The Lab

• Tara Martinez • Jessica Carmen

Transverse Myelitis: Clinical and • Deepa Deshpande • Ariel Williams
Immunologic Considerations • Yun-Sook Kim • Cidi Wee
• Mamatha Nayak • Katie Provost
• Irina Shats • Talibah Metcalf
Douglas Kerr MD/PhD
• Sonny Dike • Michele Pucak
Associate Professor, Neurology • Jenn Drummond • Emily Potter
Johns Hopkins Hospital • Sandeepa Sur
• Ann Peters
Director Johns Hopkins TM Center • Nidhi Rumpal
• Michael Levy
Director, Johns Hopkins Project
RESTORE

Collaborations/Support Outline

• Adam Kaplin • NIH

• Benjamin Greenberg
• Peter Calabresi
• Chitra Krishnan
• Mahendra Rao
• Tim Magnus
• Piotr Walczak
– R01/NINDS
• 1) Clinical Approach to Patients with Acute
– R01/NINDS Myelopathies
– R01/SAIC
• 2) Immunoablation in Aggressive MS
• Transverse Myelitis
Association • 3) Immunology and Biomarkers of TM
• ALSA
• 4) Regenerative Strategies in TM and MS
• MDA
• Families of SMA – Remyelination with glial progenitors
• Packard ALS Center
• NMSS
– Motor neuron reconstitution with ES cells

What is TM? What is TM?

• Acute inflammatory disorder of a part of the spinal cord
• Immune-mediated
• Four symptom groups: motor dysfunction (weakness,
spasticity), sensory dysfunction (numbness, paresthesias,
dysesthesias), pain, autonomic dysfunction (sexual , bowel
and bladder)
• Monophasic
• Monofocal
• In most cases, TM is likely post-infectious and
involves breakdown of immune tolerance for self
antigens
– Molecular mimicry
– Superantigen mediated T lymphocyte activation
• Why the focality?
– Unique inflammatory trigger
– Unique lymphocyte trafficking or permeability
– Unique immune response in the spinal cord

1
 Acute TM: inflammation! Results of Inflammation
Demyelination: Damage to Injury Tissue
Necrosis/Axonal Injury:
myelin producing cells damage secondary to vascular
resulting in loss of the changes and/or inflammation.
insulation around nerves Neuronal/axonal death

TM Diagnostic Criteria

• Johns Hopkins Transverse

Myelitis Center
• Established in Oct 1999
– Clinical care
– Clinical research
– Basic science research
– 1145 patients with acute
non compressive
myelopathies evaluated
• Multi-disciplinary
– Neurology, urology, rehab,
neurosurgery,
rheumatology, anesthesia,
neuroradiology, psychiatry

Myelopathy Type Number %

All 1145 100
Compressive 8 0.7
Non-Compressive, Non- 148 13
inflammatory
ischemia 108 9.8
radiation 8 0.7
Fibrocartilaginous 8 0.7
embolism Acute
Falsely negative 24 2.1
inflammatory Myelopathy
Inflammatory 952 86
Infectious 48 4
Algorithm
Rheum Disease 112 10
Associated
Confined to CNS
NMO 46 4%
ADEM 24 2%
MS 130 12%
Idiopathic 398 36%
Monophasic
Idiopathic 194 18%
Recurrent

2
Step I: Define the presence
Clinical Presentation
of myelopathy

• 4 Symptom groups
– Motor
– Sensory
– Autonomic
– Pain

Clinical: Motor Clinical: Sensory

• Motor • Paresthesias
– Weakness paraplegia • Numbness
– Legs usually > arms • Warmth
• Central cord lesion in the C spine can be arms > legs
• Try pinprick and temperature on anterior
– Tone is increased, decreased or normal
• Long-standing-increased
and posterior torso
• Acute, severe-decreased

Clinical: Autonomic Clinical: Pain

• Urinary urgency • “Burning” radicular pain

– Decreased interval from bladder fullness to evacuation
• “Tight squeezing”
– Nocturia
• Urinary retention • “banding” sensation
– “shock” like scenario
• Bowel urgency, retention
• Sexual dysfunction
• More common with intrinsic than extrinsic

3
 Step II: Rule out Step III: Define Presence of
compressive etiology Spinal Cord Inflammation

STEP V: Define extent of

Step IV inflammation

• Consider non-inflammatory causes of

myelopathy
• Consider early inflammatory myelopathy
• Repeat LP in 2-7 days

Step VA: Step VB:

Define Potential
work-up for
presence of systemic
infection inflammatory
disease

4
Step VC: Define Extent of Myelopathy as the First
Inflammation Presentation of MS
• Multiphasic by history or exam

• Imaging characteristics in the spinal cord may distinguish from TM

– lesion that occupies less than ½ the cord diameter and less than 2 rostral-
caudal spinal segments and the presence of 2 or more brain lesions

• Clinical presentation often milder: sensory >motor, always incomplete

• Oligoclonal bands much more likely in MS

• Brain MRI is often helpful in distinguishing MS from TM

• Several studies now suggest that in certain patients, treatment of patients

at high risk of developing MS may delay conversion

Genes/Environment

Trigger

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New Treatments???
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IL-6 and TM
??????

CNS Injury CNS Dysfunction

Acute TM Depression Relapse

Group Average of TM
Elevated CSF IL-6
Cytokine Elevation

5
 Quantitative Serum and CSF
IL-6 IL-6 Analysis
• Pro-inflammatory cytokine
• Stimulates HPA axis to produce cortisol.
• Induced by TNF-α
• IL6-R on neurons, astrocytes and
oligodendrocytes
• Acts through a JAK/STAT pathway to stimulate
iNOS production in the heart.
• iNOS implicated in causing CNS injury.
• Does IL6 activation stimulate further
inflammation and/or direct neural injury?

IL-6 Correlates with

Acute CSF IL-6 Correlates
CSF Nitric Oxide
with Sustained Disability
Production

Prognostic Impact of Acute IL-6 Infused Rats Become

CSF IL-6 Weak

6
 Plastic Sections of IL-6
Pathology of TM
Infused Rats

Genes/Environment
Summary Genetic Screening Genetic Screening
Trigger
Thalidomide TNF-α
IL-6
Erythropoietin
• IL-6 is markedly elevated in the CSF of
patients with acute TM STAT3
Statin
• CSF IL-6 levels correlate with long term iNOS
disability and with markers of neural injury Minocycline
• IL-6 is capable of inducing neural injury in CNS Injury CNS Dysfunction
model systems
PARP Acute TM Depression Relapse
• IL-6 acts through the JAK/STAT system Inhibitor
resulting in iNOS and PARP activation Rehabilitation Antidepressant:
Free Radical 5-HT/NE,
Adduct CRH antagonist,
Repair IL-6 antagonist

The Problem

Rebooting the Immune System: High
Dose Cyclophosphamide (Revimmune)
in MS
• MS therapies are only modestly effective
• Newer MS therapies pose significant and
largely undefined risks
• Even on the best current MS therapies,
patients continue to accrue disability (17%
increase over 2 years on natalizumab)
• Therapies must continue indefinitely

7
 Potential Solutions Results & Risks of HSCT in MS

• Clinical stabilization ~75%; variable duration

• Immunoablation with autologous stem cell • MRI:
transplantation (T cell depleted)
Decrease of T2 plaque volume.
• Rebooting the immune system without Few Active lesions.
transplantation (Revimmune Continued or ACCELERATED atrophy.
• Risks: Age >40
Poor EDSS scores
1o Progressive
Less Effective T cell depletion
• Mortality ~5 – 9%

Revimmune: Immune Ablation Advantages of Revimmune

Without Transplantation
• Revimmune eliminates mature and maturiing
WBC.
• Bone marrow Stem Cells not killed by
Revimmune.
• Aldehyde dehydrogenase
inactivates Cy.
• Immune System reconstituted from endogenous
stem cells.
“Re-booted” Immune system may be
tolerant of autoantigen.
• Revimmune is effective in eliminating mature leukocytes.
• BUT does not damage patient’s own marrow stem cells.
• Less risky treatment: Avoids most severe conditioning
procedures; Avoids HSC “mobilization”.
• Unlike HSCT, peripheral blood cells are not re-infused.
Less likely contamination.
• Much lower cost (30K vs. 300 K)
• Lower mortality (0.5% vs 5-7%)

Revimmune Protocol Side Effects of Revimmune Rx

SIDE EFFECT FREQUENCY TREATMENT

Nausea Universal Ondansetron (Zofran)

• Informed consent. Alopecia Universal Temporary
• Pre-Rx medical evaluation. Anemia, Common RBC transfusion,
Thrombocytopenia Platelet transfusion
• Cy: 50 mg/Kg/day x 4 days.
Leukopenia Universal GCSF
Hydration, Mesna, Zofran.
Loss of Immunization Universal Re-immunize at 1 yr
Anti-infection prophylaxis.
Herpes Zoster 40% Acyclovir, etc.
• gCSF 6 days later.
Febrile Neutropenia Occasional Broad Spectrum Antibiotics
• Follow daily until hematologic recovery:
Give RBC, platelets, etc. as needed. Amenorrhea Common in Older Usually temporary
women
• Re-immunize at 1 year.

8
 Hematologic Manifestations of
Rx of MS Patients with Revimmune
HiCy: Lymphocyte Counts

9 Patients Treated at JHU so far

• Marked decrease in MRI lesions.

• Clinical improvement.

• Better in patients with lower EDSS scores.

Reduced Disability After

Clinical Outcome – MSFC
Revimmune

Patient Time to Baseline (Z scores) Follow-up (Z scores)

number follow-
25 ft- 9HPT PASAT MSFC Score 25 ft- 9HPT PASAT MSFC Score
up
tim 3 time 3
(month
ed d
s)
wa walk
lk

HiCy01 24 17.4 -3.33 -3.81 -24.5 N/A -1.78 -2.59 -4.37

HiCy05 24 N/A -2.65 -1.70 -4.35 N/A -2.92 -0.968 -3.89
HiCy07 24 -0.291 0.157 -2.02 -1.57 -0.503 1.52 -2.43 -0.407
HiCy09 24 -0.488 -0.595 -1.68 -1.79 -0.625 0.499 0.0081 1.13
HiCy11 24 2.82 -0.475 -3.16 -6.46 -0.488 -0.423 -0.398 -0.333
HiCy13 18 -0.604 -2.25 -0.236 -1.88 -0.634 1.23 -0.496 1.37
HiCy14 15 -0.543 0.0685 -0.073 0.539 -0.625 0.25 0.821 1.70
HiCy15 15 -0.404 1.33 -0.805 0.93 -0.671 1.91 0.496 3.08
HiCy19 6 N/A -0.03 -1.82 -1.85 -0.537 1.82 -1.21 1.15

HyCy_07_BS HyCy_09 KG
Brain Parenchymal Volume Brain Parenchymal Volume

0.875 0.83
0.87 0.825
0.865 0.82
0.815
0.86 -1mo -1mo
0.81
0.855 0 0
3mo 0.805 3 mo
0.85
6mo 0.8 6 mo
0.845 0.795
0.84 0.79
0.835 0.785
BFV BFV

9
 Conclusions: Revimmune Stem Cells of the Brain and Spinal
Cord

Embryonic Somatic Stem Cells

– Revimmune is safe and induces rapid and sustained Stem Cells (Fetal or Adult Tissue)
clinical and radiologic improvement
– Patients with advanced disease continue to have
progression of disability while those with early disease NSC
GRPs
have an improvement in function Naturally restricted
– Planning for Phase III Trial Underway NRP GRP fate means high
efficacy and control
• Revimmune + Copaxone vs. Copaxone
with fewer side
• Multi-Center effects
APC OPC
• Blinded Examiner at baseline and 24 months

Neurons Astrocytes Oligodendrocytes

(product growth factors) (produce myelin)

Benefits of GRPs for

Treatment of Myelin Glial Restricted Precursor Cells
Disorders of the CNS (GRPs)
Cell Type Advantages Disadvantages

Function
ƒReplicate & migrate appropriately in vivo
ƒ Predictable in vivo differentiation into oligos and
Glial restricted progenitor astrocytes ƒ Tissue sourcing adequate for orphan diseases
ƒRobust myelination plus trophic support but not larger diseases
(GRP)
(2nd generation products to be derived from cell
ƒ Little risk of ectopic neurons
lines)
Q Therapeutics Preparation
ƒ Minimal in vitro manipulation – genetically stable
ƒ Readily scalable to GMP
ƒAvoids complexities of ESCs

Neural Stem Cells (NSC)
StemCells, Inc.
ƒ Tissue sourcing
ƒ Environmental malleability: less predictable
ƒ Higher proliferative potential vs GRPs survival, differentiation
ƒAvoids complexities of ESCs ƒ Less myelination
ƒ Ectopic neurons are a risk

ƒ Complex in vitro differentiation and

ƒ Unlimited self-renewal purification Immunohistochemical characterization of glial progenitor cells
ESC-derived OPC
ƒ [Efficiency of in vivo differentiation and ƒUndifferentiated cells are tumorigenic
Geron
myelination unknown] (green = A2B5+; blue= DAPI nuclear stain)
ƒEctopic neurons are a risk

Results: Robust Myelination by Results: hGRPs Mature into

Implanted hGRPs-Oligos
Oligos with Normal Compact Myelin

Ctx 1 mm
1 μm
A single injection
resulted in Treated shiverer mouse
widespread CC
myelination of the
entire forebrain LV
Striatum

• Shiverer is a mouse with a mutation that

produces defective MBP, resulting in
defective myelin. Control shiverer mouse
• Human GRPs mature into oligos that
produce normal MBP (green) and myelinate Dr. Goldman’s study has
extensive regions of shiverer mouse brain. quantitatively and
100 μm hNA/MBP qualitatively determined
• All myelin seen here is produced by human
GRPs that matured into oligos (red nuclei).
that transplant-produced 1 μm
Data from Goldman Lab, myelin is normal
Nature Medicine 2004 Data from Goldman Lab, Nature Medicine 2004

10
 Initial Indication:
Transverse Myelitis
Methodology
• Single focal lesion in spinal cord
– Acute, monophasic and monofocal lesion
– Pathology of demyelination and axonal loss • Stereotaxic injection of GRPs
– Biomarkers to define severe patients with poor – Alternatives include ES cell-derived motor neurons or
likelihood of survival neural stem cells
– Easy surgery, small target • Stable pre-treatment course
– Surrogate markers: SSEPs, DTI, MTw • Discrete baseline imaging and clinical measures
– Straightforward path to determination of clinical • Dose escalation cohorts
efficacy
• Intra-operative lesion localization
• Remyelination and axon survival
• Function recovery • Transplant into epicenter, rostral border and
– Benefits of orphan indication while establishing proof caudal border of T2 lesion
of concept for larger disease targets
• Planned IND in 2/08

DTI based Fiber Tracking in

Outcome Measures
the Human Spinal Cord

• Quantitative vibratory thresholds in feet

• Magnetization Transfer imaging
• Diffusion Tensor imaging

Susumu Mori/Peter Calabresi

Random or Directed
Embryonic Stem Cells
Differentiation of ES Cells

11
 Developmental Biology
Mature Motor Neuron
Applied to Embryonic Stem
Markers
Cells
• Function of RA
– Neuralization
– Caudalization
• Function of Shh
– ventralization

In Vivo Transplantation of ES Cell- • DAK\StemCell.wmv

Derived Motor Neurons in Paralyzed
Rats

Conclusions

• DAK\research\stem cells\BBB combined • Transplanted motor neurons reach muscle

movie.wmv given the right cues
• they form synapses with muscle
• These connections are electrically active
• Rats recover 40% of hind limb grip strength

12
 Status: Currently in Large
Pre-Clinical Timeline
Mammal Studies with hES Cells
Cohort # of Animals Length of Starting Date Ending Date End date data Benchmark
Experiment analysis

• Ricin injection for focal weakness

– Focal fulminant weakness with α-MN loss
1 6 3 weeks 4/1/07 4/21/07 5/14/07 Model established and
quantified

• Non-federally approved hES cell-derived

motor neurons 2 12 6 weeks 5/15/07 7/1/07 8/1/07 Cell survival and spinal
synapse

– Cells produced under FDA-compliant GLP

conditions 3 4 12 weeks 8/1/07 11/1/07 1/15/08 Rolipram mediated axon
extension into PNS

4 8 52 weeks 10/1/07 10/1/08 2/1/09 GDNF effect in NMJ

formation

Initial Clinical Population: Type

Clinical Timeline
Task Length of Task Starting Date Ending Date Status Benchmark
1 SMA
Conduction of focus groups 52 weeks 9/1/06 8/31/07 Underway Consensus ‘whitepaper’ on the
for clinical trial framework of clinical trial

• Universally fatal without life support

• ‘pure’ α-MN loss
Pre-pre IND with FDA 1 day 10/1/06 10/1/06 Completed Guidance from FDA on what
preclinical data will be
required

Neurosurgical colloquia to
establish surgical
technique for
52 weeks 8/1/06 7/31/07 Underway Surgical device established
and prepared for FDA
approval
• Relative lack of myelin in SC in early post-
transplantation
natal spinal cord
Pre-IND 1 day 4/1/09 4/1/09 Not yet underway Presentation of proposal with
preliminary preclinical • Developmental cues guiding axonal growth
data to FDA

still present
IND 1 day 12/1/09 12/1/09 Not yet underway Request for clearance to
proceed with clinical trial • Shorter distances to reconnect
Clinical Trial Enrollment 3 years 2/1/11 1/31/14 Not yet underway Patient enrollment

13