Analysis of Lymphatic Fluid From the Thoracic Duct of Healthy People and People With Multiple Sclerosis (MS) Before and After Ofatumumab Treatment
Phase: NA
Conditions: Multiple Sclerosis, Healthy
Sponsor: University of Pennsylvania

 
 
Research Protocol 
Analysis of lymphatic fluid from the thoracic duct of healthy 
subjects and patients with MS before and during Ofatumumab 
treatment  
 
Protocol Version 2.0 
18 Aug 2022 
 
 
 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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Principal Investigators 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Co-Investigators 
  
 
 
 
 
 
 
Research Collaborators 
Amit Bar-Or, MD, FRCPC 
Professor of Neurology 
University of Pennsylvania Department of Neurology 
Stemmler Hall Room 178A 
3450 Hamilton Walk 
Philadelphia, PA  19104 
267 591-0136 
amitbar@upenn.edu  
 
Maxim Itkin, MD 
Professor of Radiology and Pediatrics 
University of Pennsylvania Department of Radiology 
3400 Spruce St. 
Philadelphia, PA 19104 
215-823-6328 
Maxim.Itkin2@pennmedicine.upenn.edu 
 
Matthew Schindler, MD, PhD 
Instructor 
University of Pennsylvania Department of Neurology 
Perelman Center for Advanced Medicine 
3400 Civic Center Blvd, 7th Fl. South Pavilion 
Philadelphia, PA 19104 
matthew.schindler@pennmedicine.upenn.edu 
 
Harald Kropshofer, PhD 
Clinical Development Director 
Novartis Pharma AG 
WSJ-154.4.03.35  
Fabrikstrasse 12 
4052 Basel 
+41797042838 
 
Elisabetta Traggiai, PhD 
Director, Mechanistic Immunologies  
Novartis Institute of Biomedical Research 
WSJ-155.3.13.03 
4052 Basel 
+41795093398 
 
 
 
 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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Table of contents 
 
Protocol summary ................................................................................................................ 8 
1 
Introduction ....................................................................................................................... 11 
1.1 
Background ............................................................................................................ 11 
1. 
Disease .................................................................................................. 11 
 
Thoracic duct lymph in disease ............................................................. 12 
2. 
12 
3. 
CNS Lymphatic Drainage ..................................................................... 13 
1.2 
Thoracic Duct Cannulation .................................................................................... 13 
4. 
Procedural Details for ‘In-and-Out’ Catheterization or 
“Indwelling” Catheter Placement .......................................................... 14 
1.3 
Experience with Thoracic Duct Cannulation ......................................................... 15 
1.4 
Ofatumumab .......................................................................................................... 16 
2 
Objectives .......................................................................................................................... 17 
2.1 
Primary and secondary objectives ......................................................................... 18 
2.2 
Exploratory objectives ........................................................................................... 18 
3 
Study Assessments ............................................................................................................ 18 
3.1 
Detailed Immunologic Assessments ...................................................................... 19 
3.2 
Other soluble markers ............................................................................................ 21 
3.3 
Hematology Assessments ...................................................................................... 21 
3.4 
Clinical Outcomes ................................................................................................. 21 
4 
Investigational Plan ........................................................................................................... 22 
4.1 
Participant Recruitment ......................................................................................... 22 
6. 
Healthy Volunteer Recruitment ............................................................ 22 
7. 
People with Multiple Sclerosis .............................................................. 22 
8. 
Vulnerable Populations ......................................................................... 22 
4.2 
Screening Period .................................................................................................... 23 
4.3 
Procedure and Treatment Period ........................................................................... 23 
9. 
4.3.1 “In-and-out” catheter with single time point lymph fluid 
procurement ........................................................................................... 23 
10. 
4.3.2 “Indwelling” catheter with serial lymph fluid procurement ........ 23 
4.4 
Rationale for study design ..................................................................................... 24 
4.5 
Allocation to Interventional Group ........................................................................ 24 

 
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4.6 
Risk and Benefits ................................................................................................... 24 
5 
Study Population ............................................................................................................... 25 
5.1 
Inclusion criteria .................................................................................................... 25 
5.2 
Exclusion criteria ................................................................................................... 25 
5.3 
Study Procedures ................................................................................................... 26 
11. 
Screening Procedures & Visit(s) ........................................................... 26 
12. 
Visit Day 0: Thoracic Duct Cannulation and lymph fluid collection ... 27 
5.4 
Ofatumumab treatment .......................................................................................... 28 
13. 
Handling of study treatment .................................................................. 29 
14. 
Instruction for prescribing and taking study treatment ......................... 29 
15. 
Other treatment(s) ................................................................................. 31 
5.5 
Treating the patient ................................................................................................ 31 
16. 
Participant numbering ........................................................................... 31 
17. 
Drug Interruptions ................................................................................. 31 
18. 
Additional treatment guidance .............................................................. 31 
19. 
Treatment compliance ........................................................................... 31 
5.6 
Patient Monitoring ................................................................................................. 32 
20. 
‘In-and-out’ catheterization ................................................................... 32 
21. 
“Indwelling” catheter and serial sampling ............................................ 32 
22. 
Unscheduled Visit ................................................................................. 33 
5.7 
Withdrawal of informed consent ........................................................................... 33 
5.8 
Loss to follow up ................................................................................................... 33 
5.9 
Data Collection and Follow-up for Withdrawn Participants ................................. 33 
23. 
Early Termination Visits ....................................................................... 33 
5.10 
Pharmacokinetic Evaluation .................................................................................. 34 
5.11 
Safety Evaluation for Thoracic Duct Procedures .................................................. 34 
5.12 
Visit schedule and assessments ............................................................................. 34 
24. 
Visit schedule and assessment for ‘in-and-out’ cannulation ................. 34 
25. 
Visit schedule and assessment for procedure for “indwelling” 
catheter and OMB treatment ................................................................. 35 
26. 
Post procedure Visit schedule and Assessments for MS patients 
who receive 2-year OMB treatment with follow-up ............................. 37 
6 
Visit schedule and assessment ........................................................................................... 37 
6.1 
Information collected on screen failures ................................................................ 38 
6.2 
Rescreening ............................................................................................................ 38 
6.3 
Magnetic Resonance Imaging (MRI) .................................................................... 38 
7 
Statistical Analysis Plan .................................................................................................... 39 

 
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7.1 
Control of Bias and Confounding .......................................................................... 40 
7.2 
Baseline Data ......................................................................................................... 40 
7.3 
Analysis of Primary Outcome of Interest .............................................................. 40 
7.4 
Pharmacokinetic Analysis ..................................................................................... 40 
8 
Safety ................................................................................................................................. 40 
8.1 
Physical Examination (including skin) .................................................................. 41 
8.2 
Vital Signs ............................................................................................................. 41 
8.3 
Height and Weight ................................................................................................. 41 
8.4 
Laboratory evaluations .......................................................................................... 41 
8.5 
Pregnancy and assessments of fertility .................................................................. 42 
8.6 
Appropriateness of safety measurements .............................................................. 43 
8.7 
Additional assessments .......................................................................................... 44 
27. 
Biomarkers ............................................................................................ 44 
28. 
Immune cell analyses ............................................................................ 44 
9 
Study discontinuation and completion .............................................................................. 44 
9.1 
Discontinuation ...................................................................................................... 44 
29. 
Discontinuation of study treatment ....................................................... 44 
9.2 
Study completion and post-study treatment .......................................................... 45 
10 Safety monitoring and reporting ........................................................................................ 46 
10.1 
Definition of adverse events and reporting requirements ...................................... 46 
30. 
Adverse events ...................................................................................... 46 
31. 
Serious adverse events .......................................................................... 47 
32. 
SAE reporting........................................................................................ 48 
33. 
Reporting MS relapse as a SAE ............................................................ 49 
34. 
Pregnancy reporting .............................................................................. 49 
35. 
Reporting of study treatment errors including misuse/abuse ................ 49 
11 Data Collection and Database management ...................................................................... 50 
11.1 
Data collection ....................................................................................................... 50 
11.2 
Database management and quality control ............................................................ 50 
11.3 
Sample management and storage ........................................................................... 50 
12 Data analysis and statistical methods ................................................................................ 50 
12.1 
Analysis sets .......................................................................................................... 51 
12.2 
Participant demographics and other baseline characteristics ................................. 51 
12.3 
Treatments ............................................................................................................. 51 
12.4 
Analysis of the primary endpoint(s) ...................................................................... 51 
36. 
Safety endpoints .................................................................................... 51 
12.5 
Analysis of secondary endpoints ........................................................................... 51 

 
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37. 
12.5.1 Definition of secondary endpoint(s) ........................................... 51 
38. 
52 
39. 
12.5.2. Statistical model, hypothesis, and method of analysis .............. 52 
12.6 
Analysis of exploratory endpoints ......................................................................... 52 
13 Ethical Considerations and Risks ...................................................................................... 53 
13.1 
Risks ...................................................................................................................... 53 
40. 
Risks of thoracic duct cannulation procedure ....................................... 53 
41. 
Additional risks associated with indwelling catheter. ........................... 53 
42. 
Risks associated with conscious sedation. ............................................ 56 
43. 
Risks associated with fentanyl when given intravenously .................... 56 
44. 
Risks associated with midazolam when given as a single agent for 
sedation in adults: .................................................................................. 56 
45. 
Risks associated with antibiotics. .......................................................... 56 
46. 
Risk of phlebotomy. .............................................................................. 57 
47. 
Risk of radiation. ................................................................................... 57 
48. 
Risks of MRI. ........................................................................................ 57 
49. 
Loss of confidentiality. .......................................................................... 58 
50. 
Risks associated with Ofatumumab: ..................................................... 59 
13.2 
Benefits .................................................................................................................. 61 
13.3 
Risk Benefit Assessment ....................................................................................... 61 
14 Informed Consent Process / HIPAA Authorization .......................................................... 62 
15 Study Finances ................................................................................................................... 62 
15.1 
Funding Source ...................................................................................................... 62 
51. 
Conflict of Interest ................................................................................ 62 
52. 
Subject Stipends or Payments ............................................................... 62 
16 Publication Plan ................................................................................................................. 63 
17 Ethical considerations and administrative procedures ...................................................... 63 
17.1 
Regulatory and ethical compliance ........................................................................ 63 
17.2 
Responsibilities of the investigator and IRB/IEC .................................................. 63 
18 Protocol adherence ............................................................................................................ 63 
19 Thoracic Duct Complication Table ................................................................................... 64 
20 References ......................................................................................................................... 65 
 
 
 

 
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List of tables 
Table 1       Assessment Schedule............................................................................................. 34 
Table 2       Assessment schedule ............................................................................................. 35 
Table 3 
Assessment Schedule, Follow-up for patients with MS post indwelling catheter 
procedure....... ........................................................................... Error! Bookmark not defined. 
Table 4       Assessment Schedule, Follow-up for patients with MS post single catheter 
procedure .................................................................................. Error! Bookmark not defined. 
Table 5 
Laboratory Assessments ........................................................................................ 42 
Table 6 
Guidance for capturing the study treatment errors including misuse/abuse .......... 49 
 
List of abbreviations 
 
ADCC 
Antibody-Dependent Cell-mediated Cytotoxicity 
AE 
adverse event 
AESI 
adverse event of special interest 
AIDS 
Acquired Immunodeficiency Syndrome 
ALT 
alanine aminotransferase 
ARR 
Annual Relapse Rate 
AST 
aspartate aminotransferase 
BCR 
B cell receptor 
CDC 
Complement-Dependent Cytotoxicity 
CRA 
Collaborative Research Agreement 
CRF 
Case Report/Record Form (paper or electronic) 
CSF 
Cerebrospinal Fluid 
CTC 
Common Toxicity Criteria 
DMT 
Disease Modifying Therapy 
DNA 
Deoxyribonucleic Acid 
EDSS 
Expanded Disability Status Scale 
EOS 
End of Study 
FAS 
Full Analysis Set 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
Gd 
Gadolinium 
GFAP 
Glial Fibrillary Acidic Protein 
h 
hour 
HBV 
Hepatitis B Virus 
HCV 
Hepatitis C Virus 
HIV 
human immunodeficiency virus 
IDS 
Investigational Drug Service (Penn Research Pharmacy) 
Ig 
Immunoglobulin 
IRB 
Institutional Review Board 
iv 
Intravenous 

 
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LFT 
Liver function test 
MAB 
Monoclonal Antibody 
MedDRA 
Medical dictionary for regulatory activities 
mg 
milligram(s) 
ml 
milliliter(s) 
MS 
Multiple Sclerosis 
NfL 
Neurofilament Light Chain 
OMB 
Ofatumumab 
PCR 
Protein-Creatinine Ratio 
PD 
pharmacodynamic(s) 
PK 
pharmacokinetic(s) 
PML 
Progressive Multifocal Leukoencephalopathy 
PT 
Prothrombin Time 
q4 
Every 4 
RBC 
red blood cell(s) 
RMS 
Relapse Multiple Sclerosis 
s.c. 
subcutaneous 
SAE 
serious adverse event 
sCR 
serum creatinine 
TBL 
total bilirubin 
ULN 
upper limit of normal 
Penn 
University of Pennsylvania 
UTI 
Urinary Tract Infection 
WBC 
white blood cell(s) 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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Protocol summary 
IRB Number 
831944 
Full Title 
Analysis of lymphatic fluid from the thoracic duct of healthy people and 
people with MS before and after Ofatumumab treatment 
Brief title 
Immune profiles in MS patients and healthy volunteers through thoracic duct 
cannulation  
Sponsor Clinical 
Phase 
University of Pennsylvania 
3450 Hamilton Walk 
Philadelphia, PA  19104 
Investigation type 
‘In-and-out’ Cannulation: Healthy Volunteers and MS patients 
Indwelling Cannulation: Healthy Volunteers, and early MS patients before 
and after treatment with Ofatumumab (OMB) 
Methodology 
Clinical trial 
Study type 
Interventional 
Study Duration 
Approximately 4 years 
Purpose and 
rationale 
The central nervous system (CNS) contains a system of lymphatic drainage 
which has been described both based on pathology and more recently through 
in vivo imaging in humans and non-human primates. It has also been 
demonstrated that B cells traffic bi-directionally between the CNS 
compartment and the periphery – in particular the deep cervical lymph nodes 
which drain the CNS lymphatic system. From the deep cervical lymphatics, 
cells and soluble factors drain further into the thoracic duct.  
To characterize immunologically cells and soluble molecules (markers of 
inflammation/injury/repair) in the thoracic duct fluid is of great biological 
interest to understand normal immune system, changes in the immune system 
with MS and effects of treatment onto this immune system with a CD20+ B cell 
depleting drug, Ofatumumab. 
Insights from this study will have major implications both to our understanding 
of peripheral and CNS-compartmentalized biologies of MS, potentially 
identifying new druggable targets, as well as enable entirely novel assessment 
of the impact of OMB on disease-relevant immune responses in distinct 
compartments.  
Primary 
Objective(s)  
The study is designed to evaluate the safety and feasibility of collection of 
lymphoid fluid via thoracic duct cannulation in healthy people and people with 
MS.  
Secondary 
Objective 
Assess the impact of anti-CD20 OMB treatment in patients with MS on 
biological measures in the thoracic duct fluid and in the blood. 
Exploratory 
Objectives 
 
To compare immune cell profiles of blood and lymphatics between MS and 
healthy controls and in untreated MS patients before and following a B-cell 
depleting therapy (Ofatumumab). 
 
Study design 
Monocentric, single arm, open label study:  

 
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1. ‘In-and-out’ catheterization: Safety and immune-cell profile of 
lymphatic fluid in MS patients with a single time-point sampling of 
lymphatic fluids and peripheral blood compared to healthy controls. 
2. “Indwelling” catheterization: immune-biology in people with MS 
before and during/after OMB treatment within thoracic duct and 
peripheral blood via indwelling catheter and multiple time-point sampling 
compared to healthy controls (without drug treatment). 
 
All MS patients undergoing ‘in-and-out‘ or “indwelling” catheterization will 
be offered OMB treatment provided by the Research Collaborator for up 
to 2 years post-catheterization. OMB treatment is required for 
participation in the “indwelling” catheter procedure of MS patients. 
Population 
Total of 24 participants including: 
‘In-and-out’ catheterization:  Two healthy controls and six patients with 
early MS (never treated or at least 90 days after discontinued treatment with 
glatiramer acetate or interferons), who consent to the ‘In-and-out’ catheter 
procedure. MS participants can also consent to OMB treatment with 2-year 
follow-up. 
“indwelling” catheterization: Twelve patients with early MS (never treated 
or at least 90 days after discontinued treatment with glatiramer acetate or 
interferons), who consent to treatment with OMB and to the indwelling 
catheter procedure with serial sampling and up to four healthy controls (no 
drug treatment)  
 
Key Inclusion 
criteria 
• 
Written informed consent must be obtained before any assessment is 
performed 
• 
Participants must be able to understand English and be able to review and 
comprehend the informed consent form independently or with the help of 
research staff 
• 
Participants aged between 18 and 55 years of age at the time of 
screening 
• 
No use of systemic glucocorticoids in the past 4 weeks 
For subjects with MS, following additional criteria apply: 
• 
Diagnosis of MS according to the 2017 Revised McDonald criteria 
• 
Disability status at screening defined by EDSS score of 0 to 4 (inclusive) 
• 
Treatment naive (never been treated) or interferons/glatiramer acetate 
only and drugs have been discontinued at least 90 days prior to 
intervention 
• 
Neurologically stable one month prior to first study drug administration 
Key Exclusion 
criteria 
• 
History of cardiovascular disease or uncontrolled hypertension 
• 
History of diabetes mellitus 
• 
History of cancer (other than localized skin cancer) in the previous 5 years 
• 
History of systemic connective tissue disease 
• 
Chronic HBV or HCV infection 
• 
Absolute neutrophil count (ANC) <750/mm3;  
• 
Hemoglobin <10.0 g/dL 
• 
Platelet count <100,000/mm3 
• 
Prothrombin time (PT) > 2 x ULN 
• 
International normalized ration (INR) >1.5 x ULN 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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Study treatment 
where applicable 
Ofatumumab 20 mg s.c. injections will be administered s.c. on treatment day 
1, day 7 and day 14 and thereafter every 28 days. OMB also offered to MS 
patients participating undergoing “indwelling” cannulation, and will continue 
on for up to 2 years with follow-up.  
MS patients undergoing “in-and-out” may be OMB treated with 2-year post-
procedure follow-up. OMB will be provided by the Research Collaborator as 
the FDA approved formulation. Treatment will commence approximately 48 
hours after “indwelling” catheter or no sooner than 48 hours in those who 
undergo single “in-and-out” procedure, or, if present, once AEs have 
resolved.  
Efficacy 
assessment 
Biological outcomes will be described in terms of immune biology in 
healthy volunteers, people with MS, people with MS before and during early 
OMB treatment comparing central compartments (thoracic lymph) to 
peripheral (blood) 
Exploratory clinical outcomes for people with MS will be described in 
terms of: 
MRI (Gd+, T2 enlarging lesions, slowly evolving lesions, regional atrophy) 
MS relapse expressed as Annualized Relapse Rate (ARR) 
Expanded Disability Status Scale (EDSS) 
Neurofilament light chain (NfL) 
Glial fibrillary acidic protein (GFAP) 
Key safety 
assessments 
Adverse Events (AE) and Serious Adverse Events (SAE) 
Data analysis 
Analyses will be performed jointly by Penn and the Research Collaborator 
designates. Details of responsibilities and analyses will be outlined in the 
research collaboration agreement and the statistical analyses plan. 
This is an exploratory study. There is no data describing the distribution of 
populations of immune cells in the thoracic duct in healthy people, MS 
patients pre and post treatment with OMB compared to peripheral blood. 
Descriptive statistics will be used as well as nonparametric Mann Whitney U 
tests, the Wilcoxon matched pairs signed rank test or the Friedman test with 
Dunn’s multiple comparison post-test, as appropriate, to compare immune 
cell subset frequencies and antigen specific responses between thoracic 
duct and blood, between patients and controls, and prior to and following 
OMB treatment in each compartment. Correlation analyses will be analyzed 
using the nonparametric Spearman test. Differentially expressed genes 
between groups will be ranked using Smyth’s variance-moderated t-test.  
 ‘In-and-out’ thoracic duct cannulation analyses will be based on all data 
collected from untreated healthy volunteers and early MS patients 
undergoing evaluating  
1. Safety of procedure  
2. Comparing immune biology of peripheral blood and thoracic duct 
fluid 
 “indwelling” catheter intervention analyses will be based on all data collected 
from people with MS pre and post OMB and healthy controls 
1. Safety of procedure 
2. Indwelling catheter and immune profiling pre- and during OMB 
treatment for approximately 3 weeks – MS only 

 
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All patients with MS will be offered up to two years of OMB to explore 
peripheral blood and clinical measures outcomes in people with early MS 
with findings obtained from thoracic duct samples. 
Data and Safety 
Monitoring Plan 
The three Penn investigators listed above will review the safety data after 
every 4 patients for “in-and-out” and every successive participant in 
“indwelling” for the first 4 participants and every 3 participants thereafter and 
will immediately review any cases of serious AE related to the study 
procedures following a reported event. All serious AEs will be reported to the 
Research Collaborator. 
Based on their assessment of risk/benefit of the procedure, by the Sponsor 
and the Research Collaborator, the study will progress as outlined or the 
study will be terminated. 
Applicable Ethics, 
Policy and 
Procedure 
Statement 
The following research study will be conducted according to the procedures 
and stipulations described in this protocol, and in full accordance with all 
applicable University of Pennsylvania Research Policies and Procedures and 
all applicable Federal and state laws and regulations including those 
regulations governing the protection of human participants (Human 
Participants Research, 45 CFR 46, US Department of Health and Human 
Services, and the Code of Federal Regulations 21 CFR Parts 50, 54, and 
56).  
1 Introduction 
1.1 Background 
1 Disease 
Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating and 
neurodegenerative disease of the central nervous system in young adults. This disorder is a 
heterogeneous, multifactorial, immune-mediated disease that is influenced by both genetic and 
environmental factors. In most patients, reversible episodes of neurological dysfunction lasting 
several days or weeks characterize the initial stages of the disease (that is, clinically isolated 
syndrome and relapsing–remitting MS; RRMS). Over time, irreversible clinical and cognitive 
deficits develop.  
The pathological hallmark of MS is the formation of demyelinating lesions in the brain and 
spinal cord, which can be associated with neuro-axonal damage. Focal lesions are thought to be 
caused by the infiltration of immune cells, including T cells, B cells and myeloid cells, into the 
central nervous system parenchyma, with associated injury.  
In patients with MS, the most implicated T cell subset is the IL-17 expressing CD4+ T cell 
population (CD4+CD3+IL-17+ T cells). However, the effectiveness of B-cell depleting 
therapies has helped to broaden the view of MS being only a T-cell mediated disease to a view 
of an aberrant immune system that includes the interaction of multiple cell lines and signaling 
molecule located in the periphery and compartmentalized in the CNS; in particular, and as 
shown in our previous work, interactions between pro-inflammatory GM-CSF expressing B 
cells with the disease-implicated Th17 T cells are thought to be important drivers of the human 
disease  (Li et al., 2015).  

 
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Much of our understanding of immunophenotypes of cells present in the CNS of patients with 
MS is by virtue of CSF analysis – to demonstrate oligoclonal bands - which has relevant 
limitations including the very small number of cells and their fragility which make functional 
assessments extremely challenging.  
Additionally, though blood is considered a window into immune activity, recent work of the 
group demonstrated in people with HIV that studies in blood fail to capture the major 
component of HIV-specific CD8+ T cell responses resident in lymphatic tissues (Buggert et al, 
2018). Immune cells in the lymphatic channels like the thoracic duct are unique compared to 
immune cells found in peripheral blood and study of these cell can provide a new window into 
the biology of diseases like HIV and potentially also in MS.  
 
Additionally, it is now recognized that CNS cells and soluble factors can egress via lymphatics 
into deep cervical lymph nodes. The thoracic duct is an important gateway for trafficking of 
immune cells out of the CNS to the periphery through the deep draining lymph nodes and allows 
sampling of lymphatic tissues.   
2 Thoracic duct lymph in disease 
The TD represents a highly unique anatomic and physiologic compartment that is quite 
distinct from the peripheral blood. It is the major deep efferent lymphatic and, based on 
pioneering work at Penn exploring this previously inaccessible compartment in healthy 
controls and patients with HIV (Voillet V et al, JCI Insight. 2018; Buggert M et al, Sci 
Immunol. 2018; Vella LA, et al J Clin Invest 2019; Buggert M, et al Cell. 2020), is now 
known to harbor a different profile of cells compared to blood, as well as considerably higher 
concentrations of certain populations that are much more dilute in the blood.  In addition to 
the considerable fundamental immunology interest, studying the TD is of particular interest in 
multiple sclerosis. The upper part of the thoracic duct drains half of the brain in most people 
and, as such, represents a site of enrichment of brain-draining cells. Accessing this level of the 
TD provides an opportunity to study disease-relevant CNS-autoreactive cells that may not be 
present in the blood or, if present in blood, are so dilute that it has not been possible to 
characterize them. The lower part of the thoracic duct drains the gut and is major interest now 
given the implication of an important ‘gut-brain’ axis that can modulate CNS inflammation. 
While leukapheresis provides larger numbers of cells than feasible in routine blood draw, their 
profile is in essence the same as circulating blood cells, hence leukapheresis cannot be used as 
an alternate method for this research.  
 
Sampling of the cells and soluble factors in the thoracic duct will provide a unique opportunity 
to gain understanding of the following: 
1. Immune biology in healthy volunteers and comparing immune biology of lymphatic 
fluid and in blood 
2. Immune biology of people with early MS comparing immuno-profiles in blood to 
lymphatic fluids 
3. Immune biology comparing immuno-profiles before and after treatment with 
Ofatumumab, in blood and lymphatic fluids, over a period for up to 25 days. 
 

 
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3 CNS Lymphatic Drainage  
Lymphatic vessels have a key role as the afferent arm of the immune system for most organs in 
the body, thereby maintaining fluid homeostasis and acting as pathways for the passage of 
antigens and antigen-presenting cells (APCs) to regional lymph nodes.  
The CNS parenchyma has no conventional lymphatic vessels, and although a well-regulated 
Blood Brain Barrier (BBB) controls the entry of solutes into the CNS, there is still a need to 
maintain homeostasis and effective afferent pathways to lymph nodes.  
Two extracellular fluids are associated with the CNS: CSF in the ventricles and the 
Subarachnoid Space (SAS), and ISF (interstitial fluid) in the CNS parenchyma. Both fluids 
drain to cervical and lumbar lymph nodes, but by separate pathways. CSF drains along 
lymphatic vessels that pass through the cribriform plate and the dura mater, and this pathway 
allows for trafficking of immune cells.  
In contrast, ISF and solutes from the CNS parenchyma drain to lymph nodes along 100–150-
nm-thick basement membranes in the walls of cerebral capillaries and arteries. Only 15% of 
ISF draining from the cerebral hemispheres leaks into the CSF. In contrast to observations 
regarding the drainage pathways for CSF, no evidence exists showing that immune cells can 
traffic directly from the CNS parenchyma along the narrow intramural perivascular pathways 
to lymph nodes (Da Mesquite S et al., 2018). 
Drainage of CSF via this route is largely to deep cervical lymph nodes, and the lymphatic 
channels allow the trafficking of CD4+ T cells, monocytes and dendritic cells from CSF to 
cervical lymph nodes. CSF drainage to cervical lymph nodes via dural lymphatics seems to 
occur mainly at the base of the skull. Cranial and spinal nerve roots also act as pathways for 
lymphatic drainage of CSF to cervical and lumbar lymph nodes. Direct drainage pathways to 
lymph nodes for APCs in the CSF could be a major reason that CSF spaces are immunologically 
competent and demonstrate rapid inflammatory responses.  
Given the demonstration that B cells traffic bi-directionally between the CNS compartment and 
the periphery – in particular the deep cervical lymph nodes which drain the CNS lymphatic 
system, cannulating the thoracic duct provides a unique opportunity to assess, for the first time, 
the disease-relevant make-up and functional responses of CNS-draining immune cells and 
soluble factors in people with MS.  
 
1.2 Thoracic Duct Cannulation  
Cannulation of the thoracic duct is part of the interventional radiologic procedure most 
commonly performed to embolize the thoracic duct.  Historically, leaks from the thoracic duct 
or its tributaries (which result in chylothorax; leakage of lymph fluid into the pleural space), 
typically caused by trauma in adults, were surgically corrected, with high rates of morbidity and 
mortality.  The development of minimally invasive embolization of the thoracic duct following 
thoracic duct cannulation under fluoroscopy was first performed in humans in 1998, and has 
become the gold standard treatment for traumatic chylothorax (Itkin and Chen, 2011). Until 
recently, the technical success rate for catheterization of the thoracic duct was approximately 
70% to 80% (Itkin and Chen, 2011). A technical limitation of these procedures was the need to 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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cannulate lymph vessels in the feet to perform a lymphangiogram, the initial step in this 
procedure. 
The success rate of the procedure has improved following the innovation (developed by Max 
Itkin) to perform an ultrasound guided lymphangiogram following injection of dye into an 
inguinal lymph node, rather than injection of dye into lymph vessels in the feet. (Nadolski and 
Itkin, 2012)  All thoracic duct cannulation and embolization procedures have used the inguinal 
intranodal lymphangiogram technique during the past three years, and this is now the standard 
of care for clinical procedures.  
The procedure shows a low complication rate (see adverse related events chart below) of 
approximately 3%. The procedure that will be performed in this study will not include 
embolization of the thoracic duct or the use of glue, therefore none of the complications of 
embolization reported in prior series would be expected in this study.   
Additionally, Max Itkin and colleagues have shown that externalization of the thoracic duct 
catheter can be safely performed in the setting of chronic drainage of lymphatic fluid.  
 
4 Procedural Details for ‘In-and-Out’ Catheterization or “Indwelling” 
Catheter Placement 
The procedure is performed in the following manner.  Participants will have abstained from 
eating the night before the procedure.  They will come to the interventional radiology suite, and 
an intravenous catheter will be placed in a vein in the arm, antibiotics will be administered, 
consisting of either ampicillin-sulbactam, or in the event of a history of penicillin allergy, the 
combination of levofloxacin and clindamycin. The areas of the groin and abdomen will be 
prepped and draped.  Participants will be given conscious sedation with midazolam and 
fentanyl.  An ultrasound of the groin will be performed to identify suitable lymph nodes.  A 25- 
or 26-gauge spinal needle will be advanced into a lymph node and under fluoroscopic guidance 
5 mL of oil-based contrast agent (Lipiodol; Guerbet Group, Princeton, NJ) will be injected into 
the lymph node at a rate of approximately 1 mL per minute.  Lymph nodes on both sides of the 
groin will be injected as part of the intranodal lymphangiogram component of the procedure to 
enhance visualization of the lymphatic vessels.  The infusion of contrast will be stopped when 
the lymphatics are visualized at approximately the level of the L3 lumbar spine.  If the cisterna 
chyli is not visualized after the injection normal saline will be injected at the rate of 
approximately 1 mL per minute for 5 minutes to facilitate propagation of the contrast agent.   
After identification of the target lymphatic vessel (the cisterna chyli, thoracic duct, or a tributary 
of the thoracic duct), a 21- or 22-guage 15 to 20 cm Chiba needle will be passed into the vessel 
via a transabdominal approach. A 0.018 guidewire will be advanced through the needle into the 
lymph vessel and advanced into the thoracic duct.  A microcatheter will then be advanced into 
the thoracic duct over the wire.  The microcatheter will then be left in place and chyle (lymph) 
will be collected.  
For a ‘in-and-out’ catheterization procedure, up to 100 mL will be collected from the catheter 
over a 30-minute collection period at two levels within the thoracic duct and the catheter will 
then be removed and sedation will be stopped.   

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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For serial sampling, the cannulation will be done as described above followed by placement of 
an ‘indwelling’ catheter. After achieving the transabdominal access of the thoracic duct using 
the microcatheter, a guidewire will be placed through the catheter previously positioned within 
the thoracic duct and advanced into a subclavian vein. A vascular sheath will then be placed 
into a vein in the upper arm under ultrasound and fluoroscopy guidance. The wire in the 
subclavian vein will be then snared using a vascular snare through venous access sheath and 
pulled out from venous sheath. A 4 Fr catheter/sheath will then be threaded and advanced over 
the wire until its tip is in thoracic duct. The catheters from the thoracic duct will then be 
removed, leaving the 4 Fr catheter extending from the arm vein into the thoracic duct. Sedation 
will be stopped.  The procedure will take approximately 2 hours from start to completion.  The 
participant will then be monitored in the Interventional Radiology recovery area until he or she 
is able to return home.  The 4 Fr catheter/sheath will be left in place until completion of the 
procurement of lymph fluid samples (up to 21 days). Participants will be provided education on 
the standard care of an indwelling line as well as any concerning signs to contact the study team 
or on-call physician with questions. Participants will have frequent follow-up visits to identify 
any problems related to the catheter or its procedure. 
1.3 Experience with Thoracic Duct Cannulation 
The Penn Interventional Radiology team collaborating on this study has tabulated the 
complications associated with thoracic duct embolization performed for clinically indicated 
reasons (see Section 16).  They have performed 486 thoracic duct embolizations on 347 patients 
and have encountered 21 complications (complication rate of 4%). Among the 21 
complications, 16 were due to the different steps of embolization procedure (mis-embolization 
by coils or by glue) that will not be a component of the procedures conducted in this trial. One 
complication was a result of a pedal lymphangiogram that will not be performed in this study, 
and another was a complication of a thoracentesis that will also not be performed as part of this 
study. In one patient the wire was sheared during thoracic duct access, with no consequences to 
the patient. Of the remaining two complications, one involved an episode of low oxygen 
saturation and low blood pressure that may have been due to over sedation; the other was 
documented in a patient who developed acute renal failure, and was also thought to be a 
consequence of embolization (rather than cannula insertion). In the HIV study, in which only 
cannulation was done as planned in this study (i.e. without embolization or glue), none of the 
12 patients enrolled encountered complications. 
1.3.1 Indwelling catheter and drainage: 
A published meta-analysis of studies of external drainage of the thoracic duct reported on a total 
of 71 studies involving a total of 1160 patients, where this approach was used in solid organ 
transplantation, acute pancreatitis, neoplastic disease, sepsis, and autoimmune disease, for 
periods of up to and over 4 weeks (Meta analysis: Wang et. al Journal of Surgical Research. 
Vol 204. 2016). The most common complication was wound infection at the site of cannulation 
which occurred in 5-10% of participants.  Other rare complications were accumulation of lymph 
in the neck, back pain, and edema of the face or arm. There was no reported mortality related 
to thoracic duct drainage. We were surprised to find that the meta-analysis included one small 
study in patients with MS (Ring et al. Lancet, 1974) where thoracic duct drainage was assessed 
as a potential therapeutic intervention (the study reported on immunological and neurological 
outcomes but did not report on complications).  
 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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It is important to note that many of the prior studies took place between the 1960s -1980s with 
techniques of thoracic duct cannulation and drainage that involved surgical incision of the neck 
and more invasive techniques to access the duct. The modernized IR techniques have become 
more refined with lower complication rates than past surgical approaches. Important differences 
are that the majority of the prior studies described in the meta-analysis employed larger 
catheters as well as continuous drainage, unlike our proposed approach which uses considerably 
finer catheters and only intermittent sampling of smaller lymph volumes.  
 
To date, the IR team at Penn, led by Dr. Itkin has performed indwelling thoracic duct 
cannulation with drainage in 8 patients including 3 infants with anasarca and 5 adolescent 
patients with chylothorax. The duration of indwelling catheter and drainage averaged 9.9 days 
(with a range of 2 to 43 days). There were no intra-procedure complications, all 8 patients 
experienced symptomatic improvement and no line-infections developed this quite sick patient 
population. Our IR collaborators have a Penn IRB approved research protocol to perform 
thoracic duct cannulation with externalization and drainage in the context of sepsis (protocol 
:834599), though they have not yet initiated this trial.   
 
Given our proposed use of the smaller bore catheters and intermittent sampling of much smaller 
volumes of lymph fluid (50mL per sample versus the liters that are drained in the clinical 
setting), we anticipate any added risks of indwelling catheter in the generally healthy MS 
patients who do not have abnormal thoracic duct anatomy will be minimal, particularly over a 
period of several weeks. According to our IR team, the closest approximation of risk associated 
with the indwelling thoracic duct catheter in this generally healthy population is the risk 
associated with a peripherally inserted central catheter (PICC) line.  PICC line cannulation of 3 
weeks or longer is routinely done in the appropriate clinical setting. We will use a similar 4-5 
Fr line to access the thoracic duct through left arm vein. The greatest potential complication is 
infection. The risk of PICC line associated infections in the heme/onc population such as bone 
marrow transplantation patients who are much more profoundly immune compromised is low. 
In a study of oncology patients, 165 BMT patients had PICC lines for an average of 88.3 days 
and an infection rate of 0.96 infections per thousand line-days (Moturu, A et al. Infection rates 
for PICC lines in oncology patients. Journal of Vascular and Interventional Radiology, 2017). 
We anticipate an even lower risk of infection given the shorter duration of indwelling catheter 
and the healthier MS population.  
 
1.4 Ofatumumab 
Ofatumumab, a fully human monoclonal antibody (mAb) that is FDA approved for relapsing 
MS, recognizes an epitope localized close to the cell membrane on the 2 extracellular domains 
of the CD20+ molecule. CD20-binding of ofatumumab induces effective B-cell lysis primarily 
through complement-dependent cytotoxicity (CDC) and ADCC. 
Ofatumumab was evaluated in OMS112831 which was a randomized, placebo-controlled, dose-
ranging, 48-week study (24-week double-blind treatment phase, then 24-week follow-up phase) 
that examined the efficacy and safety of repeat-dose subcutaneous ofatumumab in RRMS. Two 
hundred and thirty two patients were randomized (2:1:1:1:2) to placebo, ofatumumab 3 mg, 30 
mg, or 60 mg every 12 (q12) weeks, or ofatumumab 60 mg every 4 (q4) weeks. The primary 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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endpoint was the cumulative number of new gadolinium-enhancing lesions during Weeks 0-12 
on brain magnetic resonance imaging (MRI). 
Ofatumumab reduced the mean cumulative number of new gadolinium-enhancing lesions by 
65% vs placebo during Weeks 0-12 (p < 0.001), and by ≥ 90% during Weeks 4-12 vs placebo 
in a post hoc analysis of cumulative ofatumumab doses ≥ 30 mg (p < 0.001). Dose-dependent 
decreases were maintained up to Week 48. Ofatumumab reduced cumulative few/newly 
enlarged T2 lesions vs placebo during Weeks 0-12 (60-72%; p ≤ 0.002). Between 24-48 weeks, 
new/newly enlarged T2 lesion counts were stable for all ofatumumab doses apart from the 3 mg 
dose group. 
The results also demonstrated a rapid, dose and dose frequency dependent reduction in B cell 
counts. Monthly dosing showed no signs of B-cell repletion during the inter-dosing interval. 
Both 30 mg and 60 mg q12 weeks showed approximately 75% suppression of B-cells prior to 
re-dosing. Once dosing was ceased, all treatments showed similar rate of B-cell repopulation 
over 60 weeks of follow-up. Overall, ofatumumab was safe and well tolerated in patients with 
RRMS. 
In total, 43 (64%) patients receiving placebo and 121 (74%) receiving ofatumumab (65%–81% 
across dose groups) experienced AEs during weeks 0 to 12. During weeks 12 to 24 and 24 to 
48, the proportions of patients who experienced AEs across treatment groups were 45% to 62% 
and 47% to 55%, respectively. AEs were largely mild to moderate in severity, and no patients 
died. Incidences of serious AEs (SAEs) were 3%, <1%, 4%, and <1% in weeks 0 to 12, 12 to 
24, and 24 to 48 and the individualized follow up (IFU) phase, respectively. The only SAEs to 
occur in ≥1 patient during the treatment phase were injection-related reactions (IRRs), occurring 
in 3 patients; all continued in the study, including 1 patient who reportedly experienced a 
cytokine-release syndrome within hours of the first ofatumumab (60 mg) dose. Other SAEs 
occurring in single patients were cholelithiasis and hypokalemia (both with 60 mg ofatumumab 
every 4 weeks) and angioedema and urticaria (both in the same patient receiving 3 mg 
ofatumumab). There was no pattern of SAEs in the 24-week FU phase. During the IFU, 2 (2%) 
patients, both in the ofatumumab 60 mg every 4 weeks group, reported a total of 2 SAEs: head 
injury and malignant melanoma stage IV. The latter was considered treatment related, and the 
patient recovered (as noted by the investigator). 
Two multinational Phase 3 studies of ofatumumab (ASCLEPIOS: COMB157G2301 and 
COMB157G2302) are being conducted to demonstrate efficacy, safety and tolerability for s.c. 
injection of ofatumumab 20 mg q4 weeks compared to oral teriflunomide in patients with 
relapsing MS, with about 1800 patients randomized. 
OMB received FDA approval for the treatment of Multiple Sclerosis including relapsing, active 
secondary progressive and clinically isolated syndrome in August 2020. Use of this drug in this 
study will be according to FDA label. 
 
2 Objectives 
The overall objectives of this study are to understand immune biology of the healthy, young 
immune system, to compare this to people with MS, understand composition in blood and 
thoracic duct and understand treatment effects of OMB on central and peripheral 
compartments. 

 
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2.1 Primary and secondary objectives 
To evaluate the safety and feasibility of collection of lymphoid fluids & tissue via thoracic 
duct cannulation (both “in-and-out” and “indwelling” procedures) in healthy volunteers and in 
early MS patients and understand the effects of OMB on peripheral immune markers over 2 
years compared to markers pre-OMB and shortly following OMB initiation in peripheral 
blood and thoracic duct lymph. 
2.2 Exploratory objectives 
To compare immune cell profiles of blood and lymphatics between MS and healthy controls 
and in untreated MS patients before and following a B-cell depleting therapy (Ofatumumab), 
including: 
1. to compare cellular and soluble biomarkers in thoracic duct lymph with blood measures 
in untreated MS patients 
2. to explore differences in cellular and soluble biomarkers in thoracic duct lymph and 
blood measures between healthy controls and people with MS without treatment 
3. to explore changes in immune biology in people with MS before and during OMB 
treatment 
4. compare effects of acute OMB treatment on immune biology to healthy controls 
 
3 Study Assessments 
Study measures will be assessed at a single time point for “in-and-out” catheterization and at 
multiple time points for indwelling catheter procedure combined with OMB treatment (details 
outlined below) for immunologically related endpoints. 
Fluid sampling results will be assessed in both lymph fluid collected from the thoracic duct and 
blood. Analyses will be prioritized based upon the volume of lymph fluid obtained. In addition 
to the measures listed below, other cellular or soluble markers may be measured on residual 
material.  
“In-and-out” procedure: The primary types are the percentage of patients with AE and/or SAEs 
related to study procedure (‘in-and-out’ cannulation of thoracic duct). Additional parameters 
will be explored: 
1. To generate data on cell profiles of blood and lymphatics in untreated participants with 
MS  
a. To evaluate the phenotype, function, and transcriptional profiles of immune cell sub-
populations (including T cell, B cell and innate cell subsets) 
b. To identify CNS-antigen specific responses of immune cells and profile B cell 
repertoires 
c. To quantify soluble measures of inflammation and CNS injury in lymph fluid 
compared to serum 
d. To investigate the humoral autoantibody repertoire at serological and cellular level 

 
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e. To document presence or absence of subclinical disease activity using brain MRI 
(+/- Gd) in order to better interpret immunological results (single MRI done within 
approximately one week of cannulation)  
 
“Indwelling” procedure: The primary endpoints are the percentage of patients with AE and/or 
SAEs related to study procedure (‘indwelling’ cannulation of thoracic duct). Additional 
parameters will be explored: 
5 To generate data on cell profiles of blood and lymphatics in participants with MS before 
and following a B-cell depleting therapy (OMB), and in healthy controls including: 
a. To evaluate the phenotype, function, and transcriptional profiles of immune cell sub-
populations (including T cell, B cell and innate cell subsets) 
b. To identify CNS-antigen specific responses of immune cells and profile B cell 
repertoires 
c. To quantify soluble measures of inflammation and CNS injury in lymph fluid 
compared to serum 
d. To investigate the humoral autoantibody repertoire at serological and cellular level 
e. To document presence or absence of subclinical disease activity using brain MRI 
(+/- Gd) in order to better interpret immunological results (MRIs: first done within 
a week of catheterization, second done within 1 month following removal of 
indwelling catheter. 
 
Patients with MS will be offered OMB treatment for up to 2 years free of charge, and be dosed 
according to FDA label, to evaluate clinical and biomarker outcomes in an early MS population 
and described effectiveness, safety, and tolerability. Additional testing will be including 
clinical, blood biomarkers, and imaging including standard clinical and additional research 
studies as described in detail (below). 
3.1 Detailed Immunologic Assessments 
In depth functional and phenotypic analysis of innate and adaptive immune compartments will 
be performed. The Bar-Or lab has established methods to identify phenotype and function of 
immune cell type in vitro using a different combination of stimulation, which is specific for 
different cell types present in blood circulation and thoracic duct. Specifically, the following 
analysis will be conducted:  
a) Analysis of T cell functional subsets namely: CD4 naïve, central memory, effector memory, 
terminal differentiated effector memory (TEMRA), T follicular helper cells, regulatory T cell 
and CD8 naïve, central memory, effector memory and terminal differentiated effector memory. 
Chemokine receptors, activation as well as exhaustion markers will be analyzed on the different 
T cell subsets described above upon T cell specific stimulation according to established 
protocol.  
b) Analysis of B cell functional subsets namely: transitional, naïve, IgM memory, IgG memory 
and plasma blasts/plasma cells will be enumerated. Chemokine receptors, activation markers, 
as well as antigen presentation markers (CD80, CD86, CD83) will be evaluated upon B cell 
specific stimulation according to established protocol.  
c) A recent finding has described a new population of lymphocytes co-expressing BCR and 
TCR, expanded in T1D and having a potential role in modulating autoreactive T cells. Thus we 

 
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will evaluate whether this population exist in blood and thoracic duct of healthy and MS 
patients, using the following surface markers: CD19, CD5, IgM, IgD, TCR (Ahmed et al., 
2019).  
d) Analysis of the following myeloid subsets: monocytes according to the expression of CD14 
and CD16, conventional dendritic cells and plasmacytoid dendritic cells. Chemokine receptors, 
activation markers as well as antigen presentation markers will be analysed upon myeloid 
specific activation according to established protocol.   
Overall in order to accomplish the analysis described above a combination of the following 
surface markers will be used: Phenotypic and functional assessment of mononuclear cells as 
determined by multi-parametric FACS panels well-established in the Bar-Or lab including, but 
not necessarily limited to, the following cell surface or intracellular protein markers: CD3+, 
CD4+, CD8+, CCR2, CCR5, CXCR3, CXCR4, CXCR5, CCR6, PD-1, CD57, CD127, HLA-
DR, CD38, Bcl-6, ICOS, CCR7, CD19, CD20, CD24, CD27, CD45RO, Ki67, CD95, CD28, 
CD103, CD150, CD11a, CD123, CD200, CD16, CD56, KLRG-1, LAG-3, TIGIT, Helios, IgM, 
IgD, CD80, CD86, CD83, CD69, TCR, etc. All markers will allow the identification of naive, 
central memory, effector memory, regulatory T cells and follicular helper T cells as well as 
naive and memory B cells and plasma cells. 
The adaptive and innate immune cell subsets will also be characterized using a large panel of 
soluble and intracellular functional markers of cells and cell subsets, as determined by ELISA 
or ICS: IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IL-21, IL-22, IL 23, IL 33, IL 35, and IL 
1beta, IFNa, IFNg, TGF b.; granzyme B, perforin. Specifically, we will investigate cytokine 
secretion both pro-inflammatory as well as anti-inflammatory in B and T lymphocytes.  
We will assess the magnitude and breadth of CNS antigen-specific reactivity using tetramer 
assays with established tetramer technology and/or Activation Induced Marker (AIM) assays, 
and flow cytometry in the Bar-Or lab. 
Transcriptional profiles of sorted (T cell, B cell, innate cell) immune subsets including bulk 
profiling and single cell 10X genomics. 
Repertoire analysis pre and post treatment with ofatumumab, with respect to: 1) public clones; 
2) VH and Vb gene and allele usage; 3) alterations in clonal selection through the analysis of 
somatic hypermutations); 4) integration of BCR or TCR data with scRNA sequencing profiles 
in selected samples (this would be done computationally; Prak lab). 
Functional B cell auto-antibodies repertoire analysis: for this specific end point, frozen lymph 
mononuclear cells isolated from peripheral blood as well as from the lymph fluid obtained from 
the thoracic duct will be shipped to the Research Collaborator site in Basel, Switzerland in order 
to perform a functional B cell receptor (BCR) repertoire analysis. Briefly IgM and IgG memory 
B cells will be isolated by FACS sorting and seeded at clonal level in 384 well plates. Human 
B cells will be expanded in vitro according to published protocol (Lindner et al., 2019), and a 
panel of autoantigens previously selected based on the serological and lymphatic fluid analysis 
will be used to screen for the presence of specific autoantibodies. Purified autoantibodies 
expressed in a mammalian system will be characterized for affinity and function and then 
eventually shipped back to the collaborator for further characterization and analysis.  
The Bar-Or lab has been able to establish a technique which would allow the isolation of 
cytokine secreting B cells. Thus we plan to isolate by FACS cell sorting cytokine secreting B 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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cells and perform the functional specificity analysis of the BCR with the technique established 
in Traggiai’s lab at Novartis Institute for Biomedical Research (Lindner et al., 2019). 
3.2 Other soluble markers 
A variety of other soluble markers will be examined depending on availability of fluids for 
analysis.  
1. NfL and GFAP in peripheral blood  
2. NfL and GFAP in thoracic lymphatic fluids  
3. Other fluid based biomarkers under research 
4. Potential Proteomic analysis   
5. Potential Lipid analysis   
6. Potential metabolomic analysis on fluid phase samples (in plasma and thoracic duct 
fluid) applying global metabolomics and weighted correlation network analysis 
(WGCNA) to first identify modules of correlated metabolites that change with 
treatment, followed by confirmation using targeted metabolomic assessment. Changes 
in metabolite modules and individual metabolites can be assessed in relation to changes 
in immune measurements 
7. Autoantibody profiling in serum and lymphatic fluid: autoantibody profiling will be 
conducted using a high-throughput protein microarray chip developed by the Research 
Collaborator.  This chip contains approximately 3000 self-secreted proteins. Known MS 
specific auto-antigens will be included in the printed collection.  IgM, IgG and IgA sera 
and lymphatic fluid profile on self-protein chip will give us a unique opportunity to 
investigate new potential auto-antigens and their modification in MS patients upon 
treatment. (done by Novartis) 
3.3 Hematology Assessments 
Hematology assessments include: red blood cell (RBC) count, RBC morphology, white blood 
cell (WBC) count with absolute and differential (basophils, eosinophils, lymphocytes, 
monocytes, neutrophils, and segmented neutrophils), platelet count, hemoglobin, and HCT.  
3.4 Clinical Outcomes 
This study includes the following clinical outcome assessments: 
• MS Relapse1  
• Expanded Disability Status Scale (EDSS) 
• MRI 
                                                 
1 MS relapse definition: appearance of a new neurological abnormality or worsening of previously stable or 
improving pre-existing neurological abnormality, separated by at least 30 days from onset of a preceding clinical 
demyelinating event (McDonald et al. 2001). The abnormality must have been present for at least 24 hours and 
occurred in the absence of fever (< 37.5°C) or known infection.  
Confirmation of MS relapse: the definition of a confirmed MS relapse is one accompanied by a clinically 
relevant change in the EDSS i.e. an increase of at least 0.5 points on the EDSS score, or an increase of 1 point on 
two functional scores (FSs) or 2 points on one FS, excluding changes involving bowel/bladder or cerebral FS 
compared to the previous available rating (the last EDSS rating that did not occur during a relapse).  

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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An overview of each of these assessments is provided in the sections below and the details of 
these assessments will be provided in the site manuals. 
4 Investigational Plan 
4.1 Participant Recruitment  
6 Healthy Volunteer Recruitment 
The MS center has access to different options to recruit healthy volunteers. Below is a list of 
options which will be pursued: 
a. Penn healthy volunteer pool to connect the community to research activities at Penn. 
b. A research volunteer registry for healthy volunteers outside of Penn community which 
enables direct contact with people interested in participating in research. 
Healthy volunteers identified via the above will be contacted via phone or email and, if 
interested, scheduled to come in for a screening visit, which will begin with informed consent.  
Any advertising will be IRB approved prior to use.  
7 People with Multiple Sclerosis 
Participants with MS will be recruited amongst the patients cared for within the MS Center 
practices of the University of Pennsylvania Health System. Patients will also be recruited via 
provider referral from outpatient clinic or inpatients at Hospital of the University of 
Pennsylvania. Over 5000 MS patients receive clinical care at the Penn MS center annually 
across the spectrum of disease. The clinic evaluates patients with first presentation of disease 
and provides long-term care for people with inflammatory diseases including MS.  
8 Vulnerable Populations 
No vulnerable populations will be included in this research. Penn employees and students will 
be neither targeted nor excluded from participating in this study, and appropriate protections 
are in place to ensure that these individuals are in no way coerced into participating.  During 
the Informed Consent process (which is compliant with 21 CFR 50 Subpart B, 45 CRF 46.116-
117, and federal and local laws), the study team will communicate that participation is voluntary 
and will have absolutely no effect on their employment, education, future care at Penn, or any 
other relationship with Penn. The study team will take proactive measures to ensure that 
educationally or economically disadvantaged persons are not coerced into participating. This 
will be accomplished by ensuring that each individual fully appreciates both the risks involved 
and the alternatives that exist, and can articulate a general interest in the study even prior to any 
mention of monetary compensation.  To minimize the risk of attracting people who may be 
economically coerced to participate, we have chosen not to advertise for this study. Rather, our 
team of clinicians will gauge the appropriateness for possible participation among patients 
whom they know well. We believe these proactive steps will help to minimize financial 
incentives influencing participation in the study and would be happy to implement additional 
mechanisms that may be identified. The per-visit reimbursement will be nominal and provided 
in consideration of travel to Penn and the time commitment of the participant and an individual 
who accompanies the participant. The study team will remind the potential participants that 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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their decision regarding participation in the study will have no impact on the care they receive 
at Penn at the current time or in the future. 
4.2 Screening Period 
After signing the informed consent, participants will enter the screening phase. Participant 
eligibility will be determined based on the study inclusion and exclusion criteria and baseline 
assessments.  Participants on DMT will be taken off interferons and glatiramer acetate should 
be discontinued for 90 days prior to inclusion into the study if medically appropriate. The 
screening period could thus last up to 120 days. 
4.3  Procedure and Treatment Period 
No B cell depleting therapy is provided to healthy participants.   
9 4.3.1 “In-and-out” catheter with single time point lymph fluid 
procurement 
Comprised of healthy controls and participants with MS who will undergo a single “in-and-out” 
catheterization of the thoracic duct with procurement of lymphatic fluid and peripheral blood.  
Eligible MS participants will be offered OMB treatment period to start no sooner than 48 hours 
after the ‘in-and-out’ catheter procedure. These participants will receive OMB provided by the 
Research Collaborator, as approved by the FDA, as an initial Autoinjector 20mg sc loading 
dose (20mg sc OMB starting dose, +2 to 14d after treatment initiation) and subsequent 
maintenance phase dosing consisting of 20mg s.c. ofatumumab every 4 weeks. OMB will be 
provided through the study free of charge for up to 2 years. Post-procedure follow-up study 
visits will occur per the visit schedule (Table 3). 
10 4.3.2 “Indwelling” catheter with serial lymph fluid procurement 
Comprised of healthy controls and participants with MS who will undergo thoracic duct 
cannulation and procurement of lymph fluid followed by conversion into an indwelling catheter 
with externalization. Thoracic duct lymph fluid sampling will be repeated through the external 
catheter at multiple time points as described in the visit schedule (Table 2). The external drain 
will be removed approximately 21 days later. Approximately 48 hours after the thoracic duct 
cannulation procedure, OMB will be initiated (as per FDA label, with initial Autoinjector 20mg 
sc loading dose, followed by sc injections a week later and again a week later, and subsequent 
maintenance phase dosing consisting of 20mg s.c. ofatumumab every 28 days or every 4 weeks. 
OMB will be provided free of charge through the study for up to 2 years.  Post-procedure 
follow-up 
visits 
will 
occur 
per 
the 
visit 
schedule 
(Table 
3). 
 
4.3.3.    2-year OMB Follow-up 
MS patients who undergo OMB treatment and 2-year post-procedure follow up will be treated 
with the FDA approved commercial supply medication for the treatment of MS, free of charge, 
through the study provided by the Research Collaborator. These participants will undergo 
clinical research monitoring, clinical research testing (i.e. relapse assessment, EDSS) as well as 
collections of peripheral research blood. This will allow for the study of the prolonged effects 
of OMB on the peripheral immune compartment in MS in the context of 2-year follow-up. 

 
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4.4 Rationale for study design 
The proposed study adds new dimensions into our understanding of immune biology in young 
healthy participants as well the changes seen in participants with MS. As previously published, 
we identified differences in central and peripheral immune biology in people with HIV (Buggert 
et al., 2018) including identification of subsets of cells, which were present centrally, but not 
identifiable in peripheral compartments (blood). 
In order to understand differences in immune biology between healthy individuals and 
participants with MS in the different compartments (e.g, blood and lymphatic fluids), the study 
will establish the safety and feasibility of the procedures (“in-and-out” and “indwelling” 
catheter procedures) in healthy controls and early MS, and generate understanding of the 
immune system profiles. Additionally, to understand the early treatment effects of OMB on 
peripheral blood and thoracic duct compartments. 
The study is exploratory and a limited number of participants will be exposed to the procedure. 
Based on our recent experience with HIV, the number is likely to be sufficient to describe 
differences as outlined in the objective sections. 
For MS patients, effects of B cell depletion over a 2-year period will be compared to thoracic 
duct lymph, peripheral blood, and clinical measures obtained pre- and early post-OMB 
treatment  
4.5 
Allocation to Interventional Group 
This is an open label, single arm, study including healthy controls, and people with MS for in-
and-out’ thoracic duct catheterization with or without treatment with the FDA approved anti-
CD20 MAB OMB or “indwelling” catheter procedures and treatment with OMB.  
Allocation and recruiting will be based on a convenience sample from the Penn MS clinic and 
healthy volunteer recruiting pool. People will be assigned to interventional group based on their 
eligibility of the study and their informed consent. 
Healthy controls are ascertained through described measures and will not receive any 
investigational drug. 
4.6 Risk and Benefits 
The procedure will be performed by highly experienced interventional neuroradiologist, Dr. 
Max Itkin, to minimize the risk of the procedure. There is no direct benefit to the healthy 
participants. The indirect benefit lies in the support of the understanding of immune biology in 
the healthy hence potentially supporting understanding of different diseases better.  
For people with early MS participants have no direct benefit. Indirectly they support our 
scientific understanding of MS and will benefit of being systematically monitored over 2 years 
at regular follow-ups for safety, efficacy, and deep immune profiling of early treatment with 
OMB. 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
25 
 
5 Study Population 
Six healthy volunteers and 18 MS participants will be selected from the outpatient clinic or 
inpatients at Hospital of the University of Pennsylvania meeting all eligibility criteria as 
described below. 
A 25% screen failure rate is expected, hence approximately 30-32 participants will be screened. 
Participants who discontinue within a week of indwelling catheter placement will be replaced 
(possibly requiring more patients to be screened). Participants who discontinue during the 2-
year post-procedure follow up phase will not be replaced. 
 
5.1 Inclusion criteria 
Participants eligible for inclusion in this study must meet all of the following criteria: 
1. Written informed consent must be obtained before any assessment is performed. 
2. Adult participants aged 18 to 55 years (inclusive) at Screening. 
3. Participants must be able to understand English and be able to review and comprehend the 
informed consent form independently or with the help of research staff  
 
For participants with MS following additional criteria need to be met: 
1. Diagnosis of MS according to the 2017 Revised McDonald criteria. 
2. Ability to undergo several MRIs 
3. Disability status at Screening with an EDSS score of 0 to 4 (inclusive) 
4. MS treatment history restricted to Interferons and/glatiramer acetate treatment only or 
untreated to date 
5. Neurologically stable within one month prior to interventional procedure. 
5.2 Exclusion criteria 
Participants meeting any of the following criteria are not eligible for inclusion in this study:  
1. Participants suspected of not being able or willing to cooperate or comply with study 
protocol requirements in the opinion of the investigator. 
2. Participants with primary progressive MS (Polman C et al., 2011) 
3. Participants meeting criteria for neuromyelitis optica (Wingerchuck D et al., 2015) 
4. Participants with disease duration of more than 10 years 
5. Participants who are pregnant or nursing (lactating)  
6. Participants 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 immunodeficiency syndrome 
(hereditary immune deficiency, drug-induced immune deficiency) 
7. Participants with active systemic bacterial, viral or fungal infections, or known to have 
acquired immunodeficiency syndrome (AIDS) 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
26 
 
8. Participants with neurological symptoms consistent with PML or confirmed PML 
9. Participants at risk of developing or having reactivation of syphilis or tuberculosis 
10. History of cardiovascular disease or uncontrolled hypertension 
11. History of diabetes mellitus 
12. History of cancer (other than localized skin cancer) in the previous 5 years 
13. Have received any live or live-attenuated vaccines (including for varicella-zoster virus or 
measles) within 2 months prior to first study drug administration 
14. Immunization with non-live vaccines within 2 weeks of the study procedure 
15. Participants at risk of developing or having reactivation of hepatitis: Positive results at 
Screening for serological markers for hepatitis (H) B and C indicating acute or chronic 
infection:  
a. HBs Ag and/or anti-HBc IgM and/or HB virus deoxyribonucleic acid (DNA) 
b. anti-HBs negative and Anti-HBc positive 
c. anti-HC IgG (if positive IgG, HCV-RNA PCR will be performed and if negative, 
participant can be enrolled) 
16. Use of other investigational drugs at the time of enrollment (Screening) or within the 
prior 30 days, or five elimination half-lives, or until the expected pharmacodynamic effect 
has returned to baseline, whichever is longer 
17. Absolute neutrophil count (ANC) <750/mm3;  
18. Hemoglobin <10.0 g/dL 
19. Platelet count <100,000/mm3 
20. Prothrombin time (PT) or international normalized ration (INR) >1.5 x ULN 
21. Use of systemic glucocorticoids in the 4 weeks prior to research biological sample 
acquisition 
 
5.3 Study Procedures 
All the below described study procedures and the procedure-associated medications are being 
performed for research purposes.  
11 Screening Procedures & Visit(s) 
All participants (including Healthy participants, participants with MS) will undergo screening 
evaluation.  
The screening visit(s) will occur within 120 days of the scheduled date of the study procedure.  
Participants will be screened against inclusion and exclusion criteria of the protocol. 
The screening visit(s) will include: 
• Informed consent review. Participants will review and sign the informed consent 
document before any additional screening is performed.   

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
27 
 
• Medical record review.  The participant’s complete medical history (medical diagnoses, 
surgical procedures, psychiatric treatment history, medications taken within the past 6 
months) will be obtained and reviewed.  
• Vital signs 
• Complete physical examination 
• Laboratory Tests 
• Cranial MRI for people with MS (within 4 weeks of cannulation event)  
• If receiving OMB, additional testing including neurological measurements such as 
extended disability scoring scale (EDSS). 
• If person with MS experiences at attack during screening, cannulation  
All study procedures will be obtained at the convenience of study team and study participants 
and could include single or multiple visits within the Screening window. 
Further details are listed in the Visit Schedule Tables. 
For individuals with MS who develop a relapse during screening, research biological sample 
acquisition (blood and TD) should be deferred until at least 4 weeks after the relapse-onset or 
the use of systemic steroids (whichever is longer)  
Individuals who undergo screening visits but do not complete their scheduled procedure within 
the 120-day window may undergo a second screening evaluation. 
 
12 Visit Day 0: Thoracic Duct Cannulation and lymph fluid collection 
5.3.1.1 Technique for thoracic duct cannulation: “In-and-out” procedure 
 
The procedure will take approximately 2 hours from start to completion 
1. Participants will have been instructed to abstain from eating starting at midnight the night 
before the procedure. 
2. Participants will come to the Interventional Radiology Suite at HUP and have peripheral 
IVs placed. 
3. They will be given moderate conscious sedation with midazolam and fentanyl or other 
appropriate drugs as determined by the patient status.  
4. Prophylactic antibiotics will be administered, consisting of either ampicillin-sulbactam, or 
in the event of a history of penicillin allergy, the combination of levofloxacin and 
clindamycin. 
5. The areas of the groin and abdomen and left arm will be sterilized with chlorhexidine-based 
cleanser.  
6. An intranodal lymphangiogram and thoracic duct cannulation will be performed in 
standard clinical technique. Briefly, a 25- or 26-gauge spinal needle will be advanced into 
an inguinal lymph node under ultrasound guidance and 5-10 mL of oil-based contrast agent 
(Ethiodol; Savage Laboratories, Melville, New York) will be injected into the lymph node 
at a rate of approximately 1 mL per minute under fluoroscopic guidance. The infusion of 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
28 
 
contrast will be stopped when the lymphatics are visualized at approximately the level of 
the L3 lumbar spine.  
7. If the cisterna chyli is not visualized after the injection normal saline will be injected at the 
rate of approximately 1 mL per minute for 5 minutes to facilitate propagation of the contrast 
agent.  
8. After identification of the target lymphatic vessel (the cisterna chyli, thoracic duct, or a 
tributary of the thoracic duct), a 21- or 22-guage 15 to 20 cm Chiba needle will be passed 
into the vessel via a transabdominal approach.  
9. A 0.018 guidewire will be advanced through the needle into the lymph vessel and advanced 
into the thoracic duct.  
10. A microcatheter will then be advanced into the thoracic duct over the wire. The wire will 
be removed, and contrast injected to confirm cannulation of the thoracic duct.  
11. Approximately 100mL of lymphatic fluid will be removed through the catheter and may 
occur at multiple levels within the thoracic duct. 
12. Approximately 100mL of peripheral blood will be removed through peripheral phlebotomy 
 
The thoracic duct catheter will be removed and the participant will then be monitored in the 
Interventional Radiology recovery area until he or she is able to leave. 
5.3.1.2 Technique for thoracic duct cannulation: “indwelling” procedure 
 
All procedures as described above in the “in-and-out” procedure through step 12 will occur 
and include the following additional steps: 
13. In order to externalize the thoracic duct catheter, the guidewire will be advanced though 
the microcatheter into the subclavian vein.  
14. A vascular sheath will then be placed into a vein in upper arm under ultrasound and 
fluoroscopic guidance.  
15. The wire in the subclavian vein will be then snared using a vascular snare through the 
venous access sheath and the wire will be pulled out through venous sheath to establish 
through and through access to the thoracic duct (the wire passing from the abdomen, up the 
thoracic duct and out the vein in the arm).  
16. A 5 Fr or other appropriately size catheter/sheath will then be advanced over the wire until 
its tip is well down the thoracic duct. The catheter/wire from the thoracic duct will then be 
removed, leaving the 5 Fr catheter extending from the arm vein into the thoracic duct. The 
5 Fr sheath will be then secured to the skin using monofilament sutures. 
17. The participant will then be monitored in the Interventional Radiology recovery area until 
he or she is able to leave. 
 
5.4 Ofatumumab treatment 
 
Ofatumumab is FDA approved to treat relapsing remitting MS and is available to MS patients 
commercially outside of study participation.  Participants will be informed that this drug is 
available to them regardless of study participation. Patients may incur some out of pocket 
costs in the form of co-pays, the amounts vary and are determined by insurance coverage 
plans.  

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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13 Handling of study treatment 
The Research Collaborator will provide FDA approved drug for sc OMB delivered as an auto-
injector including appropriate labelling. 
The Penn investigational drug service (IDS)pharmacy will store and dispense OMB. OMB will 
be treated as investigational medicinal product in accordance with all federal regulations by 
Penn IDS (shipment/disposition records, dispensing logs, e.g.). 
OMB must be refrigerated at 2ºC to 8ºC (36ºF to 46ºF). The product will be kept in the original 
carton to protect from light until the time of use. Do not freeze. To avoid foaming, do not shake. 
When dispensed from Penn IDS, the prescription label would indicate the federal caution label 
language of an IMP as this is hard-coded into the pharmacy’s system even though OMB will 
be approved in its use by the Food and Drug Administration (FDA) at the time of its use.  
14 Instruction for prescribing and taking study treatment 
OMB will be dispensed at scheduled visits throughout the 2-year study period. Schedule is 
defined within the visit schedule table within the protocol.  
In order to assess tolerability of the initial dose of study medication, participants will be 
assessed following administration for any reactions including injection related and post dose 
vital signs will be collected. A longer observational period may be required if these vital signs 
are not within reasonable limits of the participant's baseline values. 
Following the first dose, subsequent dose two, and three may be injected under supervision by 
clinical nurse or other HCPs to ensure compliance, appropriate timing of fluid collection and 
proper technique and safety precautions. Subjects will be instructed to not inject OMB on the 
day of a visit, unless it is after the collection of research samples 
If a participant is unable/unwilling to self-administer injections after the third dose, home-
administration may be performed by another individual (e.g. partner, relative or a healthcare 
professional) who has accompanied the participant to the site and has been trained on and 
demonstrated ability to correctly administer the s.c. injections. The participant may also 
continue to have injections at the site if this is the participant's preference.  
Participants should be instructed to record any missed doses and to inform the study staff of 
any missed s.c. injection(s). This can be done at the visits. 
Participants who miss s.c. injections or temporarily interrupt study drug without discontinuing 
from the study or withdrawing consent, will be permitted to resume study drug if determined to 
be safe and appropriate. When resuming study drug, the timing of the next s.c. injection will be 
determined based on the original study schedule as followed: 
• If one monthly injection is missed by 1 week or less, the participant should take injection 
as soon as possible. The next injection should then be administered according to the 
original schedule. 
• If one monthly injection is missed by more than 1 week, the participant should skip the 
dose and take the next dose at the time when the next injection would be due according to 
the original schedule. 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
30 
 
• If two or more consecutive monthly injections are missed, the study will be restarted based 
on clinical judgement. 
A participant injection instruction leaflet will be provided which includes detailed information, 
precautions and instructions for administering s.c. injections using the autoinjectors. This 
information should be reviewed with the participant (and his/her partner/relative as applicable) 
to ensure that they understand the correct procedure for self/home administration. 
Ofatumumab will be provided in auto-injectors for subcutaneous administration containing 20 
mg ofatumumab (50 mg/ml, 0.4 ml content). Ofatumumab (OMB) is clear to opalescent, 
colorless to pale yellow, essentially particle-free liquid.   
OMB will be supplied by the Research Collaborator and dispensed though Penn for up to 2 
years.  
 Ofatumumab is started with a loading dose at 20mg sc at Day 1, Day 7, Day 14, , followed by 
a sc injection every 28 days or every 4 weeks.  
For MS patients undergoing “in-and-out” cannulation: OMB is initiated no sooner than 48 hours 
after the procedure. 
For MS patients undergoing “indwelling” cannulation: the first injection of OMB is given 
approximately 48 hours (day 2) after the “indwelling” catheterization procedure once fluids 
have been extracted from the indwelling catheter; in case of an ongoing AE, OMB application 
could be delayed until AE is resolved based on clinical judgement.   
Afterwards, OMB is self-administered at home as sc injections every 28 days (or 4 weeks). 
Self-administration refers to patient self-treatment of the drug or by a trained caregiver 
regardless of whether dosing occurs at study site or at home. Instruction and training will be 
provided by site staff to patients/caregivers prior to treatment. 
5.4.1.1 Premedication prior to s.c. injection 
Premedication with acetaminophen and/or antihistamines (or equivalent) is optional and may 
be administered at the discretion of the Investigator. If investigators choose to administer 
premedication, it should be administered 30 to 60 minutes prior to study drug injection.  
Application of systemic corticosteroids are at the discretion of the investigator and needs to be 
recorded in the CRF. 
5.4.1.2 Concomitant therapy 
Participant will report any new medications, procedures, and significant non-drug therapies 
he/she takes starting OMB treatment. 
Each concomitant drug must be individually assessed against all exclusion criteria/prohibited 
medication.  
5.4.1.3 Treatment duration 
The planned duration of Ofatumumab treatment is up to 2 years. Participants may be 
discontinued from treatment earlier due to unacceptable adverse events, disease progression 
and/or if treatment is discontinued at the discretion of the investigator or the participant.  

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
31 
 
15 Other treatment(s) 
5.4.1.4 Prohibited medication 
Use of investigational new drugs are NOT allowed in combination with study drug. Use of 
excluded medications is not allowed while the participant is on study medication. 
5.5 Treating the patient 
16 Participant numbering 
Each participant is identified in the study by a unique Participant Number (Participant No.), that 
is assigned when the participant is first enrolled for screening and is retained as the primary 
identifier for the participant throughout his/her entire participation in the trial. Upon signing the 
informed consent form, the participant is assigned to the next sequential Participant No. 
available.  
If an enrolled participant fails to be treated for any reason, the reason will be entered on the 
Screening Participant Status CRF. If the participant is re-screened later on, a new Participant 
No. will be assigned. 
17 Drug Interruptions 
Conditions/events that may lead to study drug interruptions based on Investigator judgment and 
overall clinical assessment include: 
• reported serious adverse event; 
• emergency medical condition, unplanned hospitalization involving use of excluded 
concomitant medications; 
• abnormal laboratory value(s) or abnormal test or examination result(s) 
Should the participant interrupt the study drug for whatever reason, the re-start decision should 
be made on a case-by-case basis. Should the Investigator decide to re-initiate treatment with 
study drug, depending on the duration of the interruption, the first dose at re-start may need to 
take place at the study site to ensure observation in a similar manner as on first dose. 
The reason for interruption of treatment and date of interruption should be appropriately 
documented in the source documents as well as in the appropriate CRF. 
18 Additional treatment guidance 
5.5.1.1 Transition period including washout from previous disease modifying 
therapy (DMT) 
Participants with MS enrolled into the study will be treatment naive (i.e. never treated with 
DMT) or previously treated with interferon-B or glatiramer acetate only, with at least 90 day 
washout prior to procedure. 
19 Treatment compliance 
General instructions are provided to promote compliance e.g. by stating that compliance is 
necessary for the participant’s safety. The participant must also be instructed to contact the 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
32 
 
investigator if he/she is unable for any reason to take the study drug as prescribed. 
Compliance data will be captured in the source document at each visit. All study drug 
dispensed and returned will be recorded in the CRF.  
 
Catheter Procedure and Follow up care: All participants will receive sedating medication and 
antibiotics during the procedure to minimize infections and burden.  
5.6 Patient Monitoring 
20 ‘In-and-out’ catheterization    
5.6.1.1 Procedure Visit  
Thoracic duct cannulation procedure (described above) and procurement of up to 100 mL of 
lymph fluid (at two levels) and withdrawal of the catheter. Blood draw (up to 100 mL) by 
phlebotomy. Participants are monitoring in the IR recovery room until clinically released. 
5.6.1.2 Post-Procedure Contact 
Participants will be contacted on post-procedure Days 1 and 3. This can be done remotely as 
part of video-telemedicine visit if all is well, or in person if any concern at all.   They will 
come to clinic for final site inspection on Day 7 to ensure wound healing and absence of 
infection.  
21 “Indwelling” catheter and serial sampling  
5.6.1.3 Visits 1-3 
For participants undergoing indwelling catheter and serial sampling, thoracic duct cannulation 
procedure (described above) and procurement of up to 100 mL of lymph fluid (at two levels) 
will be done on Day 0 with conversion to an indwelling peripheral line. Blood draw (up to 
100 mL) will be done by phlebotomy. Patients will return on days 1 and 2 for additional 
thoracic duct sampling. After the sampling on day 2, and when relevant, OMB treatment will 
be initiated. 
5.6.1.4 Follow-up Visits 4-7 
Patients will return to clinic on days 4, 9, 16, and 21 after the cannulation procedure and be 
assessed for any possible adverse events. Up to 50 mL of lymph fluid will be drawn from the 
peripheral line connected to the indwelling catheter in the thoracic duct. These visits will last 
approximately 1-2 hours. At the last visit, the interventional radiology team will remove the 
catheter after collecting up to 50mL of lymph fluid. Blood draw (50 mL) by phlebotomy may 
be obtained at each visit. 
5.6.1.5 Follow-up visits 1-7 for participants receiving OMB  
Participants receiving OMB will return to clinic 1, 3, 6, 12, 18, and 24 months after starting 
OMB for visits and clinical testing. Research blood work (up to 50 mL) by peripheral 
phlebotomy may be collected at each visit. Pre- and post-procedure MRIs should not be more 
than 2 months apart. Another research brain MRI will be obtained at 24 months +/- 2 months a 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
33 
 
5.6.1.6 Study completion and discontinuation 
A patient will be considered to have completed the study when the patient has completed the 
last visit planned in the protocol. 
22 Unscheduled Visit 
An unscheduled visit (UNS) is a spontaneous visit and may take place at any time during the 
course of the clinical trial if and when the Investigator deems it necessary. The data collected 
during the UNS assessments must be recorded in the CRF. 
5.7 Withdrawal of informed consent 
Any participant may voluntarily withdraw consent to participate in the study for any reason at 
any time. Withdrawal of consent from the study is defined as when a participant: 
Does not want to participate in the study anymore 
or 
Does not want any further visits or assessments 
or 
Does not want any further study related contacts 
or 
Does not want any analyses performed on the biological material obtained 
If a participant withdraws consent after partial or complete collection of lymph fluid, we will 
ask the participant to provide their withdrawal of consent in writing.  Otherwise the material 
collected will be analyzed. 
5.8 Loss to follow up 
For participants whose status is unclear because they fail to appear for study visits without 
stating an intention to discontinue or withdraw, the investigators will show “due diligence” by 
documenting in the source documents steps taken to contact the participant, e.g. dates of 
telephone calls, emails, text messages, etc.  A patient will be considered as lost to follow-up 
until the time point of his/her scheduled end of study visit has passed. 
5.9 Data Collection and Follow-up for Withdrawn Participants 
All participants who have undergone thoracic duct cannulation will be contacted 1, 3, and 7 
days following their procedure. This will be performed on participants who withdraw their 
consent before or after completion of the procedure. For patients withdrawing early from the 
serial sampling study, they will be contacted by telephone 1, 3, and 7 days following removal 
of the catheter to assess for adverse events.  
23 Early Termination Visits   
Participants who elect not to undergo thoracic duct cannulation after undergoing screening 
evaluations will receive copies of any laboratory test results obtained during screening 
evaluations. 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
34 
 
If a participant asks to stop their participation during the course of the thoracic duct cannulation, 
the procedure will be discontinued and catheters will be removed based upon procedures felt 
by the radiologist performing the procedure to ensure the safety of the participant.  If the 
participant has received sedating medication the participant will be monitored in the post-
procedure area until the clinical staff believe it is safe for the participant to return home. 
5.10 Pharmacokinetic Evaluation  
Sampling for OMB pharmacokinetics will be done in the serial time point thoracic duct 
cannulation procedure and phlebotomy in patients pre- and post- OMB therapy. Details are 
provided elsewhere in the protocol. 
5.11 Safety Evaluation for Thoracic Duct Procedures 
The safety endpoints of the study will be those that represent complications of the study 
procedures, including adverse events associated with the use of medications that are a 
component of the study procedure, sedating agents and antibiotics.   
Complications of the thoracic duct cannulation procedure that will be queried include: failure 
to successfully cannulate the thoracic duct, pain/discomfort at the site of the insertion of 
cannulas (groin and abdomen), swelling, infection, and fevers.  Adverse events associated with 
the use of sedating agents and antibiotics that will be queried include nausea, vomiting, diarrhea, 
dizziness, confusion, rash, and fever. Participants will be asked to provide open ended responses 
to the question of whether they believe they have experienced any adverse events associated 
with study participation.   
Adverse events will be graded as mild, moderate, severe, and life threatening in severity. 
Participants who report adverse events following the procedure will be asked to report the date 
of resolution of their symptoms. 
5.12 Visit schedule and assessments 
Table lists all the assessments and indices with an X when the Visits are performed. Visit 
schedule is listed for healthy, participants undergoing single time procedure, and participants 
undergoing indwelling catheter procedure for ease of reading. 
 
24 Visit schedule and assessment for ‘in-and-out’ cannulation 
 
Table 1 
Assessment Schedule for “in-and-out” cannulation 
Period 
Screening 
Procedure 
Follow up 
Visit Name 
Screening 
Baseline 
Visit 1 
Visit 2 
(telephone 
Visit) 
Visit 3 
(telephone 
visit) 
Visit 4 
Day 
-120 to -8 
-7 to -1 
0 
1 
3 
7 
Informed consent 
X 
  
 
  
 
 
Demographics 
X 
  
 
  
 
 
Medical History 
X 
  
 
  
 
 
Inclusion / Exclusion criteria 
X 
X 
 
  
 
 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
35 
 
Period 
Screening 
Procedure 
Follow up 
Visit Name 
Screening 
Baseline 
Visit 1 
Visit 2 
(telephone 
Visit) 
Visit 3 
(telephone 
visit) 
Visit 4 
Day 
-120 to -8 
-7 to -1 
0 
1 
3 
7 
Review of System 
X 
X 
X 
X 
X 
X 
Vital Signs  
X 
X 
X 
 
 
X 
Physical Exam (include height, 
weight) 
X 
  
 
  
 
X 
MS only and chooses 
ofatumumab treatment: EDSS 
 
X 
 
 
 
 
Pregnancy Test (urine dip or 
serum) 
 
X 
 
 
 
 
Safety labs: CBC, CHEM7, 
LFTs, PT/INR 
X 
 
 
 
 
 
Peripheral phlebotomy for 
research studies 
 
X 
(Screening and/or baseline) 
  
X 
 
 
   
Lymph fluid collection for 
research studies  
 
 
X 
 
 
 
Concomitant medications  
X 
X 
X 
X 
X 
X 
Adverse Events 
X 
X 
X 
X 
X 
X 
MS only: MRI brain  
X (within 4 weeks of 
cannulation event)  
 
 
 
 
MS only - If consented to 
receive OMB through study: 
OMB safety labs (quant IgG, 
QuantiFERON gold, Hepatitis B, 
C testing, CD19 count, and JCV 
titer) 
X 
 
 
 
 
 
25 Visit schedule and assessment for procedure for “indwelling” catheter 
and OMB treatment 
 
 
Table 2  
Assessment schedule for indwelling cannulation 
Period 
Screening 
Procedure and Treatment with OMB 
Visit Name 
Screening 
Baseline 
Visit 1 
(procedure) 
Visit 
2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Day 
-120 to -8 
-7 to -1 
0 
1 
2 
4 
9 
16 
21 
Informed 
consent 
X 
  
 
  
  
  
  
  
 
Demographics 
X 
  
 
  
  
  
  
  
  
Medical 
History 
X 
  
 
  
  
  
  
  
  
Inclusion / 
Exclusion 
criteria 
X 
X 
 
  
  
  
  
  
  
Review of 
System 
 
 
X 
X 
X 
X 
X 
X 
X 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
36 
 
Period 
Screening 
Procedure and Treatment with OMB 
Visit Name 
Screening 
Baseline 
Visit 1 
(procedure) 
Visit 
2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Day 
-120 to -8 
-7 to -1 
0 
1 
2 
4 
9 
16 
21 
Washout 
Period (if 
applicable) 
X 
X 
 
  
  
  
  
  
  
MS only: 
Training for 
OMB injection 
  
  
X 
 
  
  
 
 
  
Drug 
dispensation 
  
  
 
X 
  
  
X 
X 
 
MS Relapse 
assessment 
X 
  
 
 X 
X  
X  
X 
X 
 
MS only: 
EDSS,  
X 
X 
 
  
  
  
  
  
 
MS only: MRI 
brain 
 
X 
(Within 4 
weeks of 
cannulation 
event)  
 
  
  
  
  
  
 
Xa 
 
Vital Signs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical Exam 
(may include 
height and 
weight) 
X 
  
X  
X  
 X 
 X 
X 
X 
X 
Pregnancy 
Test (urine dip 
or serum) 
 
X 
 
 
 
 
 
 
 
Safety labs: 
CBC, CHEM7, 
LFTs, PT/INR 
 
X 
 
 
 
X  
 
 
X 
Lymph fluid 
collection for 
research 
studies 
 
  
X 
X 
X 
X 
X 
X 
X 
Peripheral 
phlebotomy for 
research 
studies 
(possible) 
 X 
X 
X 
 X 
X 
X 
X 
X 
X 
MS only:  OMB 
safety labs 
(quant IgG, 
QuantiFERON 
gold, Hepatitis 
B, C testing, 
cd19 count, 
JCV titer) 
X 
  
  
  
  
  
  
  
  
Concomitant 
medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse 
Events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Treatment with 
OMB 
 
 
 
 
X 
 
X 
X 
 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
37 
 
a. 
Pre- and post-procedure MRIs should not be more than 2 months apart.  
26 Post procedure Visit schedule and Assessments for MS patients who 
receive 2-year OMB treatment with follow-up  
 
Table 3 
Assessment schedule for post-procedure follow-up for MS patients on 
treatment 
Period 
Post procedure Follow-Up for MS patients on treatment 
Visit Name 
Follow-up 
Visit 1 
Follow-up 
Visit 2 
Follow-up 
Visit 3 
Follow-up 
Visit 4 
Follow up 
Visit 5 
Follow up 
visit 6 
End of 
Study 
visit 
Day 
Post 
procedural 
visits + 1 
Month 
±14d 
Post 
procedural 
visit + 3 
Months 
±14d 
Post 
procedural 
visit + 6 
Months 
±14d 
Post 
procedural 
visit + 12 
Months 
±14d 
Post 
procedural 
visit + 18 
Months 
±14d 
Post 
procedural 
visit + 24 
Months 
±14d 
30 
days 
post 
last 
dose* 
MS Relapse 
assessment 
X 
X 
X 
X 
X 
X 
 
EDSS,  
 
 
 
X 
 
X 
 
Research MRI brain 
 
 
 
 
 
Xa 
 
Physical/Neurological 
Exam 
X 
X 
X 
X 
X 
X 
 
OMB Safety labs 
(cbc, chem7, LFT, 
JCV, IgG, CD19) 
 
 
X 
X 
X 
X 
 
Peripheral 
phlebotomy for 
research studies 
(e.g. NfL, GFAP) 
X 
X 
X 
X 
X 
X 
 
Adverse Events & 
Concomitant 
medication 
X 
X 
X 
X 
X 
X 
X 
a The ‘month 24’ MRI can be obtained within a +/- 2 month window 
* Participants who prematurely discontinue study treatment will have their EOS visit as soon as 
possible 
6 Visit schedule and assessment 
Assessment schedule lists all of the assessments and indicates with an "X" when they are 
performed. All data obtained from these assessments must be supported in the participant’s 
source documentation. Participants should be seen for all visits/assessments as outlined in the 
assessment schedule or as close to the designated day/time as possible. Missed or rescheduled 
visits should not lead to automatic discontinuation. In case a visit is performed outside of the 
schedule, subsequent visits shall be performed in keeping with the original visit schedule. In 
addition to the scheduled visits, participants may have unscheduled visits due to an acute illness 
of undetermined cause, for other reasons, or at the discretion of the Investigator. Data collected 
during unscheduled visits will be recorded in the unscheduled visits CRF. 

 
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Participants who prematurely discontinue the study for any reason should be scheduled for the 
end of study (EOS) visit as soon as possible and then enter the Safety Follow-up period 
according to the schedule in below tables. At this final visit, all dispensed investigational 
product should be reconciled, and the adverse event and concomitant medications recorded on 
the CRF.  
6.1 Information collected on screen failures 
All participants who sign informed consent but discontinue prior to Baseline Visit are 
considered screen failures. The Screening Visit Date, Demography, Informed Consent, 
Inclusion/Exclusion Criteria, Participant Rescreening, and the Screening Phase Disposition 
eCRFs must be completed. The Adverse Event eCRF and a paper SAE form must be completed 
for any SAE that occurs during the screening period. Adverse events that are not SAEs will be 
followed by the Investigator and collected only in the source data.  The Withdrawal of Informed 
consent must be documented if consent was withdrawn during the Screening period before the 
participant was enrolled. 
6.2 Rescreening 
If a patient fails on one or more laboratory (or other) assessment criteria, as part of the Screening 
process, the assessment(s) may be repeated at the discretion of the Investigator, and the patient 
may be included if criteria are then met, provided the assessments are completed within the 
Screening or Baseline time window.   
6.3 Magnetic Resonance Imaging (MRI) 
MS patients in the ‘in and out catheter’ sampling will undergo a single brain MRI (+/- Gd) 
within 4 weeks prior to the catheterization, to document presence or absence of subclinical 
disease activity, in order to better interpret immunological results. MS patients undergoing 
‘indwelling catheter’ sampling will undergo two brain MRIs (+/- Gd). The first within 4 weeks 
prior to the catheterization, and the second no more than 2 months following the initial MRI. 
Patients treated with OMB will have an additional MRI at month-24 (+/- 2 months). 
Restrictions to MRI schedule 
To avoid interferences caused by steroids (in regards to Gadolinium (Gd)-enhancing lesions) 
for the treatment of MS relapse, the following restrictions apply for this study: 
• In case of relapse, if an MRI has been scheduled within 14 days of the initiation of steroid 
treatment, MRI (with Gd enhancement) should be performed before steroid treatment is 
initiated. 
• No MRI (with Gd-enhancement) should be performed while a participant is on 
steroid therapy for relapse and within the following 14 days upon termination of steroid 
treatment. 
Because of these restrictions, MRI scheduling can be adjusted accordingly. In case a visit is 
performed outside the visit window, any subsequent visits should be performed according to 
the original visit schedule. 
Scanning 

 
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MRI scan sequences such as but not limited to will include conventional MRI measures of T1 
hypointense images (with and without contrast medium, i.e., gadolinium-DTPA), T2 weighted 
images.   
The gadolinium contrast medium may occasionally cause nausea and vomiting. Allergic 
reactions may also occur very rarely and, in extremely rare instances, can be potentially serious 
and require immediate anti-anaphylactic treatment. Any AE experience due to the contrast 
medium should be recorded on the AE eCRF. 
 
7 Statistical Analysis Plan 
This is an exploratory study. There is no data describing the distribution of populations of 
immune cells in the thoracic duct in MS patients compared to their blood. Descriptive 
statistics will be used as well as nonparametric Mann Whitney U tests, the Wilcoxon matched 
pairs signed rank test or the Friedman test with Dunn’s multiple comparison post-test, as 
appropriate, to compare immune cell subset frequencies and antigen specific responses 
between thoracic duct and blood, between patients and controls, and prior to and following 
aCD20 treatment in each compartment. Correlation analyses will we analyzed using the 
nonparametric Spearman test.  Differentially expressed genes between groups will be ranked 
using Smyth’s variance-moderated t-test. Statistical plan will be discussed further amongst 
collaborators. 
Based on our Penn collaborator’s prior work characterizing TD cells, we anticipate that our 
cohort size will generate a sufficiently powered dataset to successfully address our research 
questions including our primary outcome aiming to document anti-CD20 treatment effect on 
TD immune cell populations. As an example, in Vella et al (JCI, 2019) experiments with as 
few as n=4 and typically n=6-10 samples were sufficient to distinguish features of follicular 
T-helper (Tfh) cells and B cells in the TD from those in the blood in individuals not 
undergoing immune therapy. We expect a robust aCD20 treatment effect that we already 
know will impact the blood cells so we should be well powered to comment on aCD20 
treatment effects on TD cells.   
Power calculations were utilized to determine the effect sizes for secondary outcomes 
including comparison immune cell subset abundance differences between the compartments 
of MS patients and controls, with a goal of 80% power and a Type I error of 0.05. Our 
experimental design of 18 patients with MS and 6 healthy controls (and utilizing a Student’s t-
test), results in a Cohen’s d effect size of 1.38. We next estimated our immune subset 
abundance standard deviation as 1.5% based on our existing immune phenotyping data. This 
results in our experimental design being powered to detect immune cell subset abundance 
differences of at least 1.95%. 
Power calculations were also utilized to estimate effect sizes for gene expression changes 
within immune cell subsets, with a goal of 80% power and a False Discovery Rate of 0.05. 
We utilized a data-driven model specifically generated for power analysis in multiple-sample, 
single-cell transcriptomic studies (Schmid KT, et al. Nat Comm, 2021). With an experimental 
design of 18 patients with MS and 6 healthy controls, our experimental design is powered to 
detect gene expression changes with a magnitude log2-fold-change of at least 2. 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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7.1 Control of Bias and Confounding  
There are several potential biases that may be introduced into the study based upon selection 
of participants. There may be differences in immunologic endpoints based upon participants’ 
age, gender, race, and time from prior interferon/glatiramer acetate use, or use of other 
medication.  Given the small sample size and pilot nature of the study we will not try to 
control or adjust analyses for these potential biases. In addition, given the small sample size 
and pilot nature of the research, we will not adjust our analyses for multiple comparisons.   
7.2 Baseline Data 
Baseline and demographic characteristics will be summarized by standard descriptive 
statistics (including mean and standard deviation for continuous variables such as age and 
standard percentages for categorical variables such as gender). 
7.3 Analysis of Primary Outcome of Interest 
This is an exploratory study. There is no data describing the distribution of populations of 
immune cells in the thoracic duct in MS patients compared to their blood. Descriptive statistics 
will be used as well as nonparametric Mann Whitney U tests, the Wilcoxon matched pairs 
signed rank test or the Friedman test with Dunn’s multiple comparison post-test, as appropriate, 
to compare immune cell subset frequencies and antigen specific responses between thoracic 
duct and blood, between patients and controls, and prior to and following aCD20 treatment in 
each compartment. Correlation analyses will we analyzed using the nonparametric Spearman 
test. Differentially expressed genes between groups will be ranked using Smyth’s variance-
moderated t-test. 
7.4 Pharmacokinetic Analysis 
PK trough values at steady state for ofatumumab will be used to conduct a population 
pharmacokinetics analysis. Sampling time points are provided in the visit schedule. Samples 
for PK assessment should be taken prior to dosing at the Month 1 visit. For any later PK visits, 
if the visit coincides with the day the monthly injection is scheduled, the patient should not take 
the injection in the morning before coming to the site so that the PK sample can be drawn before 
the injection. The PK sample collection date and time must be entered on the appropriate CRF. 
A laboratory manual on sample collection, numbering, processing and shipment will be 
provided by the Research Collaborator. 
8 Safety 
Safety assessments are specified with the assessment schedule (Tables 1-3).   
Safety assessments will include:  
• Physical examination (including skin) 
• Vital signs 
• Height and Weight 
• Laboratory evaluations 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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• Adverse events 
Any new or worsening clinically relevant findings from such additional assessments meeting 
definition of an adverse event (AE) or serious AE should be recorded as AE/SAE. For details 
on AE collection and reporting, refer to Section 10.1. 
8.1 Physical Examination (including skin) 
A complete physical examination will be performed at the visits indicated and will include an 
assessment of skin, head and neck, lymph nodes, heart, lungs, abdomen, back, neurological 
function and comments on general appearance. A complete neurological examination will be 
part of the initial physical examination at Screening. 
Information for all physical examinations (including skin examination) must be included in the 
source documentation at the study site. Clinically relevant findings that are present prior to 
signing informed consent must be recorded on the appropriate eCRF that captures medical 
history. Significant findings made after signing informed consent, which meet the definition of 
an Adverse Event must be recorded on the adverse events eCRF. 
8.2 Vital Signs 
Vital signs will include sitting pulse rate (measured as radial pulse for 60 seconds), sitting 
systolic and diastolic blood pressure and body temperature (oral, or per local practice and should 
be recorded in the relevant CRF page in Celsius) which will be assessed at the visits indicated. 
After the participant has been sitting for five minutes, with back supported and both feet placed 
on the floor, systolic and diastolic blood pressure will be measured three times using an 
automated validated device, e.g. OMRON, with an appropriately sized cuff. The repeat sitting 
measurements will be made at 1 - 2 minute intervals and the mean of the three measurements 
will be used. In case the cuff sizes available are not large enough for the participant's arm 
circumference, a sphygmomanometer with an appropriately sized cuff may be used. 
For drug treatment, vital signs should be obtained 30-60 minutes before the s.c. injection and 
again approximately 60 minutes post for the Day 2 administration. If premedication is 
administered, the vital signs should be taken prior to premedication administration.  
8.3 Height and Weight 
Height in centimeters (cm) and body weight (to the nearest 0.1 kilogram (kg) in indoor clothing, 
but without shoes) will be measured. 
8.4 Laboratory evaluations 
A local laboratory will be used for analysis of all specimens collected. Abnormal laboratory 
parameters, inconsistent with the clinical presentation of MS or which cause suspicion of an 
underlying medical condition, should be repeated for confirmation. 
All abnormal lab results must be evaluated for criteria defining an adverse event and reported 
as such if the criteria are met. For those lab adverse events, repeated evaluations are mandatory 
until normalization of the result(s) or until the result is no longer considered clinically 
significant. 

 
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Table 3 
Laboratory Assessments 
Test Category 
Test Name 
Hematology 
Blood samples will be collected according to the 
schedule the respective visit schedule. 
 Hematocrit, Hemoglobin, Platelets, Red blood 
cell (RBC) count, Total WBC count and WBC 
differential counts (Basophils, Eosinophils, 
Lymphocytes, Monocytes, Neutrophils). 
Chemistry 
Blood samples will be collected according to the 
schedule as above. 
Electrolytes (Sodium, potassium, chlorine, 
bicarbonate, calcium, magnesium, phosphorus), 
random glucose, total protein, blood urea 
nitrogen, albumin, alkaline phosphatase, ALT, 
AST, y-GT, total bilirubin, conjugated bilirubin, 
creatinine, amylase, total cholesterol, 
triglycerides, high density lipoprotein and low 
density lipoprotein, C-Reactive protein. 
B-cell/T-cell sampling 
CD19+ B-cell counts and CD3+CD20+ T-cell 
counts. Samples will be collected according to 
the schedule in visit above. 
Total IgG  
Samples will be collected according to the 
schedule in visit above. 
Biomarker sampling 
Refer to visit above. 
Additional tests 
Testing of lab samples will be conducted at 
screening to determine the participant’s eligibility 
for inclusion in the study with respect to hepatitis 
viruses. Testing for syphilis and tuberculosis at 
Screening is also needed. 
A positive result for any of the following 
serological markers for hepatitis B and C as 
below is an exclusion criterion: 
• 
HBs Ag and/or anti-HBc IgM and/or HB virus 
deoxyribonucleic acid (DNA) 
• 
anti-HBs negative and anti-HBc positive 
• 
anti-HC IgG (if positive IgG, HCV RNA PCR 
will be performed and if negative, participant 
can be enrolled) 
 
Pregnancy Test 
Serum or Urine pregnancy test 
8.5 Pregnancy and assessments of fertility 
Urine pregnancy tests (UPT) will be conducted for all female subjects of childbearing potential 
at Screening visit within 1 week of thoracic duct cannulation. If UPT cannot be collected for 
any reason, a serum pregnancy test will be collected instead. 
In addition, the Investigator will review the contraception status with the participant at each 
visit to ascertain that the participant continues to comply with protocol requirements for highly 
effective contraception as shown in the below contraception guidance. 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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CONTRACEPTION GUIDANCE: 
CONTRACEPTIVES ALLOWED DURING THE STUDY INCLUDE: 
 
Highly Effective Methodsa That Have Low User Dependency Failure rate of <1% per year when used 
consistently and correctly. 
• Implantable progestogen-only hormone contraception associated with inhibition of ovulationb 
- Intrauterine device (IUD) 
- Intrauterine hormone-releasing system (IUS)c 
• Bilateral tubal occlusion 
 
Vasectomized partner 
(Vasectomized partner is a highly effective contraceptive method provided that the partner is the sole sexual 
partner of the female subject of childbearing potential and the absence of sperm has been confirmed. If not, an 
additional highly effective method of contraception should be used. Spermatogenesis cycle is approximately 90 
days.) 
 
Highly Effective Methodsa That Are User Dependent 
Failure rate of <1% per year when used consistently and correctly. 
• Combined (estrogen- and progestogen-containing) hormonal contraception associated with inhibition of 
ovulationc 
- oral 
- intravaginal 
- transdermal 
- injectable 
• Progestogen-only hormone contraception associated with inhibition of ovulationc 
- oral 
- injectable 
 
Sexual abstinence 
Sexual abstinence is considered a highly effective method only if defined as refraining from heterosexual 
intercourse during the entire period of risk associated with the study intervention. The reliability of sexual 
abstinence needs to be evaluated in relation to the duration of the study and the preferred and usual lifestyle of 
the participant.) 
 
ACCEPTABLE METHODSc 
1. Progestogen-only oral hormonal contraception where inhibition of ovulation is not the primary mode of action 
2. Male or female condom with or without spermicidee 
3. Cervical cap, diaphragm, or sponge with spermicide 
4. A combination of male condom with either cervical cap, diaphragm, or sponge with spermicide (double-
barrier methods) 
 
a  Failure rate of <1% per year when used consistently and correctly. Typical use failure rates differ from those when used 
consistently and correctly. 
b If locally required, in accordance with Clinical Trial Facilitation Group (CTFG) guidelines, acceptable contraceptive 
methods are limited to those which inhibit ovulation as the primary mode of action. 
c Considered effective, but not highly effective - failure rate of ≥1% per year. Periodic abstinence (calendar, symptothermal, 
post-ovulation methods), withdrawal (coitus interruptus), spermicides only, and lactational amenorrhea method (LAM) are 
not acceptable methods of contraception. 
8.6 Appropriateness of safety measurements 
The safety assessments included in this study are standard for the MS indication and study 
participant population and appropriate for people undergoing thoracic duct cannulation.  

 
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8.7 Additional assessments 
27 Biomarkers 
Blood samples will be drawn for analysis of exploratory biomarkers according to the schedule 
above. These specimens will be used to identify and/or verify potential markers that may be 
predictive of disease activity, disease course and/or clinical response to treatment. Analysis of 
these specimens will include NfL and GFAP. Samples for biomarker assessment should be 
taken prior to dosing at Visit 3. For any later biomarker visits, if the visit coincides with the s.c. 
injections, the participant should not take the injection in the morning before coming to the site 
so that the biomarker sample can be drawn prior to administration. The biomarker sample 
collection date and time must be entered on the appropriate eCRF. 
The details describing the collection, handling, storage and shipment requirements of samples 
will be shared amongst the collaborators and the laboratory manual, whatever is applicable. 
28 Immune cell analyses 
Detailed analyses in terms of handling of specimens, sharing of specimens between the labs, 
the work performed through the Bar-Or Lab, the Prak Lab, and the the Research Collaborator 
lab of Traggiai  (e.g. B cell functional and autoantibody testing) will be discussed amongst the 
collaborators. This will also include how specimens and the generated data are shared. 
 
9 Study discontinuation and completion 
9.1 Discontinuation 
29 Discontinuation of study treatment 
Discontinuation of study drug for a participant occurs when study drug is stopped earlier than 
scheduled in the protocol, and can be initiated by either the participant or the investigator. 
The investigator must discontinue study treatment for a given participant if he/she believes that 
continuation would negatively affect the participant's well-being. 
Study drug must be discontinued under the following circumstances: 
• Participant/guardian decision  
• Pregnancy  
• Use of prohibited treatment  
• Diagnosis of PML 
• Participants with active serious infections or reactivation (e.g. tuberculosis, hepatitis B or 
C) 
• Skin and/or mucosal reactions which raise the suspicion of severe generalized major skin 
reactions (Stevens-Johnson syndrome, or toxis epidermal necrolysis-Lyell's syndrome) 
• Hypersensitivity to the study medication 
• Any situation in which study participation might result in a safety risk to the participant 
• Protocol violation that results in a significant risk to the participant's safety 

 
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• Emergence of certain adverse events, such as malignancy (except successfully treated 
basal cell carcinoma, in situ squamous cell carcinoma and in situ carcinoma of cervix or 
uterus), liver failure or serious chronic infection (such as human immunodeficiency virus 
(HIV)) 
• Laboratory abnormalities (e.g. liver functions tests (LFTs)) and abnormal test procedure 
defined in Appendix 1  
• Severe hypoproteinemia 
• Interstitial lung disease or new onset or worsening of pulmonary symptoms, such as 
persistent cough and dyspnea, with or without associated fever, suspicious of interstitial 
lung disease 
• Non-compliance with OMB or study procedures 
• No benefit of drug treatment 
If discontinuation of OMB occurs, the investigator should make a reasonable effort to 
understand the primary reason for the participant’s premature discontinuation of study drug and 
record this information. 
Participants who discontinue study drug or who decide they do not wish to participate in the 
study further should NOT be considered withdrawn from the study UNLESS they withdraw 
their consent (see Section 6.4.3). 
If they fail to return for these assessments for unknown reasons, every effort (e.g. telephone, e-
mail, letter) should be made to contact the participant/pre-designated contact as specified 
in Section 6.4.4. This contact should preferably be done according to the study visit schedule. 
If the participant cannot or is unwilling to attend any visit(s), the site staff should maintain 
regular contact with the participant, or with a person pre-designated by the participant. This 
contact should preferably be done according to the study visit schedule for up to 9 months post 
last OMB administration. 
After OMB discontinuation, at a minimum, in abbreviated visits, the following data should be 
collected at clinic visits or via telephone/email contact: 
• New / concomitant treatments 
• Adverse Events / Serious Adverse Events 
The investigator must record the participant’s discontinuation from OMB. 
9.2 Study completion and post-study treatment 
Study completion is defined as when the last participant finishes their Study Completion visit 
and any repeat assessments associated with this visit have been documented and followed-up 
appropriately by the Investigator or, in the event of an early study termination decision, the date 
of that decision. 
A participant is considered to have completed the study when they fulfill the following criteria: 
• Participant has completed the study in its entirety according to the approved duration of 
the study  
• The participant has not stopped the study due to one or more of the following reasons:  
• Discontinuation of study treatment (Section 10.1) 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
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• Withdrawal of informed consent (Section 6.4.3) 
• Lost to follow-up (Section 6.4.4) 
End of Study (EOS) visit is mandatory for all participants. For participants that complete the 
study, the next (and last) scheduled visit is the EOS visit and should align with the overall visit 
schedule. Participants who prematurely discontinue study treatment will have their EOS visit 
as soon as possible. The Investigator provides follow-up medical care for all participants who 
are prematurely withdrawn from the study, or refers them for appropriate ongoing care. 
10 Safety monitoring and reporting 
10.1 Definition of adverse events and reporting requirements 
30 Adverse events 
An adverse event (AE) is any untoward medical occurrence (e.g. any unfavorable and 
unintended sign [including abnormal laboratory findings], symptom or disease) in a participant 
or clinical investigation participant after providing written informed consent for participation 
in the study.  
An adverse event (AE) is any symptom, sign, illness or experience that develops or worsens in 
severity during the course of the study. Intercurrent illnesses or injuries should be regarded as 
adverse events.  Abnormal results of diagnostic procedures are considered to be adverse events 
if the abnormality: 
• 
results in study withdrawal 
• 
is associated with a serious adverse event 
• 
is associated with clinical signs or symptoms 
• 
leads to additional treatment or to further diagnostic tests 
• 
is considered by the investigator to be of clinical significance 
Therefore, an AE may or may not be temporally or causally associated with the use of a 
medicinal (investigational) product. 
Adverse events may be detected when they are volunteered by the participant during or between 
visits or through physical examination findings, laboratory test findings, or other assessments. 
Adverse events must be recorded under the signs, symptoms, or diagnosis associated with them, 
and graded using the Common Toxicity Criteria (CTC) AE grade (1-4) (if the event is serious 
refer to Section 11.1.2). 
All adverse events must be treated appropriately. Treatment may include one or more of the 
following: 
• No action taken (e.g. further observation only) 
• Drug interrupted/withdrawn 
• Concomitant medication or non-drug therapy given 
• Participant hospitalization/participant's hospitalization prolonged 

 
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• Its outcome (not recovered/not resolved; recovered/resolved; recovered/resolved 
with sequelae; fatal; or unknown). 
Conditions that were already present at the time of informed consent should be recorded in 
medical history of the participant. 
Adverse events (including lab abnormalities that constitute AEs) should be described using a 
diagnosis whenever possible, rather than individual underlying signs and symptoms. 
Adverse event monitoring should be continued for at least 30 days following the last dose of 
study treatment.  
Once an adverse event is detected, it must be followed until its resolution or until it is judged to 
be permanent (e.g. continuing at the end of the study), and assessment must be made at each 
visit (or more frequently, if necessary) of any changes in severity, the suspected relationship to 
the interventions required to treat it, and the outcome. Serious adverse events that are still 
ongoing at the end of the study period will be followed up to determine the final outcome.  Any 
serious adverse event that occurs after the study period and is considered to be possibly related 
to the study intervention or study participation will be recorded and reported. 
Research staff will elicit and record all adverse events on case report forms. Information on all 
adverse events will be recorded immediately in the CRF and an AE form.  All clearly related 
signs and symptoms will be grouped under one diagnosis.  
Information about adverse drug reactions for OMB can be found in the FDA prescribing 
guidelines. For information about risks and common side effects related to the AEP 
(cholestyramine or activated charcoal), please refer to the local product label. This information 
will be included in the participant informed consent and should be discussed with the participant 
during the study as needed. Any new information regarding the safety profile of ofatumumab 
that is identified will be communicated as appropriate. New information might require an update 
to the informed consent and has then to be discussed with the participant. 
The Investigator must also instruct each participant to report any new adverse event (beyond 
the protocol observation period) that the participant, or the participant's personal physician, 
believes might reasonably be related to study treatment. This information must be recorded in 
the Investigator’s source documents; however, if the AE meets the criteria of an SAE, it must 
be reported to the Research Collaborator. 
31 Serious adverse events 
Adverse events are classified as serious or non-serious.  A serious adverse event is any AE that 
is:  
• 
fatal 
• 
life-threatening 
• 
requires a hospital admission or prolongation of existing hospitalisation  
• 
results in persistent or significant disability or incapacity 
• 
constitutes a congenital anomaly/birth defect 
• 
requires an intervention to prevent permanent impairment or damage 

 
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• 
an important medical event 
All adverse events that do not meet any of the criteria for serious will be regarded as non-serious 
adverse events.  
Serious adverse events that are still ongoing at the end of the study period will be followed up 
to determine the final outcome.  Any serious adverse event that occurs after the study period 
and is considered to be possibly related to the study intervention or study participation will be 
recorded and reported. 
Medical and scientific judgment should be exercised in deciding whether other situations should 
be considered serious reactions, such as important medical events that might not be immediately 
life threatening or result in death or hospitalization but might jeopardize the participant or might 
require intervention to prevent one of the other outcomes listed above. Such events should be 
considered as “medically significant.” Examples of such events are intensive treatment in an 
emergency room or at home for allergic bronchospasm, blood dyscrasias, or convulsions that 
do not result in hospitalization or development of dependency or abuse (please refer to the ICH-
E2D Guidelines). 
All malignant neoplasms will be assessed as serious under “medically significant” if other 
seriousness criteria are not met. 
Any suspected transmission via a medicinal product of an infectious agent is also considered a 
serious adverse reaction. 
All reports of intentional misuse and abuse of the product are also considered serious adverse 
event irrespective if a clinical event has occurred, and are to be reported as appropriate 
according to Section 10.1.3.. The Research Collaborator qualified medical personnel will be 
readily available to advise on trial related medical questions or problems. 
32 SAE reporting 
The Sponsor will be responsible for ensuring that all Adverse Events and Serious Adverse 
Events, including Suspected Unexpected Serious Adverse Reactions (SUSARs) and other 
relevant safety information are recorded and appropriately reported to the relevant health 
authorities, ethics committees and Study investigators according to Applicable Laws in the 
country where the Study is conducted.  
To ensure participant safety, every SAE, regardless of causality, shall be reported to the 
Research Collaborator as outlined in the Collaborative Research Agreement. Information about 
all SAEs is collected and recorded on Penn’s Serious Adverse Event Report Form. Each re-
occurrence, complication, or progression of the original event must be reported as a follow-up 
to that event regardless of when it occurs. The follow-up information should describe whether 
the event has resolved or continues, if and how it was treated, and whether the patient continued 
or withdrew from study participation. 
Any SAEs experienced after the 30 day period after the final study visit or after the end of 
Safety Follow-up should only be reported to the Research Collaborator if the investigator 
suspects a causal relationship to study treatment. 

 
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33 Reporting MS relapse as a SAE 
MS relapses are exempt from SAE reporting although they may meet the SAE definition on the 
basis that they are considered medically significant and are frequently associated with 
hospitalization. These events will therefore be reported on the MS relapse CRF instead of the 
SAE form. However, if, in the judgment of the Investigator, a MS relapse is unusually severe 
or medically unexpected and warrants specific notification, then an SAE form must be 
completed and submitted according to SAE reporting procedures outlined above. 
34 Pregnancy reporting 
To ensure participant safety, each pregnancy occurring after signing the informed consent and 
after receiving drug treatment must be recorded. Any reports of reports of drug exposure during 
pregnancy should be considered an SAE and be reported to Novartis as soon as it becomes 
available, but in any event within eight (8) calendar days of becoming aware of such 
information. For instances of drug exposure during pregnancy, the pregnancy should be 
followed up until after the Expected Delivery Date (EDD) to determine outcome, including 
spontaneous or voluntary termination, details of the birth, and the presence or absence of any 
birth defects, congenital abnormalities, or maternal and/or newborn complications.  
35 Reporting of study treatment errors including misuse/abuse 
Medication errors are unintentional errors in the prescribing, dispensing, administration or 
monitoring of a medicine while under the control of a healthcare professional, participant or 
consumer (European Medicines Agency (EMA) definition). 
Misuse refers to situations where the medicinal product is intentionally and inappropriately used 
not in accordance with the protocol. 
Abuse corresponds to the persistent or sporadic, intentional excessive use of a medicinal 
product, which is accompanied by harmful physical or psychological effects. 
Study treatment errors and uses outside of what is foreseen in the protocol will be recorded on 
the appropriate CRF irrespective of whether or not associated with an AE/SAE and reported to 
Safety only if associated with an SAE.  
Table 4 
Guidance for capturing the study treatment errors including  
                        misuse/abuse 
Treatment error type 
Document in Dosing 
CRF (Yes/No) 
Document in AE 
eCRF 
Complete SAE form 
Unintentional study 
treatment error 
Yes 
Only if associated with 
an AE 
Only if associated with 
an SAE 
Misuse/Abuse 
Yes 
Yes 
Only if associated with 
an SAE 
For more information on AE and SAE definition and reporting requirements, please see the 
respective sections. 

 
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11 Data Collection and Database management 
11.1 Data collection 
Designated trained investigator staff will enter the data required by the protocol into the Penn 
Electronic Case Report Forms (eCRF) and database. The Investigator/designee is responsible 
for assuring that the data recorded on CRFs is complete, accurate, and that entry and updates 
are performed in a timely manner. The Investigator must certify that the data entered are 
complete and accurate. 
All other data captured for this study will have either the CRF as source or an external 
originating source (either written or electronic) with the eCRF not being considered as source.  
All data will be recorded, handled, and stored in a way that allows its accurate reporting, 
interpretation, and verification. 
11.2 Database management and quality control 
Clinical data, imaging data, and laboratory outcomes will be kept in separate databases.   
All clinical data relevant to the study outcomes will be maintained in PennChart. Any paper 
forms will be maintained in locked filing cabinets in the study PI’s or research coordinator’s 
office. The Division of Interventional Radiology will keep records of participants who undergo 
thoracic duct cannulation procedures to monitor safety outcomes of the procedures.  
 
Laboratory outcomes of the study will be maintained in databases maintained by individual 
laboratories that will be supplied with clinical material for analysis labeled with coded 
participant identification numbers. 
 
Image data acquired during research MRI studies are automatically deidentified of Private 
Health Information (PHI) and transferred to a secure cloud-based informatics platform. 
11.3 Sample management and storage 
Biological samples will be processed locally at Penn, under the biorepository of the CNET 
(director Bar-Or) will include sample allocation and packaging to ship to the Research 
Collaborator and/or other entities to run specialized tests as outlined in the protocol. Sample 
aliquots not immediately planned for testing will be stored in appropriate conditions in the 
laboratory of Dr. Amit Bar-Or. The Research Collaborator may request additional sample 
aliquots following discussions with the primary Penn research team. At the end of the 
collaborative research agreement, all unused aliquots will remain under the control of the 
primary Penn research team. 
Consent will be obtained from the study participants to transfer samples and data outside of the 
USA to allow specialized analyses to be performed by the Research Collaborator and/or other 
entities.  
 
12 Data analysis and statistical methods 
Any data analysis carried out independently by the Investigator should be shared with the 
Research Collaborator according to the terms of the Collaborative Research Agreement (CRA). 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
51 
 
Likewise, the data generated by the Research Collaborator and/or other entities should be shared 
with the Penn research team according to the terms of the CRA. 
12.1 Analysis sets 
The Full Analysis Set (FAS) comprises all participants in the study whether or not drug 
treatment was administered.  
12.2 Participant demographics and other baseline characteristics 
Demographics, MS disease history, MRI baseline characteristics and MS medication history 
will be summarized. 
12.3 Treatments 
Exposure to investigational study medication is defined as the number of days spent on study 
treatment divided by 365 days. Intermediate treatment interruptions will be subtracted from 
drug exposure. 
Exposure to investigational study medication will be summarized with number and percentage 
of participants by time category, and with summary statistics of the number of participant years 
of exposure. 
12.4 Analysis of the primary endpoint(s) 
The primary objective of the study is exploratory. The study is designed to evaluate the safety 
and feasibility of collection of lymphoid fluid via thoracic duct cannulation in healthy people 
and people with MS. 
36 Safety endpoints 
Patients will be assessed for the following: 
1. Safety profile of “in-and-out” thoracic duct cannulation in participants (healthy controls and 
MS) and single time-point sampling of lymphatic fluids and peripheral blood 
2. Safety profile of “indwelling” thoracic duct cannulation in participants (healthy controls and 
MS) and serial sampling of lymphatic fluids and peripheral blood  
A summary of AEs and SAEs will be provided to the Research Collaborator in final study report 
as outlined in the Collaborative Research Agreement. 
12.5 Analysis of secondary endpoints 
The secondary objective of the study is also exploratory. The study will assess the impact on 
anti-CD20 ofatumumab treatment in patients with MS on biological measures in the thoracic 
duct fluid and in the blood. 
37 12.5.1 Definition of secondary endpoint(s) 
Secondary endpoints are to compare immune cell profiles of blood and lymphatics between 
MS and healthy controls and in untreated MS patients before and following a B-cell depleting 
therapy (Ofatumumab), including: 

 
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52 
 
• To evaluate the phenotype, function, and transcriptional profiles of immune cell sub-
populations (including T cell, B cell and innate cell subsets) 
• To identify CNS-antigen specific responses of immune cells and profile B cell 
repertoires 
• To quantify soluble measures of inflammation and CNS injury in lymph fluid 
compared to serum 
• To investigate the humoral autoantibody repertoire at serological and cellular level 
38  
39 12.5.2. Statistical model, hypothesis, and method of analysis 
Descriptive statistics will be used as well as nonparametric Mann Whitney U tests, the 
Wilcoxon matched pairs signed rank test or the Friedman test with Dunn’s multiple comparison 
post-test, as appropriate, to compare immune cell subset frequencies and antigen specific 
responses between thoracic duct and blood, between patients and controls, and prior to and 
following aCD20 treatment in each compartment. Correlation analyses will we analyzed using 
the nonparametric Spearman test.  Differentially expressed genes between groups will be ranked 
using Smyth’s variance-moderated t-test. Statistical plan will be discussed further amongst 
collaborators. 
12.6 Analysis of exploratory endpoints 
The following exploratory endpoints will be evaluated during the 24-month follow-up period 
of patients that have been treated with OMB, in serum and compared to earlier results in 
serum and lymph fluid. Samples collected at Penn will be shipped to the Research 
Collaborator, or other collaborating laboratories for testing. 
• 
Neurofilament light (NfL; SIMOA platform)  
• 
Glial fibrillary acidic protein (GFAP; SIMOA platform) 
• 
anti-NfL antibodies (ELISA) 
• 
Somamers (>5000 aptamer panel; SOMALOGIC) 
• 
Olink 21-panel MS (OCTAVE) 
In parallel, the clonal diversity in the B-cell receptor (BCR) reportoire of the residual B-cells 
(under treatment with OMB) in samples from the thoracic duct and peripheral blood shall be 
evaluated (collaboration with NVS, Basel). 
 
Table 6: Exploratory biomarkers to be tested  in participants receiving OMB 
No 
Measures 
Frequency 
(Months) 
Matrix 
Specifications 
1 
NfL 
1M, 3M, 6M, 
12M, 18M, 
24M 
Thoracic 
duct fluid 
&  
blood 
SIMOA (Quanterix) 
2 
GFAP 
SIMOA (Quanterix) 
3 
anti-NfL Abs 
  
4 
SOMASCAN 
SOMALOGIC (5000-
aptamer panel) 
5 
O-LINK 
OCTAVE (21-panel) 
6 
BCR clonal diversity 
1M, 6M, 12M, 
24M 
NVS, Basel 

 
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13 Ethical Considerations and Risks 
All protocol amendments will be submitted for review and approval by the University of 
Pennsylvania IRB prior to the start of any study procedures or changes in study procedures.  
Informed consent documents will be updated with any new safety information that might 
influence a participant’s willingness to enroll in the study.  An annual report will be submitted 
to the University of Pennsylvania IRB for continued renewal of the study.   
 
13.1 Risks 
Risks associated with participation in the study include those associated with the physical 
performance of the thoracic duct cannulation procedure, those associated with medications 
used during the performance of the thoracic duct cannulation procedure, those associated with 
phlebotomy, those associated with MRI (MS subjects only) and those associated with loss of 
confidentiality. 
 
40 Risks of thoracic duct cannulation procedure 
The members of the Interventional Radiology Division who perform this procedure keep 
careful records of adverse events associated with the procedure.  The most common reported 
event is mild abdominal discomfort where the abdomen is punctured, and the cannula is 
inserted that may persist for a day or two post-procedure or minor infection at the site of 
injection of the lymph node in the groin or at the site of insertion of the catheter in the 
abdomen.  The abdominal pain reported in association with the procedure often does not 
require treatment.  Pain requiring treatment is typically controlled by acetaminophen or 
ibuprofen. The interventional radiologists participating in this study have performed over 250 
thoracic duct cannulation procedures for the purposes of thoracic duct embolization.  All 
participants have been monitored and 10 serious adverse events have been reported.  Each of 
these events was associated with the embolization component of the procedure which will not 
be performed as part of this study. A list of these events is included in the table below. 
Because the procedure involves passing a catheter through the abdomen, it is theoretically 
possible that a participant could experience intraabdominal bleeding or infection.  An 
intravenous line will be maintained during the procedure, with possible adverse events of 
bruising, bleeding, infection or a blood clot at the insertion site of the intravenous line.   
41 Additional risks associated with indwelling catheter.  
Indwelling catheterization with external drainage of the thoracic duct has been successfully 
accomplished in the treatment of congenital and acquired lymphatic diseases in the inpatient 
setting. Indwelling vascular lines are commonly used in the treatment of infection. The risks 
for this procedure include infection, clotting of the line preventing sampling, bleeding, injury 
to the lymphatic drainage system and potentially other unknown risks. 
Overall, the potential risks of the proposed indwelling catheter study can be divided into three 
categories, including those associated with (i) the procedure itself including the cannulation of 
the thoracic duct and the ‘externalization’ (ie establishing indwelling antecubital access to 

 
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thoracic duct); (ii) maintaining the indwelling catheter for a period of 2-3 weeks; and (iii) any 
added risk of carrying out the procedure and maintaining the indwelling catheter in the face of 
anti-CD20 treatment with ofatumumab.    
 
(i) 
The procedure: The risks associated with the cannulation component of the 
procedure are well established based on the substantial combined clinical and 
research experience with ‘in and out’ thoracic duct catheterization at Penn, and 
described in our previously approved research protocol (Penn IRB: 831994). As 
noted by our study’s interventional radiology (IR) collaborator, Dr. Itkin, the 
additional step of externalization has not itself been viewed as adding significant 
risk and was previously approved as a ‘modification’ to the HIV research protocol 
(attached Penn IRB 823909; PIs Drs. Ian Frank and Max Itkin), to enable more 
efficient collection of thoracic duct fluid in the event that initial cannulation resulted 
in limited flow of lymph. Though externalization was approved in that protocol, the 
added procedure was never required. 
(ii) 
Potential risks associated with maintaining the thoracic duct indwelling catheter: A 
published meta-analysis of studies of external drainage of the thoracic duct reported 
on a total of 71 studies involving a total of 1160 patients, where this approach was 
used in solid organ transplantation, acute pancreatitis, neoplastic disease, sepsis, and 
autoimmune disease, for periods of up to and over 4 weeks (Meta analysis: Wang 
et. al Journal of Surgical Research. Vol 204. 2016). The most common complication 
was wound infection at the site of cannulation which occurred in 5-10% of 
participants.  Other rare complications were accumulation of lymph in the neck, 
back pain, and edema of the face or arm. There was no reported mortality related to 
thoracic duct drainage. We were surprised to find that the meta-analysis included 
one small study in patients with MS (Ring et al. Lancet, 1974) where thoracic duct 
drainage was assessed as a potential therapeutic intervention (the study reported on 
immunological and neurological outcomes but did not report on complications).  
It is important to note that many of the prior studies took place between the 1960s -
1980s with techniques of thoracic duct cannulation and drainage that involved 
surgical incision of the neck and more invasive techniques to access the duct. The 
modernized IR techniques have become more refined with lower complication rates 
than past surgical approaches. Important differences are that the majority of the prior 
studies described in the meta-analysis employed larger catheters as well as 
continuous drainage, unlike our proposed approach which uses considerably finer 
catheters and only intermittent sampling of smaller lymph volumes. To date, our IR 
team at Penn has performed indwelling thoracic duct cannulation with drainage in 8 
patients including 3 infants with anasarca and 5 adolescent patients with 
chylothorax. The duration of indwelling catheter and drainage averaged 9.9 days 
(with a range of 2 to 43 days). There were no intra-procedure complications, all 8 
patients experienced symptomatic improvement and no line-infections developed 
this quite sick patient population. Our IR collaborators have a Penn IRB approved 
research protocol to perform thoracic duct cannulation with externalization and 
drainage in the context of sepsis (protocol :834599), though they have not yet 
initiated this trial.    

 
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Given our proposed use of the smaller bore catheters and intermittent sampling of 
much smaller volumes of lymph fluid (50mL per sample versus the liters that are 
drained in the clinical setting), we anticipate any added risks of indwelling catheter 
in the generally healthy MS patients who do not have abnormal thoracic duct 
anatomy will be minimal, particularly over a period of several weeks. According to 
our IR team, the closest approximation of risk associated with the indwelling 
thoracic duct catheter in this generally healthy population is the risk associated with 
a peripherally inserted central catheter (PICC) line.  PICC line cannulation of 3 
weeks or longer is routinely done in the appropriate clinical setting. We will use a 
similar 4-5 Fr line to access the thoracic duct through left arm vein. The greatest 
potential complication is infection. The risk of PICC line associated infections in the 
heme/onc population such as bone marrow transplantation patients who are much 
more profoundly immune compromised is low. In a study of oncology patients, 165 
BMT patients had PICC lines for an average of 88.3 days and an infection rate of 
0.96 infections per thousand line-days (Moturu, A et al. Infection rates for PICC 
lines in oncology patients. Journal of Vascular and Interventional Radiology, 2017). 
We anticipate an even lower risk of infection given the shorter duration of 
indwelling catheter and the healthier MS population. 
(iii) 
Potential for added risk of carrying out the procedure, including maintaining the 
indwelling catheter, in the face of anti-CD20 treatment with ofatumumab:   Anti-
CD20 therapies have been used as front-line agents to treat B cell lymphomas for 
over 2 decades and in growing numbers of patients with a range of autoimmune 
conditions including MS. We found no studies that directly assess risks of 
catheterization or of indwelling catheters such as PICC lines in anti-CD20 treated 
patients. Several studies have attempted to isolate the added risk conferred by anti-
CD20 therapy in patients undergoing surgical interventions and have reported 
relatively minor or no obvious added risks even to fairly involved surgery. For 
example, a study assessing the safety of surgery in 133 patients with rheumatoid 
arthritis on anti-CD20 (S. Godot et al. ACR, 2013) found that complications 
including infections did not differ in relation to anti-CD20 treatment including time 
from last infusion and duration on treatment. This may reflect the relatively limited 
degree of immune compromise conferred by anti-CD20 treatment.  The role of B 
cells in host protection is largely attributed to their potential to become antibody 
secreting plasma cells and since plasma cells no longer express CD20, the anti-CD20 
agents do not directly deplete plasma cells. It is well established that pre-existing 
protective antibodies are generally not impacted by anti-CD20 monotherapy for at 
least several years of ongoing therapy. Moreover, while the ability to mount novel 
antibody responses is clearly impaired in patients who are B cell depleted, their CD4 
and CD8 T cell responses remain robust (Bar-Or et al Neurology 2020; Apsotolidis 
et al Nat Med 2021).  Among anti-CD20 treated patients, those with MS are 
relatively unique in that they tend to be generally healthier, receive lower doses (than 
cancer patients) and invariably are on anti-CD20 as a monotherapy (unlike both 
cancer and autoimmune conditions like rheumatoid arthritis where patients are 
almost always on two or more therapies that impact the immune system).  In 
particular, the approved MS dosing regimen of ofatumumab used in our study (30 
mg sc monthly), is lower than the other approved anti-CD20, ocrelizumab (600 mg 

 
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IV every 6 months) and did not appear to increase infection rates in the pivotal phase 
III ASCLEPIOS I and ASCLEPIOS MS trials (Hauser et al, NEJM, 2020).  These 
studies randomized over 1600 total MS patients 1:1 between ofatumumab and 
teriflunomide (itself not associated with more infections than placebo) and revealed 
no differences over two years of continuous treatment between ofatumumab and 
teriflunomide for either ‘any infections’ (approximately 50% for both) or ‘serious 
infections’ (approximately 2.3% for both). We do note that post-marketing studies 
of patients with MS on the anti-CD20 ocrelizumab (which has been approved for 
longer than ofatumumab) have recently shown that after several years of continuous 
treatment, patients may start dropping levels of circulating IgG which, in a small 
proportion of patients, was associated with slight increased risk of serious infections. 
Hence, while direct evidence is still lacking, and while continuous B cell depletion 
over a more extended timeframe will likely result in functional immune suppression 
in at least some patients, the observations described above are reassuring and would 
together suggest that the first few weeks of ofatumumab therapy would confer little 
to no added risk to the proposed procedure of thoracic duct cannulation, 
externalization, and maintenance as an indwelling catheter for a limited time period. 
 
42 Risks associated with conscious sedation.   
Conscious sedation will be achieved with intravenous midazolam and fentanyl, the standard 
drugs used in the clinical procedure.   
43 Risks associated with fentanyl when given intravenously 
Nausea (26.1%), vomiting (18.6%),  muscle rigidity (10.4%), hypotension (8.8%), 
hypertension (8.8%),  bradycardia (6.1%), sedation (5.3%), tachycardia (4.0%), dizziness 
(3.7%), apnea (3.5%), dyskinesia (3.2%), confusion (1.9%), visual disturbance (1.9%), 
arrhythmia (2.9%), vein pain (2.9%), bronchospasm (1.3%), laryngospasm (1.3%), rash 
(1.3%).  
44 Risks associated with midazolam when given as a single agent for 
sedation in adults:  
Hiccoughs (3.9%), nausea (2.8%), vomiting (2.6%), coughing (1.3%), over sedation (1.6%), 
headache (1.5%) and drowsiness (1.2%).  Other possible events occurring at a frequency of 
<1.0% include dyspnea, tachypnea, bradycardia, tachycardia, amnesia, anxiety, insomnia, 
ataxia, and double vision.   
45 Risks associated with antibiotics.   
The antibiotics that will be used for prophylaxis against infection will be 
ampicillin/sulbactam, and for penicillin allergic patients the combination of levofloxacin and 
clindamycin.  Single doses of antibiotics will be given, and therefore the risks are low.   
 
Risks associated with ampicillin-sulbactam include diarrhea (3%), rash (2%), and in <1% of 
recipients: itching, nausea, vomiting, fatigue, headache, abdominal distention, swelling, chills, 

 
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57 
 
tightness of the throat, elevated liver function tests, elevated renal function tests, anemia, low 
platelet counts, and low white blood cell counts. 
 
Risks associated with levofloxacin (to be administered only to participants with penicillin 
allergies) include trouble sleeping (4%), headache (6%), dizziness (3%), nausea (7%), 
diarrhea (5%), constipation (3%), abdominal pain (2%), vomiting (2%), rash (2%), swelling 
(1%), and in rare cases rupture of the tendon. 
 
Risks associated with clindamycin (to be administered only to participants with penicillin 
allergies) include: diarrhea, Clostridia difficile colitis, itching, skin rash, abnormal liver 
function tests, worsening renal functions, low white blood cell counts, low platelet counts (all 
less than 2%). 
46 Risk of phlebotomy.   
The most common risks associated with phlebotomy are bruising, bleeding, pain, and 
infection. 
47 Risk of radiation.   
Radiation is associated with an increased risk of cancer.  The dose of radiation being 
administered in this study is not believed to result in a clinically significant increased risk of 
cancer. 
48 Risks of MRI.  
The known risks associated with MRI are minimal. Implanted medical devices and metallic 
foreign fragments inside the subject's body may pose a risk if s/he were to enter the MRI 
magnet room. Therefore, questions regarding medical and work history will be asked prior to 
the subject's exam. The greatest risk is a magnetic object flying through the air toward the 
magnet and hitting the subject. To reduce this risk, we require that all people involved with 
the study remove all magnetic metal from their clothing and all magnetic metal objects from 
their pockets. No magnetic metal objects are allowed to be brought into the magnet room at 
any time except by approved personnel. In addition, once the subject is in the magnet, the 
door to the room will be closed so that no one inadvertently walks into the room. There is no 
known health risk associated with exposure to magnetic fields during an MRI. There are 
minimal risks from the loud noise associated with the MRI scanner and from the discomfort 
of lying on a hard surface. We shall provide the subject with protective earplugs as necessary 
and make every attempt to ensure his/her comfort with blankets, etc. during his/her time in the 
scanner.  
 
Gadolinium Based Contrast Agents - Retention: Traces of gadolinium may remain in the 
body long-term after contrast administration. This risk increases with the number of 
administrations, but reviews to date have not identified adverse health effects from 
gadolinium retained in the brain or bodily tissues after MRI. 
 

 
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58 
 
Gadolinium Based Contrast Agents - IV Line Placement: Multiple needle-sticks may be 
necessary if a vein cannot be properly accessed and this will be carried out with the patient’s 
permission. 
 
IV Contrast Risks: There is a rare possibility that patients could have an adverse reaction to 
the contrast agent such as rash, hives, itching, mild headache and nausea. They may also 
experience some minor discomfort and low risk of bleeding, infection and bruising associated 
with Intravenous catheter placement. 
 
Pregnancy: Gadolinium-based IV contrast agents are not approved in pregnant people and 
therefore those who are pregnant will be excluded from this trial. 
 
Some of the pulse sequences associated with this MRI are not FDA approved but are 
considered to pose no more than minimal risk. Although there are no known risks related to 
MRI on pregnant people or a fetus, there is a possibility of yet undiscovered pregnancy related 
risks. Since there is no possible benefit from participating in this protocol for a pregnant 
patient, we will exclude pregnant subjects from this study. 
 
The proposed MRIs are not clinical scans. It is possible that during the course of the research 
study, the research staff may notice an unexpected finding(s). Should this occur, the finding(s) 
will be considered by the appropriate personnel and the PI will inform the subject if 
necessary. These possible finding(s) may or may not be significant and may lead to the 
subject experiencing anxiety about his/her condition and they may lead to further work-up by 
his/her physician. 
 
The subject might find parts of the testing difficult or tiring. If the subject finds that s/he is 
becoming tired or discouraged s/he can request a break and s/he may decline to participate in 
any aspect of the testing. 
 
49 Loss of confidentiality.  
Identities of participants will be known by research and clinical personnel performing 
participant recruitment and performance of the study procedures.  Specimens obtained will be 
labeled with a participant number so that laboratory personnel handling the collected material 
will not know the identity of study participants. Clinical data abstracted from participants’ 
medical records will be identified only with a participant number.  The list linking the 
participant number and participant’s identification will be kept in the research office of Dr. 
Bar-Or in a locked cabinet with access limited to the Clinical Coordinator and Principal 
Investigator (Dr. Bar-Or). A copy of this list linking participant’s identification number and 
name will also be maintained in the research office of the Interventional Radiology Division 
so that safety data related to the study procedure can be effectively tracked.  
 
Minimization of risks. Risks of study participation will be minimized as much as possible.  
The thoracic duct cannulation procedure will be performed only by interventional radiologists 
and support staff experienced in the performance of this procedure.  The procedure will be 
halted if there is any indication that the participant is experiencing an adverse event prior to 

 
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59 
 
the completion of the procedure.  Participants will be contacted by telephone on the days 
following the procedure to whether adverse events have been encountered, and any adverse 
events can be quickly evaluated and treated.   
 
Additional precautions in place for the indwelling group due to unknown risks of this 
procedure in the ambulatory setting. As described in the study protocol, subjects will be 
assessed at multiple time points during the study to evaluate for any adverse events. Each 
subject in the indwelling group will be assessed sequentially to identify any potential 
unknown risks and the IRB and study will be updated accordingly. 
 
Alternatives to participation.  This study is solely for research purposes.  An individual may 
choose to not participate.  A decision to not participate will have no influence on the care that 
individual receives. 
50 Risks associated with Ofatumumab: 
Ofatumumab (Kesimpta) has been approved by the FDA for patients with relapsing remitting 
MS and is commonly used in the Penn Neurology clinic.  Ofatumumab has the potential for 
an increased risk of infections, including serious bacterial, fungal, and new or reactivated viral 
infections; some of these infections have been fatal in patients treated with other anti-CD20 
antibodies. In the phase 3 clinical trials the overall rate of infections and serious infections in 
patients treated with Ofatumumab was similar to patients who were treated with teriflunomide 
(51.6% vs 52.7%, and 2.5% vs 1.8%, respectively). The most common infections reported by 
Ofatumumab -treated patients in the randomized clinical relapsing MS (RMS) trials included 
upper respiratory tract infection (39%) and urinary tract infection (10%).  
Hepatitis B Virus  
Reactivation  
There were no reports of HBV reactivation in patients with MS treated with Ofatumumab. 
However, HBV reactivation, in some cases resulting in fulminant hepatitis, hepatic failure, 
and death, has occurred in patients being treated with ofatumumab for chronic lymphocytic 
leukemia (CLL) (at higher intravenous doses than the recommended dose in MS but for a 
shorter duration of treatment) and in patients treated with other anti-CD20 antibodies.  
Infection  
Ofatumumab is contraindicated in patients with active hepatitis B disease. Fatal infections 
caused by HBV in patients who have not been previously infected have occurred in patients 
being treated with ofatumumab for CLL (at higher intravenous doses than the recommended 
dose in MS but for a shorter duration of treatment). HBV screening should be performed in all 
patients before initiation of treatment with Ofatumumab. At a minimum, screening should 
include Hepatitis B surface antigen (HBsAg) and Hepatitis B Core Antibody (HBcAb) testing. 
These can be complemented with other appropriate markers as per local guidelines. For 
patients who are negative for HBsAg and positive for HB core antibody [HBcAb+] or are 

 
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carriers of HBV [HBsAg+], consult liver disease experts before starting and during treatment 
with Ofatumumab. These patients should be monitored and managed following local medical 
standards to prevent HBV infection or reactivation.  
Progressive Multifocal Leukoencephalopathy  
Progressive multifocal leukoencephalopathy (PML) is an opportunistic viral infection of the 
brain caused by the JC virus (JCV) that typically occurs in patients who are 
immunocompromised, and that usually leads to death or severe disability.  
Although no cases of PML have been reported for Ofatumumab in the RMS clinical studies, 
PML resulting in death has occurred in patients being treated with ofatumumab for CLL (at 
substantially higher intravenous doses than the recommended dose in MS but for a shorter 
duration of treatment). In addition, JCV infection resulting in PML has also been observed in 
patients treated with other anti-CD20 antibodies and other MS therapies. At the first sign or 
symptom suggestive of PML, withhold Ofatumumab and perform an appropriate diagnostic 
evaluation. Magnetic resonance imaging (MRI) findings may be apparent before clinical signs 
or symptoms. Typical symptoms associated with PML are diverse, progress over days to 
weeks, and include progressive weakness on one side of the body or clumsiness of limbs, 
disturbance of vision, and changes in thinking, memory, and orientation leading to confusion 
and personality changes.  
Vaccinations: Administer all immunizations according to immunization guidelines at least 4 
weeks prior to initiation of Ofatumumab for live or live-attenuated vaccines, and whenever 
possible, at least 2 weeks prior to initiation of Ofatumumab for inactivated vaccines.  
Ofatumumab may interfere with the effectiveness of inactivated vaccines.  
The safety of immunization with live or live-attenuated vaccines following Ofatumumab 
therapy has not been studied. Vaccination with live or live-attenuated vaccines is not 
recommended during treatment and after discontinuation until B- cell repletion. 
Injection-Related Reactions: In large phase 3 clinical trials, systemic and local injection 
reactions were reported in 21% and 11% of patients treated with Ofatumumab compared to 
15% and 6% of patients treated with teriflunomide who received matching placebo injections, 
respectively.  
Injection-related reactions with systemic symptoms observed in clinical studies occurred most 
commonly within 24 hours of the first injection but were also observed with later injections. 
Symptoms observed included fever, headache, myalgia, chills, and fatigue, and were 
predominantly (99.8%) mild to moderate in severity. There were no life-threatening injection 
reactions in the RMS clinical studies.  
Local injection-site reaction symptoms observed in clinical studies included erythema, 
swelling, itching, and pain.  

 
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Only limited benefit of premedication with corticosteroids, antihistamines, or acetaminophen 
was observed in RMS clinical studies. The first injection of Ofatumumab should be performed 
under the guidance of an appropriately trained healthcare professional. If injection-related 
reactions occur, symptomatic treatment is recommended.  
Reduction in Immunoglobulins: As expected with any B-cell depleting therapy, decreased 
immunoglobulin levels were observed. Decrease in immunoglobulin M (IgM) was reported in 
7.7% of patients treated with Ofatumumab compared to 3.1% of patients treated with 
teriflunomide in RMS clinical trials. Treatment was discontinued because of decreased 
immunoglobulins in 3.4% of patients treated with Ofatumumab and in 0.8% of patients 
treated with teriflunomide. No decline in immunoglobulin G (IgG) was observed at the end of 
the study. Monitor the levels of quantitative serum immunoglobulins during treatment, 
especially in patients with opportunistic or recurrent infections, and after discontinuation of 
therapy until B-cell repletion. Consider discontinuing Ofatumumab therapy if a patient with 
low immunoglobulins develops a serious opportunistic infection or recurrent infections, or if 
prolonged hypogammaglobulinemia requires treatment with intravenous immunoglobulins.  
Fetal Risk: Based on animal data, Ofatumumab can cause fetal harm due to B-cell 
lymphopenia and reduce antibody response in offspring exposed to Ofatumumab in utero. 
Transient peripheral B-cell depletion and lymphocytopenia have been reported in infants born 
to mothers exposed to other anti-CD20 B-cell depleting antibodies during pregnancy. Advise 
females of reproductive potential to use effective contraception while receiving Ofatumumab 
and for at least 6 months after the last dose  
 
13.2 Benefits 
This study endeavors to answer several important questions that may influence our 
understanding of immune mediated diseases including MS, its pathogenesis in such a manner 
as to influence how these diseases are treated, whether they can be cured, and whether a 
preventative vaccine or other preventative strategies can be developed. In this context, the 
research proposed may offer benefits to the community of people living with and at risk for 
MS. 
 
13.3 Risk Benefit Assessment 
Participation in this study involves risks that are more than minimal.  However, the research 
staff has considerable experience with the conduct of the interventional procedures involved 
in this study for clinically indicated purposes, and the number of reported adverse events has 
been few and generally mild in severity. There are theoretical risks of participation that are 
potentially severe or life threatening, but these have not been encountered in clinical practice.  
There will no benefits that any individual participant will receive as a consequence of their 
participation.  Despite advances in the treatment of MS, morbidity and mortality associated 
with the disease are considerable, and individuals almost always require continuous treatment 
for the remainder of their lives. This study will provide access to a unique body compartment 
that has never been evaluated in patients with MS that likely harbors a greater proportion of 
autoreactive cells than blood. In addition, the immune response directed against self-antigens 

 
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is likely to be more concentrated in this compartment than blood. Therefore, the general 
benefit related to an increase in our understanding of MS pathogenesis, with potential 
implications for new research findings that may be important in developing MS curative or 
preventative strategies outweighs the risks to individual participants. 
14 Informed Consent Process / HIPAA Authorization  
 
All subjects in this study will be provided a consent form describing this study and providing 
sufficient information for potential subjects to make an informed decision about their 
participation in this study.  The consent form will be submitted with the protocol for review 
and approval by the IRB for the study.  The formal consent of a subject, using the IRB-
approved consent form, will be obtained before that subject undergoes any study procedure.  
The subject alone must sign the consent form, which will also be signed by the investigator-
designated research professional obtaining the consent.  No surrogates will be able to provide 
informed consent on behalf of the potential subject. Subjects will be consented by the study 
Principal Investigator, or appropriate designee, in a room located within a clinic providing 
care to patients.  Healthy controls will be consented in research space used by the MS Clinical 
Research Team. Potential subjects will review the consent form in detail with the person 
designated to consent (either PI or a designee) and will have the ability to take the consent 
home for further review.  The consent form will combine consent for trial participation with 
HIPAA Authorization in a combined document.   
 
15 Study Finances 
 
15.1 Funding Source 
This study will be funded by Novartis through a research collaboration. 
51 Conflict of Interest 
All University of Pennsylvania Investigators will follow the University of Pennsylvania 
Policy on Conflicts of Interest Related to Research.  
52 Subject Stipends or Payments 
All subjects will be compensated by Greenphire ClinCard. 
 
All subjects: 
Screening visit: Transportation and parking costs (up to $50) 
 
Thoracic duct cannulation procedure: ‘In-and-out’ catheterization (single time-point 
sampling): $500 in patient stipends for completion of the study, plus additional 
transportation and parking costs (up to $50). Patient stipends include: 
• $50 after completion of the screening visit 
• $100 after completion of the baseline visit 
• $300 after the completion of the procedure 
• $50 after the completion of visit 4 

 
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Thoracic duct cannulation procedure: “Indwelling” catheter with serial sampling: $3000 in 
patient stipends for completion of serial sampling study, plus additional parking/transportation 
costs (up to $50). Patient stipends include: 
• $50 after completion of the screening visit 
• $150 after completion of the baseline visit 
• $1500 after the completion of the procedure 
• $250 each for visits 3-6  
• $300 after the completion of visit 7  
 
 
16 Publication Plan 
Results of the study will be published as a collaboration of University of Pennsylvania 
investigators and non-Penn investigators who participate in the analysis of specimens.  The 
investigators will decide who will serve as the senior investigator for individual publications 
based upon the types of analyses that are the subject of publications.  Criteria for authorship 
will follow the guidelines of the International Committee of Medical Journal Editors.    
 
17 Ethical considerations and administrative procedures 
17.1 Regulatory and ethical compliance 
This clinical study was designed and shall be implemented, executed and reported in accordance 
with the protocol, with the International Conference on Harmonisation (ICH) Harmonized 
Tripartite Guidelines for Good Clinical Practice, with applicable local regulations (including 
European Directive 2001/20/EC, US CFR 21), and with the ethical principles laid down in the 
Declaration of Helsinki. 
17.2 Responsibilities of the investigator and IRB/IEC 
Before initiating a trial, the investigator/institution must obtain approval/favorable opinion from 
the Institutional Review Board/Independent Ethics Committee (IRB/IEC) for the trial protocol, 
written informed consent form, consent form updates, participant recruitment procedures (e.g. 
advertisements) and any other written information to be provided to participants.  
 
18 Protocol adherence 
This protocol defines the study objectives, the study procedures and the data to be collected on 
study participants. Additional assessments required to ensure safety of participants should be 
administered as deemed necessary on a case by case basis. Under no circumstances including 
incidental collection is an investigator allowed to collect additional data or conduct any 
additional procedures for any purpose involving any investigational drugs under the protocol, 
other than the purpose of the study. If despite this interdiction prohibition, data, information, 

 
Protocol version 2.0 Date 18 Aug 2022 
 
 
 
 
 
 
64 
 
observation would be incidentally collected, the investigator shall immediately disclose it to the 
Sponsor and not use it for any purpose other than the study, except for the appropriate 
monitoring on study participants. 
Investigators ascertain they will apply due diligence to avoid protocol deviations. If an 
investigator feels a protocol deviation would improve the conduct of the study this must be 
considered a protocol amendment, and unless such an amendment is agreed upon by the 
Sponsor and approved by the IRB/IEC, where required, it cannot be implemented. 
 
19 Thoracic Duct Complication Table 
 
Table of Adverse Events Complicating the Thoracic Duct Cannulation Procedure at 
HUP  
Event 1 
Sex: F 
Age: 63 
Year of service:  2015 
History: Chylothorax and chylous ascites after surgery for gynecologic malignancy. 
Complication:  Embolus of lipiodol/glue to left upper lobe. No symptoms.  Hospitalized. 
Event 2 
Sex: M 
Age: not documented 
Year of service:  2009 
History:  Right chylous pleural effusion after esophagectomy. 
Complication:  Fraction of Trufill glue traveled into left SCV and into lungs causing glue 
pulmonary embolism. Patient asymptomatic. 
Event 3 
Sex: not documented 
Age: 58: 
Year of service:  2009 
History:  S/p heart transplant with chyle leak and bilateral chylous pleural effusions. 
Complication:  Patient removed sutures on feet with leakage of edema fluid. 
Event 4 
Sex: M 
Age: 51 
Year of service:  2009 
History: Recurrent chylothorax 
Complication:  Dislodgement of embolization coil into retroperitoneum.  Patient 
asymptomatic. 
Event 5 
Sex: M 
Age: not documented 
Year of service:  2009 
History:  S/p esophagectomy with right chylous pleural effusion 
Complication:  Decline in patient’s oxygen saturation and blood pressure at the end of the 
procedure. Patient sent to emergency department. 
Event 6 
Sex: F 
Age: not documented 
Year of service:  2009 
History:  Recurrent chylothorax with failed embolization procedure on two occasions. 
Complication:  Malpositioned embolization coil in retroperitoneum. 

 
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Event 7 
Sex: M 
Age: not documented 
Year of service:  2012 
History:  Bilateral chylothorax s/p CABG. 
Complication:  Pneumothorax s/p right thoracentesis. 
Event 8 
Sex: F 
Age: not documented 
Year of service:  2015 
History:  Bilateral chylothorax. 
Complication: Glue embolism into retroperitoneum and pulmonary circulation. Patient 
asymptomatic. 
Event 9 
Sex: F 
Age: not documented 
Year of service:  2013 
History:  Adenocarcinoma of lung, s/p resection with thoracic duct injury and chyle leak. 
Complications:  Sheared tip of cannula in retroperitoneum after embolism coil failed to 
migrate.  Patient asymptomatic. 
Event 10 
Sex: M 
Age: not documented 
Year of service:  2005 
History:  S/p gunshot wound to chest with chylothorax. 
Complication:  Embolism of glue to left lower lobe.  Patient asymptomatic. 
 
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