Clinical trial NCT02469896
Conditions: ALS Amyotrophic Lateral Sclerosis Lou Gehrig's Disease Motor Neuron Disease
Keywords: ALS tocilizumab biomarker
Sponsor: Barrow Neurological Institute
Start date: 2015-11-01; Completion date: 2018-07-11
Enrollment: 22 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This research study is being done to find out if tocilizumab, also known as Actemra™, can help with Amyotrophic Lateral Sclerosis (ALS). The investigators also want to find out if tocilizumab is safe to take without causing too many side effects.

Currently ALS has no cure and 2 modestly effective treatment to slow the progression of the disease. Although not the initial cause of ALS, the immune system plays a role in the death of motor neurons. The immune cells that participate in this process are stimulated by a substance called interleukin-6 (IL-6) whose effect is blocked by tocilizumab and thus, may slow the death of motor neurons and slow the disease.

Detailed description:
This is a multicenter, randomized, double-blind, placebo-controlled 16-week study evaluating the safety and tolerability of tocilizumab in subjects with ALS.

The primary objective of the study is to determine the safety and tolerability of intravenous administration of 8 mg/kg of tocilizumab every 4 weeks vs. matched intravenous placebo administered every 4 weeks over an 8 week period.

The secondary objectives of the study are to describe the expression of pro-inflammatory genes in Peripheral Blood Mononuclear Cells (PBMCs) of sporadic ALS patients, to assess the ability of tocilizumab to reduce the expression of pro-inflammatory genes in PBMCs and pro-inflammatory cytokines in the cerebrospinal fluid (CSF) of patients with sporadic ALS and to assess the CSF penetration of tocilizumab. Mean peripheral benzodiazepine receptor 28 (PBR28) uptake will be measured in the motor cortices as regions of interest (ROIs), and will be compared between pre- and post-dose, for Massachusetts General Hospital (MGH) subjects.

Approximately 5 Northeast ALS Consortium (NEALS) Centers in the US will participate in the study. Twenty-four subjects will be randomized in the study.

This study will be conducted in subjects who meet the El Escorial criteria of possible, laboratory-supported probable, probable, or definite criteria for a diagnosis of ALS. At screening, eligible subjects must be at least 18 years old, must have a slow vital capacity (SVC) ≥ 40% of predicted capacity for age, height and gender (and in the opinion of the investigator is able to comply with and complete the trial), and must provide written informed consent prior to screening. Subjects on a stable dose of riluzole and those not taking riluzole, and women of child-bearing age at screening are eligible for inclusion as long as they meet specific protocol requirements. Detailed criteria are described in the body of the protocol.

Subjects participating in the magnetic resonance imaging - positron emission tomography (MRI-PET) portion of the study (MGH only) must meet the following additional criteria.High or mixed affinity to bind translocator protein (TSPO) (Ala/Ala or Ala/Thr,) Upper Motor Neuron Burden (UMNB) Scale Score ≥25 (out of 45) at the Screening Visit.

and have the ability to safely undergo MRI-PET scans based on the opinion of the site investigator.

Subjects will be randomly assigned in a 2:1 ratio to intravenous tocilizumab 8 mg/kg or matching placebo every 4 weeks over an 8 week period.

This research study protocol allows the subject to receive up to 3 infusions of Tocilizumab. Even if the treatment is shown to be of benefit, additional infusions of Tocilizumab beyond that allowed in the protocol cannot be given to the subject while she/he is participating in this study.

Subjects will remain on randomized, placebo-controlled, double-blind treatment until the Week 8 visit. Each randomized subject will also have a Week 12 Follow-up visit and Week 16 End-of-Study visit to assess for adverse events (AEs), changes in concomitant medications, to administer the ALS Functional Rating Scale (ALSFRS-R) and selected study procedures.

Arms: arm 1: 8 subjects will receive matching IV placebo every 4 weeks for 3 months. arm 2: 14 subjects will receive 8mg/kg of IV tocilizumab every 4 weeks for 3 months.
Interventions: intervention 1: Tocilizumab intervention 2: Placebo
Intervention descriptions: intervention 1: IV Infusion intervention 2: IV Infusion

Protocol documents:
Prot_SAP_000.pdf: 
Cover Page – Protocol 
 
 
 
 
 
Study Official Title: A Phase 2 Randomized, Placebo-
Controlled Trial of Tocilizumab in Amyotrophic Lateral 
Sclerosis (ALS) 
 
 
NCT #: NCT02469896 
 
Version Date of the Document: May 30, 2017 
 


A Phase 2 Randomized, Placebo-Controlled Trial of Tocilizumab in Amyotrophic Lateral 
Sclerosis (ALS)
Regulatory Sponsor:
Barrow Neurological Institute
Funding Sponsor:
ALS Association (ALSA)
Neurological Clinical Research Institute (NCRI)
Genentech, Inc.
Barrow Neurological Foundation (BNF)
MAP ALS Partnership (ALSA, ALS ONE, ALS Finding 
a Cure) 
Study Product:
Tocilizumab (Actemra™)
Protocol Number:
TCZALS-001
IND Number:
121,214
Original Version/Date:
1.0 / 02 December 2013
Amended Version Number/Date:
8.0 / 30 May 2017
The information contained herein is confidential and proprietary in nature, and will not be
disclosed to any third party without written approval of authorized designee.
This document may be disclosed to the appropriate institutional review boards or to duly
authorized representatives of the US Food and Drug Administration or a national
regulatory authority under the condition that they maintain confidentiality.


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STATEMENT OF COMPLIANCE
This study will be conducted in compliance with the protocol, International Conference on 
Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use 
(ICH), Good Clinical Practice (GCP), and the applicable regulatory requirements, United States 
Code of Federal Regulations (CFR) Title 45 CFR Part 46 and Title 21 CFR Parts 50, 56, and 
312.


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SIGNATURE PAGE
I have read the attached protocol entitled, “A Phase 2 Randomized, Placebo-Controlled Trial 
of Tocilizumab in Amyotrophic Lateral Sclerosis (ALS)” dated 30 May 2017 (Version 8.0)
and agree to abide by all described protocol procedures.  I agree to comply with the International 
Conference on Harmonisation Tripartite Guideline on Good Clinical Practice, applicable FDA 
regulations and guidelines identified in 21 CFR Parts 11, 50, 54, and 312, local Institutional 
Review Board (IRB) guidelines and policies, and the Health Insurance Portability and 
Accountability Act (HIPAA).
Site Investigator: _______________________________________________________________
Signed:
Date:


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TABLE OF CONTENTS
STATEMENT OF COMPLIANCE ····························································································2 
SIGNATURE PAGE ··············································································································3 
LIST OF ABBREVIATIONS····································································································7 
PROTOCOL SUMMARY ·······································································································9 
1 
ETHICS/PROTECTION OF HUMAN SUBJECTS ································································· 15 
1.1 
INSTITUTIONAL REVIEW BOARD (IRB) ··············································································································15 
1.2 
ETHICAL CONDUCT OF STUDY ·························································································································15 
1.3 
SUBJECT INFORMATION AND CONSENT ··············································································································15 
2 
INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE················ 16 
2.1 
BACKGROUND INFORMATION ··························································································································16 
2.2 
RATIONALE ················································································································································ 16 
2.3 
PRELIMINARY DATA·······································································································································17 
2.3.3 Tocilizumab ··················································································································· 22 
2.4 
POTENTIAL RISKS AND BENEFITS ·······················································································································22 
2.4.1 
Potential Risks ············································································································ 22 
3 
OBJECTIVES ··············································································································· 26 
3.1 
STUDY OBJECTIVES ······································································································································26 
3.2.1 
Primary Outcome Measures ··························································································· 26 
3.2.2 
Secondary Outcome Measures························································································· 27 
4 
STUDY DESIGN············································································································ 29 
4.1 
OVERALL STUDY DESIGN AND PLAN··················································································································29 
4.2 
STUDY CENTERS ·········································································································································· 29 
4.3 
STUDY DURATION········································································································································· 29 
4.4 
PROTOCOL ADHERENCE ································································································································29 
5 
STUDY ENROLLMENT AND WITHDRAWAL···································································· 30 
5.1 
NUMBER OF STUDY SUBJECTS··························································································································30 
5.2 
INCLUSION AND EXCLUSION CRITERIA···············································································································30 
5.2.1 
Inclusion Criteria········································································································· 30 
5.2.3 IMMUNIZATION DURING TCZ THERAPY ·············································································································33 
5.3. 
TREATMENT ASSIGNMENT PROCEDURES············································································································34 
5.3.1 
Randomization Procedures····························································································· 34 
5.3.2 
Reasons for Withdrawal ································································································ 34 
5.3.3 
Handling of Withdrawals······························································································· 34 
5.3.4 
Termination of Study···································································································· 35 
6 
TREATMENTS ADMINISTERED ····················································································· 36 
6.1 
TREATMENTS ·············································································································································· 36 
6.1.1 
Study Product Description ····························································································· 36 
6.1.2 
Placebo······················································································································ 36 
6.2 
ACQUISITION ·············································································································································· 36 
6.2.1 
Formulation, Packaging, and Labeling ·············································································· 37 
6.2.2 
Product Storage and Stability ························································································· 37 
6.3 
DOSAGE, PREPARATION AND ADMINISTRATION OF STUDY INTERVENTION/INVESTIGATIONAL PRODUCT ····························37 


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6.3.1 TOCILIZUMAB STORAGE ································································································································38 
6.4 
MODIFICATION OF STUDY INTERVENTION/INVESTIGATIONAL PRODUCT FOR A SUBJECT ················································39 
6.4.1. 
Dosage Discontinuation ································································································· 43 
6.5 
ACCOUNTABILITY PROCEDURES FOR TOCILIZUMAB ······························································································44 
6.6 
PRIOR AND CONCOMITANT THERAPY ················································································································45 
6.6.1 
Prohibited Medications and Contraindications···································································· 45 
7 
STUDY SCHEDULE······································································································· 48 
7.1 
SCREENING VISIT········································································································································· 48 
7.1.1 
Screen Failures············································································································ 49 
7.2 
BASELINE VISIT ··········································································································································· 49 
7.3 
WEEK 4 (VISIT 1) ········································································································································· 50 
7.4 
WEEK 8 (VISIT 2) ········································································································································· 50 
7.5 
WEEK 12 (VISIT 3)········································································································································51 
7.6 
WEEK 16 (EOS VISIT) ···································································································································51 
7.7 
FINAL SAFETY VISIT & TELEPHONE CALL··········································································································52 
7.8 
PROTOCOL DEVIATIONS·································································································································52 
7.9 
MISSED VISITS AND PROCEDURES·····················································································································53 
8 
CLINICAL ASSESSMENTS AND OUTCOME MEASURES ···················································· 54 
8.1 
CLINICAL VARIABLES····································································································································54 
8.1.1 
Vital Signs, Height & Weight ·························································································· 54 
8.1.2 
PBMC Blood Collection, hs-CRP concentration and Cytokine Levels········································ 54 
8.1.4 
12-Lead Electrocardiogram (ECG)··················································································· 54 
8.1.5 
Chest X-Ray ··············································································································· 54 
8.1.6 
Physical Examination···································································································· 55 
8.1.7 
Neurological Examination······························································································ 55 
8.1.8 
Columbia Suicide Severity Rating Scale (C-SSRS)································································ 55 
8.1.10 
Lumbar Puncture ········································································································ 55 
8.1.11 
Adverse Events············································································································ 56 
8.1.12 
Exit Questionnaire ······································································································· 57 
8.2 
OUTCOME MEASURE·····································································································································57 
8.2.1 
ALSFRS-R················································································································· 57 
8.2.2 
Slow Vital Capacity (SVC) Testing ··················································································· 57 
8.2.3 
Hand-held Dynamometry (HHD) and Grip Strength····························································· 57 
8.2.4 
Training and Validation ································································································ 58 
8.2.5  
Neuroimaging ············································································································· 58 
9 
SAFETY AND ADVERSE EVENTS ··················································································· 61 
9.1 
DEFINITIONS OF AES, SUSPECTED ADVERSE DRUG REACTIONS & SAES·····································································61 
9.1.1 
Adverse Event and Suspected Adverse Drug Reactions·························································· 61 
9.1.3 
Serious Adverse Events ································································································· 63 
9.2 
ASSESSMENT AND RECORDING OF ADVERSE EVENTS·····························································································64 
9.2.1 
Assessment of Adverse Events ························································································· 64 
9.2.2 
Relatedness of Adverse Event to Investigational Product························································ 65 
9.2.3 
Recording of Adverse Events ·························································································· 66 
9.3 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS - REPORTABLE EVENTS·································································66 
10 DATA AND SAFETY MONITORING AND STATISTICAL ANALYSIS PLAN ···························· 69 
10.1 
DATA AND SAFETY MONITORING BOARD············································································································69 
10.2 
SAFETY MONITORING ···································································································································70 
10.2.1 
Safety Management Resource Team ················································································· 70 


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10.2.2 
Medical Monitoring······································································································ 70 
10.3 
STATISTICAL CONSIDERATIONS ·······················································································································70 
10.3.1 
Statistical Methods······································································································· 70 
10.3.2 
Analysis Populations····································································································· 72 
10.4 
MISSING DATA ············································································································································ 72 
10.4.1 
Stopping Rules ············································································································ 72 
11 DATA COLLECTION, MANAGEMENT AND MONITORING ················································ 73 
11.1 
ROLE OF DATA MANAGEMENT ························································································································73 
11.1.1 
Data Entry and Checks ································································································· 73 
11.1.2 
Data Lock Process········································································································ 73 
11.1.3 
Quality Assurance········································································································ 74 
11.2 
CLINICAL MONITORING·································································································································74 
11.3 
DATA HANDLING AND RECORD KEEPING············································································································74 
11.3.1 
Confidentiality ············································································································ 74 
11.3.2 
Study Discontinuation··································································································· 75 
11.3.3 
Retention of Records····································································································· 75 
11.3.4 
Publications················································································································ 75 
12 LITERATURE REFERENCES·························································································· 76 
13 APPENDICES ··············································································································· 78 
13.1 APPENDIX I: EL ESCORIAL WORLD FEDERATION OF NEUROLOGY CRITERIA FOR THE DIAGNOSIS OF ALS·····································78 
13.2 APPENDIX II: ALS FUNCTIONAL RATING SCALE – REVISED (ALSFRS-R) ··········································································80 
13.3 APPENDIX III: COLUMBIA-SUICIDE SEVERITY RATING SCALE (C-SSRS) BASELINE VERSION ···················································83 
13.4 APPENDIX IV: COLUMBIA-SUICIDE SEVERITY RATING SCALE (C-SSRS) SINCE LAST VISIT VERSION··········································86 
13.6 APPENDIX V: SAFETY REPORTING FAX COVER SHEET ·························································································89 


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LIST OF ABBREVIATIONS
ADR
Adverse Drug Reaction
AE
Adverse Event/Adverse Experience
ALS
Amyotrophic Lateral Sclerosis
ALSFRS-R
Amyotrophic Lateral Sclerosis Functional Rating Scale - Revised
ALT
Alanine Aminotransferase
ANC
Absolute Neutrophil Count
AST
Aspartate Aminotransferase
BiPAP
Bilevel Positive Airway Pressure
ºC
Degrees Celsius
CBC
Complete Blood Count
CC
Coordination Center
CFR
Code of Federal Regulations
CMP
Complete Metabolic Panel
CRF
Case Report Form
CSF
Cerebrospinal Fluid 
CYP450
Cytochrome P450
DM
Data Management
DPS
Diaphragm Pacing System
DSMB
Data and Safety Monitoring Board
ECG
Electrocardiogram
EDC
Electronic Data Capture
EOS
End of Study
ºF
Degrees Fahrenheit
FDA
Food and Drug Administration
GCP
Good Clinical Practice
hCG
Human Chorionic Gonadotrophin
HHD
Handheld Dynamometry
HIPAA
Health Insurance Portability and Accountability Act
HIV
Human Immunodeficiency Virus
hs- CRP
Highly sensitive C-reactive protein
ICF
Informed Consent Form
ICH
International Conference on Harmonisation
IL-6
Interleukin-6
IND
Investigational New Drug Application
IRB
Institutional Review Board


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ITT
Intent to Treat
IV
Intravenous
LP
Lumbar Puncture
LFT
Liver Function Test
mg
Milligram
mg/kg
Milligram per kilogram
mL
Milliliter
mm3
Millimeter cubed
mmHg
Millimeter of mercury
MR-PET
Magnetic Resonance Imaging - PBR28 Positron Emission Tomography
N
Number (typically refers to subjects)
NEALS
Northeast ALS Consortium
NMDA
N-methyl-D-aspartate
OHRP
Office for Human Research Protections
pg/mL
Picogram per milliliter
PA
Posterioranterior
PBMC
Peripheral Blood Mononuclear Cell
PBR28
Peripheral Benzodiazepine Receptor 28
PET
Positron Emission Tomography
PLPH
Post-LP Headache
RNA
Ribonucleic Acid
SAE
Serious Adverse Event/Serious Adverse Experience
SDT
Source Document Templates
SI
Site Investigator
sIL-6r
Soluble IL-6 Receptor
SOD1
Superoxide dismutase 1
SVC
Slow Vital Capacity
TB
Tuberculosis
TSPO
Translocator Protein
ULN
Upper Limit of Normal
UMNB
Upper Motor Neuron Burden Scale
US
United States
μg/kg
Microgram per kilogram
WOCBP
Women of Childbearing Potential


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PROTOCOL SUMMARY
Study Title
A Phase 2 Randomized, Placebo-Controlled Trial of Tocilizumab in Amyotrophic Lateral 
Sclerosis (ALS)
Version Number
8.0
Study Indication
Amyotrophic Lateral Sclerosis (ALS)
Phase of Development
2
Rationale for the Study
Despite the aggressive nature of Amyotrophic Lateral Sclerosis (ALS) and decades of research 
there has been no cure or highly efficacious therapy. In 1995, the FDA approved the only 
treatment for ALS, riluzole (Rilutek®). Although the mechanism of action is not clear, it is felt 
to act upon multiple targets to reduce excitatory neurotoxicity in motor neurons. It may do this 
through inhibition of sodium channels, by inhibiting glutamate or other excitatory 
neurotransmitter release and by N-methyl-D-aspartate (NMDA) receptor antagonism [1]. Since 
riluzole’s approval, various agents have been tested for efficacy and uniformly failed to 
demonstrate a survival benefit. 
This failure has been attributed to several factors [2]. The first and primary reason is that the 
pathophysiology of the disease is poorly understood, especially with regard to early or 
precipitating cellular triggers. There is evidence that oxidative stress, excitoxicity, mitochondrial 
dysfunction, and aberrant ribonucleic acid (RNA) processing all contribute to eventual motor 
neuron death. The second reason is that current clinical trials lack a meaningful and robust 
surrogate marker of disease activity. This results in longer, more costly trials that are not able to 
properly stratify or randomize patients. 
In this study, we hope to address these two issues by focusing on the pathophysiologic immune 
mechanisms involved in motor neuron death and by utilizing the peripheral blood mononuclear 
cells (PBMC) gene expression profile to quantify the immune response in ALS patients. If this 
profile is found to mirror disease progression, as our preliminary data suggests, this expression 


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profile could be used in a future phase 3 efficacy trial as a selection criteria as well as a surrogate 
marker of disease progression. 
We will also measure the effect of tocilizumab in reducing glial activation as measured by 
PBR28 positron emission tomography (PET) in a subset of study participants. 
Study Design
This is a multicenter, randomized, double-blind, placebo-controlled 16-week study evaluating 
the safety and tolerability of tocilizumab in subjects with sporadic ALS.
Study Objectives and Endpoints 
The primary objective of the study is to determine the safety and tolerability of intravenous 
administration of 8 mg/kg of tocilizumab every 4 weeks vs. matched intravenous placebo 
administered every 4 weeks over an 8 week period.
The primary outcome measure will be safety and tolerability of tocilizumab.
The secondary objective of the study is to describe the expression of pro-inflammatory genes in 
PBMCs of sporadic ALS patients, to assess the ability of tocilizumab to reduce the expression of 
pro-inflammatory genes in PBMCs and pro-inflammatory cytokines in the cerebrospinal fluid 
(CSF) of patients with sporadic ALS and to assess the CSF penetration of tocilizumab.
Exploratory measures will include clinical measures of disease progression such as ALSFRS-R, 
forced vital capacity, and handheld dynamometry. In addition, genotyping of the IL-6 receptor 
gene will be performed to look for polymorphisms that might affect the inflammatory state 
mediated by the IL-6 pathway.
Subjects enrolled at Massachusetts General Hospital only will have an additional exploratory 
measure.  A [11C]PBR28-PET scan will be performed at the Baseline visit (Pre-dose), and once 
at Week 8 visit (Post-dose).
Study Locations
Approximately 5 Northeast ALS Consortium (NEALS) Centers in the US will participate in the 
study. 
Number of Planned Subjects
Approximately 24 subjects will be randomized in the study.


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Study Population
This study will be conducted in subjects who meet the El Escorial criteria of possible, laboratory-
supported probable, probable, or definite criteria for a diagnosis of ALS. At screening, eligible 
subjects must be at least 18 years old, must have a slow vital capacity (SVC) 40% of predicted 
capacity for age, height and gender, and must provide written informed consent prior to 
screening.  Subjects on a stable dose of riluzole and those not taking riluzole, and women of 
child-bearing age at screening are eligible for inclusion as long as they meet specific protocol 
requirements.  Detailed criteria are described in the body of the protocol.
Treatment Groups
Subjects will be randomly assigned in a 2:1 ratio to intravenous tocilizumab 8 mg/kg or 
matching placebo every 4 weeks over an 8 week period.
This research study protocol allows the subject to receive up to 3 infusions of Tocilizumab.
Even if the treatment is shown to be of benefit, additional infusions of Tocilizumab beyond that 
allowed in the protocol cannot be given to the subject while she/he is participating in this study.
Duration of Treatment and Follow-up
Subjects will remain on randomized, placebo-controlled, double-blind treatment until the Week 8
visit.  Each randomized subject will also have a Week 12 Follow-up visit and Week 16 End-of-
Study visit to assess for adverse events (AEs), changes in concomitant medications, to administer 
the ALSFRS-R and selected study procedures.


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SCHEDULE OF ACTIVITES
1 Screening procedures must be completed within 28 days prior to Baseline Visit.
2 Vital signs include systolic and diastolic pressure in mmHg, respiratory rate/minute, heart rate/minute and temperature.  
Activity
Screening Visit1
Baseline Visit
Week   4
Week    8
Week 12
Week 16
Visit 1
Visit 2
Visit 3
EOS Visit
Written Informed Consent
X
Inclusion/Exclusion Review
X
X
Medical History
X
Demographics
X
ALS Diagnosis History
X
Physical Examination
X
X
Neurological Exam
X
X
Columbia Suicide Severity Rating Scale (C-SSRS)
X
X
X
X
X
Vital Signs2 including Height3 and Weight
X
X
X
X
X
X
Lumbar Puncture (LP)4
X4
X4
ALSFRS-R
X
X
X
X
X
Slow Vital Capacity (SVC)
X
X
X
X
X
X
Hand Held Dynamometry (HHD) and Grip Strength
X
X
X
Electrocardiogram (12-Lead ECG)
X
PA and Lateral Chest X-Ray
X
Quantiferon or T-SPOT TB Blood Test
X
Safety Labs5
X
X
X
X
X
X
PBMC Collection 
X6
X7
X
X
X
X
Plasma Biomarker Collection
X
X
X
X
X
Blood Collection for IL-6R genotype, Genentech sample
X
Concomitant Medication Review
X
X
X
X
X
X
Adverse Event Review8
X
X
X
X
X
X
Randomization9
X
Administer Study Drug
X
X
X
Exit  Questionnaire
X
[11C]PBR28-PET
X*
X**
MRI Safety Questionnaire
X
X
X
Upper Motor Neuron Burden Scale
X
X
X
Blood draw for TSPO
X***


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3 Height measured at Screening Visit only.
4 Telephone call will be made 24-48 hours post lumbar puncture (LP) to assess for adverse events (AEs) related to the LP.
5 Safety labs include Hematology (CBC with differential), ChemistryPanel, Hepatitis B panel, , Urinalysis and pregnancy test (WOCBP) as required 
throughout the study. Serum hCG will be collected at Screening Visit, and urine pregnancy tests will be collected at each subsequent visit for all 
WOCBP. 
6 Plasma Biomarkers will not be collected at the screening visit.
7  Collection at Baseline to occur prior to infusion 
8Adverse events that occur AFTER signing the informed consent form will be recorded.
9 Randomization should occur immediately prior to the Baseline Visit.
*Pre-dose [11C]PBR28-PET will be performed one time only at the Baseline visits. (-2 Weeks)
**Post-dose [11C]PBR28-PET will be performed one time only at the Week 8 visit (- 2 Weeks).
*** Blood draw for TSPO affinity testing to occur at the Screening visit, if not previously determined.


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STUDY WORKFLOW 
           
~ 50 Screened
- Study drug infusion at Baseline, Visit 1 and Visit 2. 
- Subjects who discontinue study drug early but agree to be followed will be asked to remain in the study and to attend all assigned visits until the Week 16 End-
of- study visit.  
- Subjects who discontinue study drug early and do not agree to be followed will be asked to return to the study site for a final safety assessment, and will be 
asked to have a final follow-up telephone call 28 days (+5 days) after taking their last dose of study drug.
8 weeks
8 weeks
28 days
Screening
Baseline Visit 
Week 4
Visit 1
Week 8
Visit 2
Week 12
Visit 3
Week 16
EOS Visit
Screening Period
On Active Treatment
Follow-up Period
Placebo 
Group B
N=8
Group A
N=16
Tocilizumab 
8 mg/kg 
Low inflammation group or indeterminate excluded 
High inflammation group


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1
ETHICS/PROTECTION OF HUMAN SUBJECTS
1.1
Institutional Review Board (IRB)
This study will be conducted in compliance with current Good Clinical Practices (GCP) and 
Title 21 Part 56 of the United States of America Code of Federal Regulations (CFR) relating to 
IRBs. 
1.2
Ethical Conduct of Study
The study will be conducted in accordance with GCP defined by the International Conference on 
Harmonization (ICH) and the ethical principles of the Declaration of Helsinki.
1.3
Subject Information and Consent
This study will be conducted in compliance with Title 21 Part 50 of the United States of America 
Code of Federal Regulations (CFR), Federal Regulations and ICH Guidance Documents 
pertaining to informed consent.  At the first visit, prior to initiation of any study-related 
procedures, subjects will be informed about the nature and purpose of the study, 
participation/termination conditions, and risks and benefits. Subjects will be given adequate time 
to ask questions and become familiar with the study prior to providing consent to participate. 
Subjects who wish to will give their written consent to participate in the study and will be 
provided with a copy of the fully executed consent form for their records.


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2
INTRODUCTION: 
BACKGROUND 
INFORMATION 
AND 
SCIENTIFIC 
RATIONALE
2.1
Background Information
Despite the aggressive nature of Amyotrophic Lateral Sclerosis (ALS) and decades of research,
there has been no cure or highly efficacious therapy. In 1995, the FDA approved the only 
treatment for ALS, riluzole (Rilutek®). Although the mechanism of action is not clear, it is 
believed to act upon multiple targets to reduce excitatory neurotoxicity in motor neurons. It may 
do this through inhibition of sodium channels, by inhibiting glutamate or other excitatory 
neurotransmitter release, and by N-methyl-D-aspartate (NMDA) receptor antagonism [1]. Since 
riluzole’s approval, various agents have been tested for efficacy and uniformly failed to 
demonstrate a survival benefit. 
This failure has been attributed to several factors [2]. The primary reason is that the 
pathophysiology of the disease is poorly understood, especially with regard to early or 
precipitating cellular triggers. There is evidence that oxidative stress, excitoxicity, mitochondrial 
dysfunction, and aberrant ribonucleic acid (RNA) processing all contribute to eventual motor 
neuron death. The second reason is that current clinical trials lack a surrogate marker of disease 
activity. This results in longer, more costly trials that are not able to properly target patients for 
enrollment and do not advance understanding of ALS pathophysiology.
In this study, we hope to address these two issues by focusing on the pathophysiologic immune 
mechanisms involved in motor neuron death and by utilizing the peripheral blood mononuclear 
cells (PBMC) gene expression profile to select patients for enrollment and to quantify the 
immune response in ALS patients. If this profile is found to mirror disease progression, as our 
preliminary data suggests, this expression profile could be used in a future phase 3 efficacy trial 
as surrogate marker of disease progression. 
2.2
Rationale
Tocilizumab is a humanized monoclonal antibody against the IL-6 receptor distributed by 
Genentech, Inc. under the trade name Actemra®. It was FDA approved for treatment of 
refractory moderate to severe rheumatoid arthritis. It is delivered by intravenous infusion 
monthly at a standard dose of 4-8 mg/kg [3]. We propose that tocilizumab treatment will reduce 
inflammation in ALS patients by impeding both the classical and trans-signaling IL-6 pathways.


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The anti-inflammatory approach to treat ALS by tocilizumab is based on basic science 
discoveries and information related to inflammatory signaling by the cytokine IL-6 [4-9].  IL-6 is 
one of the most important mediators of fever and acute phase responses, is capable of crossing 
the blood-brain barrier, and is secreted by macrophages in response to pathogens.  IL-6 binds to 
cell surface IL-6 receptors (IL6-R) present on numerous cell types.  In the spinal fluid and in the 
spinal cord of sporadic ALS patients, IL-6 may also transmit its inflammatory signal through a 
soluble IL6 receptor (sIL6-R) containing only the extracellular portion of the IL6-R, which then 
binds to a ubiquitous cell surface receptor called gp130 and activates numerous signaling 
pathways, including the JAK/STAT and Ras-Raf-MAPK pathways. This process is called IL-6
“trans-signaling” and can activate intracellular pathways in cell types that lack the cell surface 
IL6-R. Interestingly, many CNS cell types only respond to IL-6 via IL-6 trans-signaling [10, 11].
We have shown that the spinal fluid of ALS patients contains up to nanogram amounts of sIL6-R
but only 100 pg/mL (mean) of IL-6 protein. However, macrophages infiltrating the ALS spinal 
cord strongly express IL-6 suggesting that IL-6 signaling may be occurring in the spinal cord 
through both trans-signaling via the sIL-6R and classical signaling through the membrane 
receptor. The significance of IL-6 signaling lies in the stimulation of phagocytosis of motor 
neurons by inflammatory macrophages expressing IL-6 [5].
The study hypothesis is that tocilizumab is safe in ALS patients, penetrates the blood brain 
barrier and reduces expression of pro-inflammatory genes in patients with ALS.
2.2.1 Rationale for Dose
The dosing rationale of tocilizumab in this study is based on the FDA labeling for its use in 
rheumatoid arthritis. In general, it is administered at a dose of 8 mg/kg as an intravenous infusion 
over 1 hour every 4 weeks with the drug mixed with normal saline to a total volume of 100 mL.
2.3
PRELIMINARY DATA
2.3.1 Gene expression of pro-inflammatory cytokines
Peripheral blood mononuclear cells (PBMCs) isolated from ALS patients and treated with 
aggregated SOD1 (2 μg/mL) exhibit increased gene expression and protein levels for multiple 
cytokines [4].  Addition of tocilizumab (10 μg/mL) inhibited the expression of pro-inflammatory 
cytokines induced by aggregated SOD1 (Figure 1).
We analyzed gene expression of activated PBMCs from 10 sporadic ALS patients, 5 of whom 
were treated with tocilizumab infusions, and four healthy controls [7]. Among our sample of
sporadic ALS patients, the innate immune system was highly inflammatory in 50% of the 
patients (Group 1, mean age +/- S.E of 53.4 +/- 1.6 years) and nearly non-inflammatory in the 


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other 50% (Group 2, age 55.2 +/- 7.9 years) based on analysis of PBMC gene expression by RT-
PCR (Figure 2).  Group 1 patients were defined as those whose PBMCs show up-regulation 
(compared to healthy controls, mean age 62 +/- 10.2 years) of the transcription of inflammatory 
genes for cytokines/chemokines IL-Į IL-ȕ,/-6, IL-8, CXCL3, CXCL5, CCL20, MMP1, and 
COX-2 (p <0.05 Group 1 vs. healthy controls), whereas the “Group 2” PBMCs show normal or 
low inflammatory activation except for elevated transcription of CXCL 9, 10, 11 when compared 
to healthy controls [4].
In one Group 1 patient, we observed a reduction over 12 weeks in 
inflammatory gene expression and inflammatory biomarkers, such as serum C-reactive protein, 
IL-6 and IL-1 cytokines, after tocilizumab infusion therapy, followed by recurrence from 4 to 5
months after start of therapy, and subsequent remission of inflammation.  Serum level of 
tocilizumab in this patient was 12.3 μg/mL before infusion (trough level from prior infusion) and 
109 μg/mL after infusion, with CSF levels of 0.18 μg/mL after infusion, demonstrating that drug 
was reaching the CNS.  The patient’s rate of progression by ALSFRS-R slowed from 2.6 points 
per month to 0.4 points per month. The patient discontinued tocilizumab after 8 months of 
therapy and the disease course accelerated by ALSFRS-R measurements.
ALSFRS-R
progression slowed from 1.3 points per month to 0.75 points per month in another Group 1 
patients treated with tocilizumab for 2 months. In one Group 2 patient, the effect of tocilizumab 
infusion therapy on inflammatory gene expression was minimal, but the patient’s rate of 
progression by ALSFRS-R reversed course, changing from a 0.7 point per month decline to a 1.0 
point per month improvement over 4 months of treatment.
One other Group 2 patient 
experienced an acceleration of progression during 5 months of tocilizumab treatment.  The small 
number of patients in this pilot clinical study makes these findings difficult to generalize to the 
ALS population as a whole.  However these clinical findings merit continued study of 
tocilizumab in a larger and more carefully controlled clinical trial.  Specifically, an important 
goal of the proposed clinical trial will be to demonstrate that our gene expression profile 
biomarker of PBMCs can be used to demonstrate target engagement and a biological effect [4, 
7].


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FIGURE 1: Reduction of cytokine levels after tocilizumab treatment
Levels of IL-ȕ ,/-6, GM-CSF and TNF-D in PBMC’s from Group 1 ALS patients before 
(magenta) and after (yellow) overnight tocilizumab treatment, compared to healthy controls (black, 
null).  Drug treatment led to significantly reduced levels of cytokines released from activated 
PBMCs.


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FIGURE 2: Gene expression profiling identifies two inflammation groups in ALS


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2.3.2 PBR28 preliminary data
Imaging Neuroinflammation using [11C]PBR28-
PET
[11C]PBR28 
is 
a 
novel 
radiotracer 
of 
neuroinflammation. 
It 
binds 
to 
the 
18 
kDa 
translocator
protein (TSPO), which is highly 
expressed in activated microglia and monocytes.  We 
have shown that [11C]PBR28 uptake is increased in 
the motor cortices of symptomatic ALS patients [12].
We have administered [11C]PBR28 to more than 50 
ALS participants and 25 healthy volunteers at MGH. 
Below is a summary of our main published and 
unpublished preliminary findings to date: 
I.
[11C]PBR28 uptake is significantly increased 
in the motor cortices and subcortical regions 
in ALS participants compared to healthy 
controls [12]
(Figure 3A,B,C).
II.
The anatomical distribution of increased 
[11C]PBR28 uptake corresponds to ALS 
clinical presentation; ALS participants with 
limb-onset 
weakness 
had 
elevated 
[11C]PBR28 retention in the motor cortices, 
whereas 
participants 
with 
bulbar-onset 
weakness had increased retention in 
the brainstem [12].
III.
Increased 
[11C]PBR-28 
uptake 
strongly 
correlates with increased upper motor neuron 
dysfunction as measured by the Upper Motor 
Neuron Burden 
(UMNB) scale (Figure 3D) [12].
IV.
Using our combined Positron emission 
tomography/magnetic 
resonance 
imaging 
MR/PET scanner, we demonstrated that 
[11C]PBR-28 uptake (molecular measure of inflammation) co-localizes and correlates 
Figure 3: PBR28 PET study in 46 ALS participants and 21 
healthy volunteers (unpublished data). PBR28 uptake in the 
motor cortices is increased in ALS subjects (A) compared to 
healthy volunteers (B). Statistical maps with family-wise 
corrections for multiple testing show increased PBR28 uptake 
in the motor cortices in the ALS group (C). PBR28 uptake in 
the motor cortices is highly correlated with upper motor neuron 
burden (UMNB) (D). 


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with axonal loss (anatomical change) measured by fractional anisotropy FA and cortical 
thinning (anatomical change), providing unique in vivo evidence of the anatomical 
connection between motor neuron degeneration and the surrounding inflammation [13].
2.3.3 Tocilizumab
Tocilizumab (TCZ), formerly known as myeloma receptor antibody (MRA) is a recombinant 
humanized antihuman monoclonal antibody of the immunoglobulin IgG1 subclass directed 
against the IL-6R and produced by recombinant DNA technology. Clinical efficacy and safety 
studies of TCZ have been conducted or are ongoing in various disease areas, including adult-
onset RA, systemic-onset juvenile idiopathic arthritis and polyarticular juvenile idiopathic 
arthritis.
The half-life of TCZ is approximately 7 days. The TCZ exposures were stable over 2- years of 
treatment. The observed mean (± SD) Ctrough at 8 mg/kg IV was 15.9 ± 12.0 at
week 24 and 19.9 ± 17.0 at week 104. The observed mean (± SD) Ctrough at 4 mg/kg was 1.02 ±
6.14 at week 24 and 1.09 ± 2.77 at week 104.
The Roche clinical development program in adult RA, comprised five pivotal Phase 3 trials and 
two open-label, long-term treatment extension studies. 
Further information on TCZ can be found in the Investigator’s Brochure (IB).
2.4
POTENTIAL RISKS AND BENEFITS
2.4.1
Potential Risks
The main risks associated with the study relate to exposure to tocilizumab and lumbar puncture
(LP). Tocilizumab has both short-term and long-term risks associated with it. As with any 
intravenous (IV) drug, there is a chance for injection reactions such as anaphylaxis. These 
reactions are usually mild and responsive to antipyretics, antihistamines, and analgesics. The 
experience with tocilizumab in rheumatoid arthritis patients suggests that serious reactions occur 
at a rate of 0.1%-0.2% [3]. The most common long-term AEs  were upper respiratory tract 
infections (7%), nasopharyngitis (7%), headache (7%), hypertension (6%) and elevated liver 
functions tests (6%), reversible neutropenia, and elevated cholesterol (13%). Serious infections 
have been reported with tocilizumab use and include opportunistic infections such as 
tuberculosis. Finally, tocilizumab reverses inflammation-induced CYP450 enzyme inhibition, 
effectively increasing drug metabolism via this pathway and decreasing CYP450 metabolized 
drug exposure. Although this has been studied in the rheumatoid arthritis population, it is unclear 


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if ALS patients have clinically significant inflammation-induced CYP450 alterations [14, 15].
Unfortunately, no other anti IL-6 receptor agents are available as an alternative to tocilizumab. 
Given our hypothesis that IL-6 activity is central to the role of the inflammatory cascade in 
motor neuron death, exposure to tocilizumab is unavoidable if we wish to test the effect of 
altering IL-6 receptor activity.
Lumbar puncture poses short-term risks to patients unrelated to study drug exposure. As with 
any procedure, there is a low risk of bleeding or infection. Pain and post-lumbar puncture 
headache (PLPH) are more common risks that may impact quality of life. Pain is localized, short 
lived and responds to over-the-counter analgesics. PLPH occurs in 10-30% of cases depending 
on technique and patient population and may lead to hospitalization or the need for an 
autologous blood patch procedure [16]. This risk can be greatly reduced by the use of atraumatic 
needles. In a retrospective study of ALS patient receiving LPs for CSF biomarker studies, the use 
of atraumatic needles reduced the risk of PLPH by 50% compared to traditional cutting needles 
[17]. No patient in the atraumatic needle group required a blood patch procedure. A fundamental 
tenet of the proposed therapy is that the monoclonal antibody tocilizumab should cross the 
blood-brain barrier. There is no systematic data available to establish that the CSF contains 
sufficient concentrations of tocilizumab to be effective. A full characterization of the 
pharmacokinetics of tocilizumab would require serial sampling of blood and CSF over several 
weeks with a lumbar catheter. Instead, we propose to sample CSF from patients in this study
prior to treatment and 2.5 hours after their third infusion to estimate maximal CSF concentration
and change in cytokine levels and IL-6 receptor levels in the CSF. Given the critical importance 
of these data, LP is unavoidable.
To minimize the risk to patients, the protocol includes criteria and algorithms for interrupting or 
stopping therapy in patients with serious infections, low absolute neutrophil count (ANC), 
elevated liver enzymes, and thrombocytopenia.  It will also include criteria to exclude patients at 
risk for opportunistic infections. To minimize risk of hypersensitivity, the protocol will utilize 
pre-treatment anti-histamines as described in the site SOP, and require all infusions to occur in a 
hospital or clinic-based infusion center with experienced infusion staff and the ability to rapidly 
treat anaphylaxis.  We will exclude patients who concomitantly receive CYP450 metabolized 
drugs whose reduced exposure may pose a short-term risk. Finally, the proposed study will 
utilize atraumatic needles, standard LP procedures and exclude anticoagulated patients to 
minimize the risks of LPs.  
No socioeconomic or psychological risks are associated with the study. 
Risk of Magnetic Resonance Imaging: People who have electrically, magnetically or 
mechanically activated implants (such as heart pacemakers) or those who have clips on blood 


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vessels in their brain will not be allowed to participate in the study due to associated risks with 
MRI scanning. The MRI systems will be operated in a manner accepted by the Food and Drug 
Administration (FDA). The scanner makes loud knocking or beeping sounds as it takes images. 
Subjects will be given earplugs to help reduce this noise. If a subject notices any discomfort 
while in the MRI scanner, (s) he should notify the administrator immediately. If the discomfort 
cannot be stopped, the scanning session may be stopped. 
All credit cards and other items with magnetic strips should also be kept out of the MRI room.
The MRI has the potential, during normal routine use, to cause localized warming of the skin and 
the underlying tissues. Subjects should immediately inform the study staff if they experience 
discomfort due to warming of the skin and the procedure will be stopped.
The MRI can be stopped at any time at the subject’s request. Subjects with severe claustrophobia
are excluded from the study.
Imaging will be stopped should any untoward reaction be observed during the imaging session or 
if the subject so requests for whatever reason. Some subjects find it unpleasant or feel anxious 
when confined in the enclosed space of the scanner. If this happens, the subject will be unable to 
participate in the study.
Risk of Positron Emission Tomography Imaging: For the PET scan, no side effects from the 
radioligand [11C]PBR28. Subjects will be exposed to a small amount of radiation that will be 
significantly less than the yearly amount of radiation allowed for persons who work with 
radiation. The total amount of radiation exposure subjects will receive from participation in this 
study is equal to a whole body exposure of approximately ~3.7 milliSieverts (mSv). This amount 
of radiation is about the same as one would normally receive in 15 months from natural 
background sources from the earth and the sky.  
The radiation exposure in this study will be small and there is no evidence that it represents a 
major health risk.  If subjects have participated in other research studies in the past 12 months 
that have involved radiation exposure, they will be asked to inform the investigators or study 
staff (by writing initials on the consent form verifying that they have not been exposed to other 
radiation in the past 12 months).  If it is determined that their prior radiation exposure exceeds 
the current institutional guidelines, they will not be allowed to participate in this study.  
Subjects will be required to lie still in the PET camera several times during an approximately 1 
hour 45 minute period, and subjects may find it uncomfortable to remain still over this time. In 
order to insure that subjects do not move their heads during the imaging procedure cushions 
could be placed around the head. Subjects will be required to lie still on the imaging table with 
their heads in the scanner. This is usually well tolerated by most subjects.


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We will use [11C]PBR28 produced by the cyclotron/radiochemistry/radiopharmacy facility at the 
A.A. Martinos Center for Biomedical Imaging. We will follow the safety standards approved by 
the Radiation Safety Committee for the use of radioligands. The IV injection will be 
administered either by a physician or by a trained technician. Should there be an adverse event 
related to a study procedure, the principal investigator, co-investigator or study staff will be 
responsible for communicating with the IRB within the stipulated timeframe. At the time of 
continuing review a cumulative log of adverse events will be submitted, regardless of 
relatedness.
The PET scan can be stopped at any time at the subject’s request. Subjects with severe 
claustrophobia are excluded from the study.
Imaging will be stopped should any untoward reaction be observed during the imaging session or 
if the subject so requests for whatever reason. Some subjects find it unpleasant or feel anxious 
when confined in the enclosed space of the scanner. If this happens, the subject will be unable to 
participate in the study.
2.4.2
Known Potential Benefits
There are no specific anticipated potential benefits to subjects for participating in this study, 
although subjects may benefit from the close follow-up and detailed medical care associated with 
study participation. It is not known whether treatment with tocilizumab will be helpful or 
harmful for ALS subjects. It is possible that results from the study may yield benefits to future 
ALS patients.


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3
OBJECTIVES
3.1
Study Objectives
The study objective is to demonstrate that tocilizumab is safe and well tolerated in patients with 
sporadic ALS and determine its effect on immune markers and clinical progression in patients 
with sporadic ALS.
Successful completion of this study will support further clinical 
investigation to test the efficacy of tocilizumab in patients with sporadic ALS and define patient 
enrichment criteria and biomarkers that could be used in subsequent clinical studies.
3.1.1 Primary Objective
The primary objective of the study is to assess the short-term safety and tolerability of 
tocilizumab in patients with sporadic ALS.
3.1.1 Secondary Objectives
The secondary objectives of this study are: 
x
To describe the inflammatory gene expression profiles in PBMCs of sporadic ALS 
patients and to assess the ability of tocilizumab to reduce expression of pro-inflammatory 
genes in PBMCs of patients with sporadic ALS and a high inflammation state at baseline.
x
To assess whether tocilizumab achieves a concentration in the CSF sufficient to block IL-
6 signaling. 
x
To gather preliminary data on the concentration of key inflammatory cytokines (IL-6, IL-
8, IL-17, IL-ȕ71)-ĮV,/-6 receptor, or hs-CRP) in the plasma and CSF before and 
after treatment with tocilizumab vs. placebo.
x
To identify polymorphisms that might affect the inflammatory state mediated by the IL-6
receptor.
x
Measure the effects of tocilizumab on reducing glial activation measured by PBR28 PET 
in a subset of trial participants. 
3.2
Study Outcome Measures
3.2.1
Primary Outcome Measures
We will assess safety of tocilizumab treatment based on mortality, occurrence of SAEs, overall 
rates of AEs, clinically significant abnormal laboratory tests, and changes in vital signs. We will 
focus on events at least possibly related to study drug of moderate or severe intensity by 


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comparing participants receiving at least one dose of tocilizumab to those on placebo for time to 
mortality and time to first SAE by Cox regression, for overall rate of AEs or abnormal laboratory 
tests by negative binomial regression, and for rates of change in vital signs.
Tolerance of tocilizumab treatment will be judged on the proportion of participants remaining on 
study drug through all 3 doses and remaining on study and free from possibly drug-related and 
dose-limiting SAEs to the end of follow-up.
3.2.2
Secondary Outcome Measures
Secondary outcome measures will be descriptive and include measures of efficacy as well as 
measures of biochemical change. Efficacy outcome measures include change in the rate of 
change of ALSFRS-R, HHD, and SVC. Biochemical measures will include change in cytokine 
levels in the serum and CSF, change in PBMC pro-inflammatory gene expression, and change in 
CSF sIL-6 receptor concentrations. We will estimate the proportion of participants who 
experience a 2-fold or greater decline between baseline and the average of all follow-up 
assessments in at least 2 of the 3 pro-inflammatory genes. These measures will serve several 
purposes. First, they will be used to establish a correlation between the biochemical responses 
and drug exposure. Second, they will be used to identify the proportion of ALS patients who 
display a pro-inflammatory PBMC gene expression profile. This information will be useful when 
designing future trials, especially if an enrichment design is used. Finally, the measures will 
serve to demonstrate target engagement of the sIL-6 receptor. Mean PBR28 uptake will be 
measured in the motor cortices as regions of interest (ROIs), and will be compared between pre-
and post-dose.
3.2.3
Safety Measures
The following laboratory tests will be performed for safety:
x
Hematology with differential panel: complete blood count with differential (hematocrit, 
hemoglobin, platelet count, RBC indices, total RBC, total WBC, and WBC differential) 
x
Blood chemistry panel/Liver function tests (LFTs): alanine aminotransferase (ALT 
(SGPT)), aspartate aminotransferase (AST (SGOT)), albumin, alkaline phosphatase, 
bicarbonate, blood urea nitrogen, calcium, chloride, creatinine, glucose, magnesium, 
phosphate, potassium, sodium, total bilirubin, total protein and, carbon dioxide.
x
Urinalysis: albumin, bilirubin, blood, clarity, color, glucose, ketones, nitrate, pH, protein, 
specific gravity, urobilinogen and WBC screen 


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x
Hepatitis B panel (collected only at Screening Visit)
x
Serum human chorionic gonadotrophin (hCG) for women of childbearing potential 
(WOCBP) (collected only at Screening Visit). Urine pregnancy tests will be performed 
on all WOCBP at each subsequent visit throughout the study.
All subjects will have safety laboratory tests at the designated visits outlined in the protocol. 
These samples will be analyzed at a central laboratory.  The SI may order additional testing, if 
needed, to further assess an AE, or if there is any suspicion that a subject may be pregnant, 
throughout the course of the study.


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4
STUDY DESIGN
4.1
Overall Study Design and Plan
During the enrollment period, approximately 50 subjects will be screened from approximately 4
Northeast ALS Consortium (NEALS) Centers in the US.  Twenty-four of these subjects (or 
more, if some randomized participants never initiated treatment) will be randomly assigned in a 
2:1 ratio to intravenous tocilizumab 8 mg/kg vs. matching placebo every 4 weeks for 8 weeks (3 
doses).  After screening and randomization, subjects will undergo baseline measures, undergo LP 
and receive their first dose of study drug in clinic. Clinic visits will occur at 4 week intervals 
with total study duration of 16 weeks. A second LP will occur at the 8 week visit.
All visit windows are consecutive calendar days and are calculated from the day the participant 
starts study treatment (Day 0, the day of the Baseline Visit).
4.2
Study Centers
This study will be conducted at approximately 5 NEALS Centers in the US.  Sites will be 
selected based on recruitment record from prior trials, compliance with prior study protocols and 
regulations, clinical research expertise and availability of necessary resources.
4.3
Study Duration
Subjects will remain on randomized, placebo-controlled, double-blind treatment until the Week 8
visit.  Each randomized subject will also have a Week 12 Follow-up visit and Week 16 End-of-
Study visit to assess for AEs, changes in concomitant medications and to administer selected 
study procedures.
4.4
Protocol Adherence
Each (SI) must adhere to the protocol detailed in this document and agree that any changes to the 
protocol must be approved by the Coordination Center (CC) or their representative prior to 
seeking approval from the site IRB.  Each SI will be responsible for enrolling only those study 
subjects who have met protocol eligibility criteria.


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5
STUDY ENROLLMENT AND WITHDRAWAL
5.1
Number of Study Subjects
Twenty-four (24) subjects will be randomized in a 2:1 ratio to intravenous tocilizumab or 
matching placebo. Additional subjects may be randomized if any randomized subjects withdraw 
from the study prior to initiating study drug.
5.2
Inclusion and Exclusion Criteria
5.2.1
Inclusion Criteria
Study subjects meeting all of the following criteria will be allowed to enroll in the study: 
1. Participants with ALS diagnosed as possible, laboratory-supported probable, probable or 
definite according to the World Federation of Neurology El Escorial criteria.
2. Male or female, aged 18-75.
3. Capable of providing informed consent and complying with trial procedures.
4. High inflammatory profile of PBMC gene expression defined as having one of the 
following:
i. A three-fold up-regulation (compared to pooled control levels) of IL-6
expression
OR
ii. A two-fold up-regulation  (compared to pooled controls) of IL-6 and either IL-8
or MMP1 expression.
5. UpULJKW69&40% of predicted value for gender, height and age at the Screening Visit
and in the opinion of the investigator is able to comply with and complete the trial.
6. Women must not be able to become pregnant (e.g., post menopausal, surgically sterile, or 
using adequate birth control methods) for the duration of the study.
7. Negative tuberculosis (TB) skin test, Quantiferon or T-SPOT TB blood test at the 
Screening Visit.
8. Not taking riluzole, or on a stable dosage for at least thirty (30) days prior to the 
Screening Visit.
Riluzole. Subjects taking concomitant riluzole at study entry must be on a stable dose for 
at least 30 days prior to the Screening Visit and must continue taking the same dosage 
throughout the study, unless the SI determines that riluzole should be discontinued for 
medical reasons.    


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9. Subjects medically able to undergo lumbar puncture (LP) as determined by the 
investigator (i.e., no bleeding disorder, allergy to local anesthetics, a skin infection at or 
near the LP site, or evidence of high intracranial pressure).
10. Subjects must agree not to take live attenuated vaccines (including seasonal nasal flu 
vaccine, varicella vaccine for shingles or chickenpox, MMR or MMRV, oral polio 
vaccine and vaccines for yellow fever, measles, mumps or rubella) thirty (30) days before 
the Screening Visit, throughout the duration of the trial and for sixty (60) days following 
the subject’s last dose of study drug.
11. Geographic accessibility to the study site.
MR-PET Inclusion Criteria (MGH only):
12. High or mixed affinity to bind TSPO protein (Ala/Ala or Ala/Thr) (see section 7.1)
13. Upper Motor Neuron Burden (UMNB) Scale 6FRUH   RXW RI  DW WKH 6FUHHQLQJ
Visit.
14. Able to safely undergo PET/MRI scans based on the opinion of the site investigator. 
5.2.2
Subject Exclusion Criteria
Study subjects meeting any of the following criteria during screening evaluations will be 
excluded from entry into the study:
1. Prior use of:
x
Tocilizumab (Actemra®)
x
Cell-depleting therapies, including investigational agents or approved therapies 
(some examples are CAMPATH, anti-CD4, anti-CD5, anti¬CD3, anti-CD19 and 
anti-CD20)
x
Alkylating agents such as chlorambucil
x
Total lymphoid irradiation
x
Stem cell therapies (either investigational or approved therapies)
2. Presence of tracheostomy at Screening Visit.
3. Exposure to any anti-inflammatory agent currently under investigation for the treatment of 
patients with ALS (off-label use or investigational) within 30 days prior to the Screening 
Visit. Examples include NP001 or Lunasin. Medications that do not have an anti-
inflammatory mechanism such as mexilitine or retigabine are allowed if on a stable dose for 
30 days prior to the Screening Visit.
4. Treatment with a prohibited medication within 30 days of the ScreeningVisit:
x
Nuedexta
x
Theophylline
x
Warfarin
x
Cyclosporine


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5. Treatment with intravenous gamma globulin, plasmapheresis or Prosorba column within 6 
months of screening.
6. Presence of diaphragm pacing system (DPS) at Screening Visit. 
7. Primary or secondary immunodeficiency (history of or currently active) unless related to 
primary disease under investigation.
8. History of or active diverticulitis, diverticulosis requiring antibiotic treatment, peptic ulcer 
disease, or GI tract perforation, or chronic ulcerative lower GI disease such as Crohn’s 
disease, ulcerative colitis or other symptomatic lower GI conditions that might predispose to 
perforations.
9. Known active current or history of recurrent bacterial, viral, fungal, mycobacterial or other 
opportunistic infections (including but not limited to tuberculosis and atypical mycobacterial 
disease, Hepatitis B and C, EBV, CMV, and herpes zoster, but excluding fungal infections of 
nail beds).
10. History of severe allergic or anaphylactic reactions to human, humanized or murine 
monoclonal antibodies.
11. Presence of any of the following clinical conditions:
x
Pharmacologic or hereditary bleeding diathesis, or any other clinical condition 
that would, in the opinion of the investigator, place the patient at increased risk 
during LP.
x
Drug abuse or alcoholism within the past 12 months.
x
Unstable cardiac, pulmonary, renal, hepatic, endocrine, hematologic, or active 
infectious disease, including current or prior malignancy.
x
Rheumatic autoimmune disease, including Systemic lupus erythematsus, Mixed 
connective tissue disease, scleroderma, polymyositis, or significant systemic 
involvement secondary to RA (e.g., vasculitis, pulmonary fibrosis or Felty’s 
syndrome).
x
Evidence of active malignant disease, malignancies diagnosed within the previous 
5 years (including hematological malignancies and solid tumors, except basal and 
squamous cell carcinoma of the skin or carcinoma in situ of the cervix uteri that 
has been excised and cured, or Stage I uterine cancer), or breast cancer diagnosed 
within the previous 5 years.
x
Human immunodeficiency virus (HIV) infection or other immunodeficient state.
x
Uncontrolled hypertension defined as systolic blood pressure > 170 or diastolic blood 
pressure > 110.
x
Unstable psychiatric illness defined as psychosis or untreated major depression 
within 90 days of the Screening Visit.
12. Any major episode of infection requiring hospitalization or treatment with IV antibiotics 
within 4 weeks of screening 
13. Laboratory values: Screening alanine aminotransferase (ALT), aspartate aminotransferase 
(AST), or total bilirubin > than 1.5 times the upper limit of normal, serum creatinine > 1.6 


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mg/dL (141 μmol/L) in female patients and > 1.9 mg/dL (168 μmol/L) in male patients 
(patients with serum creatinine values exceeding limits may be eligible for the study if their 
estimated glomerular filtration rates (GFR) are >30), hemoglobin < 85 g/L (8.5 g/dL; 5.3 
mmol/L), white Blood Cells < 3.0 x 109/L (3000/mm3), absolute neutrophil count of 
<2000/mm3, absolute lymphocyte count < 0.5 x 109/L (500/mm3), platelet concentration of 
<100,000/mm3, positive Hepatitis B surface antigen (HBsAg).
14. Pregnant women or women currently breastfeeding.
15. No history of chicken pox infection by self report and no history of varicella zoster 
vaccination
16. Any reason in the opinion of the investigator that the patient may not be able to comply with 
study procedures, complete the study or is unsuitable for immunosuppressive therapy.
MR-PET Exclusion Criteria (MGH only):
17. Any contraindication to undergo MRI studies such as 
x
History of a cardiac pacemaker or pacemaker wires
x
Metallic particles in the body
x
Vascular clips in the head
x
Prosthetic heart valves
x
Claustrophobia
18. Radiation exposure that exceeds the site’s current guidelines 
19. Current use of tobacco products including cigarettes, e-cigarettes, cigars, snuff and chewing 
tobacco, or nicotine replacement products such as gum, or patch
20. Taking any other anti-inflammatory or immune modulating medications except for over the 
counter NSAIDs
21.Unwilling or unable to discontinue benzodiazepine usage (other than Lorazepam, 
Clonazepam, or Zolpidem) for one day prior to scanning
5.2.3
Immunization during TCZ therapy
Live/attenuated vaccines should not be given within 4 weeks prior to baseline and during the 
study as clinical safety has not been established. No data are available on the secondary 
transmission of infection from persons receiving live vaccines to patients receiving Tocilizumab. 
No data are available on the effectiveness of vaccination in patients receiving Tocilizumab. 
Because IL-6 inhibition may interfere with the normal immune response to new antigens, 
patients should be brought up to date on all recommended vaccinations, except for live vaccines, 
prior to initiation of therapy with Tocilizumab.


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5.3.
Treatment Assignment Procedures
5.3.1
Randomization Procedures
Each subject who meets all eligibility criteria and signs an informed consent form will be 
randomized to receive either 8 mg/kg of tocilizumab every 4 weeks or matching placebo for 8
weeks.
The randomization scheme will be developed by the study statistician and will indicate the group
assignment and the subject numbers to be used by each site. Subjects will be randomized in a 2:1 
ratio to intravenous tocilizumab 8 mg/kg or matching placebo using permuted random blocks 
stratified by site.
If a randomized subject withdraws from the study prior to initiating study 
drug, their assignment will be released and re-used.
5.3.2
Reasons for Withdrawal
A study subject will be discontinued from participation in the study if:
x
Any clinical AE (AE), laboratory abnormality, concurrent illness, or other 
medical condition or situation occurs such that continued participation in the 
study would not be in the best interest of the subject. Note, however, that any 
subject who has initiated treatment should only discontinue study drug and remain 
on study if the safety concern relates to study drug and not to study procedures.
Subjects are free to withdraw from participation in the study at any time upon request.
5.3.3
Handling of Withdrawals
A subject may choose to discontinue participation in the study at any time.  However, the SI or 
designee will encourage subjects to continue with follow-up visits, regardless of their 
compliance with study drug.  If the SI or designee is concerned about the use of a prohibited 
medication or other safety issues, then study drug may be modified or discontinued.  If a subject 
who initiated study drug permanently discontinues study drug, the SI or designee should still 
encourage subjects to follow the study protocol under the modified intent to treat principle (ITT). 
These subjects will be encouraged to follow the study visits, off drug, up to the Week 16 EOS 
Visit. Loss to follow-up should be prevented whenever possible.
Any subject who is on study drug and needs to begin the use of any prohibited medication, must 
immediately discontinue use of study drug and should not begin use of the prohibited medication
before an appropriate wash-out period occurs.  Subjects who permanently discontinue study drug 
should complete early study drug discontinuation procedures without any study drug unblinding, 


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if possible.  Subjects who must permanently discontinue study drug may continue in the ITT 
portion of the study, per protocol.
Early termination occurs when a subject withdraws consent, i.e., withdrawing his or her 
participation in future study procedures. The subject will be asked to return to the study site for a 
final safety visit. At that visit, the subject will be asked to have a final telephone call 28 days (+ 5 
days) after taking their last dose of study drug. Subjects who withdraw from the study due to 
AEs will be followed for outcome measures under the ITT protocol as noted above.  
5.3.4
Termination of Study 
This study may be prematurely terminated if, in the opinion of the principle investigator, DSMB,
or the sponsor, there is sufficient reasonable cause.  Written notification, documenting the reason 
for study termination, will be provided to the PI by the terminating party.
Circumstances that may warrant termination include, but are not limited to:
1. Determination of unexpected, significant, or unacceptable risk to subjects.
2. Enrollment is unsatisfactory.
3. Insufficient adherence to protocol requirements.
4. Data that are not sufficiently complete and/or evaluable. 
5. Plans to modify, suspend or discontinue the development of the study drug.
If the study is prematurely terminated or suspended, the sponsor will promptly inform the 
investigators/institutions, and the regulatory authority(ies) of the termination or suspension and 
the reason(s) for the termination or suspension.  The IRB/IEC will also be informed promptly 
and provided the reason(s) for the termination or suspension by the sponsor or by the 
PI/institution, as specified by the applicable regulatory requirement(s).


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6
TREATMENTS ADMINISTERED
6.1
Treatments
6.1.1
Study Product Description
Tocilizumab (Actemra®) is a recombinant humanized anti-human interleukin 6 (IL-6) receptor
monoclonal antibody of the immunoglobulin ,J*țJDPPDNDSSDVXEFODVVZLWKDW\SLFDO
H2L2 polypeptide structure. Each light chain and heavy chain consists of 214 and 448 amino 
acids, respectively. The four polypeptide chains are linked intra- and inter-molecularly by 
disulfide bonds. Tocilizumab has a molecular weight of approximately 148 kDa.
It is supplied as a sterile, preservative-free solution for intravenous (IV) infusion at a 
concentration of 20 mg per mL. It is a colorless to pale yellow liquid, with a pH of about 6.5. 
Single-use vials are available containing 80 mg per 4 mL, 200 mg per 10 mL, or 400 mg per 20 
mL of tocilizumab. Injectable solutions are formulated in an aqueous solution containing 
disodium phosphate dodecahydrate and sodium dihydrogen phosphate dehydrate (as a 15 mmol 
per L phosphate buffer), polysorbate 80 (0.5 mg per mL), and sucrose (50 mg per mL).
Tocilizumab will be provided free of charge by Genentech. The Sponsor or designee of the study 
will ensure maintenance of complete and accurate records of the receipt, dispensation, and 
disposal or return of all study drug in accordance with 21 Code of Federal Regulations (C.F.R.), 
Part 312.57 and 312.62 and Genentech requirements.
6.1.2
Placebo
A matched placebo will be used to maintain the dosage-blind.  The site pharmacy will prepare 
equivolume doses of normal saline as placebo. Administration of matching placebo will be the 
same as for subjects in the treatment group.
6.2
Acquisition
Upon receipt of the of the study treatment supplies, an inventory must be performed and a drug 
receipt log filled out and signed by the person accepting the shipment.  It is important that the 
designated study staff counts and verifies that the shipment contains all the items noted in the 
shipment inventory.  Any damaged or unusable study drug in a given shipment will be 


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documented in the study files.  The SI must notify study sponsor of any damaged or unusable 
study treatments that were supplied to the SI’s site.
6.2.1
Formulation, Packaging, and Labeling
The study drug is prepackaged in vials containing 80 mg, 200 mg or 400 mg that will be mixed 
with normal saline to a total volume of 100 mL by the site research pharmacist. The 
Investigational Pharmacist has the responsibility to ensure that the integrity of packaged study 
drug is not jeopardized prior to dispensing.  Each individual subject’s vials must be dispensed as 
provided with no further repackaging or labeling done at the investigational site, unless required 
by the institution per institutional polices.
6.2.2
Product Storage and Stability
Tocilizumab must be refrigerated at 2°C to 8°C (36°F to 46°F). It should not be frozen and 
should be protected from light by storage in the original package until time of use. Vials should 
be inspected visually for particulate matter and discoloration prior to administration. If visibly 
opaque particles, discoloration or other foreign particles are observed, the solution should not be 
used.
The SI must ensure that all investigational drug supplies are kept in a locked, safe area at 
controlled temperature (2°C to 8°C) with access limited to those directly involved in the study. 
Investigational drug supplies should not be repackaged in any way. 
6.3
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product
Tocilizumab concentrate for intravenous infusion should be diluted to 100 mL by the research 
pharmacist using aseptic technique as follows:
x
Withdraw the required amount of tocilizumab for a dose of 8 mg/kg (Equivalent
to 0.4 mL/kg).
x
Withdraw the same amount of sterile, non-pyrogenic 0.9% w/v sodium chloride 
solution from the infusion bag and discard.
x
Slowly add the withdrawn tocilizumab concentrate into the infusion bag. The final
volume in the infusion bag should total 100 mL.
x
To mix the solution, gently invert the bag to avoid foaming.
x
Allow the fully diluted tocilizumab solution to reach room temperature prior to 
infusion.
x
The infusion should be administered over 60 minutes, and must be administered 


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with an infusion set. Do not administer as an intravenous push or bolus.
Subjects should be weighed prior to each infusion, and dosage should be adjusted accordingly. 
One vial containing 400 mg tocilizumab or two vials containing 200 mg tocilizumab will be 
required for each 50 kg body weight to achieve an 8 mg/kg dose. The number of vials to be used 
depends on the patient’s body weight as follows:
1.
One 400-mg vial (or two 200-PJYLDOVLVXVHGIRUSDWLHQWVZLWKDERG\ZHLJKW
kg.
2.
Two 400-mg vials (or four 200-mg vials) are used for patients with a body weight 
>50 kg combination of the 400-mg and 200-mg vials may be used but the total dose 
should not exceed 800 mg
For individuals whose body weight is more than 100 kg, doses exceeding 800 mg per infusion 
are not recommended .
Tocilizumab solutions do not contain preservatives; therefore, unused product remaining in the 
vials should not be used.
Parenteral drug products should be inspected visually for particulate matter and discoloration 
prior to administration by the site research pharmacist, whenever solution and container permit. 
Tocilizumab is a colorless to pale, yellow liquid. If particulates or discolorations are noted, the 
product should not be used.
Fully diluted solutions are compatible with polypropylene, polyethylene and polyvinyl chloride 
infusion bags and polypropylene, polyethylene and glass infusion bottles. Tocilizumab should 
not be infused concomitantly in the same intravenous line with other drugs. No physical or 
biochemical compatibility studies have been conducted to evaluate the co-administration of 
tocilizumab with other drugs.
6.3.1
Tocilizumab Storage
Tocilizumab should not be used after the expiry date (EXP) shown on the pack.
For vials: Store between 2ºC – 8ºC, do not freeze. Keep the container in the outer carton
in order to protect from light.
For prepared infusion solution: Tocilizumab does not contain preservatives, therefore
reconstitution and dilution of the product should be performed under aseptic conditions. The
prepared infusion solution of tocilizumab is physically and chemically stable in 0.9% w/v
sodium chloride solution at 30ºC for 24 hours.


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From a microbiological point of view, the prepared infusion should be used immediately. If not 
used immediately, in-use storage times and conditions prior to use are the responsibility of the 
user and would not be longer than 24 hours at 2ºC – 8ºC, unless dilution has taken place in 
controlled and validated aseptic conditions.
6.3.2
Tocilizumab Overdosage
There are limited data available on overdoses with Tocilizumab. One case of accidental overdose 
was reported in which a patient with multiple myeloma received a dose of 40 mg/kg. No adverse 
drug reactions were observed. No serious adverse drug reactions were observed in healthy 
volunteers who received single doses of up to 28 mg/kg, although all 5 patients at the highest 
dose of 28 mg/kg developed dose-limiting neutropenia.
In case of an overdose, it is recommended that the patient be monitored for signs and symptoms 
of adverse reactions. Patients who develop adverse reactions should receive appropriate 
symptomatic treatment.
6.4
Modification of Study Intervention/Investigational Product for a Subject
Any dosage adjustment, including the reason for and dates of adjustment, will be documented in 
the CRF for each subject requiring this manipulation. In consultation with the Medical Monitor, 
the SI or licensed physician Sub-Investigator may reduce the dosage of study drug or discontinue 
the study drug in its entirety for AEs (AEs) thought to be related to the study drug or for other 
reason during the trial (the reason for, and dates of suspension or dose reduction must be 
documented). 
Opportunistic Infections and Serious Infections 
Tociluzimab should not be administered in patients with active infection. The effects of TCZ on 
CRP, neutrophils, and the signs and symptoms of infection should be considered when 
evaluating a patient for a potential infection. 
Vigilance for timely detection of serious infection is recommended for patients receiving 
biologic agents for treatment of moderate to severe RA as signs and symptoms of acute 
inflammation may be lessened due to suppression of the acute phase reaction. Patients must be 
instructed to contact their physician immediately when any symptoms suggesting infection 
appear, in order to assure rapid evaluation and appropriate treatment. 
If a patient develops a serious infection, administration of TCZ is to be interrupted until the 
infection is controlled. The clinician should consider the benefit-risk before resuming treatment 
with tocilizumab.


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Gastrointestinal Perforations  
Timely diagnosis and appropriate treatment may reduce the potential for complications of 
diverticulitis and thus reduce the risk of GI perforations. Therefore, patients should be made 
aware of the symptomatology potentially indicative of diverticular disease, and they should be 
instructed to alert their healthcare provider as soon as possible if these symptoms arise.  
Discontinuation of TCZ is recommended for patients who develop GI perforations.
Demyelinating Disorders 
The impact of treatment with TCZ on demyelinating disorders is not known; events were rarely 
reported.  Patients should be closely monitored for signs and symptoms potentially indicative of 
central demyelinating disorders.  Physicians should exercise caution in considering the use of 
TCZ in patients with pre-existing or recent onset demyelinating disorders. Treatment with 
tocilizumab should be interrupted during assessment of a potential demyelination event and only 
resumed if the benefit of continuing study drug is favorable.  
Hematologic Abnormalities and Bleeding Events
Decreases in neutrophil and platelet counts have been observed following treatment with TCZ in 
combination with MTX. In addition, there may be an increased risk of neutropenia in patients 
who have previously been treated with a TNF antagonist.
The risk mitigation strategies for neutropenia and thrombocytopenia are summarized below. For 
patients with concomitant medications associated with hematologic toxicity, the reduction or 
interruption of the suspected medication is recommended prior to modifying TCZ.
Low Absolute Neutrophil Count (ANC)
 (cells per mm3)
Recommendation
ANC 
greater 
than 1000 
Maintain dose 
ANC 500 to 1000 
Interrupt study drug dosing and check Complete Blood Count (CBC) weekly.  
When ANC greater than 1000 cells per mm3 resume tocilizumab at 4 mg per 
kg and increase to 8 mg per kg the next infusion if ANC remains greater 
than 1000. 
ANC less than 
500 
Discontinue study drug
Patients withdrawn from tocilizumab treatment due to a reduced neutrophil count should be 
monitored for signs of infection, with treatment as deemed appropriate by the sponsor or 
designee, and should have a repeat white blood cell count with differential performed weekly 


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until the ANC is above 1000 cells/mm3 (1.0 x 109/L). If the ANC does not return to above 1000 
cells/mm3 (1.0 x 109/L) within 2 months (or sooner if deemed necessary by the sponsor or 
designee), a hematology referral is recommended.
Low Platelet Count
Lab Value 
 (cells per mm3) 
Recommendation
Greater than 
100,000 
Maintain dose 
50,000 to 
100,000
Interrupt tocilizumabdosing and check CBC weekly. When platelet count is 
greater than 100,000 cells per mm3 resume study drug at 4 mg per kg and 
increase to 8 mg per kg if platelets remain greater than 100,000 
Less than 50,000 
Discontinue tocilizumab
Patients withdrawn from tocilizumab treatment due to a reduced platelet count should have a 
repeat platelet count performed weekly until the count is above 100,000 cells/mm3 (100 x 109/L). 
If the platelets do not return to above 100,000 cells/mm3 (100 x 109/L) within 2 months (or 
sooner if deemed necessary by the sponsor or designee), a hematology referral is recommended.
Elevated Liver Enzymes and Hepatic Events
Elevations in ALT and AST have been observed during treatment with the study
medications
Liver Enzyme Abnormalities  
Lab Value 
Recommendation 
Greater than 1 to 3x 
Upper limit of normal 
(ULN)  
Dose modify concomitant disease modifying anti-rheumatic drugs 
(DMARD)if appropriate
For persistent increases in this range, reduce tocilizumab
dose to 4 mg/kg or interrupt tocilizumab until ALT/AST
have normalized. Check Complete Metabolic Panel (CMP) every 2 
weeks until ALT/AST normal.
Restart with 4 mg/kg or 8 mg/kg, once ALT/AST normal and after 
discussion with medical monitor.  
Greater than 3 to 5x ULN 
(confirmed by repeat 
testing)  
Check CMP every 2 weeks until normal. If still greater than 3x ULN 
after 4 weeks, discontinue study drug 
Greater than 5x ULN  
Discontinue study drug 


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Patients withdrawn from tocilizumab treatment due to elevated liver function tests should have 
repeat tests performed, as clinically appropriate, until levels return to baseline.  If the patient’s 
liver function tests have not returned to baseline within 6 months (or sooner, if deemed necessary 
by the sponsor or designee), an ultrasound and/or liver biopsy should be considered.
Cardiovascular Events and Elevated Lipids
Patients with RA have an increased risk for cardiovascular disorders, therefore, risk factors for 
cardiovascular disease (eg, hypertension, hyperlipidemia) should be managed as part of their 
standard of care.  See section on Drug Interactions.  
)RUSDWLHQWVZLWK /'/ FKROHVWHUROPJG/ LWLVVWURQJO\ UHFRPPHQGHGWKDWLQYHVWLJDWRUV
advise therapeutic lifestyle changes that may include initiation lipid-lowering agents. Lipid-
lowering agents should also be considered for patients with lower LDL cholesterol levels as part 
of their therapeutic lifestyle changes depending on their overall risk as defined in NCEP ATP III 
or other national guidelines.
Malignancies
The impact of immunosuppression on the development of malignancies is not known, however 
an increased rate of some malignancies, notably lymphoma, has been observed in RA patients. 
Although no imbalance of malignancies was observed in controlled clinical trials of TCZ, 
malignancies have been identified as a concern for other biologics.  It is recognized that 
identification of such events in TCZ-treated patients may require a longer period of surveillance.  
TCZ should be discontinued in patients with malignancies (with the exception of local basal or 
squamous cell carcinoma of the skin that is completely excised with free margins).
Hypersensitivity or Anaphylaxis
An infusion/dose reaction is defined as an adverse event occurring during and within 24 hours 
after the infusion or subcutaneous injection of tocilizumab. This may include hypersensitivity 
reactions or anaphylactic reactions. 
Signs of a possible hypersensitivity reaction include but are not limited to: 
x
fever, chills, pruritus, urticaria, angioedema, and skin rash.
x
cardiopulmonary reactions, including chest pain, dyspnea, hypotension or hypertension.
Healthcare professionals administering TCZ infusions  should be trained in the appropriate 
administrative procedures, be able to recognize the symptoms associated with potential 
anaphylactic or hypersensitivity reactions, and have the appropriate medication available for 
immediate use in case of anaphylaxis or hypersensitivity reaction during or after administration 


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of TCZ.  Healthcare professionals should also instruct patients to seek medical attention if they 
experience symptoms of a hypersensitivity reaction outside of the clinic.
If a patient has symptoms of anaphylaxis or serious hypersensitivity, or requires an interruption 
of the study drug because of symptoms of anaphylaxis or hypersensitivity, administration of TCZ 
must be discontinued permanently and the patient should be withdrawn from the study.  The 
patient should be treated according to the standard of care for management of the 
hypersensitivity reaction.  A blood sample for the presence of anti-tocilizumab antibodies should 
be obtained at time of event and at least 6 weeks after the last dose*.
*6 weeks for the RA indication. For other indications consultation with the clinical 
pharmacology group will be required to assess how many weeks after the last dose testing should 
be conducted. 
Viral Reactivation
Though rarely reported within the TCZ program due to exclusion criteria at study entry,
reactivation of viral and other serious infections (e.g. EBV or TB) has been observed with
biologic therapies for RA, including TCZ.
Drug Interaction
The formation of CYP450 enzymes may be suppressed by increased levels of cytokines (eg, IL-
6) during chronic inflammation. Therefore, it is expected that for molecules that antagonize 
cytokine activity, such as TCZ, the formation of CYP450 enzymes could be normalized. Given 
its long elimination half-life (t1/2), the effect of tocilizumab on CYP450 enzyme activity may 
persist for several weeks after stopping therapy.
6.4.1. Dosage Discontinuation
Subjects who meet the following criteria should discontinue study medication:


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6.4.1.1. Tocilizumab-Specific Criteria
x
Anaphylaxis or hypersensitivity reaction or requires an interruption of the study drug 
because of symptoms of anaphylaxis or hypersensitivity (tocilizumab should be 
permanently discontinued from these patients)
x
ALT or AST value > 5X ULN or persistent elevation > 3X ULN
x
Platelet count (cells/mm3) < 50,000
x
ANC (cells/mm3) < 500
6.4.1.2. General Criteria
x
AE
x
Sponsor termination
x
Protocol deviation/Lost to follow-up
x
Patient request
x
Death
x
Inability of subject to comply with study requirements
x
Determination by the Medical Monitor or Site Investigator that it is no longer safe for 
the subject to continue therapy
All serious AEs (SAEs) that occur in a subject who has discontinued early must be recorded and 
reported to the CC within 24-hours of awareness.  
Study subjects who discontinue study drug prematurely (early termination from study) and 
decide to not remain in the ITT portion of the study will be encouraged to return for a Final 
Safety Visit and participate in a Follow-Up Telephone Call 28 days (+ 5 days) after the last dose 
of study drug. 
All subjects who discontinue study drug early and choose to remain in the ITT portion of the 
study will be encouraged to follow the study visits, off drug, up through the last visit (Week 16
EOS Visit).  
SAEs will be followed for resolution for 28 days (+5 days) after a subject’s last dose of study 
drug, regardless if they prematurely discontinued study drug or completed 4 weeks of treatment. 
6.5
Accountability Procedures for tocilizumab
At the completion of the study, there will be a final reconciliation of drug shipped, drug 
consumed, and drug remaining.  This reconciliation will be logged on the drug reconciliation 
form, signed and dated.  Any discrepancies noted will be investigated, resolved, and documented 


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prior to return or destruction of unused study drug.  Drug destroyed on site will be documented 
in the study files.
6.6
Prior and Concomitant Therapy
Throughout the study, SIs may prescribe concomitant medications or treatments including 
edaravone (Radicava®), deemed necessary to provide adequate supportive care provided that the 
medications are licensed in the United States. Study subjects should not receive other 
experimental agents during the study. This includes marketed agents at experimental dosages 
that are being tested for the treatment of ALS and experimental procedures such as stem cell 
therapies or diaphragm pacing devices. All concomitant medications and/or treatments and 
significant non-drug therapies including supplements and assistive devices, received by a subject 
should be recorded on the appropriate source document and eCRF at each study visit.
Any investigational pharmacotherapy being used or evaluated for the treatment of ALS is 
prohibited beginning 30 days prior to the Screening Visit and throughout the study.  This 
includes, but is not limited to, the following:
x
Pioglitazone
x
Arimoclomol
x
Olanzapine
x
Nuedexta
x
NP001
x
Ibudilast
x
Fingolimod
x
GSK 1223249
x
Rasagiline
x
Memantine
x
Ezogabine
6.6.1
Prohibited Medications and Contraindications
Prohibited Medications
Throughout the course of the trial, study subjects should not be treated with the following 
medications.  If an investigator learns that a patient has begun therapy with any of these 
medications, this should be reported to the CC immediately and action should be taken to 
discontinue the prohibited medication.  If, for safety or health reasons, or by subject choice, the 
prohibited medication cannot be stopped, then the study medication should be stopped.
Prohibited medications include:


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x
Nuedexta
x
Theophylline
x
Cyclosporine
x
Warfarin
x
Live vaccines
Pregnancy & Nursing Mothers
There are no adequate and well-controlled studies in pregnant women.  Subjects or partners of 
male subjects should not become pregnant during the study or within 6 months after stopping 
study drug.
If a female subject becomes pregnant, study treatment must be discontinued 
immediately. 
It is not known whether tocilizumab is excreted in human milk or absorbed systemically after 
ingestion. Caution should be exercised; therefore, no subject should nurse her infant while 
participating in this study.
MR-PET (MGH only): Additionally, subjects should withhold taking benzodiazepines for 24 
hours prior to scanning. Lorazepam, Clonazepam, and Zolpidem are allowed under the protocol 
within this timeframe.
6.7
Blinding of Study Drug
The Randomization ID will be used to identify the subject's electronic case report forms 
(eCRFs), laboratory tests, study medication and all communications.  
Study Subjects, Site Investigators, Coordinators, Clinical Evaluators (and all other study site 
staff), Study Monitors, Project Management, Data Management personnel, and the Sponsor will 
be blinded to treatment group assignment throughout the study. 
Only the Biostatistician who develops the randomization schedule, the Research Pharmacists at 
the Central Pharmacy and the Site Pharmacist (if applicable) storing and distributing the study 
drug at each site will be unblinded to the individual drug assignments in this study.
6.7.1. Emergency Unblinding
An emergency unblinding procedure will allow the Site Investigator (SI) the option of learning 
the treatment assignment for an individual subject if clinical circumstances require it.  For 
unblinding, the SI must contact the Medical Monitor (MM).  The SI should attempt to contact the 
MGH Coordination Center (CC) prior to completing emergency unblinding if time allows. If this 


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is not possible, the site must inform the MGH CC as soon as possible regarding the emergency 
unblinding and the circumstances surrounding it.  The SI must document the reason for 
unblinding in the subject’s source documents. 
Rarely should such an extreme action be taken.  Experimental medications can usually be 
withdrawn without the need for unblinding.  Sites should take care to unblind only those study 
staff member(s) and other persons whose knowledge of the treatment assignment is necessary for 
the clinical safety of the subject.  Sites should take all reasonable measures to keep study staff 
(especially those completing any Final Safety Visit procedures), MGH CC staff and the subject 
blinded to treatment assignment if possible.  
In the event that emergency disclosure of treatment assignment is necessary, the study subject 
will be withdrawn from further participation in the trial.  All adverse events (AEs) resulting in 
emergency unblinding will be followed for resolution.  


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7
STUDY SCHEDULE
No study procedures should be performed prior to the signing of the informed consent form
(ICF). All subjects will sign an ICF prior to undergoing any study tests or procedures. The slow 
vital capacity (SVC) should be performed first at the visits so as not to fatigue the subject with 
other testing, however the order of testing will be at the discretion of each SI.
Visit windows are consecutive calendar days and the target visit dates are calculated from the 
Baseline Visit.  
7.1
Screening Visit
The following procedures will be performed at an office visit to determine the subject’s 
eligibility for the study:
1. Obtain written informed consent from subject
2. Assess inclusion and exclusion criteria
3. Obtain medical history and demographics
4. Obtain ALS diagnosis history
5. Measure vital signs including height and weight
6. Perform neurological examination
7. Perform physical examination
8. Perform slow vital capacity (SVC)
9. Perform 12-lead electrocardiogram (ECG)
10. PA and Lateral Chest X-Ray
11. Collect blood samples for baseline clinical laboratory assessments (CBC with differential 
and chemistry panel), Quantiferon or T-SPOT TB test (or TB skin test), Hepatitis B panel
and serum pregnancy test (for woman of childbearing potential [WOCBP])
12. Collect urine sample for urinalysis
13. Collect blood samples for PBMC collection
14. Review and document concomitant medications and therapies
15. Assess and document AEs (AEs) after subject signs informed consent form (ICF)
16. Tentatively schedule the Baseline Visit  
Pregnancy Test: Serum pregnancy test will be done at the Screening Visit for women of child
bearing potential (WOCBP).  Thereafter, urine pregnancy testing will be performed on an as 
needed basis throughout the study.
MR-PET (MGH only):
17. Blood Draw for TSPO Binding Affinity Test (**If not previously determined)


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18. Upper Motor Neuron Burden Scale.
19. MRI Safety Questionnaire
Binding affinity: Venous blood will be collected and sent to the Partners Healthcare Center for 
Personalized Genetic Medicine to genotype for the Ala147Thr TSPO polymorphism in the TSPO 
gene (rs6971) [18] . About 10% of human subjects show low binding to PBR28.  High and 
mixed affinity binders (Ala/Ala or Ala/Thr) will be eligible, whereas the low affinity binders 
(Thr/Thr) will be ineligible to enroll.
7.1.1
Screen Failures
Any subject who signs consent will be considered enrolled in the study.  If a subject fails 
screening, at a minimum, the following information should be captured and entered in the 
Electronic Data Capture (EDC) System:
o
Inclusion and Exclusion Criteria
o
Demographics
o
Reason for screen failure
7.2
Baseline Visit
This visit will take place within 28 days of the Screening Visit.  The following procedures will 
be performed:
1. Randomize subject (approximately 24 hours prior to the subject’s Baseline visit)
2. Review inclusion and exclusion criteria 
3. Measure vital signs including weight
4. Administer C-SSRS
5. Administer ALSFRS-R questionnaire 
6. Perform SVC [Note: Baseline Visit SVC is not exclusionary, even if below 40%]
7. Perform Hand-held Dynamometry (HHD) and Grip Strength 
8. Collect blood samples for clinical laboratory assessments
9. Collect blood samples for cytokine levels, hs-CRP concentration and PBMC collection
10. Collect blood sample for IL-6 receptor genotyping
11. Collect a blood sample for Genentech
12. Collect urine sample for urinalysis
13. Review and document concomitant medications and therapies
14. Assess and document AEs
15. Perform LP for CSF collection
16. Schedule next Study Visit
17. Administer 1st dose of study drug after all baseline study procedures are complete. The 
subject should be observed at the site for 60 minutes after the infusion by an appropriate 


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healthcare staff member according to the site’s institutional/state regulations to assess 
medical status and any immediate reaction to the study drug.
Post-Lumbar Puncture Follow-up Telephone Call 
Subjects will receive a phone call 24-48 hours after their LP to assess and document AEs. 
MR-PET (MGH only) (* -2 Week window for these procedures to be conducted):
1. Pre-dose neuroimaging study (MRI/PET) will be performed one time
2. Upper Motor Neuron Burden Scale.
3. MRI Safety Questionnaire
7.3
Week 4 (Visit 1)
This visit will take place 28 ± 3 days after the Baseline Visit.  The following procedures will be 
performed:
1. Measure vital signs including weight
2. Administer C-SSRS
3. Administer ALSFRS-R questionnaire 
4. Perform SVC
5. Collect blood samples for clinical laboratory assessments 
6. Collect urine sample for urinalysis
7. Collect blood samples for cytokine levels, hs-CRP concentration and PBMC collection
8. Review and document concomitant medications and therapies
9. Assess and document AEs
10. Administer 2nd dose of study drug.  The subject should be observed at the site for 30
minutes after the infusion by an appropriate healthcare staff member according to the 
site’s institutional/state regulations to assess medical status and any immediate reaction to 
the study drug.
11. Schedule next Study Visit 
7.4
Week 8 (Visit 2)
This visit will take place 56 ± 3 days after the Baseline Visit. The following procedures will be 
performed:
1. Measure vital signs including weight
2. Administer C-SSRS
3. Administer ALSFRS-R questionnaire 
4. Perform SVC
5. Perform HHD and Grip Strength


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6. Collect blood samples for clinical laboratory assessments 
7. Collect urine sample for urinalysis
8. Collect blood samples for cytokine levels, hs-CRP concentration and PBMC collection
9. Review and document concomitant medications and therapies
10. Assess and document AEs
11. Administer 3rd dose of study drug.  The subject should be observed at the site for 30 
minutes after the infusion by an appropriate healthcare staff member according to the 
site’s institutional/state regulations to assess medical status and any immediate reaction to 
the study drug.
12. Perform 2nd LP CSF collection 3 hours +/- 30 minutes after the start of study drug 
infusion.
13. Schedule next Study Visit 
Post-Lumbar Puncture Follow-up Telephone Call 
Subjects will receive a phone call 24-48 hours after their LP to assess and document AEs that 
may be directly related to the LP. 
MR-PET (MGH only) (* -2 Week window for these procedures to be conducted):
1. Post-dose neuroimaging study (MRI/PET) will be performed one time
2. Upper Motor Neuron Burden Scale.
3. MRI Safety Questionnaire
7.5
Week 12 (Visit 3)
This visit will take place 84 ± 3 days after the Baseline Visit. The following procedures will be 
performed:
1. Measure vital signs including weight
2. Administer C-SSRS
3. Administer ALSFRS-R questionnaire 
4. Perform SVC
5. Collect blood samples for clinical laboratory assessments 
6. Collect urine sample for urinalysis
7. Collect blood samples for cytokine levels, hs-CRP concentration and PBMC collection
8. Review and document concomitant medications and therapies
9. Assess and document AEs
10. Schedule next Study Visit 
7.6
Week 16 (EOS Visit)
This visit will take place 112 ± 3 days after the Baseline Visit. The following procedures will be 
performed:


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1. Perform neurological examination
2. Perform physical examination
3. Measure vital signs including weight
4. Administer C-SSRS
5. Administer ALSFRS-R questionnaire 
6. Perform SVC
7. Perform HHD and Grip Strength
8. Collect blood samples for clinical laboratory assessments
9. Collect urine sample for urinalysis
10. Collect blood samples for cytokine levels, hs-CRP concentration and PBMC collection
11. Review and document concomitant medications and therapies
12. Assess and document AEs
13. Exit questionnaire
7.7
Final Safety Visit & Telephone Call
Subjects who withdraw consent and do not agree to be followed for ITT will be asked to come in 
for a Final Safety Visit and will be asked to have a final Follow-Up Telephone Call 28 (+ 5) days 
after the subject’s last dose of study drug.
1. The final visit should be scheduled as soon as possible. The following will be performed:
2. Perform neurological examination
3. Perform physical examination
4. Measure vital signs including weight
5. Administer C-SSRS
6. Administer ALSFRS-R questionnaire 
7. Perform SVC
8. Perform HHD and Grip Strength
9. Collect blood samples for clinical laboratory assessments
10. Collect urine sample for urinalysis
11. Collect blood samples for cytokine levels, hs-CRP concentration and PBMC collection
12. Review and document concomitant medications and therapies
13. Assess and document AEs
This phone call will take place 28 days after the subject’s last dose of study drug.  The following 
procedures will be performed:
14. Review and document concomitant medications and therapies
15. Assess and document AEs
7.8
Protocol Deviations
A protocol deviation is any non-compliance with the clinical trial protocol, Good Clinical 
Practice (GCP), or Manual of Procedures requirements.  The noncompliance may be either on 


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the part of the subject, the SI, or the study site staff.  As a result of deviations, corrective actions 
are to be developed by the site and implemented promptly.
All deviations from the protocol must be addressed in the subject’s source documents.  Protocol 
deviations must be sent to the local IRB per their guidelines and entered in the Protocol 
Deviations Log in the Electronic Data Capture (EDC) System.
7.9
Missed Visits and Procedures 
Missed visits and any procedures not performed (not attempted) for reasons other than illness, 
injury or progressive disability (i.e. subject is physically unable to perform test) will be reported 
as protocol deviations.
Procedures or visits not performed due to illness, injury or disability, including procedures that 
were attempted but failed (i.e. blood samples unable to be drawn after multiple attempts, or 
weight unable to be obtained due to subject immobility) will not be reported as protocol 
deviations unless there is a patient safety concern.
In the event of illness, injury or disability, every attempt will be made to complete each visit.  In 
the event that the visit is not completed, a phone visit will be performed to review concomitant 
medications, perform the ALSFRS-R, and record AEs.
Study drug compliance that is outside the limits set in the study operations manual will be 
reported as a protocol deviation. 
Details and specific instructions regarding protocol deviations, including any exceptions to this 
standard procedure, are found in the study operations manual.


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8
CLINICAL ASSESSMENTS AND OUTCOME MEASURES
8.1
Clinical Variables
Assessments will be performed at designated time-points throughout the study for clinical 
evaluation.  In addition to the assessments evaluated below, subjects will provide information on 
their demographics, past medical history, including ALS, as well as concomitant medication 
usage.
8.1.1
Vital Signs, Height & Weight
Vital signs will be obtained after the subject has been in a seated position for several minutes.  
Vital signs, including systolic and diastolic blood pressure, pulse rate (radial artery)/minute, 
respiratory rate/minute, temperature and weight will be assessed at specified visits.  Height will 
be measured and recorded at the Screening Visit only.  
8.1.2
PBMC Blood Collection, hs-CRP concentration and Cytokine Levels
Blood will be collected to measure the expression of inflammatory genes in peripheral blood 
mononuclear cells (PBMCs).  Plasma cytokine levels, including IL-6, IL-17A, IL-ȕ IL-8,
MMP1, TNF-ĮV,/-6 receptor and hs-CRP, will also be assessed. These samples will be sent to 
Dr. Robert Bowser’s laboratory at the Barrow Neurological Institute (BNI) for analysis.  
In addition, a blood sample will be sent to Genentech, Inc. in California at baseline and again at 
the time of a hypersensitivity, anaphylaxis or drug discontinuation event. All samples will be 
labeled with a code.  The code will not include any identifiable information.
8.1.4
12-Lead Electrocardiogram (ECG)
A standard 12-lead ECG will be performed at the Screening Visit only.
Tracings will be 
reviewed by a site cardiologist and a copy of the tracings will be kept on site as part of the source 
documents.
8.1.5
Chest X-Ray
A standard PA and Lateral Chest X-Ray will be performed at the Screening Visit to assess for 
evidence of active pulmonary tuberculosis (TB). A copy of the report will be kept on site as part 
of the source documents.


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8.1.6
Physical Examination
A physical examination will be performed and recorded at the Baseline Visit.  The following 
systems will be examined: head/neck, eyes, ears, nose/throat, cardiovascular, lungs, abdomen, 
musculoskeletal, central nervous system, extremities, and skin.  
8.1.7
Neurological Examination
A complete neurological examination will be performed and recorded at the Baseline Visit.
8.1.8
Columbia Suicide Severity Rating Scale (C-SSRS)
The US FDA recommends the use of a suicidality assessment instrument that maps to the 
Columbia Classification Algorithm for Suicide Assessment (C-CASA)[19]. The C-CASA was 
developed to assist the FDA in coding suicidality data accumulated during the conduct of clinical 
trials of antidepressant drugs. One such assessment instrument is the Columbia Suicide Severity 
Rating Scale (C-SSRS). The C-SSRS involves a series of probing questions to inquire about 
possible suicidal thinking and behavior.
At the Baseline Visit, the C-SSRS Baseline version will be administered.  This version is used to 
assess suicidality over the subject’s lifetime and specifically for the previous 6 month time 
period.
At all other study visits, the Since Last Visit version of the C-SSRS will be administered. This 
version of the scale assesses suicidality since the subject’s last visit.
8.1.9
IL-6 Genotyping
Blood will be collected and sent to the Hawkins lab at Wake Forest. DNA will be isolated and 
the IL-6 receptor gene will be sequenced. Common polymorphisms, including one previously 
described in patients with asthma, will be identified and be entered into the EDC record of each 
patient.
8.1.10 Lumbar Puncture
Lumbar punctures will be performed and CSF will be collected at the Baseline Visit and Visit 2. 
All patients will receive the Baseline Visit LP prior to receiving the first dose of study drug.
Patients will receive the visit 2 LP 2 hours +/- 30 minutes after completion of the Visit 2 study 
drug infusion administration. The SI will discuss all potential LP risks to the subjects including:
o
Local pain at injection site;
o
Reaction to anesthetic agents;
o
Bleeding at needle entrance site; 


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o
Infection at needle entrance site; and,
o
Post-LP low-pressure headache.  
Extensive experience with research LP in Alzheimer’s disease reveals a very low incidence of 
complication, including the incidence of PLPH3.  Fewer than 2.6% of patients in a memory 
disorder clinic developed PLPH, and only a single patient in a cohort of over 1000 had a 
headache lasting more than 5 days [20]. No other local or generalized complications occurred.  
The procedure must be performed by the SI or another licensed practitioner with experience and 
training in performing LPs, and who is listed on the site delegation log. LPs will be performed 
with an atraumatic Sprotte needle to reduce the risk of PLPH. CSF samples will be sent to Dr. 
Bowser’s laboratory at BNI to determine the concentration of inflammatory cytokines (IL-6, IL-
ȕDQGsIL-6 receptor) in the CSF before and after treatment with tocilizumab vs. placebo. In 
addition, CSF will be sent to the Hawkins lab at Wake Forest for measurement of sIL-6 receptor 
levels.
Remaining CSF samples will be stored in a sample repository.  These samples will be used for 
future research in motor neuron diseases.  All samples will be labeled with a code.  The code will 
not include any identifiable information.  
Any research performed on the samples is for research purposes only.  Although genetic 
information may be analyzed, no genetic information will be given to the subject nor will the 
information that may be obtained be placed in a subject’s medical records. 
There is no scheduled date on which the samples will be destroyed.  Samples may be stored for 
research until they are used, damaged, decayed or otherwise unfit for analysis. Subjects have the 
option of declining participation in this portion of the study at any time by withdrawing their 
consent to have their sample used.  However, it will not be possible to destroy samples that may 
have already been used.
8.1.11 Adverse Events
Adverse events will be documented at each study visit, including the Screening Visit once the 
informed consent form has been signed by the subject, and at all study visits. Information on 
adverse effects of study medication and on inter-current events will be determined at each visit 
by direct questioning of the subjects, review of concomitant medications, and vital sign results. 


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8.1.12 Exit Questionnaire
An exit questionnaire will be completed by subjects and Site Investigators at the End Of Study 
Visit (Week 16). This will include questions regarding blindedness, LP and infusion experiences, 
and overall views of the quality of the trial. 
8.2
Outcome Measure
8.2.1
ALSFRS-R
The ALSFRS-R is a quickly administered (5 minutes) instrument used to determine subjects' 
assessment of their capability and independence in 12 functional activities, each rated on an 
ordinal scale (ratings 0-4).  All 12 activities are relevant in ALS. Initial validity was established 
by documenting that in ALS patients, change in ALSFRS-R scores correlated with change in 
strength over time, was closely associated with quality of life measures, and predicted survival. 
The test-retest reliability is greater than 0.88 for all test items. The advantages of the ALSFRS-R
are that the categories are relevant to ALS, it is a sensitive and reliable tool for assessing 
activities of daily living function in those with ALS, and it is quickly administered.  With 
appropriate training the ALSFRS-R can be administered with high inter-rater reliability and test-
retest reliability.  The ALSFRS-R can be administered by phone with good inter-rater and test-
retest reliability.  The equivalency of phone versus in-person testing, and the equivalency of 
study subject versus caregiver responses have also recently been established.
Therefore, if 
necessary, the ALSFRS-R may be given to the study subject over the phone. All ALSFRS-R
evaluators must be NEALS certified.
8.2.2
Slow Vital Capacity (SVC) Testing
The vital capacity (VC) (percent of predicted normal) will be determined, using the upright slow 
VC method. The VC can be measured using conventional spirometers that have had a calibration 
check prior to subject testing.  A printout from the spirometer of all VC trials will be retained. 
All VC evaluators must be NEALS certified. Three VC trials are required for each testing 
session, however up to 5 trials may be performed if the variability between the highest and 
second highest VC is 10% or greater for the first 3 trials with variability being calculated as 
[(Highest VC-Second highest VC)/ Highest VC] x 100. Only the 3 best trials are recorded on the 
CRF. The highest VC recorded is utilized for eligibility. 
8.2.3
Hand-held Dynamometry (HHD) and Grip Strength
Hand Held Dynamometry (HHD):
Hand held dynamometry (HHD) will be used as a
quantitative measure of muscle strength for this study.  Six proximal muscle groups will be 
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extension, hip flexion, knee flexion, and knee extension), all of which have been validated 
against maximum voluntary isometric contraction (MVIC) testing [21]. In addition, wrist 
extension, first dorsal interosseous contraction and ankle dorsiflexion will be measured
bilaterally; these muscles are often affected in ALS.  Mean and standard deviation for each 
muscle group will be established from the initial values for each subject in this trial, so that 
strength determinations can be converted to Z scores and averaged to provide an HHD 
megascore for both upper and lower extremities.   
Grip Strength:  Bilateral hand grip will be measured using a study approved dynamometer to test 
the maximum isometric strength of the hand and forearm muscles.  
8.2.4
Training and Validation
All evaluators must be NEALS certified to perform the ALSFRS-R, SVC and HHD; specific 
certification requirements are outlined in the study operations manual. Repeat NEALS 
certification will be required on a bi-annual basis for all outcome measures. It is strongly 
preferred that a single evaluator performs all measures throughout the study, if possible. NEALS 
certification is required for all evaluators prior to performing any study tests.
8.2.5
Neuroimaging
MGH Subjects will undergo Magnetic Resonance Imaging (MRI) / PBR28 Positron Emission 
Tomography (PET) twice during the course of the study. The goal of these scans is to measure 
activated microglia in study participants before treatment and after several weeks of treatment. 
The first scan will occur at the Baseline visit (Pre-dose), after subject eligibility has been 
confirmed by TSPO affinity binding assay. The second scan will occur at the Week 8 (Post-
dose). Neuroimaging will be performed at the MGH Martinos Center for Biomedical Imaging.  
Subjects will be asked to lie still in a supine position for the duration of the study, which will 
take approximately 90 min. 
Each PET scan will include one administration of up to 15 mCi of [11C]PBR28 (which is 
equivalent to ~3.7 mSv), injected intravenously with a slow bolus over a 30 s period. The 
catheter will be flushed post-injection with 0.9% saline solution. Dynamic data will be collected 
over approximately 90 minutes in list mode, and framed post-collection. 
 
8.2.5.1 Imaging Data Acquisition and Proccessing (MGH Only)
Simultaneous PET-MRI Data Acquisition
The proposed studies will benefit from our experience using a unique PET-MRI scanner called 
BrainPET. This scanner consists of a dedicated brain avalanche photodiode-based PET scanner 


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that operates in the bore of a 3T whole body MR scanner. Our center has over seven years’ 
experience using this scanner and has successfully completed over 200 human studies. Our data 
suggest that high quality PET data can be acquired fully simultaneously with conventional and 
advanced MRI data without substantial artifacts. The simultaneous acquisition of PET and MRI 
data reduces scan time, which minimizes the discomfort experienced by our ALS patient 
population. In addition, the simultaneous PET/MRI acquisition allows us to apply our novel 
MRI-based methods for generating attenuation correction maps, which enable PET motion 
correction and improve image quality [22, 23].
PET Data
Up to 15 mCi of [11C]PBR28 or ~5 mCi (4.8 ± 0.4 mCi) of [11C]PBR28 will be injected 
intravenously with a slow bolus over a 30 second period [24]. The catheter will be flushed post-
injection with 0.9% saline solution. Dynamic data will be collected over approximately 90 
minutes in list mode, and framed post-collection. The PET list-mode data will first be sorted in 
the line-of-response (LOR) space and motion corrected using MRI-derived motion estimates for 
each individual frame [22, 23]. Next, the data will be rebinned in the sinogram space, generating 
prompt and random coincidences sinograms. The head attenuation map (μ-map) will be obtained 
using a recently implemented MR-based attenuation correction method [22, 23]. The scatter 
sinogram will be obtained using a calculated method based on the single scatter estimation 
method[25]. The images will be reconstructed using the Ordinary Poisson Ordered Subset 
Expectation Maximization (OP-OSEM) 3D algorithm from prompt and expected random 
coincidences, normalization, attenuation and scatter coincidences sinograms using 16 subsets and 
six iterations [26]. The reconstructed volume consists of 153 slices with 256×256 pixels 
(1.25×1.25×1.25 mm3). Regional uptake of the tracer will be estimated as described below.
MRI Data
A high resolution structural volume will be collected for the purposes of anatomical localization, 
ROI definition, and structural analyses. A multi-echo MPRAGE pulse sequence will be utilized, 
with an isotropic voxel of 1 mm. Cortical reconstruction and volumetric segmentation will be 
performed with the Freesurfer image analysis suite (http://surfer.nmr.mgh.harvard.edu/). A dual-
echo ultrashort echo time MRI sequence (DUTE) will be collected to determine attenuation 
correction for PET data. Other routine exploratory MRI sequences will be acquired including:  a) 
Magnetic Resonance Spectroscopy (MRS) to measure NAA, Choline, Creatine, and MyoInositol 
in the motor cortices; and b) Diffusion tensors imaging (DTI) to detect changes in the 
corticospinal tract in correlation with PBR28 uptake.


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8.2.5.2 Imaging Analysis Plan
Region of interest (ROI) analyses will be conducted to detect differences in brain uptake of 
PBR28 (Pre-dose compared to Post-dose) as described in [12, 13].
Standard uptake volumes (SUV) images for PBR28 will be created for radioactivity in the field 
of view 60–90 minutes post-radioligand injection. To account for motion that may have occurred 
between MP-RAGE acquisition and the 60–90 minute post-injection time point corresponding to 
the PET frame of interest, SUV60–90 min will be generated in a two-step procedure. First, a 
SUV60–90 min image will be created for each subject using an attenuation correction map 
computed from the native MP-RAGE. Subsequently, a new attenuation map will be created 
based on the registration of the native MP-RAGE with the SUV60–90 min image obtained in the 
first reconstruction using FreeSurfer3s spmregister. A final SUV60–90 min will then be 
reconstructed based on this new attenuation correction image well registered with the 60–90 min 
PET data. Individual SUV60–90 min images will then be registered to MNI (Montreal 
Neuroogical Institute) space, spatially smoothed (6 mm full width at half maximum), and 
intensity-normalized to a mean of 1 (SUVR60–90 min) in order to account for differences in 
global signal across subjects, as previously described [12]. Average SUVR60–90 min in 
predetermined ROIs for the left and right precentral gyri for both the gray matter and the 
underlying white matter will be derived from the automatic T1 segmentation in Freesurfer (20)
will be estimated from [11C]-PBR28 SUVR in the ROIs. 
8.2.6 Upper Motor Neuron Burden (UMNB) Scale
The Upper Motor Neuron-Burden (UMNB) Scale assesses pathological UMN signs on 
examination.  The UMNB measures the following deep tendon (scores 0–4) and pathological 
reflexes (present — 1 or absent — 0): biceps, brachioradialis, triceps, knee jerk, ankle jerk, 
Hoffman, Babinski, and jaw jerk. The total UMNB score ranges from 0 to 45, with 0 
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9
SAFETY AND ADVERSE EVENTS
The AE definitions and reporting procedures provided in this protocol comply with all applicable 
United States Food and Drug Administration (FDA) regulations and International Conference on 
Harmonization (ICH) guidelines.  The SI will carefully monitor each subject throughout the 
study for possible AEs.  All AEs will be documented on CRFs designed specifically for this 
purpose.  It is also important to report all AEs, especially those that result in permanent 
discontinuation of the investigational product being studied, whether serious or non-serious.
9.1
Definitions of AEs, Suspected Adverse Drug Reactions & SAEs
9.1.1
Adverse Event and Suspected Adverse Drug Reactions
An AE is any unfavorable and unintended sign (including a clinically significant abnormal 
laboratory finding, for example), symptom, or disease temporally associated with a study, use of 
a drug product or device whether or not considered related to the drug product or device. 
Adverse drug reactions (ADR) are all noxious and unintended responses to a medicinal product 
related to any dose.  The phrase “responses to a medicinal product” means that a causal 
relationship between a medicinal product and an AE is at least a reasonable possibility, i.e., the 
relationship cannot be ruled out.  Therefore, a subset of AEs can be classified as suspected 
ADRs, if there is a possible causal relationship to the medicinal product.
Examples of AEs include: new conditions, worsening of pre-existing conditions, clinically 
significant abnormal physical examination signs (e.g., skin rash, peripheral edema), or clinically 
significant abnormal test results (e.g., lab values or vital signs), with the exception of outcome 
measure results, which are not being recorded as AEs in this trial (they are being collected, but 
analyzed separately). Stable chronic conditions (e.g., diabetes, arthritis) that are present prior to 
the start of the study and do not worsen during the trial are NOT considered AEs.  Chronic 
conditions that occur more frequently (for intermittent conditions) or with greater severity, 
would be considered as worsened and therefore would be recorded as AEs.
AEs are generally detected in two ways:
Clinical Æ symptoms reported by the subject or signs detected on examination.


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Ancillary Tests Æ abnormalities of vital signs, laboratory tests, and other diagnostic 
procedures (other than the outcome measures, the results of which are not being captured 
as AEs).
For the purposes of this study, symptoms of progression/worsening of ALS, including ‘normal’ 
progression, will be recorded as AEs.    
The following measures of disease progression will not be recorded as AEs even if they worsen 
(they are being recorded and analyzed separately): vital capacity results and ALSFRS-R results. 
If discernible at the time of completing the AE log, a specific disease or syndrome rather than 
individual associated signs and symptoms should be identified by the SI and recorded on the AE 
log.  However, if an observed or reported sign, symptom, or clinically significant laboratory 
anomaly is not considered by the SI to be a component of a specific disease or syndrome, then it 
should be recorded as a separate AE on the AE log.  Clinically significant laboratory 
abnormalities, such as those that require intervention, are those that are identified as such by the 
SI.
Subjects will be monitored for AEs from the time they sign consent until completion of their 
participation in the study (defined as death, consent withdrawal, loss to follow up, early study 
termination for other reasons, or following completion of the entire study).  
An unexpected AE is any AE, the specificity or severity of which is not consistent with the 
current Investigator’s Brochure.  An unexpected, suspected adverse drug reaction is any 
unexpected AE for which, in the opinion of the SI or Sponsor, there is a reasonable possibility 
that the investigational product caused the event.
9.1.2
Adverse Events of Special Interest
Adverse events of special interest (non-serious and serious) are required to be reported by the 
Investigator to the CC within 24 hours after learning of the event. The CC must forward this 
information to Genentech Drug safety within 24 hours of receipt (see Section 9.3 for reporting 
instructions).  Non-serious and serious AEs of special interest for this study include the 
following:
x
Infections including all opportunistic infections and non-serious infections as
defined by those treated with IV anti-infectives.
x
Suspected transmission of an infectious agent by the medicinal product (STIAMP). Any 
organism, virus, or infectious particle (e.g., prion protein transmitting transmissible 
spongiform encephalopathy), pathogenic or non-pathogenic, is considered an infectious 


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agent. A transmission of an infectious agent may be suspected from clinical symptoms 
or laboratory findings that indicate an infection in a patient exposed to a medicinal 
product. This term applies only when a contamination of the study drug is suspected.
x
Myocardial infarction/acute coronary syndrome.
x
GI perforation and related events.
x
Malignancies.
x
Hypersensitivity reactions.
x
Demyelinating disorders.
x
Stroke.
x
Bleeding events.
x
Hepatic events.
Guided questionnaires have been prepared for the AEs of special interest.  
The notification of AEs of special interest (including non-serious events of special interest) will 
follow the established procedures for AEs and SAEs in the study (i.e., documented and reported 
to the CC within 24 hoursGuided questionnaires have been prepared for the AEs of special 
interest and will be sent to the investigator(s) to obtain more detailed information, as necessary.  
The documentation and reporting requirements for those AEs of special interest will be further 
described in a separate document (Actemra Events of Special Interest Guidance Document).
9.1.3
Serious Adverse Events
A SAE is defined as an AE that meets any of the following criteria:
o
Results in death.
o
Is life threatening: that is, poses an immediate risk of death as the event occurred.
x
This criterion applies if the study subject, in the view of the SI or Sponsor, is at 
immediate risk of death from the AE as it occurred.  It does not apply if an AE 
that hypothetically might cause death if it were more severe.
o
Requires inpatient hospitalization or prolongation of existing hospitalization.
x
Hospitalization for an elective procedure (including elective PEG tube/g-
tube/feeding tube placement) or a routinely scheduled treatment is not an SAE by 
this criterion because an elective or scheduled “procedure” or a “treatment” is not 
an untoward medical occurrence.
o
Results in persistent or significant disability or incapacity.
x
This criterion applies if the “disability” caused by the reported AE results in a 
substantial disruption of the subject’s ability to carry out normal life functions.
o
Results in congenital anomaly or birth defect in the offspring of the subject (whether the 
subject is male or female).  


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o
Necessitates medical or surgical intervention to preclude permanent impairment of a 
body function or permanent damage to a body structure.
o
Important medical events that may not result in death, are not life-threatening, or do not 
require hospitalization may also be considered SAEs when, based upon appropriate 
medical judgment, they may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition.  Examples of such 
medical events include serious infections, blood dyscrasias or convulsions that do not 
result in inpatient hospitalization, or the development of drug dependency or drug abuse.
An inpatient hospital admission in the absence of a precipitating, treatment-emergent, clinical 
AE may meet criteria for "seriousness" but is not an adverse experience, and will therefore, not 
be considered an SAE.  An example of this would include a social admission (subject admitted 
for other reasons than medical, e.g., lives far from the hospital, has no place to sleep).
A serious, suspected adverse drug reaction is an SAE for which, in the opinion of the SI or 
Sponsor, there is a reasonable possibility that the investigational product caused the event.
The SI is responsible for classifying AEs as serious or non-serious.
9.2
Assessment and Recording of Adverse Events
The SI will carefully monitor each subject throughout the study for possible AEs.  All AEs will 
be documented on CRFs designed specifically for this purpose.  All AEs will be collected and 
reported in the electronic data capture (EDC) system and compiled into reports for 
monthlyreviewing by the Medical Monitor. The Medical Monitor shall promptly review all 
information relevant to the safety of the investigational product, including all SAEs.  Special 
attention will be paid to those that result in permanent discontinuation of the investigational 
product being studied, whether serious or non-serious.
9.2.1
Assessment of Adverse Events
At each visit (including telephone interviews), the subject will be asked if they have had any 
problems or symptoms since their last visit in order to determine the occurrence of AEs. If the 
subject reports an AE, the Investigator will probe further to determine:
x
Type of event
x
Date of onset and resolution (duration)
x
Severity (mild, moderate, severe)
x
Seriousness (does the event meet the above definition for an SAE)
x
Causality, relation to investigational product and disease


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x
Action taken regarding investigational product
x
Outcome
9.2.2
Relatedness of Adverse Event to Investigational Product
The relationship of the AE to the investigational product should be specified by the SI, using the 
following definitions:
1.  Not Related:         
Concomitant illness, accident or event with no reasonable 
association with treatment.
2.  Unlikely:
The reaction has little or no temporal sequence from 
administration of the investigational product, and/or a more likely 
alternative etiology exists.
3.  Possibly Related:
The reaction follows a reasonably temporal sequence from 
administration of the investigational product and follows a known 
response pattern to the suspected investigational product; the 
reaction could have been produced by the investigational product 
or could have been produced by the subject’s clinical state or by 
other modes of therapy administered to the subject. (Suspected 
ADR)
4.  Probably Related:
The reaction follows a reasonably temporal sequence from 
administration of investigational product; is confirmed by 
discontinuation of the investigational product or by re-challenge; 
and cannot be reasonably explained by the known characteristics 
of the subject’s clinical state. (Suspected ADR)
5.  Definitely Related:
The reaction follows a reasonable temporal sequence from 
administration of investigational product; follows a known or 
expected response pattern to the investigational product; and is 
confirmed by improvement on stopping or reducing the dosage of 
the investigational product, and reappearance of the reaction on 
repeated exposure. (Suspected ADR)


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9.2.3
Recording of Adverse Events
All clinical AEs are recorded in the AE Log in the subject’s study binder. The site should fill out 
the AE Log and enter the AE information into the Electronic Data Capture (EDC) system within 
48 hours of the site learning of a new AE or receiving an update on an existing AE.  
Please Note: SAEs must be reported to the CC within 24 hours of the site learning of the SAE.
This applies regardless of whether the subject is taking study drug or not. Infusion reactions,
gastrointestinal perforations, tuberculosis, invasive fungal infections, bacterial, viral and other 
infections
due to opportunistic pathogens, elevated liver enzymes, neutropenia or 
thrombocytopenia, whether classified as SAEs or not, must also be reported within 24 hours. 
Entries on the AE Log (and into the EDC) will include the following: name and severity of the 
event, the date of onset, the date of resolution, relationship to investigational product, action 
taken, and primary outcome of event.  
9.3
Adverse Events and Serious Adverse Events - Reportable Events
The following are considered reportable events and must be reported to the CC within 24 hours 
of the site being notified of the event. The CC must then report the events to Genentech Drug 
safety within 24 hours of learning of the event, regardless of relationship to study drug:
x
SAEs.
x
Non-serious and serious AEs of special interest.
x
Pregnancies.
The Investigator must report new significant follow-up information for these events to the CC 
within 1 working day after becoming aware of the information. The CC must send this 
information to Genentech Drug Safety within 1 working day of receipt.  New significant 
information includes the following:
x
New signs or symptoms or a change in the diagnosis.
x
Significant new diagnostic test results.
x
Change in causality based on new information.
x
Change in the event’s outcome, including recovery.
x
Additional narrative information on the clinical course of the event.
Investigators must also comply with local requirements for reporting SAEs to the local health 
authority and IRB/EC.
All serious adverse events (SAEs) for which there is a reasonable possibility the experience may 
have been caused by Tocilizumab (this applies to both expected and unexpected events) should 


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be recorded on a MedWatch 3500A Form and emailed to the NCRI Project Manager and the 
Medical Monitor (contact information found in the study manual) .
The Coordination Center will then forward this form within 24 hours of receipt to:
Genentech Drug Safety
Tel:
(888) 835-2555
Fax:
(650) 225-4682 or (650) 225-4683
The Coordination Center will use the safety reporting fax sheet in Appendix V. 
MedWatch 3500A Reporting Guidelines:
In addition to completing appropriate patient demographic and suspect medication information, 
the report should include the following information within the Event Description (section 5) of 
the MedWatch 3500A form:
x
Treatment regimen (dosing frequency, combination therapy)
x
Protocol description (and number, if assigned)
x
Description of event, severity, treatment, and outcome if known
x
Supportive laboratory results and diagnostics
x
Investigator’s assessment of the relationship of the adverse event to each investigational 
product and suspect medication
Follow-up information:  
Additional information may be added to a previously submitted report by any of the following 
methods:
x
Adding to the original MedWatch 3500A report and submitting it as follow-up
x
Adding supplemental summary information and submitting it as follow-up with the 
original MedWatch 3500A form 
x
Summarizing new information and faxing it with a cover letter including subject 
identifiers (i.e. D.O.B. initial, subject number), protocol description and number, if 
assigned, suspect drug, brief adverse event description, and notation that additional or 
follow-up information is being submitted  (The patient identifiers are important so that 
the new information is added to the correct initial report)


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Occasionally Genentech may contact the reporter for additional information, clarification, or 
current status of the subject for whom and adverse event was reported.  For questions regarding 
SAE reporting, you may contact the Genentech Drug Safety representative noted above.
The following are also considered reportable events and must be reported to the CC within 24 
hours of the site being notified of the event. 
o
Dosage Changes (Dose Management)
o
Investigational Product Suspension, Reduction or Re-challenge
o
Investigational Product Discontinuation
o
Key Study Events:
o
Subject Final Disposition
o
Feeding Tube Placement
o
Permanent Assisted Ventilation (PAV)*
o
Tracheostomy
o
Mortality
o
Pregnancy
o
Diaphragm Pacing System (DPS) device implantation
* Permanent Assisted Ventilation (PAV) is defined as more than 22 hours daily of non-invasive 
mechanical ventilation for more than one week (7 days).  The date of onset of PAV is the first 
day of the seven days.
STUDY CLOSE-OUT
Any study report submitted to the FDA by the Sponsor or designee should be copied to 
Genentech. This includes all IND annual reports and the Clinical Study Report (final study 
report). Additionally, any literature articles that are a result of the study should be sent to 
Genentech. 


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10
DATA AND SAFETY MONITORING AND STATISTICAL ANALYSIS PLAN
10.1
Data and Safety Monitoring Board
An independent Data and Safety Monitoring Board (DSMB) will be assembled for the trial. The 
DSMB receives the blinded and unblinded summary reports of the frequency of all clinical AEs
and safety laboratory tests for planned periodic meetings approximately every 3 months 
throughout the study. The DSMB can ask to receive the SAE reports more frequently. Meetings 
will be held via teleconference. In addition, the DSMB Chair may call ad hoc meetings. 
Summaries of SAEs and enrollment will be provided approximately monthly to the DSMB by 
the Study Biostatistician. Serious infusion reactions or deaths occurring within 24 hours of 
dosing, infusion reactions, tuberculosis, invasive fungal infections, bacterial, viral and other 
infections due to opportunistic pathogens, elevated liver enzymes, neutropenia or 
thrombocytopenia, and any severe unexpected SAEs are considered events of interest and will be 
reported to the DSMB prior to their meeting on a quarterly basis.  As necessary, the DSMB can 
review the frequencies of clinical and laboratory abnormalities.  Recommendations for 
modification or termination of the trial based on safety data will be made by the DSMB to the 
PIs and Steering Committee.  The DSMB will review safety data throughout the trial and may 
stop the trial for safety if they determine that there is a significant difference in the rate of a 
particular AE that would indicate a risk that is greater than the possible benefit of the study drug.  
A notable increase in the frequency of any AE should be examined by the DSMB although it 
may not lead to a recommendation by the DSMB. There will not be early stopping for efficacy in 
this trial. 
Prior to each DSMB meeting, the CC will provide an update to the DSMB on enrollment, data 
quality (missing data) and protocol adherence.  The CC will be responsible for communication 
with the DSMB.  
Complete information can be found in the Data and Safety Monitoring Board Charter.
If 2 or more serious AEs are deemed to be definitely, probably, or possibly related to study drug 
occur, the DSMB will review these events promptly and make recommendations about potential 
changes to the study including possible changes to protocol, updates to the informed consent 
form, or even ending the study early.


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10.2
Safety Monitoring
10.2.1 Safety Management Resource Team 
The CC Safety Management Resource Team (SMaRT) is comprised of physicians, Project 
Managers, Data Managers, and Systems personnel.  
On a periodic basis, the SMaRT group will review blinded reports of AEs, SAEs, abnormal 
laboratory measurements and protocol deviations as detailed in the study’s Safety Management 
Plan.
10.2.2 Medical Monitoring
The designated Medical Monitor for the study is identified in the study operations manual. Site 
personnel should contact the Medical Monitor for assistance with the following:
x
Medically-related protocol questions
x
Safety concerns, including AEs and SAEs
x
Protocol deviations
x
Unblinding questions
x
Protocol eligibilty questions
x
Dose changes
10.3
Statistical Considerations
10.3.1 Statistical Methods
Given the difficulty with generalizing the safety and tolerability of tocilizumab to ALS patients, 
it is important to assess this as a primary outcome measure if a future phase 3 trial is to be 
considered. We will assess safety of tocilizumab treatment based on mortality, occurrence of 
SAEs, overall rates of AEs, clinically significant abnormal laboratory tests, and changes in vital 
signs. We will focus on events at least possibly related to study drug of moderate or severe 
intensity by comparing participants receiving at least one dose of tocilizumab to those on placebo 
for time to mortality and time to first SAE by Cox regression, for overall rate of AEs or 
abnormal laboratory tests by negative binomial regression, and for rates of change in vital signs. 
Tests will be two-tailed at alpha = 0.10 to increase power. With 16 participants randomized to 
tocilizumab and followed for safety outcomes, the study will have 80% power to detect adverse 
events expected to occur in at least 10% of patients. Although under-powered for comparing 
rates of rare events, the study will have 80% power to detect treatment differences in events 
expected to occur, for example, in 50% of placebo participants if tocilizumab increases the 
hazard 4.2-fold.


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Tolerance of tocilizumab treatment will be judged on the proportion of participants remaining on 
study drug through all 3 doses and remaining on study and free from possibly drug-related and 
dose-limiting SAEs to the end of follow-up. The study will have 80% power to declare 
tocilizumab treatment tolerable based on non-inferiority to 50% tolerance with a one-sided 95% 
exact confidence bound if the true tolerance rate is at least 81%
We will assess successful target engagement by comparing rates of qualitative gene expression 
response in PBMCs between the two treatment groups. We will estimate the proportion of 
participants who experience a 2-fold or greater decline between baseline and the average of all 
follow-up assessments in at least 2 of the 3 pro-inflammatory genes. The study will have 80% 
power for this comparison if no more than 5% of placebo participants experience such a change 
and at least 30% of tocilizumab participants do. Assuming that at least 80% of participants are 
truly in a high inflammatory state given our enrollment criteria, that would correspond to roughly 
6% vs. 38% response among susceptible patients randomized to placebo or tocilizumab, 
respectively. We will have greater power to detect quantitative changes in expression of pro-
inflammatory genes. Serial measures of pro-inflammatory gene expression levels will be log-
transformed and analyzed in repeated-measures ANOVAs with unstructured covariance over 
time. With an expectation of rapid change in gene expression, we will compare treatments on the 
basis of change from a shared baseline to the mean of all follow-up assessments using linear 
contrasts. The study will have 80% power to detect a difference in expression equal to an effect 
size of 1.11 based on two-tailed testing at or an effect size of 1.50 based on two-tailed tests at 
alpha = 0.017, applying a Bonferroni correction for multiple comparisons over 3 inflammatory 
genes.
Clinical responses to tocilizumab treatment, measured as change in ALSFRS-R, SVC, and HHD 
megascores will be analyzed in linear mixed models with shared baseline and random 
participant-specific intercepts and slopes. If we conservatively estimate power for detecting 
treatment-dependent differences in 16-wk change from a simple two-group t-test, the study will 
have 80% power for effect sizes of 0.76. With a standard deviation for ALSFRS-R rate of change 
equal to 1.58 units/month based on the follow-up schedule of this trial and variance component 
estimates from the Ceftriaxone trial, the study would have 80% power if tocilizumab reduces
decline in ALSFRS-R by 2.0 units/month based on a two-tailed at alpha = 0.05. Power will be 
greater using the proposed linear mixed model.
Change in average [11C]-PBR28 SUV60-90 min in pre-defined regions of interest focused on the 
left and right precentral gyri for both the gray matter and the underlying white matter will be 
compared between participants receiving tocilizumab vs. placebo in a shared-baseline repeated-
measures ANOVA with unstructured within-person covariance.


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10.3.2 Analysis Populations
The primary analyses, whether for safety, tolerability, target engagement, efficacy, or PET/MRI, 
will use an as-treated sample that includes all participants classified according to the treatment 
actually received. This choice of an analysis sample reflects our interest in assessing mechanisms 
of action and potential efficacy rather than clinical effectiveness. For the purposes of planning 
future trials, a secondary efficacy analysis will use an intent-to-treat (ITT) sample that includes
all randomized participants grouped according to their randomized treatment assignment.
10.4
Missing Data
Analyses will attempt to avoid bias due to intolerance by including all participants who initiate 
treatment regardless of their compliance with their assigned treatment. Every effort will be made 
to obtain follow-up information for all subjects whether or not they continue on treatment. 
10.4.1 Stopping Rules
The DSMB will review safety data throughout the trial and may stop the trial for safety.  Any 
unexpected death or death unrelated to disease progression will lead to prompt review by the 
DSMB.  Two or more of the same SAE deemed probably or definitely related to study drug by 
SIs will lead to prompt review by the DSMB. In addition, if there is one death in the treatment 
group deemed probably or definitely related to study drug by the DMSB, the study will be 
stopped immediately.


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11
DATA COLLECTION, MANAGEMENT AND MONITORING
11.1
Role of Data Management
Data Management (DM) is the development, execution and supervision of plans, policies, 
programs, and practices that control, protect, deliver, and enhance the value of data and 
information assets.
All data will be managed in compliance with NEALS policies, and applicable Sponsor and 
regulatory requirements. Site personnel will collect, transcribe, correct, and transmit the data 
onto source documents, Case Report Forms (CRFs), and other forms used to report, track and 
record clinical research data.  Clinical sites will be monitored to ensure compliance with data 
management requirements and Good Clinical Practices.  DM is responsible for developing, 
testing, and managing clinical data management activities.
11.1.1 Data Entry and Checks
The site personnel are instructed to enter information into the Electronic Data Capture (EDC) 
System within 5 days of a visit.  Data collection is the responsibility of the staff at the site under 
the supervision of the SI.  During the study, the SI must maintain complete and accurate 
documentation for the study.
The EDC includes password protection. An edit checking and data clarification process will be 
put in place to ensure accuracy and completeness of the database.  Logic and range checks as 
well as more sophisticated rules will be built into the EDC to provide immediate error checking 
of the data entered.  The system has the capability to automatically create electronic queries for 
forms that contain data that are out of range, out of window, missing or not calculated correctly.  
The sites will only have access to the queries concerning their subjects.  
11.1.2 Data Lock Process
The application will have the ability to lock the database to prevent any modification of data 
once the study is closed.  Once this option is activated, every user will have Read-Only access to 
the data. The database can only be locked after each SI has signed off on their subjects and all 
queries have been resolved.  


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11.1.3 Quality Assurance
Protocol procedures are reviewed with the SI and associated personnel prior to the study to 
ensure the accuracy and reliability of data. Each SI must adhere to the protocol detailed in this 
document and agree that any changes to the protocol must be approved by the CC prior to 
seeking approval from the site IRB.  Each SI will be responsible for enrolling only those study 
subjects who have met protocol eligibility criteria.
11.2
Clinical Monitoring
Study Monitors will visit each study site to review source documentation materials, informed 
consent forms, and confirm entered data and that data queries have been accurately completed, 
and again at a study close-out visit.  Study Monitors will also verify that SAEs and protocol 
deviations have been reported appropriately, as required.  The Study Monitors will also review 
clinical facilities, resources and procedures for evaluating study subjects and study drug 
dispensing.  Subsequently, the Study Monitors will provide monitoring reports to the Project 
Manager and, if requested, will provide reports of protocol compliance to the Study Principal 
Investigator and the Steering Committee.  Completed informed consent forms from each subject 
must be available in the subject’s file and verified for proper documentation.  A document 
outlining the monitoring plan is provided to each Study Monitor.
11.3
Data Handling and Record Keeping
The SI is responsible to ensure the accuracy, completeness, legibility, and timeliness of the data 
reported.  All source documents should be completed in a neat, legible manner to ensure accurate 
interpretation of data.  Dark ink is required to ensure clarity of reproduced copies.  When making 
changes or corrections, cross out the original entry with a single line, and initial and date the 
change.  Do not erase, overwrite, or use correction fluid or tape on the original. 
Source document templates (SDTs) will be provided for use and maintained for recording data 
for each subject enrolled in the study.  Data reported in the eCRF derived from source documents 
should be consistent with the source documents and discrepancies should be explained.  The CC
will provide guidance to SIs on making corrections to the source documents and eCRFs.
11.3.1 Confidentiality
Study subject medical information obtained by this study is confidential, and disclosure to third 
parties other than those noted below is prohibited. Upon the subject’s permission, medical 
information may be given to his or her personal physician or other appropriate medical personnel 


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responsible for his or her welfare. All local and federal guidelines and regulations regarding 
maintaining study subject confidentiality of data will be adhered to.
Data generated by this study must be available for inspection by representatives of the Office for 
Human Research Protections (OHRP), the sponsor, all pertinent national and local health and 
regulatory authorities, the CC or their representative, Study Monitoring personnel, and the IRBs.
11.3.2 Study Discontinuation 
The study can be terminated at any time.  Reasons for terminating the study may include the 
following:
x
The incidence or severity of AEs in this or other studies indicates a potential health 
hazard to study subjects.
x
Study subject enrollment is unsatisfactory.
x
Data recording is inaccurate or incomplete.
x
Sponsor withdraws funding.
11.3.3 Retention of Records
US FDA regulations (21 CFR 312.62[c]) require that records and documents pertaining to the 
conduct of this study and the distribution of investigational drug, including CRFs (if applicable), 
consent forms, laboratory test results, and medical inventory records, must be retained by the 
Site Investigator (SI) for two years after marketing application approval.  If no application is 
filed, these records must be kept for two years after the investigation is discontinued and the US 
FDA and the applicable national and local health authorities are notified.  The Coordination 
Center or their representative will notify the Site Investigators of these events. The Site
Investigators should retain all study documents and records until they are notified in writing by 
the Sponsor or their representative. 
11.3.4 Publications
The Principal Investigator, Shafeeq Ladha, MD, and the study Steering Committee will be 
responsible for publications of results from this trial. Their responsibilities will include the 
following:
x
Analyze and interpret data gathered in this study, and write publications from these data.
x
Submit manuscripts to selected journals and address peer reviewers’ comments.
x
Submit abstracts to selected meetings and present data at the meetings.
x
Determine authorship on the basis of the Uniform Requirements for Manuscripts.


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12
LITERATURE REFERENCES
1.
Bellingham, M.C., A review of the neural mechanisms of action and clinical 
efficiency of riluzole in treating amyotrophic lateral sclerosis: what have we 
learned in the last decade? CNS Neurosci Ther, 2011. 17(1): p. 4-31.
2.
Cudkowicz, M.E., et al., Toward more efficient clinical trials for amyotrophic 
lateral sclerosis. Amyotrophic Lateral Sclerosis. 11(3): p. 259-65.
3.
Actemra [package insert]. 2013, Genentech, Inc.: San Fransicso, CA.
4.
Mizwicki, M.T., et al., Tocilizumab attenuates inflammation in ALS patients 
through inhibition of IL6 receptor signaling. Am J Neurodegener Dis, 2012. 1(3): 
p. 305-15.
5.
Liu, G., et al., Neuronal phagocytosis by inflammatory macrophages in ALS 
spinal cord: inhibition of inflammation by resolvin D1. Am J Neurodegener Dis, 
2012. 1(1): p. 60-74.
6.
Graves, M.C., et al., Inflammation in amyotrophic lateral sclerosis spinal cord and 
brain is mediated by activated macrophages, mast cells and T cells. Amyotroph 
Lateral Scler Other Motor Neuron Disord, 2004. 5(4): p. 213-9.
7.
Fiala, M., et al., Tocilizumab infusion therapy normalizes inflammation in sporadic 
ALS patients. Am J Neurodegener Dis, 2013. 2(2): p. 129-39.
8.
Appel, S.H., et al., The microglial-motoneuron dialogue in ALS. Acta Myol, 2011. 
30(1): p. 4-8.
9.
Sarantopoulos, A., et al., Tocilizumab treatment leads to a rapid and sustained 
increase in Treg cell levels in rheumatoid arthritis patients: comment on the
article by Thiolat et al. Arthritis Rheumatol, 2014. 66(9): p. 2638.
10.
März P, et al., Role of Interleukin-6 and soluble IL-6 Receptor in Region Specific 
Induction of Astrocytic Differentiation and Neurotrophin Expression. Glia, 1999. 
26: p. 191-200.
11.
März P, C.J.-C., Gadient RA, Patterson P, Stoyan T, Otten U, Sympathetic 
Neurons can produce and respond to Interleukin-6. Proc Natl Acad Sci 1998. 95:
p. 3251-3256.
12.
Zürcher, N.R., et al., Increased in vivo glial activation in patients with amyotrophic 
lateral sclerosis: Assessed with [11C]-PBR28. NeuroImage: Clinical, 2015. 7: p. 
409-414.
13.
Alshikho, M.J., et al., Glial activation colocalizes with structural abnormalities in 
amyotrophic lateral sclerosis. Neurology, 2016. 87(24): p. 2554-2561.
14.
Zhang, X., et al., Disease-drug-drug interaction studies of tocilizumab with 
cytochrome P450 substrates in vitro and vivo. Clin Pharmacol Ther 2009. 85: p. 
0009-9236.
15.
Schmitt, C., et al., Disease-drug-drug interaction involving tocilizumab and 
simvastatin in patients with rheumatoid arthritis. Clin Pharmacol Ther, 2011. 
89(5): p. 735-40.


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16.
Hammond, E.R., et al., Needle type and the risk of post-lumbar puncture 
headache in the outpatient neurology clinic. J Neurol Sci, 2011. 306(1-2): p. 24-8.
17.
Quinn, C., et al., Post-lumbar puncture headache is reduced with use of 
atraumatic needles in ALS. Amyotroph Lateral Scler Frontotemporal Degener, 
2013.
18.
Owen, D.R., et al., An 18-kDa Translocator Protein (TSPO) polymorphism 
explains differences in binding affinity of the PET radioligand PBR28. J Cereb 
Blood Flow Metab, 2012. 32(1): p. 1-5.
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Posner, K., et al., Columbia Classification Algorithm of Suicide Assessment (C-
CASA): classification of suicidal events in the FDA's pediatric suicidal risk 
analysis of antidepressants. Am J Psychiatry, 2007. 164(7): p. 1035-43.
20.
Zetterberg, H., et al., Low incidence of post-lumbar puncture headache in 1,089 
consecutive memory clinic patients. Eur Neurol, 2010. 63(6): p. 326-30.
21.
Beck, M., et al., Comparison of maximal voluntary isometric contraction and 
Drachman's hand-held dynamometry in evaluating patients with amyotrophic 
lateral sclerosis. Muscle Nerve, 1999. 22(9): p. 1265-70.
22.
Catana, C., et al., Toward implementing an MRI-based PET attenuation-
correction method for neurologic studies on the MR-PET brain prototype. Journal 
of nuclear medicine : official publication, Society of Nuclear Medicine, 2010. 
51(9): p. 1431-8.
23.
Catana, C., et al., MRI-assisted PET motion correction for neurologic studies in 
an integrated MR-PET scanner. Journal of nuclear medicine : official publication, 
Society of Nuclear Medicine, 2011. 52(1): p. 154-61.
24.
Debruyne, J.C., et al., PET visualization of microglia in multiple sclerosis patients 
using [11C]PK11195. European journal of neurology : the official journal of the 
European Federation of Neurological Societies, 2003. 10(3): p. 257-64.
25.
Watson, C.C., New, faster, image-based scatter correction for 3D PET. IEEE 
Transactions on Nuclear Science, 2000. 47(4): p. 1587-1594.
26.
Brown, A.K., et al., Radiation dosimetry and biodistribution in monkey and man of 
11C-PBR28: a PET radioligand to image inflammation. Journal of nuclear 
medicine : official publication, Society of Nuclear Medicine, 2007. 48(12): p. 
2072-9.
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Fischl, B., FreeSurfer. NeuroImage, 2012. 62(2): p. 774-81.


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13
APPENDICES
13.1 APPENDIX I: EL ESCORIAL WORLD FEDERATION OF NEUROLOGY 
CRITERIA FOR THE DIAGNOSIS OF ALS
Information obtained from the web site: www.wfnals.org.
The diagnosis of Amyotrophic Lateral Sclerosis [ALS] requires: 
A - The presence of:
(A:1)
evidence of lower motor neuron (LMN) degeneration by clinical, electrophysiological or 
neuropathologic examination, 
(A:2)
evidence of upper motor neuron (UMN) degeneration by clinical examination, and 
(A:3)
progressive spread of symptoms or signs within a region or to other regions, as 
determined by history or examination, together with
B - The absence of:
(B:1)
electrophysiological and pathological evidence of other disease processes that might 
explain the signs of LMN and/or UMN degeneration, and 
(B:2)
neuroimaging evidence of other disease processes that might explain the observed 
clinical and electrophysiological signs. 
CLINICAL STUDIES IN THE DIAGNOSIS OF ALS
A careful history, physical and neurological examination must search for clinical evidence of 
UMN and LMN signs in four regions [brainstem, cervical, thoracic, or lumbosacral spinal cord] 
(see Table A1) of the central nervous system [CNS]. Ancillary tests should be reasonably 
applied, as clinically indicated, to exclude other disease processes. These should include 
electrodiagnostic, neurophysiological, neuroimaging and clinical laboratory studies.  Clinical 
evidence of LMN and UMN degeneration is required for the diagnosis of ALS.  The clinical 
diagnosis of ALS, without pathological confirmation, may be categorized into various levels of 
certainty by clinical assessment alone depending on the presence of UMN and LMN signs 
together in the same topographical anatomic region in either the brainstem [bulbar cranial motor 
neurons], cervical, thoracic, or lumbosacral spinal cord [anterior horn motor neurons]. The terms 
Clinical Definite ALS and Clinically Probable ALS are used to describe these categories of 
clinical diagnostic certainty on clinical criteria alone:
A. Clinically Definite ALS is defined on clinical evidence alone by the presence of UMN, as 
well as LMN signs, in three regions. 


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B. Clinically Probable ALS is defined on clinical evidence alone by UMN and LMN signs in at 
least two regions with some UMN signs necessarily rostral to (above) the LMN signs.
C. Clinically Probable ALS - Laboratory-supported is defined when clinical signs of UMN and 
LMN dysfunction are in only one region, or when UMN signs alone are present in one region, 
and LMN signs defined by EMG criteria are present in at least two limbs, with proper 
application of neuroimaging and clinical laboratory protocols to exclude other causes.
D. Clinically Possible ALS is defined when clinical signs of UMN and LMN dysfunction are 
found together in only one region or UMN signs are found alone in two or more regions; or 
LMN signs are found rostral to UMN signs and the diagnosis of Clinically Probable -
Laboratory-supported ALS cannot be proven by evidence on clinical grounds in conjunction with 
electrodiagnostic, neurophysiologic, neuroimaging or clinical laboratory studies. Other diagnoses 
must have been excluded to accept a diagnosis of Clinically Possible ALS.
Table A1
Brainstem
Cervical
Thoracic
Lumbosacral
Lower motor 
neuron signs
weakness,
atrophy,
fasciculations 
jaw, face, 
palate,
tongue, larynx 
neck, arm, hand,
diaphragm 
back,
abdomen 
back, abdomen, 
leg, foot 
Upper motor
neuron signs
pathologic spread 
of reflexes, clonus, 
etc. 
clonic jaw
gag reflex
exaggerated 
snout reflex
pseudobulbar 
features
forced yawning
pathologic 
DTRs
spastic tone 
clonic DTRs 
Hoffman reflex
pathologic 
DTRs
spastic tone
preserved reflex 
in weak wasted 
limb 
loss of 
superficial 
abdominal 
reflexes
pathologic 
DTRs
spastic tone 
clonic DTRs -
extensor plantar 
response
pathologic DTRs
spastic tone
preserved reflex in 
weak wasted limb 


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13.2 APPENDIX II: ALS FUNCTIONAL RATING SCALE – REVISED (ALSFRS-R)
ALSFRS-R
QUESTIONS:
SCORE:
1. Speech
4 = Normal speech processes
3 = Detectable speech disturbances
2 = Intelligible with repeating
1 = Speech combined with nonvocal communication
0 = Loss of useful speech
2. Salivation
4 = Normal
3 = Slight but definite excess of saliva in mouth; may have nighttime drooling
2 = Moderately excessive saliva; may have minimal drooling
1 = Marked excess of saliva with some drooling
0 = Marked drooling; requires constant tissue or handkerchief
3. Swallowing
4 = Normal eating habits
3 = Early eating problems – occasional choking
2 = Dietary consistency changes
1 = Needs supplemental tube feeding
0 = NPO (exclusively parenteral or enteral feeding)
4. Handwriting
4 = Normal
3 = Slow or sloppy; all words are legible
2 = Not all words are legible
1 = No words are legible but can still grip a pen 
0 = Unable to grip pen


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5a. Cutting Food and Handling Utensils (patients without gastrostomy)
4 = Normal
3 = Somewhat slow and clumsy, but no help needed
2 = Can cut most foods, although clumsy and slow; some help needed
1 = Food must be cut by someone, but can still feed slowly
0 = Needs to be fed
5b. Cutting Food and Handling Utensils (alternate scale for patients with gastrostomy)
4 = Normal
3 = Clumsy, but able to perform all manipulations independently
2 = Some help needed with closures and fasteners
1 = Provides minimal assistance to caregivers
0 = Unable to perform any aspect of task
6. Dressing and Hygiene
4 = Normal function
3 = Independent, can complete self-care with effort or decreased efficiency
2 = Intermittent assistance or substitute methods
1 = Needs attendant for self-care
0 = Total dependence
7. Turning in Bed and Adjusting Bed Clothes
4 = Normal function
3 = Somewhat slow and clumsy, but no help needed
2 = Can turn alone, or adjust sheets, but with great difficulty
1 = Can initiate, but not turn or adjust sheets alone
0 = Helpless
8. Walking
4 = Normal
3 = Early ambulation difficulties
2 = Walks with assistance
1 = Nonambulatory functional movement only
0 = No purposeful leg movement


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9. Climbing Stairs
4 = Normal
3 = Slow
2 = Mild unsteadiness or fatigue
1 = Needs assistance
0 = Cannot do
R-1. Dyspnea
4 = None
3 = Occurs when walking
2 = Occurs with one or more of the following: eating, bathing, dressing
1 = Occurs at rest, difficulty breathing when either sitting or lying
0 = Significant difficulty, considering using mechanical respiratory support
R-2 Orthopnea
4 = None
3 = Some difficulty sleeping at night due to shortness of breath, does not routinely use more than 
two pillows
2 = Needs extra pillow in order to sleep (more than two)
1 = Can only sleep sitting up
0 = Unable to sleep without mechanical assistance
R-3 Respiratory Insufficiency
4 = None
3 = Intermittent use of BiPAP
2 = Continuous use of BiPAP during the night
1 = Continuous use of BiPAP during the night and day
0 = Invasive mechanical ventilation by intubation or tracheostomy
Evaluator’s Initials: _____ _____ ______


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13.3 APPENDIX III: COLUMBIA-SUICIDE SEVERITY RATING SCALE (C-SSRS) 
BASELINE VERSION
Information obtained from: http://www.cssrs.columbia.edu/SUICIDAL IDEATION 
Ask questions 1 and 2. If both are negative, proceed to “Suicidal Behavior” section. If the answer 
to question 2 is “yes”, ask questions 3, 4 and 5. If the answer to question 1 and/or 2 is “yes”, 
complete “Intensity of Ideation” section below. 
Lifetime: 
Time 
He/She Felt 
Most 
Suicidal 
1. Wish to be Dead 
Subject endorses thoughts about a wish to be dead or not alive anymore, or wish to fall asleep and not wake up. 
Have you wished you were dead or wished you could go to sleep and not wake up? 
If yes, describe: 
Yes No 
ƑƑ
2. Non-Specific Active Suicidal Thoughts 
General, non-specific thoughts of wanting to end one’s life/commit suicide (e.g., “I’ve thought about killing myself”)
without thoughts of ways to kill oneself/associated methods, intent, or plan. 
Have you actually had any thoughts of killing yourself? 
If yes, describe: 
Yes No 
ƑƑ
3. Active Suicidal Ideation with Any Methods (Not Plan) without Intent to Act 
Subject endorses thoughts of suicide and has thought of at least one method during the assessment period. This is different 
than a specific plan with time, place or method details worked out (e.g., thought of method to kill self but not a specific 
plan). Includes person who would say, “I thought about taking an overdose but I never made a specific plan as to when, 
where or how I would actually do it…and I would never go through with it.” 
Have you been thinking about how you might do this? 
If yes, describe:
Yes No 
ƑƑ
4. Active Suicidal Ideation with Some Intent to Act, without Specific Plan 
Active suicidal thoughts of killing oneself and subject reports having some intent to act on such thoughts, as opposed to “I 
have the thoughts but I definitely will not do anything about them.” 
Have you had these thoughts and had some intention of acting on them? 
If yes, describe: 
Yes No 
ƑƑ
5. Active Suicidal Ideation with Specific Plan and Intent 
Thoughts of killing oneself with details of plan fully or partially worked out and subject has some intent to carry it out. 
Have you started to work out or worked out the details of how to kill yourself? Do you intend to carry out this plan? 
If yes, describe: 
Yes No 
ƑƑ
INTENSITY OF IDEATION 
The following features should be rated with respect to the most severe type of ideation (i.e., 1-5 from above, 
with 1 being the least severe and 5 being the most severe). Ask about time he/she was feeling the most 
suicidal. 
_____ _________________________________________________ 
Most Severe Ideation: 
Type # (1-5) Description of Ideation 
Most 
Severe 
Frequency 
How many times have you had these thoughts? 
(1) Less than once a week (2) Once a week (3) 2-5 times in week (4) Daily or almost daily (5) Many times each day 
____ 
Duration 
When you have the thoughts, how long do they last? 
(1) Fleeting - few seconds or minutes (4) 4-8 hours/most of day 
(2) Less than 1 hour/some of the time (5) More than 8 hours/persistent or continuous 
(3) 1-4 hours/a lot of time 
____ 


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Controllability 
Could/can you stop thinking about killing yourself or wanting to die if you want to? 
(1) Easily able to control thoughts (4) Can control thoughts with a lot of difficulty 
(2) Can control thoughts with little difficulty (5) Unable to control thoughts 
(3) Can control thoughts with some difficulty (0) Does not attempt to control thoughts 
____ 
Deterrents 
Are there things - anyone or anything (e.g., family, religion, pain of death) - that stopped you from 
wanting to die or acting on thoughts of committing suicide? 
(1) Deterrents definitely stopped you from attempting suicide (4) Deterrents most likely did not stop you 
(2) Deterrents probably stopped you (5) Deterrents definitely did not stop you 
(3) Uncertain that deterrents stopped you (0) Does not apply 
____ 
Reasons for Ideation 
What sort of reasons did you have for thinking about wanting to die or killing yourself? Was it to 
end the pain or stop the way you were feeling (in other words you couldn’t go on living with this 
pain or how you were feeling) or was it to get attention, revenge or a reaction from others? Or both? 
(1) Completely to get attention, revenge or a reaction from others (4) Mostly to end or stop the pain (you couldn’t go 
on 
(2) Mostly to get attention, revenge or a reaction from others living with the pain or how you were feeling) 
(3) Equally to get attention, revenge or a reaction from others (5) Completely to end or stop the pain (you couldn’t go 
on 
and to end/stop the pain. living with the pain or how you were feeling) 
(0) Does not apply 
____ 
SUICIDAL BEHAVIOR 
(Check all that apply, so long as these are separate events; must ask about all types) 
Lifetime 
Actual Attempt: 
A potentially self-injurious act committed with at least some wish to die, as a result of act. Behavior was in part thought 
of as method to kill oneself. Intent does not have to be 100%. If there is any intent/desire to die associated with the act, 
then it can be considered an actual suicide attempt. There does not have to be any injury or harm, just the 
potential for injury or harm. If person pulls trigger while gun is in mouth but gun is broken so no injury results, this is 
considered an attempt. 
Inferring Intent: Even if an individual denies intent/wish to die, it may be inferred clinically from the behavior or 
circumstances. For example, a highly lethal act that is clearly not an accident so no other intent but suicide can be 
inferred (e.g., gunshot to head, jumping from window of a high floor/story). Also, if someone denies intent to die, but 
they thought that what they did could be lethal, intent may be inferred. 
Have you made a suicide attempt? 
Have you done anything to harm yourself? 
Have you done anything dangerous where you could have died? 
What did you do? 
Did you______ as a way to end your life? 
Did you want to die (even a little) when you_____? 
Were you trying to end your life when you _____? 
Or did you think it was possible you could have died from_____? 
Or did you do it purely for other reasons / without ANY intention of killing yourself (like to relieve 
stress, feel better, get sympathy, or get something else to happen)? (Self-Injurious Behavior without suicidal 
intent) 
If yes, describe: 
Has subject engaged in Non-Suicidal Self-Injurious Behavior? 
Yes No 
ƑƑ
Total # of 
Attempts 
______ 
Yes No 
ƑƑ


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Interrupted Attempt: 
When the person is interrupted (by an outside circumstance) from starting the potentially self-injurious act (if not for that, 
actual attempt would have occurred). 
Overdose: Person has pills in hand but is stopped from ingesting. Once they ingest any pills, this becomes an attempt 
rather than an interrupted attempt. Shooting: Person has gun pointed toward self, gun is taken away by someone else, or 
is somehow prevented from pulling trigger. Once they pull the trigger, even if the gun fails to fire, it is an attempt. 
Jumping: Person is poised to jump, is grabbed and taken down from ledge. Hanging: Person has noose around neck but 
has not yet started to hang - is stopped from doing so. 
Has there been a time when you started to do something to end your life but someone or something 
stopped you before you actually did anything? 
If yes, describe: 
Yes No 
ƑƑ
Total # of 
interrupted 
______ 
Aborted Attempt: 
When person begins to take steps toward making a suicide attempt, but stops themselves before they actually have 
engaged in any self-destructive behavior. Examples are similar to interrupted attempts, except that the individual stops 
him/herself, instead of being stopped by something else. 
Has there been a time when you started to do something to try to end your life but you stopped 
yourself before you actually did anything? 
If yes, describe: 
Yes No 
ƑƑ
Total # of 
aborted 
______ 
Preparatory Acts or Behavior: 
Acts or preparation towards imminently making a suicide attempt. This can include anything beyond a verbalization or 
thought, such as assembling a specific method (e.g., buying pills, purchasing a gun) or preparing for one’s death by 
suicide (e.g., giving things away, writing a suicide note). 
Have you taken any steps towards making a suicide attempt or preparing to kill yourself (such as 
collecting pills, getting a gun, giving valuables away or writing a suicide note)?
If yes, describe: 
Yes No 
ƑƑ
Suicidal Behavior: 
Suicidal behavior was present during the assessment period? 
Yes No 
ƑƑ
Answer for Actual Attempts Only 
Most 
Recent 
Attempt 
Date: 
Most 
Lethal 
Attempt 
Date: 
Initial/First 
Attempt 
Date: 
Actual Lethality/Medical Damage: 
0. No physical damage or very minor physical damage (e.g., surface scratches). 
1. Minor physical damage (e.g., lethargic speech; first-degree burns; mild bleeding; sprains). 
2. Moderate physical damage; medical attention needed (e.g., conscious but sleepy, somewhat 
responsive; second-degree burns; bleeding of major vessel). 
3. Moderately severe physical damage; medical hospitalization and likely intensive care 
required (e.g., comatose with reflexes intact; third-degree burns less than 20% of body; 
extensive blood loss but can recover; major fractures). 
4. Severe physical damage; medical hospitalization with intensive care required (e.g., 
comatose without reflexes; third-degree burns over 20% of body; extensive blood loss 
with unstable vital signs; major damage to a vital area). 
5. Death 
Enter 
Code 
______ 
Enter 
Code 
______ 
Enter Code 
______ 
Potential Lethality: Only Answer if Actual Lethality=0 
Likely lethality of actual attempt if no medical damage (the following examples, while having 
no actual medical damage, had potential for very serious lethality: put gun in mouth and 
pulled the trigger but gun fails to fire so no medical damage; laying on train tracks with 
oncoming train but pulled away before run over). 
0 = Behavior not likely to result in injury 
1 = Behavior likely to result in injury but not likely to cause death 
2 = Behavior likely to result in death despite available medical care 
Enter 
Code 
______ 
Enter 
Code 
______ 
Enter Code 
______ 


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13.4 APPENDIX IV: COLUMBIA-SUICIDE SEVERITY RATING SCALE (C-SSRS) 
SINCE LAST VISIT VERSION
Information obtained from: http://www.cssrs.columbia.edu/
SUICIDAL IDEATION 
Ask questions 1 and 2. If both are negative, proceed to “Suicidal Behavior” section. If the answer to 
question 2 is “yes”, ask questions 3, 4 and 5. If the answer to question 1 and/or 2 is “yes”, complete 
“Intensity of Ideation” section below.  
Since 
Last 
Visit  
1. Wish to be Dead 
Subject endorses thoughts about a wish to be dead or not alive anymore, or wish to fall asleep and not wake up. 
Have you wished you were dead or wished you could go to sleep and not wake up?  
If yes, describe: 
Yes No 
Ƒ     Ƒ  
2. Non-Specific Active Suicidal Thoughts  
General, non-specific thoughts of wanting to end one’s life/commit suicide (e.g., “I’ve thought about killing myself”) without 
thoughts of ways to kill oneself/associated methods, intent, or plan during the assessment period.  
Have you actually had any thoughts of killing yourself? 
If yes, describe: 
Yes No 
Ƒ     Ƒ 
3. Active Suicidal Ideation with Any Methods (Not Plan) without Intent to Act 
Subject endorses thoughts of suicide and has thought of at least one method during the assessment period. This is different than a 
specific plan with time, place or method details worked out (e.g., thought of method to kill self but not a specific plan). Includes 
person who would say, “I thought about taking an overdose but I never made a specific plan as to when, where or how I would 
actually do it…and I would never go through with it.”  
Have you been thinking about how you might do this? 
If yes, describe: 
Yes No 
Ƒ     Ƒ 
4. Active Suicidal Ideation with Some Intent to Act, without Specific Plan  
Active suicidal thoughts of killing oneself and subject reports having some intent to act on such thoughts, as opposed to “I have the 
thoughts but I definitely will not do anything about them.”  
Have you had these thoughts and had some intention of acting on them? 
If yes, describe: 
Yes No 
Ƒ     Ƒ  
5. Active Suicidal Ideation with Specific Plan and Intent 
Thoughts of killing oneself with details of plan fully or partially worked out and subject has some intent to carry it out.  
Have you started to work out or worked out the details of how to kill yourself? Do you intend to carry out this plan? 
If yes, describe: 
Yes No 
Ƒ Ƒ   
INTENSITY OF IDEATION 
The following features should be rated with respect to the most severe type of ideation (i.e., 1-5 from above, with 1 
being the least severe and 5 being the most severe).  
Most Severe Ideation: _____ _________________________________________________  
Type # (1-5) Description of Ideation  
Most 
Severe  
Frequency 
How many times have you had these thoughts? 
(1) Less than once a week (2) Once a week (3) 2-5 times in week (4) Daily or almost daily (5) Many times each day  
____ 
Duration 
When you have the thoughts, how long do they last?  
(1) Fleeting - few seconds or minutes (4) 4-8 hours/most of day  
(2) Less than 1 hour/some of the time (5) More than 8 hours/persistent or continuous  
(3) 1-4 hours/a lot of time  
____ 


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Controllability 
Could/can you stop thinking about killing yourself or wanting to die if you want to?  
(1) Easily able to control thoughts (4) Can control thoughts with a lot of difficulty  
(2) Can control thoughts with little difficulty (5) Unable to control thoughts  
(3) Can control thoughts with some difficulty (0) Does not attempt to control thoughts  
____ 
Deterrents 
Are there things - anyone or anything (e.g., family, religion, pain of death) - that stopped you from wanting 
to die or acting on thoughts of committing suicide?  
(1) Deterrents definitely stopped you from attempting suicide (4) Deterrents most likely did not stop you 
(2) Deterrents probably stopped you (5) Deterrents definitely did not stop you  
(3) Uncertain that deterrents stopped you (0) Does not apply  
____ 
Reasons for Ideation 
What sort of reasons did you have for thinking about wanting to die or killing yourself? Was it to end the 
pain or stop the way you were feeling (in other words you couldn’t go on living with this pain or how you 
were feeling) or was it to get attention, revenge or a reaction from others? Or both?  
(1) Completely to get attention, revenge or a reaction from others (4) Mostly to end or stop the pain (you couldn’t go on  
(2) Mostly to get attention, revenge or a reaction from others living with the pain or how you were feeling)  
(3) Equally to get attention, revenge or a reaction from others (5) Completely to end or stop the pain (you couldn’t go on  
and to end/stop the pain living with the pain or how you were feeling)  
(0) Does not apply  
____ 
SUICIDAL BEHAVIOR 
(Check all that apply, so long as these are separate events; must ask about all types) 
Since Last 
Visit 
Actual Attempt: 
A potentially self-injurious act committed with at least some wish to die, as a result of act. Behavior was in part thought of as 
method to kill oneself. Intent does not have to be 100%. If there is any intent/desire to die associated with the act, then it can be 
considered an actual suicide attempt. There does not have to be any injury or harm, just the potential for injury or 
harm. If person pulls trigger while gun is in mouth but gun is broken so no injury results, this is considered an attempt.  
Inferring Intent: Even if an individual denies intent/wish to die, it may be inferred clinically from the behavior or circumstances. 
For example, a highly lethal act that is clearly not an accident so no other intent but suicide can be inferred (e.g., gunshot to head, 
jumping from window of a high floor/story). Also, if someone denies intent to die, but they thought that what they did could be 
lethal, intent may be inferred.  
Have you made a suicide attempt? 
Have you done anything to harm yourself?  
Have you done anything dangerous where you could have died? 
What did you do? 
Did you______ as a way to end your life? 
Did you want to die (even a little) when you_____? 
Were you trying to end your life when you _____? 
Or did you think it was possible you could have died from_____?  
Or did you do it purely for other reasons / without ANY intention of killing yourself (like to relieve stress, 
feel better, get sympathy, or get something else to happen)? (Self-Injurious Behavior without suicidal intent)  
If yes, describe: 
Has subject engaged in Non-Suicidal Self-Injurious Behavior?  
Yes No 
ƑƑ
Total # of 
Attempts 
______  
Yes No 
ƑƑ
Interrupted Attempt: 
When the person is interrupted (by an outside circumstance) from starting the potentially self-injurious act (if not for that, actual 
attempt would have occurred).  
Overdose: Person has pills in hand but is stopped from ingesting. Once they ingest any pills, this becomes an attempt rather than 
an interrupted attempt. Shooting: Person has gun pointed toward self, gun is taken away by someone else, or is somehow 
prevented from pulling trigger. Once they pull the trigger, even if the gun fails to fire, it is an attempt. Jumping: Person is poised 
to jump, is grabbed and taken down from ledge. Hanging: Person has noose around neck but has not yet started to hang - is 
stopped from doing so.  
Has there been a time when you started to do something to end your life but someone or something 
stopped you before you actually did anything?  
If yes, describe: 
Yes No 
ƑƑ
Total # of 
interrupted  
______  


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Aborted Attempt: 
When person begins to take steps toward making a suicide attempt, but stops themselves before they actually have engaged in 
any self-destructive behavior. Examples are similar to interrupted attempts, except that the individual stops him/herself, instead 
of being stopped by something else.  
Has there been a time when you started to do something to try to end your life but you stopped 
yourself before you actually did anything?  
If yes, describe: 
Yes No 
ƑƑ
Total # of 
aborted 
______  
Preparatory Acts or Behavior: 
Acts or preparation towards imminently making a suicide attempt. This can include anything beyond a verbalization or thought,
such as assembling a specific method (e.g., buying pills, purchasing a gun) or preparing for one’s death by suicide (e.g., giving 
things away, writing a suicide note).  
Have you taken any steps towards making a suicide attempt or preparing to kill yourself (such as 
collecting pills, getting a gun, giving valuables away or writing a suicide note)?  
If yes, describe: 
Yes No 
ƑƑ
Suicidal Behavior: 
Suicidal behavior was present during the assessment period?  
Yes No 
ƑƑ
Suicide: 
Yes No 
ƑƑ
Answer for Actual Attempts Only 
Most Lethal 
Attempt 
Date: 
Actual Lethality/Medical Damage: 
0. No physical damage or very minor physical damage (e.g., surface scratches).  
1. Minor physical damage (e.g., lethargic speech; first-degree burns; mild bleeding; sprains).  
2. Moderate physical damage; medical attention needed (e.g., conscious but sleepy, somewhat responsive; second-degree burns; 
bleeding of major vessel).  
3. Moderately severe physical damage; medical hospitalization and likely intensive care required (e.g., comatose with reflexes 
intact; third-degree burns less than 20% of body; extensive blood loss but can recover; major fractures).  
4. Severe physical damage; medical hospitalization with intensive care required (e.g., comatose without reflexes; third-degree 
burns over 20% of body; extensive blood loss with unstable vital signs; major damage to a vital area).  
5. Death 
Enter Code  
______ 
Potential Lethality: Only Answer if Actual Lethality=0 
Likely lethality of actual attempt if no medical damage (the following examples, while having no actual medical damage, had 
potential for very serious lethality: put gun in mouth and pulled the trigger but gun fails to fire so no medical damage; laying on 
train tracks with oncoming train but pulled away before run over).  
0 = Behavior not likely to result in injury  
1 = Behavior likely to result in injury but not likely to cause death 
2 = Behavior likely to result in death despite available medical care 
Enter Code  
______ 


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13.6 APPENDIX V: SAFETY REPORTING FAX COVER SHEET
SAFETY REPORTING FAX COVER SHEET
Investigator Sponsored Trials
SAE FAX No: (650) 225-4682
Alternate Fax No: (650) 225-4683
Study Number
(Genentech study number)
Principal Investigator
Site Name
Reporter name
Reporter Telephone #
Reporter Fax #
Initial Report Date
(DD/MON/YYYY)
____ / _____ /_____
Follow-up Report Date
(DD/MON/YYYY)
____ / _____ /_____


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Subject Initials
(Please enter a dash if the patient 
has no middle name)
____ - ____ -____
PLEASE PLACE MEDWATCH REPORT or IND SAFETY REPORT BEHIND THIS COVER 
SHEET
Please contact Genentech Safety for any questions regarding SAE or IND Safety reporting 
at (888) 835-2555
Page 1 of ___


Cover Page – Statistical Analysis Plan 
 
 
 
 
 
Study Official Title: A Phase 2 Randomized, Placebo-
Controlled Trial of Tocilizumab in Amyotrophic Lateral 
Sclerosis (ALS)
 
 
NCT #: NCT02469896 
 
Version Date of the Document: May 30, 2017 
 


Ladha (PI) Tocilizumab Trial: Statistical Analysis Plan
Version 1.0, 01 Mar 2019
Page 1 of 20
 
 
STATISTICAL ANALYSIS PLAN (SAP)
Title
A Phase 2 Randomized, Placebo-Controlled Trial of
Tocilizumab in Amyotrophic Lateral Sclerosis (ALS)
Regulatory Sponsor
Shafeeq Ladha, MD
Current Protocol Version
8.0
Current Protocol Date
30 May 2017
Statistical Analysis Plan
Version
1.0
Statistical Analysis Plan
Date
01 Mar 2019
SAP APPROVAL SIGNATURES
Shafeeq Ladha, MD
Principle Investigator and Sponsor
Date
Eric A. Macklin, PhD
Study Biostatistician
Date
Eric A. Macklin 
Digitally signed by Eric A. Macklin 
DN: cn=Eric A. Macklin, o=Massachusetts General Hospital, ou=MGH 
Biostatistics Center, email=emacklin@mgh.harvard.edu, c=US 
Date: 2019.03.14 23:14:34 -04'00' 
3/11/2019


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Version 1.0, 01 Mar 2019
Page 2 of 20
 
 
SAP REVISION HISTORY
Version
Date
Description of Changes
1.0
01Mar2019
Initial version


Ladha (PI) Tocilizumab Trial: Statistical Analysis Plan
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Page 3 of 20
 
 
Table of Contents
1. Introduction........................................................................................................................... 5
2. Study Design .......................................................................................................................... 5
2.1
Overview......................................................................................................................... 5
2.2
Study Objectives ............................................................................................................. 5
2.3
Study Populations............................................................................................................ 5
2.4
Participant Flow and Site Approval ................................................................................ 6
2.5
Treatment Allocation....................................................................................................... 6
2.6
Treatment Administration ............................................................................................... 6
2.7
Allocation Concealment.................................................................................................. 6
2.8
Schedule of Assessments ................................................................................................ 7
3. General Statistical Considerations....................................................................................... 8
3.1
Statistical Software.......................................................................................................... 8
3.2
Summary Statistics.......................................................................................................... 8
3.3
Precision.......................................................................................................................... 8
4. Analysis Samples................................................................................................................... 8
5. Study Endpoints .................................................................................................................... 8
5.1
Safety Endpoints ............................................................................................................. 8
5.2
Tolerability Endpoint ...................................................................................................... 9
5.3
Efficacy Endpoints.......................................................................................................... 9
5.4
Effect Modifiers ............................................................................................................ 10
6. Measurement Definitions.................................................................................................... 10
6.1
Target Engagement Measures ....................................................................................... 10
6.1.1
Expression of pro-inflammatory genes in PBMCs.................................................... 10
6.1.2
Concentration of CRP by R&D Systems ELISA ...................................................... 11
6.1.3
Concentration of pro-inflammatory cytokines by MSD V-PLEX............................. 12
6.1.4
Concentration of sIL-6R, IL-6, and gp130 by R&D Systems ELISA....................... 13
6.1.5
IL6R genotyping for Asp358Ala polymorphism....................................................... 13
6.1.6
Brain glial activation measured by [11C]-PBR28 PET.............................................. 14
6.2
Clinical Progression Measures...................................................................................... 14
6.2.1
ALSFRS-R................................................................................................................ 14
6.2.2
SVC........................................................................................................................... 14
6.2.3
Handheld dynamometry............................................................................................ 14
6.2.4
Grip strength ............................................................................................................. 15
7. Power Calculations.............................................................................................................. 15
8. Participant Characterization.............................................................................................. 16
8.1
Baseline Characteristics ................................................................................................ 16
8.2
Participant Disposition.................................................................................................. 16
9. Statistical Analyses.............................................................................................................. 16
9.1
Safety Measures ............................................................................................................ 16
9.1.1
Mortality ................................................................................................................... 16
9.1.2
Treatment-emergent Adverse Events........................................................................ 16
9.1.3
Suicidality ................................................................................................................. 17
9.1.4
Safety Labs................................................................................................................ 17
9.1.5
Anthropometrics and Vital Signs .............................................................................. 17
9.2
Study Drug Tolerance ................................................................................................... 17
9.3
Target Engagement Measures ....................................................................................... 17


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9.3.1
Primary Analysis....................................................................................................... 17
9.3.2
Secondary Analysis................................................................................................... 18
9.3.3
Subgroup Analyses.................................................................................................... 18
9.4
Efficacy Measures......................................................................................................... 19
9.4.1
Primary Analysis....................................................................................................... 19
9.4.2
Secondary Analysis................................................................................................... 19
9.5
Additional Considerations............................................................................................. 19
9.5.1
Multiplicity Adjustments .......................................................................................... 19
9.5.2
Missing Data ............................................................................................................. 19
10.
References........................................................................................................................ 20


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Page 5 of 20
 
 
1. Introduction
This statistical analysis plan (SAP) defines the outcome measures and analysis samples and
specifies the planned analyses of data from Dr. Shafeeq Ladha's Tocilizumab trial. The SAP
supplements the clinical protocol. Please refer to the clinical protocol for details on the rationale
for the intervention, eligibility criteria, conduct of the trial, clinical assessments and the timing of
their use in the trial, definitions and reporting of adverse events, data management conventions,
and regulatory oversight and compliance procedures. In case of discrepancies between the SAP
and the clinical protocol concerning matters of data analysis, the SAP is authoritative. On all
other matters, the clinical protocol is authoritative.
2. Study Design
2.1
Overview
This is a multicenter, double-blind, placebo-controlled, two-arm, parallel-group, phase 2
randomized controlled trial to examine the safety and tolerability of tocilizumab in sporadic ALS
patients. Trial participation includes a screening visit, randomization to 8 mg/kg tocilizumab or
placebo administered every 4 weeks by intravenous infusions, 8 weeks of follow-up on study
drug, and 4 weeks of follow-up after last dose of study drug. A subset of participants receive pre-
and post-treatment MR-PET scanning to measure glial activation. The trial is registered at
Clinicaltrials.gov as study NCT02469896 (see https://clinicaltrials.gov/ct2/show/NCT02469896).
2.2
Study Objectives
The primary objective of the trial is to determine whether 8 mg/kg tocilizumab administered
every 4 weeks by intravenous infusions for 8 weeks is safe and tolerable. Safety will be assessed
by the occurrence of severe adverse events (SAEs), rates of adverse events (AEs) classified to
MedDRA system organ class and preferred term, frequency of clinically significant abnormal
laboratory tests, and changes in vital signs. Tolerability will be assessed by the proportion of
participants who complete all three infusions and remain on study and free from any possibly or
definitely drug-related and dose-limiting AEs to the week 16 visit.
Secondary objectives include assessing the ability of tocilizumab to reduce the expression of pro-
inflammatory genes in peripheral blood mononuclear cells (PBMCs), to reduce concentrations of
pro-inflammatory cytokines in plasma and cerebrospinal fluid (CSF), to reduce concentration of
soluble IL-6 receptor (sIL-6R) in the CSF, to reduce glial activation measured by [11C]-PBR28
PET, and to slow progression of clinical measures of function and strength. The trial also aims to
test whether the Asp358Ala polymorphism of the IL6R gene (SNP rs2228145) modifies effects
of tocilizumab on expression of pro-inflammatory genes in PBMCs and cytokine and C-reactive
protein (CRP) levels in the plasma and CSF.
2.3
Study Populations
Individuals eligible for trial participation as an ALS participant are men or women age 18 to 75
years who meet the El Escorial criteria of possible, laboratory-supported probable, probable, or
definite criteria for a diagnosis of ALS, have either a 3-fold up-regulation of IL-6 gene
expression or a 2-fold up-regulation of both IL-6 and either IL-8 or MMP1 in PBMCs relative to
a pooled healthy control sample, have a slow vital capacity (SVC) at least 40% of that expected


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based on age, sex, and height, and are free of conditions that contraindicate use of tocilizumab.
Detailed inclusion and exclusion criteria are specified in the clinical protocol.
Participants will be recruited from approximately 5 clinical sites in the Northeast ALS (NEALS)
Consortium located across the US.
2.4
Participant Flow and Site Approval
After a site receives local IRB approval and is activated, site staff may recruit participants.
Individuals with sporadic ALS must provide written informed consent to initiate screening. After
being determined eligible, approximately 24 ALS participants will be randomized to receive 8
mg/kg tocilizumab or placebo in a 2:1 ratio, stratified by clinical site. The first dose of study
drug is administered by intravenous infusion at the baseline visit. If tolerant, participants receive
two additional study drug infusions at 4-week intervals over 8 weeks. After baseline, participants
complete in-clinic evaluations at weeks 4, 8, 12, and 16. Participants are contacted by telephone
24 to 48 hours after the lumbar punctures (LPs) at baseline and week 8 to assess for AEs.
Participants who discontinue study drug should remain on study following the normal schedule
of assessments. Patients withdrawing consent are asked to delay withdrawing consent until after
they return for a final safety visit and after a final telephone call scheduled 28 days after their last
dose of study drug.
Detailed descriptions of study procedures and timing are specified in the clinical protocol.
2.5
Treatment Allocation
Prior to the baseline visit, eligible ALS participants are randomly allocated to receive 8 mg/kg
tocilizumab or placebo in a 2:1 ratio according to a permuted-block randomization schedule,
stratified by site. The randomization schedule was prepared by computer program by the
unblinded study statistician.
2.6
Treatment Administration
Study drug will be administered as a 100 mL intravenous infusion over 60 min. An unblinded
research pharmacist at each site will prepare study drug infusion bags. Tocilizumab will be
mixed in 0.9% sodium chloride solution at 8 mg/kg, up to a maximum 800 mg. Placebo
participants will receive 0.9% sodium chloride solution.
2.7
Allocation Concealment
The randomization schedule is known only by the unblinded study statistician who generated the
schedule and by unblinded research pharmacists at each site. Concealment of the true treatment
allocation of specific participants is facilitated by the equivalent appearance of infusion bags
containing tocilizumab (active arm) and those containing only saline (placebo arm). Plasma and
CSF sIL-6R levels will be determined by a central lab not otherwise involved in the trial and sent
only to the unblinded statistician prior to locking the trial database. Although no other
biomarkers are expected to be unblinding, levels of CRP and pro-inflammatory cytokines in
plasma and CSF will be handled similarly. Clinical members of the Steering Committee, site
investigators and other site staff, clinical coordination and data management staff, the medical
monitor, and all participants are blinded to participant treatment allocations. The Data and Safety
Monitoring Board (DSMB) members are provided treatment-specific information in order to


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monitor the trial but such information is masked by use of coded values to identify the treatment
groups. The DSMB may request the true treatment identities.
2.8
Schedule of Assessments
Activity
Screening
Visit1
Baseline
Visit
Week 4
Week 8
Week 12
Week 16
Visit 1
Visit 2
Visit 3
EOS Visit
Written Informed Consent
X
 
 
 
 
 
Inclusion/ExclusionReview
X
X
 
 
 
 
Medical History
X
 
 
 
 
 
Demographics
X
 
 
 
 
 
ALS Diagnosis History
X
 
 
 
 
 
PhysicalExamination
X
 
 
 
 
X
Neurological Exam
X
 
 
 
 
X
Columbia Suicide Severity Rating Scale (C-SSRS)
 
X
X
X
X
X
Vital Signs2 including Height3 and Weight
X
X
X
X
X
X
Lumbar Puncture (LP)
 
X4
 
X4
 
 
ALSFRS-R
 
X
X
X
X
X
Slow Vital Capacity (SVC)
X
X
X
X
X
X
Hand Held Dynamometry (HHD) and Grip Strength
 
X
 
X
 
X
Electrocardiogram (12-Lead ECG)
X
 
 
 
 
 
PA and Lateral Chest X-Ray
X
 
 
 
 
 
Quantiferon or T-SPOT TB Blood Test
X
 
 
 
 
 
Safety Labs5
X
X
X
X
X
X
PBMC Collection
X
X6
X
X
X
X
Plasma Biomarker Collection
 
X
X
X
X
X
Blood Collection for IL-6R genotype, Genentech sample
 
X
 
 
 
 
Concomitant Medication Review
X
X
X
X
X
X
Adverse Event Review7
X
X
X
X
X
X
Randomization8
 
X
 
 
 
 
Administer Study Drug
 
X
X
X
 
 
Exit Questionnaire
 
 
 
 
 
X
[11C]-PBR28 PET
 
X9
 
X10
 
 
MRI Safety Questionnaire
X
X
 
X
 
 
Upper Motor Neuron Burden Scale
X
X
 
X
 
 
Blood draw for TSPO
X11
 
 
 
 
 
1 Screening procedures must be completed within 28 days prior to Baseline Visit.
2 Vital signs include systolic and diastolic pressure in mmHg, respiratory rate/minute, heart rate/minute and
temperature.
3 Height measured at Screening Visit only.
4 Telephone call will be made 24-48 hours post lumbar puncture (LP) to assess for adverse events (AEs) related to
the LP. Baseline LP occurs prior to the first infusion. Week 8 LP should occur 2.5 to 3.5 hours post-infusion.
5 Safety labs include hematology (CBC with differential), chemistry panel, hepatitis B panel, urinalysis. For women
of child-bearing potential, pregnancy will be tested by serum hCG at the Screening Visit and by urine pregnancy test
at each subsequent visit.
6 Collection at Baseline to occur prior to infusion
7 Adverse events that occur after signing the informed consent form will be recorded.
8 Randomization should occur immediately prior to the Baseline Visit.


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9 Pre-dose [11C]-PBR28 PET will be performed one time only at the Baseline visits. (-2 Weeks)
10 Post-dose [11C]-PBR28 PET will be performed one time only at the Week 8 visit (- 2 Weeks).
11 Blood draw for TSPO affinity testing to occur at the Screening visit, if not previously determined.
3. General Statistical Considerations
3.1
Statistical Software
All statistical analyses will be performed using SAS (SAS Institute, NC, USA) or R (R
Foundation for Statistical Computing, Vienna, Austria).
3.2
Summary Statistics
Data will be summarized with respect to disposition, demographics, pre-treatment
characteristics, safety outcomes, tolerability, and efficacy outcomes. Summary statistics for
continuous variables will include the number of subjects, the mean, median, standard deviation,
and range. For categorical data, summaries will include counts and percentages.
3.3
Precision
Results will generally be reported to 3 significant figures. Percentages will generally be reported
to 0.1 percentage points. P-values will be reported to two digits when greater than or equal to
0.095, to three digits when greater than or equal to 0.00095 and less than 0.095, and as <0.001
for all smaller values.
4. Analysis Samples
The following analysis samples will be used for testing safety, tolerability, and efficacy
endpoints:
x
Safety and Tolerability (ST) Sample: Participants who are eligible, randomized, and initiate
at least the first infusion of study drug, classified according to the actual treatment received.
Observations made after premature permanent discontinuation of study drug are included in
this sample, should such participants remain on study.
x
Efficacy As-treated (AT) Sample: Participants who are eligible, randomized, and initiate at
least the first infusion of study drug, classified according to the actual treatment received.
Observations made after premature permanent discontinuation of study drug are included in
this sample, should such participants remain on study.
x
Efficacy Modified Intent-to-treat (mITT) Sample: Participants who are eligible, randomized,
and initiate at least the first infusion of study drug, classified according to their randomized
treatment assignment. Observations made after premature permanent discontinuation of study
drug are included in this sample, should such participants remain on study.
5. Study Endpoints
5.1
Safety Endpoints
The following safety endpoints will be evaluated:
x
Time to mortality


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x
Time to first treatment-emergent SAE
x
Proportion of participants experiencing and number of unique events of each type of
treatment-emergent adverse event (TEAE) and serious TEAE classified by MedDRA system
organ class and preferred term
x
Proportion of participants experiencing and number of unique events of TEAEs classified by
seriousness, severity, relatedness to study drug, action taken with study drug, and outcome,
summarized across all MedDRA terms
x
Proportion of participants experiencing and number of unique events of each type of
treatment-emergent adverse event (TEAE) and serious TEAE classified by MedDRA system
organ class and preferred term
x
Instances of treatment-emergent laboratory abnormalities judged to be clinically significant
by assay
x
Mean change from baseline and percent change from baseline in weight, systolic blood
pressure, and diastolic blood pressure
x
Proportion of participants reporting any treatment-emergent suicidal ideation
x
Maximum post-baseline suicidal ideation planning, frequency, duration, controllability,
deterrents, and reasons
x
Proportion of participants reporting any post-baseline suicidal behavior, actual attempts, non-
suicidal self-injurious behavior, interrupted attempts, aborted attempts, preparatory acts or
behaviors, or completed suicide
x
Maximum post-baseline suicidal behavior lethality and potential lethality
Reported proportions will use as their denominator all participants in the ST sample.
5.2
Tolerability Endpoint
Participants will be judged tolerant of study drug if they complete all three infusions and remain
on study and free from any possibly or definitely drug-related and dose-limiting AEs to the week
16 visit. Tolerability will be summarized as the proportion of participants in a treatment group
who are tolerant of study drug. Reported proportions will use as their denominator all
participants in the ST sample.
5.3
Efficacy Endpoints
Secondary efficacy endpoints include measures of target engagement and measures of clinical
progression. Assessments of target engagement will focus on changes from baseline to the week
12 visit, the end of the study drug exposure interval. Assessments of possible effects on clinical
progression will focus on mean rates of change over the 16-week follow-up interval.
Target engagement measures:
x
proportion of participants who experience a 2-fold or greater decline between baseline and
the average of all follow-up assessments in relative expression of at least 2 of the following 3
pro-inflammatory genes measured in PBMCs: IL-6, IL-8, and MMP1 (primary target
engagement measure)


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x
relative expression levels of the following pro-inflammatory genes in PBMCs by Applied
Biosystems quantitative polymerase chain reaction (qPCR): IL-6, IL-8, and MMP1
x
concentrations of CRP in plasma and CSF by R&D Systems enzyme-linked immunosorbent
assay (ELISA)
x
concentrations of the following pro-inflammatory cytokines in plasma and CSF by Meso
Scale Discovery (MSD) V-PLEX multiplexed sandwich immunoassay: IL-ȕ IL-6, IL-8, IL-
17A/F, and TNF-Į
x
concentrations of the following analytes in plasma and CSF by R&D Systems sandwich
ELISA: sIL-6R, IL-6, and gp130
x
brain glial activation measured by [11C]-PBR28 PET
Clinical progression measures:
x
ALS Functional Rating Scale-Revised (ALSFRS-R) total score
x
Slow vital capacity (SVC) as percent of predicted normal volume
x
Handheld dynamometry (HHD) of the following maneuvers: left and right shoulder flexion,
left and right elbow flexion and extension, left and right wrist extension, left and right first
dorsal interosseous, left and right hip flexion, left and right knee flexion and extension, and
left and right ankle dorsiflexion
x
Grip strength of left and right hands
5.4
Effect Modifiers
IL6R genotype for the Asp358Ala polymorphism (SNP rs2228145, A > C) will be investigated
as a potential modifier of tocilizumab target engagement. IL6R genotype will be analyzed using
an additive model for penetrance, with participants carrying more copies of the C allele expected
to have higher sIL-6R levels and greater sensitivity to treatment with tocilizumab.
6. Measurement Definitions
6.1
Target Engagement Measures
6.1.1
Expression of pro-inflammatory genes in PBMCs
PBMCs will be separated from whole blood using BD Vacutainer Cell Preparation Tubes (CPT
Sodium Citrate tubes, catalog #362761). Sites will ship CPT tubes on ice to Dr. Robert Bowser’s
laboratory at Barrow Neurological Institute (BNI) for analysis on the day collected. PBMCs be
isolated and frozen -80°C. RNA will be extracted from isolated PBMCs using a Qiagen RNeasy
extraction kit (catalog #74106) using the manufacturer’s protocol.
Expression levels of RNA for three pro-inflammatory genes (IL-6, IL-8, and MMP1) and one
reference gene (GAPDH) will be estimated by quantitative real-time polymerase chain reaction
(qPCR). The analysis will use an Applied Biosystems Quantstudio 6 qPCR system, Thermo
Fisher TaqMan Gene Expression Assay system reverse transcription reagent and amplification
solution (catalog #4331182) and TaqMan Fast Advanced Master Mix (catalog # 4444556). The
primer and probe set used for each gene are as follows (Thermo Fisher catalog #4453320):


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Gene
Primer and Probe Set
IL-6
Assay ID Hs00985639_m1
IL-8
Assay ID Hs00174103_m1
MMP1
Assay ID Hs00899658_m1
GAPDH
Assay ID Hs99999905_m1
Each run of study samples will include RNA from pooled PBMCs from healthy controls for
reference. Each study sample and the pooled control sample will be run in triplicate on each
plate. A No Template control reaction will also be included in each run where nuclease-free
water replaces the cDNA template. Complementary DNA (cDNA) templates will be synthesized
for each sample from 0.5 ȝg of total RNA using a Thermo Fisher High-Capacity RNA-to-cDNA
Kit (catalog #4387406).
For each qPCR run, the reaction set up and volume for each well of a 96-well plate will include:
Component
Volume
2X TaqMan Fast Advanced Master Mix
10 ȝL
20X TaqMan Gene Expression Assay
1 ȝ/
Nuclease-free water
7 ȝ/
cDNA template from each sample
2 ȝ/
The TaqMan Fast qPCR protocol specifications will be:
A quantification cycle (Cq) value will be obtained from each well to determine relative
expression using standard ǻǻCt, log2-fold change calculations. Specifically, replicate Cq values
will be averaged for each gene, and the difference of each pro-inflammatory gene mean Cq from
the reference gene mean Cq will be calculated separately for study samples and the pooled
control sample ǻCq). The negative difference of differences (-ǻǻCq) calculated as the pooled
control ǻCq minus the sample ǻCq, the log-base 2 of the fold-change between study and control
samples, will be the measure of gene expression used for analysis. Higher values indicate greater
relative expression of pro-inflammatory genes.
6.1.2
Concentration of CRP by R&D Systems ELISA
Plasma and CSF samples will be frozen and stored at sites at -70°C or colder prior to being
shipped to Dr. Robert Bowser’s laboratory at BNI for analysis. At the end of the study, all
samples will be assayed as a single batch. Measurement of CRP will be performed using the
R&D Systems Human C-Reactive Protein DuoSet ELISA kit (catalog #DY1707) following the
qPCR Step
Temp
Time
Incubation with uracil-N-glycosylase
Polymerase activation
PCR denaturation
PCR annealing and extension
50°C
95°C
95°C
60°C
120 s
20 s
1 s
20 s


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manufacturer’s instructions. Briefly, plasma will be diluted 1:200 in Tris-buffered saline (TBS).
CSF will be diluted 1:100 in TBS. Diluted samples will be incubated in the 96-well plate
previously coated with 2.0 μg/mL capture antibody for 1.5 hrs. After washes, 100 μL of 90
ng/mL detection antibody will be added per well for 1.5 hrs, wells washed, streptavidin-HRP
added for 20 min, and tetramethylbenzidine reaction product generated for 15 min. Standard
curves will be generated using purified CRP protein in the following dilution series:
Specimen
Standard concentration sequence (ng/mL)
Plasma
25, 8.33, 2.78, 0.926, 0.309, 0.103, 0.034,0
CSF
1.0, 0.5, 0.25, 0.125, 0.063, 0.031, 0.016, 0
The geometric mean of replicate concentration estimates will be used for analysis.
Concentrations of CRP in plasma and CSF will be reported as ȝJP/ and ng/mL, respectively.
6.1.3
Concentration of pro-inflammatory cytokines by MSD V-PLEX
Plasma and CSF samples will be frozen and stored at sites at -70°C or colder prior to being
shipped to Dr. Robert Bowser’s laboratory at BNI for analysis. At the end of the study, all
samples will be assayed as a single batch. The concentration of the following cytokines will be
assayed: IL-ȕ IL-6, IL-8, IL-17A/F, and TNF-Į. Measurements for IL-ȕ IL-6, IL-8, and
TNF-ĮZLOO be performed using an MSD V-PLEX Proinflammatory kit (catalog #K15053).
Measurements for IL-17 will be performed using an MSD V-PLEX IL-17A/F kit (catalog #
K151WN). Plasma samples will be diluted 1:2. CSF samples will be used undiluted. A total
sample volume of 50 μl will be assayed per well. Samples will be analyzed in duplicate.
Standard curves will be generated using the following concentrations for each pure protein:
Cytokine
Standard concentration sequence (pg/mL)
IL-ȕ
576, 144, 36, 9, 2.25, 0.563, 0.141, 0
IL-6
764, 191, 47.8, 11.9, 2.98, 0.746, 0.187, 0
IL-8
621, 155, 38.8, 9.7, 2.43, 0.606, 0.152, 0
IL-17
5430, 1360, 339, 84.8, 21.2, 5.3, 1.33, 0
TNF-Į
369, 92.3, 23.1, 5.77, 1.44, 0.36, 0.0901, 0
Lower limit of detection (LLOD) ranges and lower limits of quantitation (LLOQ) are the
following:
Cytokine
LLOD
(pg/mL)
LLOQ
(pg/mL)
IL-ȕ
0.01-0.17
0.646
IL-6
0.05-0.09
0.633
IL-8
0.03-0.14
0.591
IL-17
0.930
7.57
TNF-Į
0.01-0.13
0.690


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The geometric mean of replicate concentration estimates will be used for analysis.
Concentrations will be reported as pg/mL.
6.1.4
Concentration of sIL-6R, IL-6, and gp130 by R&D Systems ELISA
Plasma and CSF samples will be frozen and stored at sites at -70°C or colder prior to being
shipped to BNI, inventoried, and sent to Dr. Carolanne Milligan's laboratory at Wake Forest
School of Medicine for analysis. At the end of the study, all samples will be assayed as a single
batch. The concentrations of the following proteins will be assayed by R&D Systems sandwich
ELISA: sIL-6R, IL-6, and gp130.
Protein
Assay
Control(s)
sIL-6R
Catalog #DR600
lot #P185493 (first six plates)
lot #P191955 (last two plates)
IL-6
Catalog #Q6000B
lot #P186912
gp130
Catalog #DGP00
lot #P161984
Catalog #QC05
lot #1279937 (low control)
lot #1279938 (medium control)
lot #1279939 (high control)
Catalog #QC193
lot #732-180404
Catalog #QC23
lot #P180985
All samples, controls, and standards will be run in triplicate following kit instructions. For sIL-
6R, plasma samples will be diluted 1:100 or more as needed and CSF sample will be assayed
undiluted. For IL-6, all plasma and CSF samples will be assayed undiluted. For gp130, plasma
samples will be diluted 1:100 and CSF sample will be diluted 1:9. The following standard
concentration series will be used for IL-6: 0, 0.75, 1.5, 3, 6, 12, and 30 pg/mL.
For sIL-6R and gp130, plates will be read five minutes after the stop solution is added on a
BioTek Epoch plate reader (BioTek Instruments, Winooski, VT) set to 450 nm, with wavelength
correction at 570 nm. For IL-6, relative light units (RLUs) will be determined using a Victor 3
plate reader (PerkinElmer Life Science, Boston, MA) with the luminometer set for a lag time of
1.0 sec and a read time for wells of 0.5 sec.
If the three replicates have a coefficient of variation (CV) greater than 15%, the most disparate
value will be eliminated, and the average of the remaining two wells will be used. If the CV is
still above 15% after removing any outlier, then the sample will be re-run.
Concentrations will be estimated from OD values fit to a four-parameter logistic curve for sIL-
6R and gp130 assays and from RLU values fit to a quadratic curve for IL-6 assay using PRISM
software (GraphPad Software, San Diego, CA). The mean of replicate concentration estimates
will be used for analysis.
6.1.5
IL6R genotyping for Asp358Ala polymorphism
Blood for determining IL6R genotype will be frozen and stored at sites at -70°C or colder prior to
being shipped to BNI, inventoried, and sent to Dr. Carolanne Milligan's laboratory at Wake
Forest School of Medicine for analysis. At the end of the study, all samples will be assayed as a
single batch.


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DNA will be purified from whole blood using Qiagen chemistry on the Gentra AutoPure
platform. Absorption ratio at 260/280 nm will be used to determine DNA concentration and
purity. A 9 ng sample of DNA will be genotyped according to manufacturer’s instructions using
a commercially available and validated Applied Biosystems TaqMan SNP genotyping assay
(catalog # 4351379) for the IL6R Asp358Ala polymorphism (SNP rs2228145 A>C, assay ID
C    16170664_10, lot #P160407-000 C10). The genotype at that locus will be read using an
allelic discrimination protocol on an Applied Biosystems qPCR (model 7300, Foster City, CA).
Each gene expression plate will be run with three control samples that had been fully sequenced
and are known to carry one of the three genotypes, and three no-template controls.
Each participant will be scored for the IL6R Asp358Ala polymorphism as the count of the minor
C allele, with CC homozygous participants expected to be more sensitive to treatment with
tocilizumab.
6.1.6
Brain glial activation measured by [11C]-PBR28 PET
Glial activation will be estimated in a subset of participants by magnetic resonance positron
emission tomography (MR-PET) using the [11C]-PBR28 ligand. The subset excludes individuals
homozygous for the T/T allele of the Ala147Thr TSPO polymorphism (rs6971) associated with
low affinity for [11C]-PBR28. Glial activation will be quantified as a mean standardized uptake
value (SUV) using PET images acquired from 60 to 90 minutes post-injection of approximately
430 MBq [11C]-PBR28. FreeSurfer v6.0 (https://surfer.nmr.mgh.harvard.edu) will be employed
to circumscribe a region of interest (ROI) defined anatomically as the precentral gyrus and the
anterior portion of the paracentral lobule, bilaterally. SUV of the ROI will be normalized to the
SUV of the whole brain and expressed as a SUV ratio (SUVR) to control for inter-individual
variability in the global [11C]-PBR28 PET signal.
6.2
Clinical Progression Measures
6.2.1
ALSFRS-R
The ALSFRS-R (Cedarbaum et al. 1999) is a 12-item clinician-completed interview scale for
assessing participants’ function in four domains: bulbar, fine motor, gross motor, and respiratory.
Each item is scored from 0 to 4 with higher scores indicating greater function. The ALSFRS-R
total score is the sum of all items (range 0 to 48).
6.2.2
SVC
Slow vital capacity (SVC) is the maximum volume of air that can be slowly exhaled after slow,
maximal inhalation. Trained technicians coach participants through 3 to 5 maneuvers using an
EasyOne Plus spirometer (ndd Medical Technologies, Inc., Andover, MA). Assessments will be
analyzed if at least two acceptable maneuvers are recorded. The maximum volume expired is
converted to percent of predicted normal. Normal values are calculated based on sex, age, and
height using equations published by Knudsen et al. (1983). Higher values indicate better
pulmonary function.
6.2.3
Handheld dynamometry
Handheld dynamometry (HHD) will be used by trained technicians to estimate isometric strength
using a MicroFET2 HHD (Hoggan Scientific, Salt Lake City, UT). Nine upper and lower
extremity muscles or muscle groups will be examined: shoulder flexion, elbow flexion, elbow
extension, wrist extension, first dorsal interosseous contraction, hip flexion, knee flexion, knee


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extension, and ankle dorsiflexion. Each muscle or muscle group will be measured twice or three
times bilaterally. The average of the two highest measurements will be analyzed.
Two measures will be derived from HHD data: (1) upper and lower extremity megascores at each
visit, and (2) time from baseline to first loss of measurable strength in any of the 18 muscles or
muscle groups.
To calculate megascores, the mean and standard deviation of each muscle or muscle group,
without regard to laterality, will be calculated from the baseline assessment of all participants in
the AT sample. Maneuvers that cannot be completed by a participant due to weakness will be
scored as zero kg. Strength estimates of each bilateral muscle or muscle group will be converted
to Z scores by subtracting the relevant mean and dividing by the relevant standard deviation. Z
scores for all upper extremity measurements (shoulder flexion, elbow flexion, elbow extension,
wrist extension, and first dorsal interosseous contraction) and all lower extremity measurements
(hip flexion, knee flexion, knee extension, and ankle dorsiflexion) will be averaged to yield
upper and lower extremity megascores. Larger values indicate greater strength.
6.2.4
Grip strength
Grip strength of the left and right hand will be measured using a study-approved Jamar hand
dynamometer (Performance Health, Warrenville, IL or Lafayette Instrument Company,
Lafayette, IN). At each assessment, two trials will be completed with each hand. The average
isometric strength recording of the stronger hand, measured in kg of force, will be analyzed.
7. Power Calculations
With 16 participants randomized to tocilizumab and followed for safety outcomes, the study will
have 80% power to detect adverse events expected to occur in at least 10% of patients. Although
under-powered for comparing rates of rare events, the study will have 80% power to detect
treatment differences in events expected to occur, for example, in 50% of placebo participants if
tocilizumab increases the hazard 4.2-fold.
The study will have 80% power to declare tocilizumab treatment tolerable based on non-
inferiority to 50% tolerance with a one-sided 95% exact confidence bound if the true tolerance
rate is at least 81%
The study will have 80% power to detect a significant treatment-dependent difference in the
proportion of participants who experience a 2-fold or greater decline between baseline and the
average of all follow-up assessments in at least 2 of the 3 pro-inflammatory genes if no more
than 5% of placebo participants experience such a change and at least 30% of tocilizumab
participants do. Assuming that at least 80% of participants are truly in a high inflammatory state
given our enrollment criteria, this condition would correspond to roughly 6% vs. 38% response
among susceptible patients randomized to placebo or tocilizumab, respectively. We will have
greater power to detect quantitative changes in expression of pro-inflammatory genes. The study
will have 80% power to detect a significant difference in expression equal to an effect size of
1.50 based on two-tailed tests at alpha = 0.017, applying a Bonferroni correction for multiple
comparisons over 3 inflammatory genes.
If we conservatively estimate power for detecting treatment-dependent differences in 16-wk
change in ALSFRS-R, SVC, and HHD megascores using a simple two-group t-test, the study
will have 80% power for effect sizes of 0.76. With a standard deviation for ALSFRS-R rate of


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change equal to 1.58 units/month based on the follow-up schedule of this trial and variance
component estimates from the Ceftriaxone trial, the study would have 80% power if tocilizumab
reduces decline in ALSFRS-R by 2.0 units/month based on a two-tailed at alpha = 0.05.
8. Participant Characterization
8.1
Baseline Characteristics
Each analysis sample will be summarized overall and by treatment group for the following
characteristics: randomization site; age, sex, race, ethnicity, El Escorial diagnosis, years since
ALS symptom onset, delay between symptom onset and ALS diagnosis, site of ALS symptom
onset, use of riluzole at baseline, and baseline levels of IL-6, IL-8, and MMP1 gene expression,
CRP and cytokines in plasma and CSF by MSD V-PLEX, sIL-6R, IL-6, and gp130 in plasma
and CSF by R&D System ELISA, ALSFRS-R total score, SVC, HHD upper and lower extremity
megascores, and grip strength.
8.2
Participant Disposition
The number of participants who consented and were screened, randomized, completed scheduled
follow up, and prematurely withdrew will be summarized overall and by treatment group.
Reasons for screen failure and for withdrawal from study will be tabulated and quantified as a
percentage of consented and randomized participants, respectively.
9. Statistical Analyses
9.1
Safety Measures
9.1.1
Mortality
If any mortality is observed, time to mortality will be estimated starting at date of treatment
initiation by Kaplan-Meier product-limit estimates in the ST sample. Association with treatment
will be tested by log-rank test. The hazard ratio for treatment with tocilizumab vs. placebo will
be estimated by Cox regression with 90% confidence bounds.
9.1.2
Treatment-emergent Adverse Events
Time to first treatment-emergent SAE will be estimated starting at date of treatment initiation by
Kaplan-Meier product-limit estimates in the ST sample. Association with treatment will be tested
by log-rank test. The hazard ratio for treatment with tocilizumab vs. placebo will be estimated by
Cox regression with 90% confidence bounds.
The incidence of TEAEs and serious TEAEs will be summarized for the ST sample by the
number of events of a given classification experienced by participants in each treatment group
and by the number and proportion of participants experiencing such an event in each treatment
group in the ST sample. TEAEs will be summarized in aggregate across all MedDRA terms and
separately by MedDRA system organ class and preferred term. For each type of TEAE or serious
TEAE, the association with tocilizumab will be tested by Poisson regression for counts of events
per participant and by Fisher's exact test for the proportion of participants experiencing the given
type of event. Association of tocilizumab with the overall TEAE rate will be tested by negative
binomial regression.


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TEAEs will also be summarized by tabulating treatment-specific frequency distributions for the
following characteristics: (a) seriousness, (b) severity, (c) relatedness to study drug, (d) action
taken with study drug, and (g) outcome. For each level of a given TEAE characteristic,
summaries will include the number of events of a given classification and the number and
proportion of participants for which that level of a characteristic was the worst they experienced
(treating any unknown characteristic as not worst). For each characteristic, the association with
tocilizumab will be tested by exact Cochrane-Armitage test for the proportion of participants
experiencing a given level of the characteristic
9.1.3
Suicidality
The proportion of participants in the ST sample who report any post-baseline suicidal ideation or
any post-baseline suicidal behavior will be summarized by treatment group. Suicidal behaviors
will include: actual attempts, non-suicidal self-injurious behavior, interrupted attempts, aborted
attempts, preparatory acts or behaviors, and completed suicide. The most severe ideation,
maximal frequency, maximal duration, minimal controllability, minimal deterrents, maximal
reasons for ideation, maximal actual lethality or medical damage, and maximal potential lethality
will be summarized as means, standard deviations, medians, and ranges by treatment group.
9.1.4
Safety Labs
The absolute level and the absolute change from baseline for each safety laboratory assay will be
summarized as means, standard deviations, medians, and ranges at each visit by treatment group
among participants in the ST sample. The proportion of participants with abnormal and clinically
significant safety lab levels will be summarized by treatment group by visit and at any post-
baseline visit. For each safety lab, the association of clinically significant safety lab
abnormalities with tocilizumab will be tested by Fisher's exact test.
9.1.5
Anthropometrics and Vital Signs
The absolute level and the percent change from baseline for weight, systolic blood pressure, and
diastolic blood pressure will be summarized as means, standard deviations, medians, and ranges
at each visit by treatment group among participants in the ST sample. Associations with
tocilizumab will be tested in a shared-baseline, linear mixed model with fixed terms for month
since baseline and treatment x month interaction and random participant-specific intercepts and
slopes with unstructured covariance.
9.2
Study Drug Tolerance
The number of completed infusions of study drug among participants in the ST sample will be
summarized by treatment group. The proportion of participants classified as tolerant of study
drug will be summarized for each treatment group. Associations with tocilizumab will be tested
by Fisher's exact test. The relative risk of intolerance will be estimated with exact confidence
bounds.
9.3
Target Engagement Measures
9.3.1
Primary Analysis
The primary analysis of target engagement measures will compare the proportion of participants
in the AT sample who experience a 2-fold or greater decline between baseline and the average of
all follow-up assessments in at least 2 of the 3 pro-inflammatory genes. Participants lost to


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follow-up prior to any follow-up blood collection will be assumed to have not experienced a 2-
fold or greater decline. Associations with tocilizumab will be tested by Fisher's exact test. The
relative risk of intolerance will be estimated with exact confidence bounds. The following SAS
code specifies the analysis, with trt a 0/1 indicator variable indicating treatment with 4/mg/kg
tocilizumab and response a 0/1 indicator variable indicating a 2-fold or greater decline in at least 2
of the 3 pro-inflammatory genes:
proc freq data=xxx; 
tables trt * response / fisher; 
exact relrisk(method=score column=1); 
A relative risk greater than 1 significant at two-tailed p < 0.05 would indicate successful target
engagement.
9.3.2
Secondary Analysis
Secondary analysis of target engagement measures will use data from the AT sample in a shared-
baseline, repeated-measures mixed model of log-transformed gene expression, CRP, cytokine,
sIL-6R, or gp130 levels with fixed terms for visit (5 levels: baseline and weeks 4, 8, 12, and 16),
treatment group (2 levels: placebo and 4 mg/kg tocilizumab) x post-baseline visit (4 levels) and
random participant-specific visits with unstructured covariance. The interaction between fixed
terms for treatment group and visit will be restricted to post-baseline visits by including a
numeric indicator variable (0 pre-treatment, 1 post-treatment) in the interaction. Data from any
early termination visits will be assigned to the next scheduled post-baseline visit. The following
SAS code specifies the model:
proc mixed data=xxx method=reml; 
class id trt visit; 
model Value = visit post*trt*visit; 
random visit / subject=id type=un; 
Treatment-dependent differences in the change from baseline to the average of all follow-up
visits and to week 16 specifically will be estimated by linear contrasts and tested using a two-
tailed Wald-test at alpha = 0.05. The following SAS code specifies the linear contrasts (with the
sort order for treatment group being placebo first and 4 mg/kg tocilizumab second, and the sort
order for visit being chronological):
estimate "3|TCZ vs PLB|dWeek 16" post*trt*visit 0 0 0 0 -1 0 0 0 0 1 / cl; 
estimate "3|TCZ vs PLB|dWk 4-16" post*trt*visit 0 -1 -1 -1 -1 0 1 1 1 1 / cl divisor=4; 
The estimates and their 95% confidence bounds will be back-transformed for reporting as a
treatment-dependent ratio of post-baseline and week 16 concentration relative to baseline.
Additional contrasts will be used to estimate treatment- and visit-specific concentrations with
back-transformation for reporting on their original scale of measurement.
9.3.3
Subgroup Analyses
Differences in the effect of tocilizumab on target engagement measures dependent on IL6R
genotype will be estimated by adding minor allele count, minor allele count x visit, and minor
allele count x treatment x post-baseline visit interaction terms to the primary target engagement
model.


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9.4
Efficacy Measures
9.4.1
Primary Analysis
The primary analysis of clinical efficacy measures will use data from the mITT sample in a
shared-baseline, random-slopes mixed model with fixed terms for month from treatment
initiation and treatment group (2 levels: placebo and 4 mg/kg tocilizumab) x month from
treatment initiation interaction and random participant-specific intercepts and slopes with
unstructured covariance. Data from any early termination visits will be at their observed follow-
up time. The following SAS code specifies the model and the linear contrast used to estimate the
treatment-dependent difference in rate of change per month:
proc mixed data=xxx method=reml; 
class id trt; 
model Value = month trt*month; 
random intercept month / subject=id type=un; 
estimate "3|TCZ vs PLB|Slope/mn" intercept 0 month 0 trt*month -1 1 / cl; 
9.4.2
Secondary Analysis
A secondary analysis of clinical efficacy measures will use the same shared-baseline, repeated-
measures mixed model described for secondary analysis of target engagement measures with
modification of the visit term to reflect the schedule of assessment for each measure (ALSFRS-R
and SVC = 5 levels [baseline and weeks 4, 8, 12, and 16]; HHD and grip strength = 3 levels
[baseline and weeks 8 and 16]). The same estimates of treatment- and visit-specific changes from
baseline and treatment-dependent differences in change from baseline will be used.
9.5
Additional Considerations
9.5.1
Multiplicity Adjustments
Tests of treatment-dependent differences in time to mortality, time to first treatment-emergent
SAE, overall TEAE rate, and distribution of TEAE characteristics will be declared significant
based on two-tailed p < 0.10 without adjustment for multiple comparison. Tests of treatment-
dependent differences in other safety outcomes will be declared significant based on two-tailed p
< 0.05 without adjustment for multiple comparison. These criteria increase the probability of
type 1 errors but also increase power for detecting true differences should any exist. Both
unadjusted and step-down Bonferroni-adjusted p-values will be reported for secondary analyses
of target engagement measures. Both unadjusted and step-down Bonferroni-adjusted p-values
will be reported for clinical efficacy measures.
9.5.2
Missing Data
Baseline values for target engagement and clinical efficacy endpoints will be determined from
the last non-missing data collected prior to the first dose of study medication. The planned mixed
models yield estimates that are unbiased conditional on the observed scores under a missing at
random assumption. Sensitivity analyses may be pursued to impute missing values or otherwise
construct models for unobserved outcomes if more than 20% of participants are missing follow-
up data for any reason.


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10. References
Cedarbaum JM, Stambler N, Malta E, Fuller C, Hilt D, Thurmond B, Nakanishi A. The
ALSFRS-R: a revised ALS functional rating scale that incorporates assessments of
respiratory function. BDNF ALS Study Group (Phase III). J Neurol Sci. 1999 Oct 31;169(1-
2):13-21.
Knudson RJ, Lebowitz MD, Holberg CJ, Burrows B. Changes in the normal maximal expiratory
flow-volume curve with growth and aging. Am Rev Respir Dis. 1983 Jun;127(6):725-34.