing MS. Two courses of treatment with anti-
CD52 monoclonal antibody, alemtuzumab, have also been reported to be associated with prolonged 
freedom from clinical and radiological disease activity. In a report from 5 years of extension of the pivotal 
clinical trials of alemtuzumab in relapsing MS (CARE-MS studies), more than 60% of patients who only 
received two courses of alemtuzumab, did not need retreatment. However, both these approaches have 
high morbidity and are even associated with mortality, are reserved for cases with extremely severe 
disease and patients who have accumulated disability and their disease activity could not be controlled 
with other approaches. 
In an ideal situation, a short course of treatment with a safe disease modifying therapy would have long-
term or permanent effects on immune tolerance and stop the disease activity. 
 
B-cell depleting antibodies and relapsing MS 
B-cell depleting anti-CD20 monoclonal antibodies, rituximab and ocrelizumab, have been shown to reduce 
the disease activity and using the most objective measure (Gd-enhancing lesions on the MRI), they 
effectively stop the disease. The short-term use of these medications has been safe and aside from 
preventable or manageable infusion reactions, no short-term serious side effects have been associated 
with them. However, based on the current practice and the medication label, these antibodies are 
supposed to be infused either every six months or at intervals trying to keep the peripheral blood depleted 
of B-cells. Long-term B-cell depletion has known and predictable side effects (such as 
hypogammaglobulinemia) and possible unpredictable adverse effects (e.g. increased risk of infection or 
cancer). For example, in two pivotal clinical trials of ocrelizumab in RRMS, the rate of upper respiratory 
infection and nasopharyngitis was mildly elevated, compared to the interferon beta-1a. However, across 
the clinical trials (in both RRMS and PPMS), the rate of developing the first neoplasm seemed to be twice 
as high as the comparator groups (0.4 per 100 patient-years of exposure versus 0.2 per 100 patient-years 
of exposure to other agents, including placebo and interferon beta-1a). It is conceivable that longer 
duration of exposure to these medications may be associated with increased number of cases 


Version: 2.0 
Date: 3/8/2019 
7 
 
experiencing serious side effects, including infections and perhaps cancer. Long-term use of these 
medications is also very costly. However, there is evidence that even short-term treatment with B-cell 
depleting agents can result in prolonged suppression of disease activity. For example, long term follow-
up of patients who participated in a phase II clinical trial of ocrelizumab demonstrated that the relapse 
rate did not increase after the B-cell repopulation and only one patient in the ocrelizumab group had a 
Gd-enhancing lesion 18 months after the 4th and final ocrelizumab infusion, similar to the 12 month data 
following only one cycle of rituximab infusion in the phase two trial. Our colleagues have reported 
cessation of disease activity in four patients with neuromyelitis optica for a long period of time after they 
stopped treatment with rituximab. Based on these data and experiences, we believe that even a short 
course of treatment with anti-CD20, B-cell depleting antibodies can result in prolonged drug-treatment-
free cessation of disease activity, at least in a subset of patients. With treatment with anti-CD20 therapies 
for a limited period (instead of long-term treatment), we probably avoid the side effects of prolonged lack 
of B-cells in the circulating blood. If a short course treatment with these medications has similar efficacy 
to continuous treatment, aside from lower probably of experiencing side effect, it puts much smaller 
financial burden on patients, their families and the health system. This strategy would put an end to the 
controversy of whether to start people with MS on a safe, but less efficacious medication (escalation 
approach) or on a potent, but with potential or unknown long-term side effects (induction approach). If 
temporary B-cell depletion stops the disease activity in a large proportion of patients, its safety and 
inexpensiveness allow us to use it almost universally in most people with MS. The next step would be 
trying to find biomarkers that might be associated with this long-term suppression of the disease activity. 
Anecdotal and early phase clinical trials evidence of long-term efficacy of short-term B-cell 
depletion 
The principal investigator (BN) has reviewed clinic charts of patients with highly active relapsing MS who 
were treated with rituximab and found four cases that stopped this medication after one or two courses 
without starting another DMT. There was no return of radiological disease activity even several years after 
cessation of treatment with rituximab (unpublished data).  
The long-term efficacy data in a phase II trial of ocrelizumab in relapsing MS reported only one patient 
(among more than 180 patients with follow-up data) developed enhancing lesions on the MRI, 18 months 
after the last ocrelizumab infusion. These results demonstrate that reinfusion of anti-CD20 antibodies 
every six months is probably unnecessary in most MS patients.  
B-cell tolerance defects in MS and other autoimmune diseases 
 Defects in early B-cell tolerance checkpoints are associated with many autoimmune diseases including 
MS, type 1 diabetes (T1D), rheumatoid arthritis (RA), pediatric systemic lupus erythematosus (SLE) and 
systemic sclerosis (SS), and result in large numbers of circulating autoreactive B-cells in the blood of these 
patients. Early B-cell tolerance checkpoints include a central B-cell selection step in the bone marrow (BM) 
that is vastly dependent on sensing self-antigen binding to B-cell receptors (BCRs) and most likely Toll-like 
receptors (TLR),  
whereas the peripheral B-cell tolerance checkpoint appears to require functional regulatory T cells (Treg) 
to prevent the accumulation of autoreactive mature naïve B-cells. In line with these observations, 
polymorphisms in PTPN22, BLK, LYN, CSK, BANK1 identified by GWAS studies as susceptibility genes for 
RA, SLE, or T1D but not MS, encode components regulating BCR signaling and likely result in the impaired 
central B-cell tolerance characteristic of T1D, RA, and SLE. In contrast, people with MS do not display gene 


Version: 2.0 
Date: 3/8/2019 
8 
 
polymorphisms linked to the BCR signaling pathway and most of these patients establish normal central 
B-cell tolerance. However, people with MS suffer from an impaired peripheral B-cell tolerance checkpoint, 
resulting in the accumulation of autoreactive mature naïve B-cells in their blood potentially linked to 
alterations of their Treg function and IFN-gamma and IL-17 production. Interestingly, IL-17 production by 
T cells and T follicular helper (Tfh) development is favored by IL-6, a pro-inflammatory cytokine that is 
secreted with GM-CSF by B-cells from people with MS. Since anti-B-cell therapy normalizes IL-6 production 
in newly generated B-cells, this regiment may therefore also decrease IL-17 production by T cells including 
Tregs and circulating Tfh in treated people with MS in remission. In addition, B-cells have been shown to 
support the maintenance of memory T cells and their elimination may therefore favor the elimination of 
self-antigen specific T cells secreting inflammatory cytokines. Moreover, transitional B-cells enhanced 
after anti-B-cell therapy may also secrete increased amount of IFN-alpha, which has been shown to 
improve the condition of people with MS. We hypothesize that long-term suppression of disease activity 
in MS is the result of anti-B-cell therapy eliminating autoreactive B-cells in patients’ blood and that newly 
generated B-cells after B-cell repopulation will not contain autoreactive clones associated with 
autoimmunity, an observation that may correlate with a normalized Treg compartment that no longer 
secrete inflammatory cytokines. Hence, a short two course of B-cell depletion may correct the peripheral 
B-cell tolerance defect seen in most people with relapsing MS and this normalization may be responsible 
for the long-term suppression of disease activity. 
Specific aims and study overview 
Aim1: To determine whether two courses of treatment with ocrelizumab is associated with long-term 
suppression of disease activity in people with highly active early relapsing MS.  
We hypothesize that only two courses of treatment with ocrelizumab in subjects with early relapsing MS 
will stop the disease activity, as measured by new T2 or Gd-enhancing lesions on brain and spinal cord 
MRI, even long after B-cell repopulation in the blood. In an open label unblinded trial, we will enroll 10 
patients with relapsing MS and with at least two Gd-enhancing lesions on the brain and spinal cord MRI 
and treat them with two courses of ocrelizumab and follow them clinically and radiologically for at least 
two and a half years.    
Aim 2: To test if two courses of treatment with ocrelizumab corrects the B-cell tolerance defects in 
patients with relapsing MS. We hypothesize that B-cells repopulating after a two course-treatment with 
ocrelizumab will NOT contain an elevated proportion of autoreactive clones observed pre-treatment. In 
the patients recruited in aim 1, we will harvest pre-treatment and post-repopulation peripheral B-cells 
and assess early B-cell tolerance checkpoints by testing the reactivity of recombinant antibodies cloned 
from single B-cells. 
Risk/benefit assessment   
Known potential benefits 
With stopping ocrelizumab after two courses, participants will be less likely to suffer from 
immunosuppressive effects of this medication. The long-term safety of this medication is also not known 
and with limiting the exposure of participants to only two courses of treatment, they will not be exposed 
to unknown risk associated with long term use of this medication. 


Version: 2.0 
Date: 3/8/2019 
9 
 
Known Potential Risks 
By limiting the participants to two courses of treatment with ocrelizumab, participants are at higher risk 
of return of MRI or clinical disease activity. However, it is well within the standard practice to start patients 
with safer, but less efficacious disease modifying treatments and if the disease activity continued, to 
escalate to higher efficacy medications. Even in the case of more effective medications such as 
alemtuzumab, the standard practice is treatment with only two courses of the medication and 
retreatment if the disease activity returns. In this study, we will restart the treatment with ocrelizumab in 
patients who experience the return of the disease activity, a practice similar to escalation or retreatment 
which are standards of care.  
Loss of privacy is another risk of participating in this study. Several mechanisms will help prevent loss of 
privacy. Access to the data will be limited to study personnel and the database will be password-
protected. Furthermore, each member of the study team will be limited to only the data they need to 
perform their tasks so that privacy is protected as much as possible. Only study personnel who need to 
access the database will be given such access. 
The risks associated with the study are minimal, and an adequate protection plan is in place; as such, the 
potential benefits exceed the potential risks. The participation of patients is completely voluntary; the 
alternative to the patients is to not participate in the study. Lack of participation will in no way 
compromise the relationship they have with their neurologists or the ongoing care they receive for their 
MS. 
Study objectives 
Primary objective  
 To determine whether two courses of treatment with ocrelizumab is associated with long-term 
suppression of disease activity in people with highly active, relapsing MS. 
Secondary objective  
To determine whether these two courses of treatment with ocrelizumab correct the B-cell tolerance 
defects in patients with relapsing MS. 
Investigational plan 
Study design 
This is an open label, single-arm study of anti-CD20 antibody, ocrelizumab in people with relapsing MS. 
Patients who fulfill the eligibility criteria will undergo blood draw for collection of peripheral blood 
mononuclear cells (PBMC). Participants will receive two infusions of ocrelizumab (300 mg) two weeks 
apart and then six months later (one infusion of 600 mg of ocrelizumab), according to the medication 
label and the standard of care. Baseline brain, cervical and thoracic MRI will be obtained about 12 weeks 
after the first course of treatment. Participants will be followed with clinical visits at least twice a year 
(or more frequently, if needed) and brain MRI (every six months) and cervical and thoracic spine MRI, at 
least annually, up to two and a half years after the baseline MRI.  
 
The trial design is depicted in the following diagram: 


Version: 2.0 
Date: 3/8/2019 
10 
 
 
 
 Rationale of study design 
The reason we chose a single arm, open label study, as opposed to a blinded controlled study is because 
the latter does not help with answering our research question. Our research question is if two courses of 
treatment with anti-CD20 antibodies have long lasting effects on the suppression of the disease activity. 
One thought regarding a controlled trial design would be a non-inferiority trial with the control group 
continuing the use of anti CD20 treatment (every 6 months), similar to the current practice. However, as 
we mentioned above, continued use of B-cell depleting antibodies is extremely capable in suppressing 
the disease activity and in our opinion, there is no equipoise about the ability of continued B-cell 
depletion in completely suppressing the disease activity. However, as we argued earlier, continued use 
of these antibodies have known and unknown long-term side effects and a three-year trial will not be 
able to demonstrate the long-term safety. Also, the cost of continuous treatment is not comparable to 
the treatment with only two courses of antibody infusion. 
End of Study Definition 
A participant is considered to have completed the study if he or she has completed all phases of the 
study including the last visit or the last scheduled procedure shown in the diagram above. 
Study Population  
Participants will be recruited at the Johns Hopkins MS Center. We will recruit  about 10 patients with 
relapsing MS who are naïve to DMTs or have been on injectable DMTs (interferons or glatiramer 
acetate) or have been off of natalizumab, fingolimod or dimethyl fumarate for at least three months and 
have at least 2 Gd-enhancing lesions on the brain and/or spinal cord MRI on a scan obtained in three 
months prior to enrollment.  
 
 


Version: 2.0 
Date: 3/8/2019 
11 
 
Inclusion criteria: 
- 
Age 18 years and older.  
- 
Diagnosis of RRMS based on revised McDonald criteria 
- 
At least two Gd-enhancing lesions on the brain or spinal cord MRI done in the prior three months 
- 
Naïve to disease modifying therapies or using an injectable therapy (interferons or glatiramer 
acetate); or, if history of receiving natalizumab, fingolimod, dimethyl fumarate and 
teriflunomide, no exposure for past three months   
- 
Expanded Disability Status Scale (EDSS) score at the time of screening =<3 
Exclusion criteria: 
- 
Contraindication to treatment with anti-CD20 antibodies, including being seropositive for HBsAg 
or HIV antibody or T-spot (or Quantiferon-Gold) positive 
- 
Ever received B-cell depleting antibodies (rituximab, ocrelizumab, ofatumumab), alemtuzumab, 
daclizumab, mitoxantrone or hematopoietic stem-cell transplant 
- 
Female who are pregnant, nursing or unwilling to use contraception up to six months after the 
second course of the infusion (or 12 months after the first infusion) 
- 
Treatment with steroids in the past 30 days 
- 
Clinically unstable medical or psychiatric disorders at the time of screening that require acute 
treatment as determined by the PI 
SCREEN FAILURES 
Screen failures are defined as participants who consent to participate in the clinical trial but do not 
subsequently receive the study intervention or enter in the study. A minimal set of screen failure 
information is required to ensure transparent reporting of screen failure participants, to meet the 
Consolidated Standards of Reporting Trials (CONSORT) publishing requirements. Minimal information 
includes demography, screen failure details, eligibility criteria, and any serious adverse event (SAE). 
Intervention 
Investigational treatments 
The intervention in this study is the two courses of infusions of ocrelizumab, an FDA approved medication 
for relapsing MS. The patients will receive ocrelizumab 300 mg IV infusion on days one and 15, according 
to the standard infusion protocols. The protocols include pre-medicating with acetaminophen 650 mg 
orally, diphenhydramine 50 mg orally and methylprednisolone 125 mg IV. The anti-CD20 antibody infusion 
will be repeated in six months (single infusion of ocrelizumab 600 mg). These infusions are standard of 
care and are done according to the medication label. However, the infusions will not be repeated after 
these two courses of infusion (the first two infusion of 300 mg ocrelizumab two weeks apart is considered 
one course and the infusion of 600 mg of ocrelizumab six months later is considered the second course).  
Treatment arms 
This is an open label, single arm, unblinded interventional study.  
Treatment assignment  
Patients who meet eligibility criteria are offered to participate in the study. This is an open label, unblinded 
interventional study, in which all participants receive essentially the same intervention. 


Version: 2.0 
Date: 3/8/2019 
12 
 
Study 
intervention 
discontinuation 
and 
participant 
discontinuation/withdrawal 
Discontinuation of study intervention  
The study intervention is treatment with two courses of ocrelizumab. So, discontinuation from study 
intervention equates to not completing two courses of treatment with ocrelizumab or restarting the 
ocrelizumab or starting another MS disease modifying treatment after two courses of treatment with 
ocrelizumab for reasons other than breakthrough disease activity. All patients started on treatment will 
be followed in the study. Participants are free to withdraw from participation in the study at any time 
upon request. If a participant is unable to obtain insurance authorization for ocrelizumab within 90 days 
of screening visit (absent an administrative delay or other reason that is expected to be easily resolvable), 
the participant will be deemed a screen failure and will be replaced.   
Lost to follow-up 
A participant will be considered lost to follow-up if he or she fails to return for 3 consecutive scheduled 
visits and is unable to be contacted by the study staff.  
The following actions must be taken if a participant fails to return to the clinic for a required study visit: 
• 
The study staff will attempt to contact the participant and reschedule the missed visit within 1 
month of the missed visits, counsel the participant on the importance of maintaining the assigned visit 
schedule and ascertain if the participant wishes to and/or should continue in the study. Attempts to 
reschedule the missed visit will continue up to 3 months after the target visit date.  Beyond 3 months, the 
visit will be considered missed and the visit window for the next standard of care clinic visit will open. 
• 
Before a participant is deemed lost to follow-up, the investigator or designee will make every 
effort to regain contact with the participant (where possible, 3 telephone calls and at least 2 attempts to 
reach each of their emergency contacts and, if necessary, a certified letter to the participant’s last known 
mailing address or local equivalent methods). These contact attempts should be documented in the 
participant’s medical record or study file.  
• 
Should the participant continue to be unreachable (3 consecutive visits missed and no response 
to repeated attempts to contact), he or she will be considered to have withdrawn from the study with a 
primary reason of lost to follow-up. 
Schedule of events 
 Study visits will be conducted at the Johns Hopkins Outpatient Center (JHOC). Aside from obtaining 
informed consent for participation in the study and collecting peripheral blood mononuclear cells (PBMCs) 
and the NeuroQoL, all other procedures are part of the routine clinical and standard of care at Johns 
Hopkins MS Center. The only deviation from the standard of care in this study is stopping the ocrelizumab 
infusions after two courses. Study procedures are depicted in the following table (procedures in bold font 
are standard of care): 
Tests and 
assessments 
Screening 
visit 
Infusion 
visits 
 (month 0) 
Month 3 
(baseline) 
Month 6 
Month 12 
Month 18  
Month 24 
Month 30 
Informed Consent  
X 
 
 
 
 
 
 
 
Verifying eligibility   
X 
 
 
 
 
 
 
 


Version: 2.0 
Date: 3/8/2019 
13 
 
Medical history 
X 
 
X 
x 
x 
x 
x 
x 
Physical 
examination and 
vital signs 
assessment 
X 
 
X 
x 
x 
x 
x 
x 
Neurological 
examination 
(EDSS) 
X 
 
X 
x 
x 
x 
x 
x 
Blood work to 
assess eligibility 
for receiving 
ocrelizumab and 
its safety after the 
infusions 
X 
 
X 
X 
X 
 
 
 
OCT 
X 
 
X 
 
X 
 
X 
 
Brain MRI  
 
 
x 
 
x 
x 
x 
x 
Cervical/thoracic 
MRI 
 
 
x 
 
x 
 
x 
 
NeuroQoL 
X 
 
X 
x 
x 
x 
x 
x 
Lymphocyte 
subset analysis  
 
 
X 
X 
X 
X 
 
 
Blood sample 
collection for 
storage and future 
analyses 
X 
 
X 
X 
X 
X 
 
 
Blood draw and 
collection to be sent 
to Yale 
X 
 
 
 
 
X (or 
months 
24) 
 
 
Ocrelizumab 
infusions 
 
X 
2 infusions two 
weeks apart 
 
X 
1 infusion 
 
 
 
 
Side effects 
assessment  
 
 
X 
x 
x 
x 
x 
x 
   
Changes in the study visits and procedures due to response to the CIVD-19 pandemic: 
Considering the recent events (March 2020) surrounding the COVID-19 pandemic and JHU response, and 
the fact that our study participants can be particularly vulnerable to the infection with the novel 
coronavirus, we will implement the following changes in our study protocol to reduce  the immediate 
hazard to our participants related to the risk of exposure to COVID-19: 
- 
Follow-up visits, including Month 3, Month 6, Month 12 Month 18, Month 24 and Month 30 in-
person visits can be replaced with telemedicine visits with the study PI.  
- 
The medical history, symptom evaluation and side effects assessment components of the study 
visits will be conducted through the telemedicine visits.  
- 
The physical examination, vital signs assessment, neurological examination, OCT and research 
blood sample collection for future analysis will be paused temporarily, as they would require an 
in-person visit and would pose a risk to the participants and the staff. They will be resumed as 
soon as possible, after the resumption of normal research activities at the JHU. 
- 
The NeuroQoL quality of life assessment will be delivered to particpants electronically. Secure 
Redcap surveys will be sent to the participants’ email address. Results will be printed and filed in 
the study binders as usual. 


Version: 2.0 
Date: 3/8/2019 
14 
 
- 
The blood work to assess and monitor safety and adverse effects of the therapy, including 
lymphocyte subset analysis, will continue to be ordered and results will be reviewed electronically 
by the PI. 
- 
Infusions of ocrelizumab: Ocrelizumab may theoretically increase the risk of infection with the 
novel coronavirus. Delaying the ocrelizumab infusions will be determined on a case-by-case basis 
by the PI. 
- 
MRI Procedures: MRI studies may be delayed because of the delays in the ocrelizumab infusions.  
Screening visit: PI or designee will explain the study consent to study participants, and the study visit 
assessments will occur after the study participants sign the consent form. Screening visit procedures 
include review of eligibility criteria, physical exam, neurological examination, collecting vitals, blood work 
for assessing eligibility (liver function tests, complete blood count, Hepatitis- B surface antigen, Hepatitis-
B core antibody, HIV antibody, lymphocyte subset including CD19+ B-cells, total IgG and IgM and urine 
pregnancy test) and blood draw (for collecting PBMCs), and completion of Neuro‐QoL adult item banks. 
Study participants will be enrolled into the study after the study physician confirms participants’ eligibility 
to move forward with the study.  
 
Obtaining the B-cell depleting antibody (ocrelizumab) and all the subsequent study visits and MRIs are 
part of routine and standard clinical care. If the B-cell depleting medication is not approved by the 
participants’ insurance policy, the patient will be excluded from the study.  
MRI procedures: MRI scans will be performed at one of the Hopkins Radiology facilities at 3T. 
Conventional T2/FLAIR/T1-weighted images as well as post-contrast T1-weighted images of the brain will 
be obtained at months 3 (baseline), 12, 18, 24 and 30. Conventional T2/STIR/T1-weighted images as well 
as post-contrast T1-weighted images of the cervical and thoracic spinal cord will be obtained at months 3 
(baseline), 12 and 24. Similar to the standard of care, brain, cervical or thoracic spine MRIs may be done 
as needed if the patient develop symptoms that could be suggestive for an MS relapse (based on the 
evaluation by a study neurologist). The MRI scans will be read by an attending radiologist at Johns Hopkins 
University, and these readings will be used for making decisions regarding the presence of new T2 or Gd-
enhancing lesions suggestive for re-emergence of the radiological disease activity.  
 
Outcomes: 
Primary outcome: The primary endpoint (the failure event) of this study is the time to return of disease 
activity after the third month post-first-infusion, objectively demonstrated by development of new T2 
hyperintense lesions or Gd-enhancing lesions on the MRI or a clinical relapse. A relapse is defined as new 
or worsening neurologic symptom(s) with an objective change on the EDSS of at least 1.5 points for 
participants with baseline EDSS scores of 0 or 0.5 and at least 1-point change for participants with EDSS 
of 1 or more, as determined by the examining neurologist. Symptoms must have been attributable to MS, 
last ≥48 hours, been present at normal body temperature, and preceded by at least 30 days of clinical 
stability. If a new or worsening neurological symptom did not fulfil the above-mentioned criteria, but was 
associated with a corresponding new or enhancing lesion on the MRI that is done in 30 days after the start 
of that symptom, it is still considered a clinical relapse.  


Version: 2.0 
Date: 3/8/2019 
15 
 
 
Secondary outcome:  
Disability: six-month confirmed worsening or improvement of disability: We will report the 
proportion of patients who experience one step worsening or improvement of EDSS that is confirmed on 
a clinical visit six months later.  
 
Quality of life measures: We will administer Neuro-QoL in the computer-adaptive test (CAT) format 
before the infusions and in all the subsequent clinic visits.  
Assessment of T and B-cell phenotypes and function at baseline and 18-24 months post-B-cell 
depletion 
The assessment T and B-cell phenotypes and function and analysis of the central and peripheral B-cell 
tolerance checkpoints, as well as measurement of cytokines will be done at Yale University. 30 milliliters 
of blood will be drawn from the participants before start of the treatment with ocrelizumab (at the 
screening visit) and 12 or 18 months after the second course of ocrelizumab infusion (month 18 or 24 of 
the study) and will be sent to Yale University for the analyses explained below.  
We will investigate the T and B-cell phenotypes in the blood samples from the participants in the study, 
pre-treatment and after they repopulate their peripheral B-cells likely at either 18- or 24-months time 
points. We will follow the frequencies of CD3+CD4+CD25hiCD127loFOXP3+ Tregs, circulating 
CD3+CD4+CXCR5+PD-1+ Tfh cells increased in several autoimmune diseases, and other CD4+ and CD8+ 
naïve and memory T cell subpopulations. We will also assess the production of several cytokines including 
IL-2, IL-4, IL-6, IL_10, IL-17, Il-21, IFN-gamma and TNF-alpha by Tregs and other T cell subsets after 
activating PBMCs with phorbol-12-myristate-13-acetate (PMA) and ionomycin for 4 hours in the presence 
of GolgiStop (BD Biosciences) and intracellular staining with specific monoclonal anti-cytokine antibodies 
as previously reported by our laboratory. Hence, we will determine if the abnormal production of IL-17 
and IFN-gamma previously identified in Tregs from people with MS or their elevated Tfh frequencies are 
corrected after two courses of anti-B-cell deletion. In addition, we will assess in vitro the suppressive 
function of Tregs from people with MS before and after B-cell deletion. Indeed, Tregs from people with 
MS have been reported to display decreased suppressive function and we will therefore be able to 
determine if ocrelizumab can normalize Treg function and if this feature is associated with long-term 
disease suppression. 
We will also characterize various B-cell subpopulations in the blood of people with MS before and after 
treatment with ocrelizumab. We will follow the frequencies of CD19+CD10+CD27-IgMhiCD21lo transitional 
B-cells that recently emigrated from the bone marrow, CD19+CD10-CD27-IgM+CD21+ mature naïve B-cells, 
CD19+CD10-CD27+IgM+CD21+ circulating marginal zone/ IgM memory B-cells, CD19+CD10-CD27+IgM-
IgG+CD21+ and CD19+CD10-CD27+IgM-IgA+CD21+ conventional IgG+ and IgA+ isotype switched B-cells as 
well as unconventional CD19+CD10-CD27-CD21lo CD11c+ memory B-cells that express T-bet, contain 
autoreactive clones and are generated after TLR activation in the presence of IFN-gamma and IL-21. The 
impact of anti-B-cell therapy on B-cell activation will also be determined by following CD69 expression 
that we previously reported to be increased on B-cells from people with MS. The expression of other B-
cell activation markers such as CD80, CD86, TACI and FAS will also be assessed by flow cytometry on each 


Version: 2.0 
Date: 3/8/2019 
16 
 
B-cell subsets described above. We will also obtain a preview on the peripheral B-cell selection in MS by 
characterizing their proliferative homeostasis history by following the expression of proliferation marker 
Ki67 and using the detection of Kappa recombination excision circles (KREC) by quantitative PCR in freshly 
isolated B-cell subpopulations from healthy controls and people with MS before and after anti-B-cell 
deletion as previously described. Indeed, we previously reported that mature naïve B-cells from people 
with MS have undergone increased homeostatic expansion in the periphery that may result from the 
amplification of autoreactive clones. Altogether, these studies will determine the impact of anti-B-cell 
therapy on the phenotype and function of T and B-cells in people with MS. 
Analysis of the central and peripheral B-cell tolerance checkpoints 
Single CD19+CD10hiCD21-/loIgMhiCD27- new emigrant/transitional and CD19+CD10-CD21+IgM+CD27- 
mature naive B-cells will be isolated from the blood of these patients before and after treatment and 
frequencies of polyreactive, HEp-2 reactive and anti-nuclear clones will be determined as previously 
described. In brief, Ig heavy and light chain genes will be amplified by RT-PCR and 20-30 immunoglobulin 
genes from single new emigrant/transitional and mature naïve B-cells will be cloned and expressed in 
vitro. Recombinant antibodies will be tested for HEp-2 reactivity and polyreactivity by ELISAs and anti-
nuclear reactivity using indirect fluorescence assay on HEp-2 slides as previously described. Recombinant 
antibodies will be considered polyreactive when they recognized all three antigens tested individually  
(dsDNA, insulin, and LPS) using supernatants tested at 1μg/ml antibody concentrations and three 1:4 
dilutions in PBS as reported. In HEp-2 and polyreactivity ELISAs, ED38 recombinant antibody will be used 
as positive control. Hence, we will determine if B-cell depletion can correct the impaired removal of 
developing autoreactive B-cells in the periphery of people with MS and is associated with disease 
suppression. 
Safety and adverse effect monitoring: Potential participants will have lab work done (liver function 
tests, complete blood count, Hepatitis- B surface antigen, Hepatitis-B core antibody, HIV antibody, 
lymphocyte subset including CD19+ B-cells, total IgG and IgM and urine pregnancy test) as part of routine 
clinical care to assess for study eligibility and as the baseline for future comparison. Patients who are 
positive for Hepatitis-B surface antigen (HBsAg) or Hepatitis-B core antibody (HBcAb) will be excluded 
from participation.  
Ocrelizumab has been extensively used in people with MS. In this small study, with limited and short-term 
use of B-cell depleting antibodies, we do not expect to see unanticipated side effects worth reporting, 
however, the most important safety consideration in this setting is the return of disease activity after B-
cell reconstitution. Unlike patients who stop medications such as natalizumab and fingolimod and may 
experience severe return of the disease activity, rebound phenomenon has never been described in 
association with cell depleting antibodies such as alemtuzumab, rituximab or ocrelizumab. For example, 
most patients who receive alemtuzumab have severe MS, uncontrolled with other DMTs. Most patients 
only receive two courses of treatment with alemtuzumab and after lymphocyte reconstitution, there has 
been no reports of severe return of disease activity (rebound). Similar to this situation, we do not 
anticipate that patients with B-cell reconstitution after B-cell depletion therapy will experience rebound. 
Although a subgroup of patients may have return of disease activity, the disease activity will be mild and 
non-disabling.  


Version: 2.0 
Date: 3/8/2019 
17 
 
 To comply with rules and regulations of performing clinical studies, we will record all safety data during 
the study. Upon signing the informed consent form, each participant will be given the names and 
telephone numbers of study staff for reporting adverse events and medical emergencies. 
Adverse event (AE):  An AE is the development or worsening of any undesirable symptom, sign or medical 
condition occurring after starting the study drug even if the event is not considered related to study 
medication. Medical conditions or diseases present before starting study drug are only considered AEs if 
they worsen after initiating study medication. At each study encounter, the occurrence of AEs should be 
sought by non‐directive and directive questioning. AEs may also be collected when they are volunteered 
by the subjects during or between encounters. 
 
Serious adverse event (SAE): An SAE is defined as an event which: 
- is fatal or life-threatening 
- results in persistent or significant disability 
- constitutes a birth defect/congenital abnormality  
- requires inpatient hospitalization for at least 24 hours or prolongation of existing hospitalization for at 
least 24 hours 
- is medically significant 
Data safety monitoring committee (DSMB): DSMB is not required for a small study using a medication that 
is FDA-approved for the condition, however, we will have the following treatment stop and study 
termination rules: 
 
Treatment stop rules: Stopping the study is equivalent to restarting the B-cell depleting antibody (i.e. 
ocrelizumab) or another DMT, if there is medical contraindication to the continued use of ocrelizumab 
(e.g. development of allergic or severe infusion reactions during the last infusion). The followings are the 
criteria for restarting the DMT (stopping the study).  
1. A clinical relapse, affecting optic nerves (with worst visual acuity of 20/50 or less) or causing motor 
weakness, ataxia, bladder or bowel symptoms. Pseudo-relapses are not uncommon among patients with 
MS. When in doubt, the PI will use MRI (similar to the standard of care) to confirm development of new 
lesions corresponding to patient’s symptoms (based on the possible localization of the symptoms).   
2. Development of two new T2 hyperintense lesions or two gadolinium enhancing lesions on any MRI after 
the baseline MRI. The baseline MRI was defined the MRI that is obtained three months after the first 
ocrelizumab infusion.   
 
Data management 


Version: 2.0 
Date: 3/8/2019 
18 
 
We will use REDCap (Research Electronic Data Capture) [https://projectredcap.org/], a secure web 
application to collect data, create the study database and access the data for analysis. Study coordinator 
will enter the data required by the protocol into the Electronic Case Report Forms (CRFs). The PI will assure 
that the data entered into CRFs are complete and accurate.                                                      
 
Sample size 
We selected to recruit 10 participants in this open-label, non-controlled study. We limited the number of 
participants for two reasons: The pre- and post-infusion analysis of B-cell tolerance is expensive and time-
consuming, and we could accommodate the analysis of only 20 samples (10 pre and 10 post-infusions 
samples) during the proposed time of the study. We also wanted to make sure that we would observe a 
prolonged drug-free absence of disease activity in a subset of participants, before recruiting a larger group 
of patients for a potential larger study.  
The sample size is supported by our previous studies of central and peripheral B-cell checkpoints in 
patients with autoimmune diseases and healthy control subjects in which we had found that the 
frequency of autoreactive mature naïve B-cells was 48.9±3.06% in patients and 20.1±0.9% in healthy 
donors.  Assuming that the frequency of autoreactive cells among post-treatment repopulated B-cells will 
be similar to normal controls, our proposed sample size of 10 subjects per group before and after 
treatment would give us more than 90% power to detect a significant difference with an α of 0.05 with a 
2-sided Student’s t-test. The analysis of each individual is very labor intensive and costly but fortunately, 
significant findings on the establishment of B-cell tolerance can be obtained with a relatively small number 
of subjects. 
 
Statistical analysis 
We will describe the demographic, clinical characteristics and MRI data of all participants before 
treatment with the B-cell depleting agent. We will report the proportion of participants who experience 
the primary endpoint of the study (return of the disease activity) and the time to failure event, using a 
Kaplan-Meyer curve. Proportion of patients with clinical relapse, new MRI activity or disability progression 
will separately be reported. Quality of life data will be analyzed using linear mixed effect models.  
To look at the association between long-term response to B-cell depletion therapy and autoreactivity of 
B-cells at different stages of development, we will use logistic regression and Cox proportional hazards 
models with failure or time-to-failure event as the outcome variable and the above-mentioned proportion 
of auto-reactive B-cells at different stages of development and the change in proportions before and after 
treatment as the predictor variables. Failure event as defined in the Aim 1 is the return of disease activity 
after the third month post-first-infusion, objectively demonstrated by development of new T2 
hyperintense lesions or Gd-enhancing lesions on the MRI or a clinical relapse that is confirmed with an 
objective change in the neurological examination or a corresponding new or enhancing lesion on the MRI 
that is obtained after the onset of symptoms.  


Version: 2.0 
Date: 3/8/2019 
19 
 
Using paired t-test, we will compare the frequency of autoreactive mature naïve B-cells, Treg and Tfh cells 
and cytokine production before treatment with ocrelizumab and after the B-cell repopulation.  
  
Ethical considerations 
Regulatory and ethical compliance 
The Investigators will ensure that this study is conducted in full conformance with the principles of the 
“Declaration of Helsinki” which affords the greater protection to the individual. It is the mission of the 
physician to safeguard the health of the people. The study physicians’ knowledge and conscience are 
dedicated to the fulfilment of this mission. The clinical study will fully adhere to the principles outlined in 
“Guideline for Good Clinical Practice” ICH Tripartite Guidelines for greater protection to the subject. 
The study will be submitted to JHU IRB for approval prior to enrolling study participants into the study. 
The IRB will be notified annually about study progress, and all important updates on the study or medically 
important events associated with the study will be submitted to IRB as the study progresses. 
The investigator will establish secure safeguards of confidentiality of research data as described in the 
current revision of the International Ethical Guidelines for Biomedical Research involving Human Subjects. 
The Health Insurance Portability and Accountability Act (HIPAA), also known as “The Privacy Rule”, has set 
new standards and regulations to protect patients from inappropriate disclosures of their “protected 
health information” (PHI) that could cause harm to their insurability, employability and/or their privacy. 
PHI pertains to any information that can be used to identify an individual which is created, used, or 
disclosed in the course of providing a health care service, such as diagnosis or treatment. HIPAA does 
allow for researchers to access and use PHI when necessary to conduct research. The study records will 
be identified using subject’s study number to protect the privacy of the study participants. The study team 
will follow the JHU IRB recommendations to protect the privacy of study participants. Study consent will 
also include the privacy language mandated by the Institution’s IRB. Vulnerable populations (including 
fetuses, neonates, pregnant women, children and prisoners) will not be involved in this study. All 
members of the research teams at JHU have received required training in protection of human subjects 
in research and will receive refresher courses at intervals based on state and federal policies.   
Informed consent procedures 
The study investigators, or a person designated by the investigators will explain the IRB approved study 
consent to study participants and obtain signed informed consent from each subject. The study 
participants will be given ample time to review the consent, and are encouraged to ask questions during 
the consent process. After the subject and representative, have orally consented to participation in the 
trial, the witness’ signature on the form will attest that the information in the consent form was accurately 
explained and understood. The study participants are encouraged to ask questions about the study during 
the study participation period. The investigator or designee must also explain to the subjects that they 
are completely free to refuse to enter the study or to withdraw from it at any time, for any reason. The 
study participants will be notified about the new study information during the study participation period, 
and will be re-consented when applicable. A copy of the signed informed consent form will be given to 
the subjects for their records. 


Version: 2.0 
Date: 3/8/2019 
20 
 
Publication of the study protocol and results 
The key design elements of this protocol will be posted in a publicly accessible database such as 
clinicaltrials.gov. 
Protocol adherence 
Investigators ascertain they will apply due diligence to avoid protocol deviations. Any change or addition 
to the protocol can only be made in a written protocol amendment. Only amendments that are required 
for patient safety may be implemented prior to IRB approval.