Note: Descriptions are shown in the official language in which they were submitted.
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
COMPOSITION FOR NEURONAL REGENERATION COMPRISING MYELIN-SPECIFIC ANTIBODIES
AND
COMPLEMENT PROTEINS
FIELD OF THE INVENTION
This invention relates to compositions and their methods of use in promoting
the
growth and/or regeneration of neurological tissue within the central nervous
system
(CNS).
BACKGROUND
CNS damage
Approximately 1,100 new spinal cord injuries occur each year in Canada; over
10,000
per year occur in the United States. These numbers are five times higher if
one also
includes patients suffering brain damage involving inhibition to neural growth
following traumatic brain injury. The number of patients with chronic spinal
cord
injuries in North America is in the order of 300,000. Again, this number is
five times
higher if one includes patients suffering from brain damage involving
inhibition to
neural growth following traumatic brain injury.
Spinal cord injuries often result in a permanent loss of voluntary movement
below the
site of damage. Mostly young and otherwise healthy persons become paraplegic
or
quadriplegic because of spinal cord injuries. There are an estimated 200,000
quadriplegics in the United States. Given the amount of care required, it is
not difficult
to envision how health care costs associated with caring for patients with
central
nervous system (CNS) damage is well over $10 billion a year for North America.
The CNS (the brain and the spinal cord) is comprised of neurons and glia, such
as
astrocytes, microglia, and oligodendrocytes. Neurons typically have two types
of
processes: dendrites, which receive synaptic contact from the axons of other
neurons;
and axons, through which each neuron communicates with other neurons and
effectors.
The axon of a CNS neuron is wrapped in a myelin sheath.
In higher vertebrates, axons within the CNS possess a limited capacity for
repair after
injury. Axotomized neurons of the adult mammalian CNS fail to exhibit
substantial
1
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
axonal regeneration, in contrast to neurons within the embryonic or neonatal
CNS or
within the adult peripheral nervous system (PNS) (Saunders et al., (1992)
Proc. R Soc.
Lond. B. Biol. 250:171-180; Schwab and Bartoldi (1996) Physiol. Rev. 76:319-
370;
Steeves et al., (1994) Prog. Brain Res. 103:243-262). In fact, complete CNS
axonal
disruption is likely to preclude recovery. Although axotomized fibers proximal
to the
neuronal cell body initiate regenerative growth, this is subsequently aborted
within a
short distance (1-2 mm) and is often followed by retrograde degeneration.
Although
CNS axons will not regrow in the environment of the adult spinal cord,
peripheral nerve
grafts into the CNS provide a favorable environment through which CNS axons
will
anatomically regenerate (May et al., Cajal's Degeneration and Regeneration of
the
Nervous System, History of Neuroscience Series #5 (NY and Oxford: Oxford Univ.
Press, 1991) at 769). These findings indicate that adult CNS neurons retain
intrinsic
growth properties and, given favorable environmental conditions, are capable
of
successfully reactivating growth programs.
Current treatments of spinal cord injuries
A number of current therapies exist for the treatment of spinal cord injuries.
Interventional therapies, including opiate antagonists, thyrotropin-releasing
hormone,
local cord cooling, dextran infusion, adrenergic blockade, corticosteroids,
and
hyperbaric oxygen have been utilized, but are of questionable clinical value.
Peripheral nerve transplants have been suggested as bridges across CNS lesions
(David
and Aguayo (1981) Science 214:931-933; Houle (1991) Exp. Neurol. 113:1-9;
Richardson et al., (1984) J. Neurocytol. 13:165-182; Richardson et al., (1980)
Nature
284:264-265; Xu et al., (1995) Exp. Neurol. 138:261-276; Ye and Houle (1997)
Exp.
Neurol. 143:70-81). Olfactory ensheathing cell transplants have been used
recently to
promote the regeneration of injured corticospinal projections in the rat (Li
et al., (1997)
Science 277:2000-2002). A recent study (Cheng et al., (1996) Science 273:510-
513)
employed a combinatorial approach that extended earlier work (Siegal et al.,
(1990)
Exp. Neurol. 109:90-97): after transection of the adult rat spinal cord,
peripheral grafts
were used to connect white matter tracts to central gray matter in such a way
as to direct
regenerating fibers out of an inhibitory environment and into the more
permissive gray
2
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
matter.
US Patents No. 5650148 and 5762926 describe a method for treating damage to
the
CNS by grafting donor cells into the CNS that have been modified to produce
molecules such as neurotrophins.
The use of transplanted neural cells is also of limited clinical value:
although axons will
be able to grow into the transplanted tissue, they will not be able to grow
out of the
transplanted tissue back into the CNS due to inhibitory matter in the CNS.
This review of current methods of treating spinal cord injuries indicates that
a need
remains for a means of promoting regrowth, repair, and regeneration of neurons
in the
mammalian CNS in both the acute and chronic situations.
Myelin
It has been suggested that the failure of CNS axons to regenerate after injury
is
associated with the presence of myelin. The myelin sheath wrapping an axon is
composed of compacted plasma membranes of Schwann cells and oligodendrocytes.
Although its composition resembles that of any other plasma membrane in that
it
contains lipids, proteins, and water, the relative proportions and
dispositions of these
components are unique to myelin. Myelin in the CNS is produced by
oligodendrocytes
and is characterized by the expression of myelin basic protein (MBP). MBP is
only
associated with myelin and is one of the first proteins expressed at the onset
of
myelination of CNS axonal fibers. Galactocerebroside (GaIC) is the major
sphingolipid
produced by oligodendrocytes. GaIC comprises approximately 15 percent of the
total
lipid in human myelin and is highly conserved across species. Although GaIC is
expressed on the surface of oliogodendrocyte cell bodies, it is expressed in
greater
concentration on the surface of myelin membranes (Ranscht et al., (1982) Proc.
Natl.
Acad. Sci. USA 79:2709-2713).
There is growing evidence that the presence of CNS myelin can retard or
inhibit the
regenerative growth of some severed CNS axons (Schwab and Bartoldi (1996)
Physiol.
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
Rev. 76:319-370), including a number of examples from widespread vertebrate
families
(Schwegler et al., (1995) J. Neurosci. 15:2756-2767; Steeves et al., (1994)
Prog. Brain
Res. 103:243-262). Both the lower vertebrate CNS (e.g. lamprey) and the
developing
CNS of higher vertebrates (e.g. birds and mammals) exhibit substantial axonal
regeneration after injury (Davis and McClellan (1994).1. Comp. Neurol. 344:65-
82;
Hasan et al., (1993) J. Neurosci. 13:492-507; Hasan et al., (1991) Restor.
Neurol.
Neurosci. 2:137-154; Iwashita et al., (1994) Nature 367:167-170; Saunders et
al.,
(1992) Proc. R. Soc. Lond B. Biol. 250:171-180; Treherne et al., (1992) Proc.
Natl.
Acad. Sci. USA 89:431-434; Varga et al., (1995) Eur. J. Neurosci. 7:2119-
2129). The
common phenotype for all these positive examples of regeneration is either a
CNS that
lacks compact myelin (lamprey) or incomplete myelin development (embryonic
chick,
neonatal opossum and rat) at the time of injury. The developmental appearance
of
myelin temporally correlates with the loss of regeneration by injured CNS
axons. In
addition, the robust growth of transplanted fetal neurons in the adult CNS
(Bregman et
al., (1993) Exp. Neurol. 123:3-16; Li and Raisman (1993) Brain Res. 629:115-
127;
Yakovleff et al., (1995) Exp. Brain Res. 106:69-78) may be partially
attributed to either
a lack of receptors for myelin inhibitors at that stage of their
differentiation and/or an
ability to override any inhibitory signals from myelin.
Specific molecules associated with myelin have been identified as putative
mediators of
this inhibitory activity, including myelin-associated glycoprotein (MAG)
(McKerracher
et al., (1994) Neuron. 13:805-811; Mukhopadhyay et al., (1994) Neuron. 13:757-
767)
and NI35/250, an as yet unidentified myelin-derived protein (Bandtlow and
Schwab
(1991) Soc. Neurosci. Abstr. 17:1495; Caroni and Schwab (1988) J. Cell Biol.
106:1281-1288; Caroni and Schwab (1988) Neuron 1:85; Crutcher (1989) Exp.
Neurol.
104:39-54; Savio and Schwab (1989) J. Neurosci. 9:1126-1133; Schwab and Caroni
(1988) J. Neurosci. 8:2381-2393); IN-1 (Brosamle, et al, (1998) Abst. Soc
Neurosci.,
24:1559; NI-35/250 (Huber et al., (1998) Abst. Soc Neurosci., 24:1559; NI-
220/250
(van der Haar et al., (1998) Abst. Soc Neurosci., 24:1559; arretin, Janani et
al., (1998)
Abst. Soc Neurosci., 24:1560; and NOGO (Chen et al., ( 1998) Abst. Soc
Neurosci.,
24:1776.
4
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
Experimental attempts to functionally block myelin-associated inhibition
involving
NI35/250, by using an anti-NI35/250 antibody, IN-1, have facilitated some
anatomical
regeneration of corticospinal axons (Bregman et al., (1995) Nature 378:498-
SO1;
Carom and Schwab (1988) Neuron. 1:85-96; Schnell and Schwab (1990) Nature
343:269-272).
The immunological disruption of mature myelin within the avian spinal cord
(Keirstead
et al., (1995) J. Neurosci. 15:6963-6974), and the delay of onset of CNS
myelination
during normal avian or mammalian neurodevelopment (Keirstead et al., (1992)
Proc.
Natl. Acad Sci. (USA) 89:11664-11668; Keirstead et al., (1997) Brain. Res.
Bull.
44:727-734; Varga et al., (1995) Eur. .l. Neurosci. 7:2119-2129) have also
facilitated
CNS axonal re-growth and/or sprouting.
The presence of certain components located or embedded in myelin that are
inhibitory
to the regeneration of axonal growth after injury makes it desirable to
transiently
remove myelin and its inhibitory components to promote the repair of injured
adult
spinal cord. Adult spinal cord can be demyelinated in vivo via drugs (e.g.
ethidium
bromide); however, these drugs have non-specific deleterious effects on other
cell types
in the central nervous system (e.g., astrocytes). In addition, myelin-
deficient strains of
mice and rats are readily available, but are of limited experimental value due
to a
shortened life span: most do not survive beyond a couple of weeks after birth.
Consequently, there is a need for improved methods of disrupting myelin in
vivo in
order to enhance regeneration of neurological tissue. The present invention
provides
methods that address this need.
Complement
The complement system is the primary humoral mediator of antigen-antibody
reactions.
It consists of at least 20 chemical and immunologically distinct serum
proteins capable
of interacting with one another, with antibody, and with cell membranes (see,
for
example, J. Klein, Immunology: The Science of Self Nonself Discrimination (New
York: John Wiley & Sons, 1982) at 310 346). The principal actors in this
system are
5
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
11 proteins, designated C 1 to C9, B, and D, which are present normally among
the
plasma proteins. These proteins are normally inactive, but they can be
activated in two
separate ways: the classical pathway or the alternate pathway.
The classical pathway is activated by an antigen-antibody reaction: when an
antibody
binds with an antigen, a specific reactive site on the constant portion of the
antibody
becomes activated, which in turn binds directly with the C1 molecule of the
complement system. This sets into motion a cascade of sequential reactions,
beginning
with the activation of the C 1 proenzyme. Only a few antigen-antibody
combinations
are required to activate many molecules in this first stage of the complement
system.
The C1 enzymes then activate successively increasing quantities of enzymes in
the later
stages of the complement system. Multiple end-products are formed, which cause
important effects that help to prevent damage by an invading organism or
toxin,
including opsonization and phagocytosis, lysis, agglutination, neutralization
of viruses,
chemotaxis, activation of mast cells and basophils, and inflammatory effects.
The complement system can also be activated by an alternate pathway without
the
intermediation of an antigen-antibody reaction. Certain substances react with
complement factors B and D, forming an activation product that activates
factor C3,
setting off the remainder of the complement cascade; thus, essentially all the
same final
products of the system are formed as in the classical pathway, causing the
same effects.
Since the alternate pathway does not involve an antigen-antibody reaction, it
is one of
the first lines of defense against invading microorganisms.
Since components of both the classical pathway and the alternative pathway of
the
complement system act locally to activate C3, this is the pivotal component of
complement. C3 is a 195 kD protein, which comprises two disulfide bridged
chains of
105 and 75 kD. The enzymatically active C4-C2 complex, activated in the
classical
pathway by the binding of C 1 q to an antigen-antibody complex, cleaves C3
into two
fragments, C3a and C3b. The larger fragment, C3b, binds covalently to the
surface of a
target cell where it acts as a protease to catalyze the subsequent steps in
the complement
cascade. It is also recognized by specific receptor proteins on macrophages
and
6
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
neutrophils that enhance the ability of these cells to phagocytose the target
cell. In
particular, membrane-immobilized C3b triggers a further cascade of reactions
that leads
to the assembly of membrane attack complexes from the late components.
Complement fixation by cell-surface binding antibodies has been shown to
compromise
the ionic homeostasis of many different cells in vitro within minutes of
activation
(Mayer (1972) Proc. Natl. Acad. Sci. USA 69:2954-2958; Morgan (1989) Biochem.
J.
264:1-14).
Use of Complement with Myelin-Specific Antibodies
After attachment of a specific complement-fixing antibody to a myelin surface
antigen,
serum complement forms a membrane attack complex through an enzymatic cascade
resulting in a rapid influx of extracellular calcium (Dyer and Benjamins
(1990) J. Cell
Biol. 111:625-633) and subsequent cytoskeletal re-arrangement (Dyer and
Matthieu
(1994) J. Neurochem. 62:777-787). In vivo, this would make the disrupted
myelin
processes a target for phagocytosis by subsequent microglia, as well as by any
invading
macrophages.
The in vitro application of serum complement with myelin-specific antibodies
has been
shown to suppress myelin elaboration in purified oligodendrocyte cultures
(Dorfman et
al., (1979) Brain Res. 177:105-114; Dubios-Dalcq et al., (1970) Pathol. Eur.
5:331-
347; Dyer and Benjamins (1990) J. Cell Biol. 111:625-633; Fry et al., (1974)
Science
183:540-542; Hruby et al., (1977) Science 195:173-175).
In vivo myelin disruption has been shown in the guinea pig optic nerve using
anti-GaIC
serum and complement (Sergott et al., (1984) J. Neurol. Sci. 64:297-303);
myelin
disruption was observed within 1 to 2 hours of treatment.
The Chick Model
In the avian model, the onset of myelination in the embryonic chick spinal
cord at E13
coincides with the transition from a permissive to a restrictive period for
the functional
repair of transected spinal cord. The first appearance of chick
oligodendrocytes on the
7
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
tenth and eleventh embryonic day of development (E10-El l) precedes the
initial
formation of myelin by 2-3 embryonic days and is characterized by the
expression of
galactocerebroside (GaIC), the major sphingolipid produced by
oligodendrocytes.
In the mature avian spinal cord, after spinal cord transection, immunological
disruption
of local spinal cord myelin facilitated regeneration by brainstem-spinal
neurons
(Keirstead et al., (1995) J. Neurosci. 15:6963-6974; Keirstead et al., (1997)
Brain Res.
Bull., 44: 727-734). The immunological disruption of myelin was transient,
produced
by an intraspinal infusion of both serum complement and a myelin-specific,
complement-fixing antibody (e.g. GaIC antibodies). Such treatment resulted in
the
regeneration of up to 20% of mature brainstem-spinal axons.
This background information is provided for the purpose of making known
information
believed by the applicant to be of possible relevance to the present
invention. No
admission is necessarily intended, nor should be construed, that any of the
preceding
information constitutes prior art against the present invention. Publications
referred to
throughout the specification are hereby incorporated by reference in their
entireties in
this application.
SUMMARY OF THE INVENTION
The purpose of the present invention is to provide a means of promoting
regrowth,
repair, and regeneration of neurons in the mammalian CNS. Accordingly, the
invention
provides compositions and methods of use for promoting regrowth, repair,
and/or
regeneration of neurons in the CNS of a mammalian subject, such as a human, in
both
chronic and acute disorders
One embodiment of the present invention provides a composition comprising
therapeutically effective amounts of the following:
(a) one or more complement-fixing antibodies or fragments thereof, which
specifically bind to an epitope of myelin; and
(b) one or more complement proteins or fragments thereof;
wherein the binding of said antibodies to myelin causes transient disruption
and/or
transient demyelination of myelin. The antibodies may be monoclonal and/or
8
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
polyclonal. The complement proteins or fragments thereof may be derived from a
species different from that species to which it is administered. In a
preferred
embodiment, the complement proteins or fragments thereof are human. The
complement component may be a physically distinct component from the antibody
component, or it may be covalently or noncovalently attached directly to the
antibody
component, such that binding of the antibody to the surface of the myelin
triggers the
endogenous immune system attack. One or more growth factors may be added (in
an
appropriate sequence) to facilitate regrowth and regeneration.
In a specific embodiment, the epitope of myelin is a myelin sheath epitope,
such as
galactocerebroside (GaIC), 04, Myelin Oligodendrocyte Glycoprotein (MOG), or
Myelin Associated Glycoprotein (MAG), NOGO, NI22, NI-35/250, or arretin, or
fragments thereof. In a preferred embodiment, the epitope of myelin is GaIC.
Another
preferred embodiment is MOG.
In a preferred embodiment, the complement proteins or fragments thereof
include the
C3 component or a fragment, variant, analog, or chemical derivative thereof.
In a
preferred embodiment, the component C3b is used.
In another embodiment of the present invention, the composition further
comprises
neurotrophins and growth factors, such as NT-3, CNTF, FGF-1, BDNF, PDGF, GDNF,
CT-1, or BMP.
The present invention also relates to the use of these compositions to promote
regrowth,
repair, and/or regeneration of neurons in a subject by the transient
disruption and/or
transient demyelination of myelin.
In one embodiment ofthe present invention, the compositions are used in
subjects
requiring neuron repair and/or regeneration due to neuron dysfunction. This
neuron
dysfunction may be a result of acute or chronic injury to the CNS. It may also
be a
result of degenerative disease, such as Alzheimer's or Parkinson's disease.
9
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
In another embodiment of the present invention, the compositions are used in
subjects
to generate an environment within the CNS that is relatively permissive to
growth of
transplanted cells.
The present invention also relates to a method of promoting regrowth, repair,
and
regeneration of neurons in mammalian CNS, wherein the damage resulted from
either a
chronic or acute disorder. The method entails delivery of one or more
complement-
fixing antibodies or fragments thereof, which specifically bind to an epitope
of myelin
and delivery of one or more complement proteins or fragments thereof,
delivered either
together or separately to effect transient disruption and/or transient
demyelination of
myelin in the neuronal zone requiring regeneration.
Various other objects and advantages of the present invention will become
apparent
from the detailed description of the invention.
TABLES AND FIGURES
Table 1 presents rubrospinal neuronal cell counts obtained from individual
control and
experimental animals with retrograde Fluorogold labeling from the lumbar cord
of an
adult rat.
Figure 1 presents (A) Photomicrograph of a transverse section of spinal cord
of an adult
rat at the level of T 10 left side hemisection lesion, stained with cresyl
violet. All lesions
were assessed and always resulted in severing the funiculi through which the
rubrospinal tract traverses. The contralateral dorsal (dh) and ventral (vh)
horns were
always left undamaged; the central canal (cc) is labeled for orientation. (B)
Assessment
of visible Fluorogold diffusion in the control treated and immunologically
disrupted
hemisected spinal cord. Difr'usion of the retrograde tracer was measured at
the light
microscope level at the time points indicated after injection into the lumbar
spinal cord
(see methods for details). Immunological demyelination did not significantly
affect the
diffusion of the tracer.
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
Figure 2 shows electron photomicrographs of transverse sections through the
dorsolateral funiculus after continuous intraspinal infusion of immunological
reagents
for 7 days. (A) Within one spinal segment (<2mm) of the infusion site, large
regions of
naked, demyelinated axons were visible. Some axons were observed to be
associated
with monocyte cells (M, e.g. infiltrating macrophage) and or endogenous
microglia,
some of which also contained myelin ovoids (arrow) or myelin debris. (B) On
other
grids, monocytes and invading polymorphonucleocytes (PMN) could also be seen
in
close association with demyelinated axons. Macrophages and/or microglia were
identified on the basis of their high density endoplasmic reticulum (arrow-
heads), and
"finger-like" processes. Some monocytes have laid down basal lamina components
such as collagen (Col), which distinguishes them from astrocytes. Mufti-lobed
nuclei
are characteristic of PMNs and facilitate their identification. (C) Example of
myelin-
disruption. This is often observed 4-8mm (1-2 spinal segments) from the
immunological infusion site where the axons were still associated with myelin;
however, the myelin lamellae were disrupted (delaminated). Some regions of
coherence
in the myelin wrapping did persist (arrows). (D) Example of the appearance of
axons
within the dorsolateral funiculus after a control infusion of Guinea-pig
complement
alone. Some non-specific damage of myelin sheathes occurred, especially within
one
spinal segment of the infusion site; however, the compact nature of the myelin
remained
intact. Original magnification x 4000 (A, B, D), x10000 (C).
Figure 3 presents demonstrations of regeneration of rubrospinal neurons after
left-side
thoracic hemisection and subsequent immunological myelin suppression
treatment.
Panels A and B are photomicrographs of rubrospinal neurons from the same
experimentally-treated animal (14 days infusion of serum complement with anti-
GaIC);
A is from the uninjured Red nucleus while B is from the injured Red nucleus.
Panels C
and D are also from same control-treated animal (14 days infusion of serum
complement only): C is the uninjured Red nucleus and D is the injured Red
nucleus.
Fluorogold injection within the rostral lumbar cord 28 days after injury
resulted in the
retrograde labeling of uninjured rubrospinal neurons (A and C) as well as
those
rubrospinal neurons that had regenerated from the injured Red nucleus (B and
D). (E)
and (F) Axotomized rubrospinal neurons were retrograde labeled at the time of
injury
11
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
with the first label RDA (solid arrow heads) and subsequently 28 days later
with the
second label FG (open arrow heads). Double-labeled (RDA+FG) cells are
indicated by
an asterisk and represent those rubrospinal neurons that had regenerated after
immunological myelin-suppression treatment. Scale bar = 100~m.
Figure 4 shows a relative quantitative assessment of regeneration of
rubrospinal
neurons after thoracic injury and immunological treatment. Regeneration was
assessed
by counting FG-labeled cells in alternating tissue sections: those with both
multipolar
neuronal morphology and FG labeling were deemed to be positive. Percentage
regeneration was calculated by comparison of the retrograde labeled cell
counts from
the injured Red nucleus with the control uninjured Red nucleus within the same
animal.
For each animal, the degree of lesion was assessed. Filled bar: myelin
suppressed;
hatched bar: pooled control treated groups. Data shown ~ s.d.
Figure 5 demonstrates effects of removal of a single complement protein on
immunological demyelination. (A) Control uninjured spinal cord. Electron
photomicrographs of transverse sections through the dorsolateral funiculus
indicating
the ultrastructure of adult myelin sheaths. (B) 7 day infusion with myelin-
specific
antibody and human complement sera results in a profound myelin suppression.
(C)
The removal of the C3 component of complement results in a lack of myelin-
removal,
indicating the fundamental role of this protein in either (i) opsonization, or
(ii) the
propagation of the cascade to the lytic membrane attack complex (MAC), the
final lytic
pathway complex. It is believed that it is a fundamental and essential
requirement of a
myelin specific cell surface binding antibody to activate the classical
complement
pathway for effective transient demyelination.
Figure 6 shows a relative quantitative assessment of regeneration of lateral
vestibulospinal neurons after thoracic injury and delayed immunological
treatment.
Immunological demyelination treatment was delayed for 1 or 2 months after
injury as
indicated. Regeneration was assessed by counting FG-labeled cells in
alternating tissue
sections: those with both multipolar neuronal morphology and FG labeling were
deemed to be positive. Percentage regeneration was calculated by comparison of
the
12
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
retrograde labeled cell counts from the injured lateral vestibulospinal
nucleus with the
control uninjured lateral vestibulospinal nucleus within the same animal. For
each
animal, the degree of lesion was assessed. Filled bar: myelin suppressed; open
bar:
pooled control treated groups. Data shown t s.d.
Figure 7 presents A) Drawing of a dorsal view of the rat central nervous
system,
indicating the relative origins and course of the rubrospinal tract (RN) and
lateral
vestibular tract (LVe). Also illustrated (solid line) is the left-side
thoracic hemisection
lesion (~ T10, line), the immunological infusion site (~ T11, vertical
hatching), and the
site of the Fluorogold injection (~L1, diagonal hatching). B) composite
photomicrograph of parasagittal sections through the lower thoracic and
rostral lumbar
spinal cord (T9- L1, rostral is up). Some Fluorogold diffusion can be clearly
emanating
from the injection site as an intense white "halo", however, this staining
rapidly
decreased with distance from the site of injection and none was ever visible
rostral to
T11, the immunological infusion site (i.e. no diffusion to or above the lesion
at T10,
thus no evidence for any "false" positive retrograde labeling of brainstem-
spinal
projections). C) photomicrograph of a transverse section of spinal cord at the
level of
T10 left side hemisection lesion, stained with cresyl violet. All lesions were
assessed
and always resulted in severing the funiculi through which the rubrospinal and
lateral
vestibulospinal tracts traverse. The contralateral dorsal (dh) and ventral
(vh) horns were
always left undamaged; the central canal (cc) is labeled for orientation. D
and E) Non-
specific fluorescence associated with blood cells within the lesion and pump
implantation sites indicating the degree of damage associated with the lesion
and
cannula implantation, respectively. Specific Fluorogold fluorescence labeling
was never
observed at the level of the cannula implantation or hemisection injury.
Figure 8 shows regeneration of lateral vestibulospinal neurons after left-side
thoracic
hemisection and subsequent immunological myelin suppression treatment. Panels
A and
B are photomicrographs of lateral vestibulospinal neurons from the same
experimentally-treated animal (14 days infusion of serum complement with anti-
GaIC);
A is of the injured lateral vestibular nucleus and B is from the uninjured
lateral
vestibular nucleus and. Panels C and D are also from same control-treated
animal ( 14
13
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
days infusion of serum complement only); where C is the injured lateral
vestibulospinal
nucleus and D is the uninjured lateral vestibulospinal nucleus. Fluorogold
injection
within the rostral lumbar cord 28 days after injury resulted in the retrograde
labeling of
uninjured lateral vestibulospinal neurons (B and D) as well as those lateral
vestibulospinal neurons that had regenerated from the injured lateral
vestibulospinal
nucleus (A and C), please see results for further details. Panel E is a
drawing of a
transverse section through the midbrain indicating the location of the lateral
vestibular
nucleus (LVe), SpVe = spinal vestibular nucleus, MVe = medial vestibular
nucleus, 4V
= 4~' ventricle, FN = facial nerve tract, 7 = 7~' cranial (facial) nucleus,
PFI =
paraflocculus. Scale bar = 1001tm
Figure 9 shows relative quantitative assessment of regeneration of rubrospinal
and
lateral vestibulospinal neurons after thoracic injury and immunological
treatment.
Regeneration was assessed by counting FG-labeled cells in alternating tissue
sections;
1 S those with both multipolar neuronal morphology and FG labeling, were
deemed to be
positive. Percentage regeneration was calculated by comparison of the injured
nucleus
with the contralateral (uninjured) nucleus within the same animal. For each
animal the
degree of lesion was assessed. Filled bars, experimental; open bars, pooled
control
groups.
Figure 10 shows a quantitative assessment of regeneration of descending
brainstem-
spinal axons after chronic lateral hemisection & delayed immunological
treatment.
Percentages of retrogradely labeled red nucleus (red) and lateral vestibular
(green)
neurons vs. Contralateral uninjured, after control (PBS, Ab, GpC) treatment
(open bars)
or immunological disruption/demyelination (filled bars). Expressed as
percentage
labeled cells in the injured nucleus vs. Uninjured contralateral.
Figure 11 shows the factors known to be involved in the classical and
alternative
pathways involved in immunological demyelination.
Figure 12 shows the effects of removal of a single complement protein from the
composition on immunological demyelination. Electron photomicrographs are of
14
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
transverse sections through the dorsolateral funiculus indicating the
ultrastructure of
adult myelin sheaths. (A) demonstrates the effect of 7-day infusion with
myelin-specific
antibody and human complement sera results in a profound myelin suppression.
(B)
The removal of C3 component of the complement results in a lack of myelin
removal.
(C) The removal of Factor B from the complement indicates that the alternative
pathway is not involved in immunological demyelination. (D) The removal of C4
component of the complement results in a lack of myelin removal, indicating
that the
Classical Pathway is required for immunological demyelination. (E) The removal
of C6
component indicates that this component is not necessary to be included in the
composition for immunological demyelination.
Figure 13 presents the effects of removal of a single complement protein, C5,
from the
composition on immunological demyelination. The electron photomicrograph is of
a
transverse section through the dorsolateral funiculus indicating the
ultrastructure of
adult myelin sheaths. The removal of CS component indicates that this
component is
not necessary to be included in the composition for immunological
demyelination.
DETAILED DESCRIPTION OF THE INVENTION
The following terms and abbreviations are used throughout the specification
and in the
claims:
The term "antibodies or fragments thereof' includes recombinant, chimeric, and
affinity
modified forms made by techniques of molecular biology well known in the art;
"CNS" refers to the central nervous system;
The term "complement protein or fragment thereof' (C) refers to any of 13
whole serum
proteins or any of more than 20 intermediates and complexes of the complement
system, the primary humoral mediator of antigen-antibody reactions, and
includes
variants, analogs, and chemical derivatives thereof.
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
The term "composition" is used to indicate more than one component. The
elements of
the composition can be mixed together, however, it is not necessary that they
be
combined in the same solution. In an alternative embodiment, they do not need
to be
packaged, stored or even mixed together. The elements (antibody-type and
complement-type) can be delivered to the site of nerve damage sequentially, or
at the
same time. The need for a therapeutically effective temporal sequence is
understood by
one skilled in the art. The concept of at least one complement fixing antibody
or
fragment thereof, plus at least complement protein or active fragment thereof
equates
with the concept of the composition. These elements are delivered to the site
of damage
to form a complex with an appropriate epitope present in myelin to be
transiently
demyelinated. Thus, the first two types of elements (of which there can be
more than
one member of each type of element, for example, two or more antibody or
component
proteins or fragments) are delivered to the site targeted for transient
demyelination to
form a complex in situ, in vivo with the epitope(s) on myelin.
The term "demyelination" refers to the removal or breakdown of myelin in
neurological
tissue. Demyelination consists of the removal of the myelin sheath, such as
that
surrounding neurons or neuronal projections (e.g., the axons). This process
may be
chemical or immunological in both the experimental and pathological states.
This
invention effects transient demyelination in order to promote repair and
regrowth.
The term "disruption" refers to delamination or disruption of the three-
dimensional
conformation of myelin;
The term "dysfunction" when used to describe the therapeutic use of the
invention
encompasses any type of trauma to the nervous system and resulting loss of
function.
Such trauma can arise from either physical injury or disease;
The term "Fab" means an antibody fragment that is obtained by cleaving an
antibody in
the hinge region yielding two Fab fragments, each having the heavy and light
chain
domains of the antibody, along with an Fc region;
16
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
The term "Fc" means the constant region of the antibody, which may activate
complement;
The term "Fv fragment" means a heterodimer of the heavy and light chain
variable
domain of an antibody. These variable domains may be joined by a peptide
linker or by
an engineered disulphide bond;
Growth factors are extracellular polypeptide signaling molecules that
stimulate a cell to
IO grow or proliferate. Examples are epidermal growth factor (EGF) and
platelet-derived
growth factor (PDGF). Most growth factors have other actions besides the
induction of
cell growth or proliferation. Growth factors can be divided into broad- and
narrow-
specificy classes. The broad-specificity factors, like PDGF and EGF affect any
classes
of cells. At the opposite extreme lie narrow-specificity factors. In intact
animals
15 proliferation of mot cell types depends on a specific combination of growth
factors
rather than a single growth factor. Thus a fairly smal number of growth factor
families
may serve, in different combinations, to regulate selectively the
proliferation of each of
the many types of cells in a higher animal.
20 Fibroblast Growth Factor (FGF) is any one of a group of proteins, usually
intracellular,
that have important angiogenic function and enhance would healing and tissue
repair.
Over-activity of these factors has been associate with neoplasia.
Neurotrophic factors are a family of substances that promote growth and
regeneration
25 of neurons. While growth factors elsewhere in the body promote and support
cell
division, neurons cannot divide; but they can regenerate after injury and
neurotrophic
factors promote this regeneration. They also promote the growth of axons and
dendrites,
the neuron branches that form connections with other neurons.
30 "GaIC" refers to galactocerebroside;
"MAG" refers to myelin-associated glycoprotein;
17
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
"MBP" refers to myelin basic protein;
"MOG" refers to myelin oligodendrocyte glycoprotein;
The term "neurological tissue" refers to neurons and other cells typically
situated in the
region of the nervous system, such as the spinal cord of the CNS;
"PNS" refers to the peripheral nervous system;
The term "recombinant antibodies or fragments thereof' collectively includes
chimeric
or recombinant forms of the antibodies or fragments thereof wherein the Fc
domain is
substituted for an Fc domain of another species or isotype, affinity modified
forms of
the antibodies or fragments thereof wherein the binding sites are altered,
avidity
modified forms of the antibodies or fragments thereof wherein the hinge
regions are
altered, immunoreactive fragments thereof, and combinations thereof; and
The term "regeneration of neurological tissue" includes the regrowth of
neurons that
results in the reformation of neuronal connections, both anatomically and/or
functionally.
The present invention resides in the unexpected discovery that a combination
of both
antibody, which binds an epitope on a myelin-producing glial cell, and
complement can
be used for disruption and demyelination of the myelin sheath, such that
repair and
regeneration of mammalian neurological tissue is enhanced. The composition of
this
invention is valuable as a therapeutic agent in cases in which there is injury
or disease
of the mammalian nervous system such that there is a need to facilitate
neuronal
plasticity and the regrowth of neural connections. The neurological tissue is
exposed to
the myelin disrupting composition, according to the invention, as soon as
possible
following the injury, trauma, or disease.
The present invention provides compositions and methods of their use for
promoting
18
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
regeneration of neurological tissue in a mammalian subject, such as a human,
with a
nervous system dysfunction by contacting the neurological tissue with a
therapeutically
effective amount of a composition comprising a complement fixing antibody,
which
binds to myelin, and complement. Uses of the composition in the field of
veterinary
medicine are also an embodiment of the present invention.
The compositions of the present invention are comprised of one or more
antibodies or
fragments thereof, which bind myelin, and one or more serum complement
proteins or
fragments thereof.
Antibodies
The antibodies used in this invention can be any antibodies or fragments
thereof that
specifically bind to myelin, wherein said antibodies activate the complement
system.
The preferred antibodies of the present invention specifically bind a myelin
sheath
epitope, such as galactocerebroside (GaIC), 04, Myelin Oligodendrocyte
Glycoprotein
(MOG), or Myelin Associated Glycoprotein (MAG). Other preferred epitopes are
NOGO (formerly NI 35/250) and NI220 and arretin.
Generation of Antibodies
The antibodies of the present invention, or fragments thereof, can be:
a) naturally occurring;
b) antibodies obtained from disease states such as B-cells from multiple-
sclerosis patients;
c) produced by recombinant DNA technology;
d) produced by biochemical or enzymatic fragmentation of larger
molecules;
e) produced by methods resulting from a combination of a) to c); or
f) produced by any other means for producing antibodies.
Human antibodies can be generated by a number of techniques known to those
skilled
in the art, including the use of insect cells and transgenic plants such as
tobacco or corn
seed (framer, C.L., CropTech Development Corp; Reno, J., NeoRx - IVC's IV
Annual
Conference: Sept 9-12, S.F., U.S.A.)
19
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
The antibodies of the present invention can also be made by traditional
technqiues such
as monoclonal or polyclonal, although monoclonal antibodies are preferred. In
general,
antibodies may be obtained by injecting the desired immunogen into a wide
variety of
vertebrates or invertebrates in accordance with conventional techniques. While
rodents,
particularly mice, are preferred, other species may be employed, such as
members of
the bovine, ovine, equine, porcine, or avian families. Immunization of these
animals can
be readily performed and their lymphocytes, particularly splenocytes, may be
obtained
for fusions.
Immunization protocols are well known and can vary considerably yet remain
effective
(Goding, Monoclonal Antibodies: Principles and Practice (2nd ed.) (Academic
Press,
1986). Isolated proteins, synthetic peptides, and bacterial fusion proteins
which contain
antigenic fragments of the myelin molecule may be used as immunogens.
Preferably the
immunogen of peptides or recombinant proteins will be enriched for proteins or
fragments thereof containing the epitopes to which antibody-producing B cells
or
splenocytes are desired.
Once the proteins or peptides thereof have been purified to the extent
desired, they may
be suspended or diluted in an appropriate physiological carrier for
immunization, or
may be coupled to an adjuvant. Immunogenic amounts of antigenic preparations
enriched in myelin, or antigenic portions thereof, are injected, generally at
concentrations in the range of 1 ug to 100 mg/kg of host. Administration may
be by
injection, such as intramuscularly, peritoneally, subcutaneously, or
intravenously.
Administration may be one or a plurality of times, usually at one to four week
intervals.
Immunized animals are monitored for production of antibody to the desired
antigens,
then the spleens are removed and splenic B-lymphocytes isolated and fused with
a
myeloma cell line or transformed. The B-lympocytes can also be isolated from
the
blood. The transformation or fusion can be carried out in conventional ways,
the fusion
technique being described in an extensive number of patents, such as U.S.
Patent Nos.
4,172,124; 4,350,683; 4,363,799; 4,381,292; and 4,423,147. The manner of
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
immortalization is not critical, but the most common method is fusion with a
myeloma
fusion partner. Other techniques of immortalization include EBV
transformation,
transformation with bare DNA, such as oncogenes or retroviruses, or any other
method
that provides for stable maintenance of the cell line and production of
monoclonal
antibodies. The general process for obtaining monoclonal antibodies has been
described
(Kohler and Milstein (1975) Nature 256:495-497). Human monoclonal antibodies
may
be obtained by fusion of the spleen cells with an appropriate human fusion
partner, such
as WI-L2, described in European Application No. 82.301103.6. A detailed
technique
for producing mouse X-mouse monoclonal antibodies has been taught (0i and
Herzenberg (1980) in Mishell and Shiigi (eds.) SelectedMethods in Cellular
Immunology 351-372). The resulting hybridomas are screened to isolate
individual
clones, each of which secretes a single antibody species to the antigen.
The immortalized cell lines may be cloned and screened in accordance with
conventional techniques, and antibodies in the cell supernatants detected that
are
capable of binding to myelin. The appropriate immortalized cell lines may then
be
grown in vitro or injected into the peritoneal cavity of an appropriate host
for
production of ascites fluid. Immortalized hybridoma cell lines can be readily
produced
from a variety of sources. Alternatively, these cell lines may be fused with
other
neoplastic B-cells, where such other B-cells may serve as recipients for
genomic DNA
coding for the antibody.
The monoclonal antibody secreted by the transformed or hybrid cell lines may
be of any
of the classes or subclasses of immunoglobulins, such as IgM, IgD, IgA,
IgG,.~, or IgE.
As IgG is the most common isotype utilized in diagnostic assays, it is often
preferred.
To circumvent the possible antigenicity in a human host of a monoclonal
antibody
derived from an animal other than human, chimeric antibodies may be
constructed. For
example, the antigen binding fragment of an immunoglobulin molecule (variable
region) may be connected by peptide linkage to at least part of another
protein not
recognized as foreign by humans, such as the constant portion of a human
immunoglobulin molecule. This can be accomplished by fusing the animal
variable
21
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
region exons with human kappa or gamma constant region exons. Various
techniques
are known to the skilled artisan, such as those described in PCT 86/01533,
EP171496,
and EP173494.
As an alternative method of producing antibodies, US Patent No. 5627052
describes
methods of producing proteins that replicate the binding characteristics and
desired
function of particular antibodies. An example of application of this method
includes the
isolation and characterization of a human B-lymphocyte cell, producing a
specific anti-
myelin antibody, for example from the blook of a patient with Multiple
Sclerosis.
Antibody Engineering
The antibodies may be used intact, or as fragments, such as Fv, Fab, and
F(ab')2 as long
as there is an Fc region present to bind complement. Such antibody fragments
provide
better diffusion characteristics in vivo than the whole antibody, due to their
smaller size.
The means for engineering antibodies by recombinant DNA and chemical
modification
methods are considered well-known in the art.
The antibodies may be fragmented to obtain highly immunoreactive F(ab')2,
F(ab'), and
Fab fragments using the enzyme pepsin by methods well known in the art (see
Colcher
et al., (1983) Cancer Res. 43:736-742).
Due to the development of molecular cloning technqiues, it is now possible to
produce
human monoclonal antibody fragments quickly by paning phage display libraries
against predefined antigenic specificities. For exemplary techniques see:
Pistillo et al.,
Human Immunology, 57(1):19-26, 1997 Sep 15).
Antibodies or fragments thereof are also made into recombinant forms by
techniques of
molecular biology well known in the art (see Rice et al., (1982) Proc. Natl.
Acad Sci.
USA 79:7862-7865; Kurokawa et al., (1983) Nucleic Acids Res. 11:3077-3085; Oi
et
al., (1983) Proc. Natl. Acad. Sci. USA 80:825-829; Boss et al., (1984) Nucleic
Acids
Res. 12:3791-3806; Boulianne et al., (1984) Nature (London) 312:643-646;
Cabily et
al., (1984) Proc. Natl. Acad. Sci. USA 81:3273-3277; Kenten et al., (1984)
Proc. Natl.
22
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
Acad. Sci. USA 81:2955-2959; Liu et al., (1984) Proc. Natl. Acad. Sci. USA
81:5369-
5373; Mornson et al., (1984) Proc. Natl. Acad Sci. USA 81:6851-6855; Neuberger
et
al., (1984) Nature (London) 312:604-608; Potter et al., (1984) Proc. Natl.
Acad. Sci.
USA 81:7161-7165; Neuberger et al., (1985) Nature (London) 314:268-270; Jones
et
al., (1986) Nature (London) 321:522-525; Oi et al., (1986) BioTechniques 4:214-
221;
Sahagan et al., (1986) J. Immunol. 137:1066-1074; Sun et al., (1986) Hybridoma
S
(Supp. 1):517-S20; and Sun et al., (1987) Proc. Natl. Acad. Sci. USA 84:214-
218).
More specifically, the antibodies and fragments thereof may be altered to a
chimeric
form by substituting antibody fragments of another species, e.g., human
constant
regions (Fc domains) for mouse constant regions by recombinant DNA techniques
known in the art as described in the above cited references. These Fc domains
can be of
various human isotypes, i.e., IgG,, IgGz, IgG3, IgGa, or IgM.
In addition, the antibodies and fragments thereof may be altered to an
affinity modified
form, avidity modified form, or both, by altering binding sites or altering
the hinge
region using recombinant DNA techniques well known in the art as described in
the
above cited references.
The recombinant antibody forms may also be fragmented to produce
immunoreactive
fragments F(ab')z, F(ab'), and Fab in the same manner as described.
Antibody fragments may also include Fv fragments, the smallest functional
modules of
antibodies required to maintain the binding and specificity of the whole
antibody. Fv
fragments are heterodimers composed of a variable heavy chain and a variable
light
chain domain. Proteolytic digestion of antibodies can yield isolated Fv
fragments, but
the preferred method of obtaining Fvs is by recombinant technology (See Skerra
and
Pluckthun (1988) Science 240:1038-1041).
Fvs can be noncovalently-associated VH and VL domains, although these tend to
dissociate from one another. Stable Fvs can be produced by making recombinant
molecules in which the VH and VL domains are connected by a peptide linker so
that the
23
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
antigen-combining site is regenerated in a single protein. These recombinant
molecules
are termed single chain Fvs (scFvs). The means for preparing scFvs are known
in the
art (See: Raag and Whitlow (1995) FASEB 9:73; Bird et al., (1988) Science
242:423-
426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
Alternatively,
the two variable domains may be joined and stabilized by an engineered
disulphide
bond; these are termed disulfide Fvs (dsFvs) (Reiter and Pastan (1996) Clin.
Cancer
Res. 2:245-252).
The Fc domain of an antibody is required for the activation of complement. Fv
fragments, which lack the Fc domain, cannot activate complement. In order for
Fv
fragments to be useful in the present invention, they would have to be
designed with a
novel activator of the complement cascade. As an example, the Fv fragment
could be
designed to include the CH2 domain of an IgG antibody. As an alternative
example, a
wholly synthetic molecule may be linked to the Fv fragment to activate
complement, or
an activator of complement familiar to those in the field may be linked to the
Fv
fragment.
The antibody may also be modified by the addition of such molecules as
polyethylene
glycol (as described in U.S. Patent 5766897) as to prolong its biological half
life,
potency, or the diffusion of the molecule in situ (U. S. Patent 5747446,
Chinol et al., 98
Brit. J. Cancer, 78:189-197; Francis et al., 98, Intl J. Hematol. 68:1-18).
Labeling of Antibodies or Fragments:
The antibodies of this invention, or fragments thereof, may be used without
modification or may be modified in a variety of ways, for example, by
labeling.
Labeling is intended to mean joining, either covalently or non-covalently, a
label which
directly or indirectly provides for a means of detection of the antibody to
enable
monitoring of the progress of therapeutic treatment using the composition.
A label can comprise any material possessing a detectable chemical or physical
property. A wide variety of labels is known, including radionuclides, enzymes,
enzyme
substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly
haptens),
24
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
fluorescers, chromophores, luminescers, and magnetic particles. These labels
are
detectable on the basis of either their own physical properties (eg.,
fluorescers,
chromophores and radioisotopes), or their reactive or binding properties (eg.,
enzymes,
substrates, cofactors and inhibitors). These materials are well known to one
skilled in
the art. U.S. Patent 4,671,958 teaches methods that can be used for labelling
antibodies
or attaching complement to antibodies.
Complement
The complement portion of the composition may be comprised of one or more
complement proteins, fragments, variants, analogs, and/or chemical
derivatives.
A fragment of a complement protein refers to any subset of the C molecule. For
example, fragments of C3 include C3b, iC3b, C3a, C3c, C3dg, and C3d.
A "variant" of a complement protein or fragments thereof refers to a molecule
substantially similar to either the entire protein or a fragment thereof,
which possesses
biological activity that is substantially similar to a biological activity of
the complement
protein or fragments thereof. A molecule is said to be "substantially similar"
to another
molecule if both molecules have substantially similar structures or if both
molecules
possess a similar biological activity.
Variants of C3b, for example, include C3b dimers, and higher oligomers. When C
activation occurs at the cell surface, multiple cycles of enzyme reactions
result in the
deposition on the surface of C3b in multimeric form. C3b dimers or higher
oligomers
indeed have higher affinity for the cell than do C3b monomers.
Variants of complement protein or fragments thereof are produced by chemical
or
recombinant means well-known in the art. Such variants include, for example,
deletions from, or insertions or substitutions of, amino acid residues within
the amino
acid sequence. For example, at least one amino acid residue may be removed and
a
different residue inserted in its place. Substantial changes in functional or
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
immunological properties are made by selecting substitutions that are less
conservative,
ie. that differ more significantly in their effect on maintaining (a) the
structure of the
peptide backbone in the area of the substitution, for example, as a sheet or
helical
conformation, (b) the charge or hydrophobicity of the molecule at the target
site, or (c)
the bulk of the side chain. The substitutions that in general are expected to
induce
greater changes are those in which (a) glycine and/or proline is substituted
by another
amino acid or is deleted or inserted; (b) a hydrophilic residue, e.g., Beryl
or threonyl, is
substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,
phenylalanyl, valyl,
or alanyl; (c) a cysteine residue is substituted for (or by) any other
residue; (d) a residue
having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is
substituted for (or
by) a residue having an electronegative charge, e.g., glutamyl or aspartyl; or
(e) a
residue having a bulky side chain, e.g., phenylalanine, is substituted for (or
by) one not
having such a side chain, e.g., glycine.
Most deletions, insertions, and substitutions are not expected to produce
radical changes
in the characteristics of the protein molecule; however, when it is difficult
to predict the
exact effect of the substitution, deletion, or insertion in advance of doing
so, one skilled
in the art will appreciate that the effect will be evaluated by routine
screening assays.
For example, a change in the immunological character of the protein molecule,
such as
binding to a given antibody, is measured by an immunoassay such as a
competitive type
immunoassay.
An "analog" of a complement protein or fragment thereof refers to a non-
natural
molecule substantially similar to either the entire protein or a fragment
thereof.
A "chemical derivative" of a complement protein or fragment thereof contains
additional chemical moieties that are not normally part of the protein or
fragment.
Covalent modifications of the peptides are included within the scope of this
invention.
Such modifications may be introduced into the molecule by reacting targeted
amino
acid residues of the peptide with organic derivatizing agents that are capable
of reacting
with selected side chains or terminal residues, as is well-known in the art
(T. E.
Creighton Proteins: Structure and Molecule Properties (San Francisco: W. H.
Freeman,
26
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
1983) at 70-86 ).
The complement portion of the composition may be a physically distinct
component
from the antibody component. Alternatively, the complement proteins or
fragments
thereof, may be covalently or noncovalently attached directly to the antibody
component, such that binding of the antibody to the surface of the myelin
triggers the
endogenous immune system attack.
The complement components may be fractions that have been purified as well as
those
that have been enriched in the proteins which comprise the complement system.
Such
preparations should take into account the relative lability of complement and
provide a
sufficient combination of factors to allow complete activation of the
complement
cascade to allow transient demyelination to occur.
The complement portion of the composition may be comprised of one or more
complement proteins, fragments, variants, analogs, and/or chemical
derivatives. It
should be noted, however, that the C3 component of complement plays a
fundamental
role either in opsonization or in the propagation of the cascade to the lytic
MAC. In a
preferred embodiment, the C3 component or a fragment, variant, analog, or
chemical
derivative thereof should be included in the complement portion of the
composition. In
situations targeted for demyelination, the C3 component should certaintly be
present for
optimal results. In situations targeted for regeneration, it is less
certaintly required.
The invention also includes modifications of the complement proteins.
Modifications
(which do not normally alter primary sequence) include in vivo, or in vitro
chemical
derivatization of polypeptides, e.g., acetylation, or carboxylation. Also
included are
modifications ofglycosylation, e.g., changing glycosylation patterns, e.g.,
those made
by modifying the glycosylation patterns of a polypeptide during its synthesis
and
processing or in further processing steps, e.g., by exposing the polypeptide
to enzymes
which affect glycosylation, e.g., mammalian glycosylating or deglycosylating
enzymes.
Such modifications can be used to improve, for example, bioavailability and
stability of
the complement proteins for use in the compositions of the present invention.
27
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
The complement portion of the composition may be derived from a subject's own
serum, from the serum of a donor, or from the pooled sera of a number of
donors, such
as those available commercially, which are produced to consistent, approved
standards.
The complement components may be derived from species different from that
species
to which it is administered due to the fact that the compositions are
introduced directly
to the neural tissue (e.g., intrathecally).
I O In one embodiment of the present invention, the complement portion is a
depleted
complement, wherein one or more components of the normal complement have been
removed. Example V demonstrates that removal of certain components allows the
complement portion of the composition to act according to the present
invention.
While, removal of the C3 protein or the C4 protein results in a composition
with
1 S reduced efficacy, the use of complement depleted in Factor B, CS or C6
protein resulted
in effective demyelination. In each case, after removal of the complement
component,
the resulting composition is tested in vivo, for efficient demyelination
ability. The
advantages to the use of depleted complement rather than whole complement, are
the
reduction of potential side effects and the capacity to tailor the composition
to each
20 situation. In this way, the composition of the present invention can be
designed to meet
the requirements of treatment of various disorders and individuals.
In a related embodiment of the present invention, the complement portion is
prepared in
combination with one or more inhibitors of one or more components of the
normal
25 complement. For example, several regulatory proteins of the complement
system have
been identified. Their primary functions are to regulate the activity of C3/CS
convertases for prevention of excessive complement activation and autolytic
destruction
of host tissues. These complement regulators are either soluble plasma
proteins or
integral membrane proteins expressed on a variety of cell types. The former
include
30 C4b binding protein (C4bp) and Factor H. The latter include the C3b/C4b
receptor
(Complement receptor 1, CRI, CD35), membrane cofactor protein (MCP, CD46), and
decay accelerating factor (DAF, CD55). These proteins possess many structural
28
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
similarities. Each is composed of multiple short consensus repeats (SCRs) of
approximately 60 amino acids in length having conserved cysteine, glycine and
proline
residues. The genes encoding these proteins have been localized to chromosome
1 and
are collectively known as the regulators of complement activation (RCA) gene
cluster
(Hourcade, D. et al., 1989, Adv. Immunol. 45:381). In addition to its role in
regulating
complement activation, erythrocyte CRl also functions as a receptor for
circulating
immune complexes to promote their clearance from plasma (Cornacoff, J. et al.,
1983,
J. Clin. Invest. 71:236).
U.S. Patent No. 5,851,528 discloses chimeric proteins in which a first
polypeptide
which inhibits complement activation is linked to a second polypeptide which
inhibits
complement activation, and the polynucleotides encoding such chimeric
proteins. The
invention also features a method of reducing inflammation characterized by
excessive
complement activation by administering the chimeric protein of the invention
to a
patient afflicted with such a condition. A worker skilled in the art would be
able to
incorporate the chimeric proteins of U.S. Patent No. 5,851,528 in the
compositions of
the present invention in order to selectively inhibit one or more of the
complement
components.
Other Factors
The composition may optionally include other chemicals or drugs such as growth
factors and neurotrophins. It is known that the beneficial effects of blocking
CNS
myelin-associated inhibitors on axonal regeneration can be augmented by the
concomitant application of neurotrophins, such as NT-3 (Bregman et al., (
1995) Nature
378:498-501; Schnell et al., (1994) Nature 367:170-173). FGF-1 can also be
used
(Chang et al., 1996, supra).
PCT Publication Number WO 93/06116, suggests the use of GDNF for preventing
and
treating nerve damage and nerve related diseases, such as Parkinson's disease,
by
implanting, into the brains of patients, cells that secrete GDNF. PCT
Publication
Number WO 93/08828, teaches the intravenous administration of certain nerve
growth
factors in the treatment of neuronal damage associated with ischemia, hypoxia
or
29
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
neurodegeneration. PCT Publication Number WO 90/07341 states that nerve growth
factor (NGF) has been demonstrated to be a neurotropic factor for the
forebrain
cholinergic nerve cells that die during Alzheimer's disease. NGF increases
growth of
said nerve cells in culture. Further, experiments in animals demonstrate that
NGF
prevents the death of cholinergic nerve cells of the forebrain after traumatic
injury and
that NGF can reverse cognitive losses that occur with aging. European Patent
Application EP 0450386 A2, suggests the use of Brain Derived Neurotrophic
Factor
(BDNF) from recombinantly derived biologically active forms for treatment of
Alzheimer's disease. BDNF promotes the survival of motor neurons in several
species
(Henderson, et al., (1993) Nature 363:266-270), and also promotes the survival
of
cholinergic neurons of the basal forebrain following frimbrial transections
(Knusel, et
al., (1992) J. Neurosci.12: 4391-4402).
The preceding prior art teaches that administration of nerve growth factors
may be an
effective treatment for neurodegenerative disorders. However, they fail to
address the
difficulty associated with the unsupportive or inhibitory environment of the
CNS, which
would tend to antagonize the effects of these neuronal growth factors. The
compositions of the present invention are designed to reduce the inhibitory
components
of the CNS to, thereby, facilitate axonal growth. Therefore, the addition of
trophic
factors, as described above, in combination with the compositions of the
present
invention is an even more effective tool to treat neurodegenerative disorders
due to
disease or traumatic damage of nervous tissues. Thus, another embodiment is
the
preparation of compositions of the present invention in combination with
neurotrophic
factors in any amount, concentration or level deemed to be effective in the
treatment of
neurological diseases in a manner familiar to one skilled in the art.
It is known that transplantation of cells, for example peripheral nerve
Schwann cells
(Keirstead et al., (1999) Exp. Neurol. 159:225-236), enhances axonal
regeneration and
neurite outgrowth in injured spinal cord and brain (Li and Raisman (1994) J.
Neurosci.
14:4050-4063; Montero-Menei et al., (1992) Brain Res. 570:198-208). This
technique
enhances regeneration of injured spinal cord axons by providing scaffolding
for growth
and support and cell filled guidance channels which induce ingrowth and
elongation of
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
regenerating CNS axons (Smith and Stevenson (1988), Exp. Brain Res.; Xu et
al.,
(1995) Exp. Neurol. 134:261-272). In addition, it is known to someone skilled
in the art
that Schwann cells express a variety of cell adhesion molecules including N-
CAM, L1,
and N-cadherin, synthesize extracellular matrix molecules and release trophic
factors
including the neurotrophins NGF, BDNF, and NT3 (Venstrom and Reichardt (1993)
FASEB J. 7:996-1003; Guenard et al., (1993) 5:401-411). Therefore, an even
greater
level of axonal regeneration can result from a therapy combining neurotrophic
support
and transplantation (Keirstead et al. (1999) Expt. Neurol.).
In another embodiment the compositions of the present invention may
additionally
comprise neurotrophic factors, and may optionally be administered prior to,
simultaneously with, or subsequent to Schwann cell transplantation. The
demyelination
renders the environment more permissive to cellular growth following Schwann
cell
transplantation. The composition shall be prepared and delivered by a method
previously known to someone skilled in the art, for example by injection,
transplantation, or perfusion.
In another embodiment, the compositions of the present invention can be
prepared
comprising Tumour Necrosis Factor (TNF), which is used in a quantity and
purity
sufficient to facilitate regeneration of CNS axons in mammals, particularly
humans.
In another embodiment, the compositions of the present invention can be
prepared
comprising mononuclear phagocytes. U.S. Patent No. 5, 800,812 demonstrates
that
allogenic mononuclear phagocytes can be effective in the promotion of axonal
regeneration following injury or disease of the CNS.
The composition of this invention can be used in combination with cell
grafting
techniques such as those taught in U.S. Patent No. 5,750,103, which provides a
method
for grafting a cell in the brain of a mammalian subject comprising allowing
the cell to
attach to the surface of a support matrix in vitro, preferably by culturing
the cell with
the matrix, such that the cell is not encapsulated by the matrix, and
implanting the
support matrix with the attached cell into the brain. The techniques include
support
31
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
matrices made of glass or other silicon oxides, polystyrene, polypropylene,
polyethylene, polyacrylamide, polycarbonate, polypentene, acrylonitrile
polymer,
nylon, amylases, gelatin, collagen, natural or modified polysaccharides,
including
dextrans and celluloses (e.g. nitrocellulose), hyaluronic acid, extracellular
matrix, agar,
or magnetite. Preferred support matrices are beads, porous or nonporous, in
particular
microbeads having a diameter from about 90 to about 150 Vim.
The composition of the present invention may be used with methods that employ
cells
of many different types, preferably either cells of neural or paraneural
origin, such as
adrenal chromaffin cells. Also useful are cell lines grown in vitro. Cells not
of neural or
paraneural origin, such as fibroblasts, may also be used following
transfection with
DNA encoding a neuropeptide or an enzyme or set of enzymes which results in
production of neurotransmitter, or a neuronal growth factor.
A number of different cell types are useful. Typically, a cell will be
selected based on
its ability to provide a missing substance to the recipient brain. Missing
substances can
be neurotransmitters or other neurally-active molecules, the absence of which
results in
neurological disease or dysfunction. It is important that the transplanted
cell not grow as
a tumor once it is inserted into the recipient.
One source of donor cells is established neural cell lines. Many neuronal
clones exist
which have been used extensively as model systems of development since they
are
electrically active with appropriate surface receptors, specific
neurotransmitters,
synapse forming properties and the ability to differentiate morphologically
and
biochemically into normal neurons. Neural lines may express a tremendous
amount of
genetic information that corresponds to the genetic expression seen in CNS
neurons.
Such cells are described in the following references: Kimhi, Y. et al., Proc.
Natl. Acad.
Sci. USA 73: 462-466 (1976); In: Excitable Cells in Tissue Culture, Nelson, P.
G. et al.,
eds., Plenum Press, New York, 1977, pp. 173-245); Prasad, K. M. et al., In:
Control of
Proliferation of Animal Cells, Clarkson, B. et al., eds., Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, N.Y., 1974, pp. 581-594); Puro, D. G. et al., Proc.
Natl.
Acad. Sci. USA 73: 3544-3548 (1976); Notter, M. F. et al., Devel. Brain Res.
26: 59-68
32
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
(1986); Schubert, D. et al., Proc. Natl. Acad. Sci. USA 67: 247-254 (1970);
Kaplan, B.
B. et al., In: Basic and Clinical Aspects of Molecular Neurobiology, Guffrida-
Stella, A.
M. et al., eds., Milano Fondozione International Manarini, 1982)).
The composition of the present invention may be used with another important
source of
potential graft material which are cells engineered by somatic cell
hybridization, a
process which can immortalize single neurons. Fusing cells which differ in the
expression of specific genes allows for the exploration of the mechanisms
controlling
gene expression while chromosome alterations occur at rates to generate
genetically
different cell lines. Hybrid cells can be formed which retain the properties
of
differentiated cells. Hybrids derived from fusion of sympathetic ganglia and
neuroblastoma cells can synthesize dopamine (Greene, L. A. et al., Proc. Natl.
Acad.
Sci. USA 82: 4923-4927 (1975) while brain cell hybrids express choline
acetyltransferase (Minna, J. D. et al., Genetics 79: 373-383 (1975)).
Therefore,
embryonic precursors to dopaminergic neurons from the CNS can be fused with
mitotic
cells to incorporate both genomes into a single one that loses extra
chromosomes over
time and results in a new hybrid line. It is within the skill of the art to
produce such
hybrid neural or paraneural cells without undue experimentation, screen them
for the
desired traits, such as dopamine secretion, and select those having the best
potential for
transplantation.
The composition of the present invention may be used with cells for
transplantation
derived from another source of cells which is the adrenal medulla. This neural
crest-
derived tissue has been involved in clinical trials to treat Parkinson's
disease. Adult
monkey adrenal medulla can be cultured in vitro for at least about three weeks
as single
cells (Hotter, M. F. et al., Cell Tiss. Res. 244: 69-76 (1986)). Similarly,
retinal pigment
epithelial cells secrete dopamine and other factors and may be used for brain
implants
according to the present invention (Li, L. et al., Exp. Eye Res. 47: 771-785
(1988); Lui,
G. M. et al., Proc. Int'1. Soc. Eye Res. 6: 172 (1990); Li, L. et al., Inv.
Ophthal. Vis. Sci.
31(Suppl): 595 (1990, abstr. 2915-13); Sheedlo, H. J. et al., ibid., abstr.
2916-14;
Fektorovich, E.G. et al., ibid. (abstr. 2917-15); Song, M-K et al., supra).
33
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
Cells transplanted into the mammalian brain have shown survival in the absence
of
added growth factors. However, an additional embodiment is directed to
transplantation
of cells attached to a support matrix combined with the treatment, either in
vitro prior to
transplant, in vivo after transplant, or both, with the appropriate
growth/differentiation
factor and the composition of the present invention.
Grafted glial cells may play an important role in functional recovery of
neurons and
may be an important source of trophic factors (Doering, L. C. et al., J.
Neurolog. Sci.
63: 183-196 (1984); Gumple, J. et al., Neurosci. Lett. 37: 307-311 (1984)).
Therefore,
another embodiment involves co-culture of neural or paraneural cells with
glial cells,
their co-incubation with a support matrix, followed by implantation of the
support
matrix carrying both cell types into the CNS following or concurrent with use
of the
composition of the present invention.
In additional embodiments of the present invention involves use of the
composition
with olfactory ensheathing glia (Ramon-Cueto , et al (1998) J Neurosci.,
18:3803-15),
which are helper cells found only in nerves that carry odor sensations to the
brain.
Olfactory ensheathing glia share some characteristics with Schwann cells,
including
some of their growth-promoting properties, but they also express traits that
resemble
astrocytes, a helper cell in the CNS that can inhibit axon growth. Unlike
either cell
type, EG also migrate extensively within the CNS.
In additional embodiments of the present invention involves use of the
composition
with cells which are not of neural or paraneural origin, but which have been
altered to
produce a substance of neurological interest. A preferred cell type is a human
foreskin
fibroblast which is easily obtained and cultured, and survives upon
transplantation into
the rat brain using the methods of the present invention. For use in the
present
invention, the cells are genetically altered, using methods known in the art,
to express
neuronal growth factors, neurotransmitters, neuropeptides, or enzymes involved
in brain
metabolism. (See, for example, Gage, F. H. et al., Neuroscience 23: 795-807
(1987);
Rosenberg, M. B. et al., Science 242: 1575-1578 (1988); Shimohama, S. et al.,
Mol.
Brain Res. 5: 271-278 (1989).
34
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
Standard reference works setting forth the general principles of recombinant
DNA
technology and cell biology nclude Watson, J. D., et al., Molecular Biology of
the
Gene, Volumes I and II, Benjamin/Cummings Publishing Co., Inc., Menlo Park,
Calif.
(1987); Darnell, J. E. et al., Molecular Cell Biology, Scientific American
Books, Inc.,
New York, N.Y. (1986); Lewin, B. M., Genes II, John Wiley & Sons, New York,
N.Y.
(1985); Old, R. W. etal., Principles of Gene Manipulation: An Introduction to
Genetic
Engineering, 2nd Ed., University of California Press, Berkeley, Calif. (1981);
Maniatis,
T., et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,
Cold
Spring Harbor, N.Y. (1982)); Sambrook, J. et al.(Molecular Cloning: A
Laboratory
Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)
and
Albers, B. et al., Molecular Biology of the Cell, 2nd Ed., GarlandPublishing,
Inc., New
York, N.Y. (1989).
The transplanted cells may be attached to, or mixed with, a support matrix,
which are
frozen and stored in a frozen state using methods well known in the art.
Following
thawing, the matrix-bound cells are implanted into a recipient brain.
The cells may be xenogeneic (=heterologous, i.e., derived from a species
different from
the recipient), allogeneic (=homologous, i.e., derived from a genetically
different
member of the same species as the recipient) or autologous, wherein the
recipient also
serves as the donor.
Materials of which the support matrix can be comprised include those materials
to
which cells adhere following in vitro incubation, and on which cells can grow,
and
which can be implanted into a mammalian brain without producing a toxic
reaction, or
an inflammatory or gliosis reaction which would destroy the implanted cells or
otherwise interfere with their biological or therapeutic activity. Such
materials may be
synthetic or natural chemical substances or substances having a biological
origin. The
matrix-materials include, but are not limited to, glass and other silicon
oxides,
polystyrene, polypropylene, polyethylene, polyvinylidene fluoride,
polyurethane,
polyalginate, polysulphone, polyvinyl alcohol, acrylonitrile polymers,
polyacrylamide,
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
polycarbonate, polypentene, nylon, amylases, gelatin, collagen, natural and
modified
polysaccharides, including dextrans and celluloses (e.g. nitrocellulose),
agar, and
magnetite. Either resorbable or non-resorbable materials may be used. Also
intended are
extracellular matrix materials, which are well-known in the art (see below).
Extracellular matrix materials may be obtained commercially or prepared by
growing
cells which secrete such a matrix, removing the secreting cells, and allowing
the cells
which are to be transplanted to interact with and adhere to the matrix.
To improve cell adhesion, survival and function, the solid matrix may
optionally be
coated on its external surface with factors known in the art to promote cell
adhesion,
growth or survival. Such factors include cell adhesion molecules,
extracellular matrix,
such as, for example, fibronectin, laminin, collagen, elastin,
glycosaminoglycans, or
proteoglycans (see: Albers, B. supra, pp. 802-834) or growth factors, such as,
for
example, NGF. Alternatively, if the solid matrix to which the implanted cells
are
attached is constructed of porous material, the growth- or survival-promoting
factor or
factors may be incorporated into the matrix material, from which they would be
slowly
released after implantation in vivo.
Alternatively, cells growing on, or mixed with, resorbable matrices, such as,
for
example, collagen, can be implanted in sites of neurological interest other
than the
brain, in order to promote neuronal regrowth or recovery. For example, cells
attached to
the matrix of the invention may be implanted into the spinal cord, or placed
in, or
adjacent to, the optic nerve.
The matrix material on which the cells to be implanted grow, or with which the
cells are
mixed, may be an endogenous product of the implanted cells themselves. Thus,
for
example, the matrix material may be extracellular matrix or basement membrane
material which is produced and secreted by the very cells to be implanted.
The composition of the present invention may be used with a number of methods
for
treating various human neurological diseases, wherein it would be useful to
transplant
cells into the CNS. The composition can be used either prior to, or concurrent
with
36
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
cellular transplantation. For example, Parkinson's Disease can be treated
according to
the present invention by implanting dopamine-producing cells in the
recipient's
striatum, either concurrent or following use of the composition. Alzheimer's
disease
involves a deficit in cholinergic cells in the nucleus basalis. Thus,
according to the
invention, a subject having Alzheimer's disease or at risk therefor may be
implanted
with cells producing acetylcholine. Huntington's disease involves a gross
wasting of
the head of the caudate nucleus and putamen, usually accompanied by moderate
disease
of the gyrus. A subject suffering from Huntington's disease can be treated by
implanting
cells producing the neurotransmitters gamma amino butyric acid (GABA),
acetylcholine, or a mixture thereof. According to the present invention, the
support
matrix material to which such cells are attached is preferably implanted into
the caudate
and ptamen.
Epilepsy is not truly a single disease but rather is a symptom produced by an
underlying
abnormality. One skilled in the art will appreciate that each epileptic
subject will have
damage or epileptic foci which are unique for the individual. Such foci can be
localized
using a combination of diagnostic methods well-known in the art, including
electroencephalography, computerized axial tomography and magnetic resonance
imaging. A patient suffering from epilepsy can be treated according to the
present
invention by implanting the support matrix material to which GABA-producing
cells
are attached into the affected site. Since blockers of glutamate receptors and
NMDA
receptors in the brain have been used to control experimental epilepsy, cells
producing
molecules which block excitatory amino acid pathways may be used according to
the
invention. Thus implantation of cells which have been modified as described
herein to
produce polyamines, such as spermidine, in larger than normal quantities may
be useful
for treating epilepsy.
The composition of the present invention is intended for use with any mammal
which
may experience the beneficial effects of the methods of the invention.
Foremost among
such mammals are humans, although the invention is not intended to be so
limited, and
is also applicable to veterinary uses.
37
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
The composition can also be used with certain agents identified herein as "CNS
neural
growth modulators" (CNGMs), and particularly to a class of neural cell
adhesion
molecules as defined herein, to promote neurite outgrowth in the central
nervous system
(CNS). In general, neurons in the adult central nervous system have been
considered
incapable of regrowth, due to inhibitory molecular cues present on glial
cells. The
agents and methods of the present invention can be used to overcome this
inhibition and
promote CNS neurite outgrowth.
Such agents may be selected from any cell adhesion molecule which is capable
of
modulating or promoting CNS neurite outgrowth, and particularly to molecules
belonging to the immunoglobulin superfamily. More particularly, the molecules
are
selected from the members of the immunoglobulin superfamily which mediate Ca
2+ -
independent neuronal cell adhesion, including L1, N-CAM and myelin-associated
glycoprotein. The invention also contemplates fragments of these molecules,
and
analogs, cognates, congeners and mimics of these molecules which have neurite-
promoting activity. Particularly preferable structural motifs for these
fragments and
analogs include domains similar to the fibronectin type III homologous repeats
particularly repeats 1-2) and immunoglobulin-like domains (particularly
domains I-II,
III-IV and V-VI).
The composition of the present invention may be used with ectopic expression
of CNS
neural growth modulators (CNGMs) or neural cell adhesion molecules on
difFerentiated
astrocytes in vivo. The inhibitory action of astroglial and oligodendroglial
cells may be
overcome, at least in part, by the neurite outgrowth promoting properties of
the agents
defined herein, and as particularly illustrated by the activity of ectopically
expressed
L1. Expression ofLl by astrocytes seems also to compensate for inhibitory
effects
exerted by oligodendrocytes.
As indicated earlier, the present invention extends to the promotion of neural
growth in
the CNS, including such growth as is desired to regenerate structures lost due
to injury
or illness, as well as those structures and tissues exhibiting incomplete or
immature
formation.
38
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
The composition of the present invention may be used in combination with CNGM-
secreting cells for the modulation of neural outgrowth, regeneration, and
neural survival
in the CNS. As such, certain soluble CNGMs and fragments thereof, and cognate
molecules thereof are also within the invention.
In another embodiment the composition of the present invention can be prepared
in
combination with inhibitors of myelination such as metalloproteases (U. S.
Patent No.
6,025,333), inhibitors of apoptosis and/or necrosis (U.S. Patent Nos.
6,004,579,
6,015,665, and 6,046,007), inhibitors of proinflammatory cytokines, activators
antiinflammatory cytokines, (U.S. Patent No. 5,650,396), antiinflammatory
cytokines,
activators and generators of antioxidants (U.S. Patent Nos. 5,747,532,
5,747,459, and
5,667,776), or any combination thereof. The compositions of the present
invention may
also be administered in conjunction with methods that increase the production
of
phagocytosis (U.S. Patent No. 5,800,812).
In a preferred embodiment, the composition is comprised of a GaIC-specific
monoclonal antibody and human serum complement.
In another preferred embodiment, the composition is comprised of a MOG-
specific
monoclonal antibody and human serum complement.
Uses
The compositions of the present invention can be used to promote regrowth,
repair,
and/or regeneration of neurons in the CNS of a subject by stimulating
transient
immunological disruption of myelin or transient demyelination of axons.
Preferably,
the transient demyelination process of the present invention occurs in the
CNS, most
preferably in the spinal cord.
The subject may be any mammal. In a preferred embodiment, the subject is
human.
The compositions of the present invention can be used to promote regrowth,
repair,
and/or regeneration of dysfunctional neurons in the CNS that have been damaged
as a
39
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
result of injury, such as a spinal cord injury. The method can be used
following
immediate or chronic injury.
The compositions of the present invention can also be used to promote
regrowth, repair,
and/or regeneration of dysfunctional neurons in the CNS that have been damaged
as a
result of disease, such as degenerative diseases including Alzheimer's and
Parkinson's
disease.
The compositions. of the present invention can also be used to generate an
environment
within the mammalian CNS that is relatively permissive to growth of
transplanted cells.
For example, if PNS cells are transplanted into a site in the CNS that has
been damaged,
axons will be able to grow into the transplanted tissue but will be unable to
grow out of
this tissue back into the CNS due to the inhibitory effects of myelin. The
compositions
of the present invention can be used to disrupt the myelin in the CNS to allow
the axons
to extend into this area.
Preparations and Administration
Methods of using the compositions of the present invention comprise
administering a
therapeutically effective amount of such a composition to the subject. As used
herein,
the term "therapeutically effective amount" refers to an amount of composition
sufficient to effectively and transiently disrupt and/or demyelinate the CNS
so that
repair and regeneration of neurological tissue and neuronal connections is
enhanced.
Generally, the therapeutic composition is administered at a range from about
0.03 mg
antibody to about 0.6 mg antibody in a 20% to 30% complement solution per kg
body
weight. Preferably, the range is from 0.05 mg antibody to 0.4 mg antibody in a
20% to
30% complement solution per kg body weight. Most preferably, the range is from
0.1
mg antibody to 0.3 mg antibody in a 20% to 30% complement solution per kg body
weight. The exact ratio of antibody to complement will vary depending on the
circumstances; however, since the amount of complement activated is directly
proportional to the number of bound antibody molecules, it is possible to
administer
relatively high concentrations of complement in excess of the relative
concentration of
antibody. In addition, the particular concentration of antibody administered
will vary
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
with the particular dysfunction and its severity, as well as with such factors
as the age,
sex, and medical history of the patient. Those of skill in the clinical arts
will know of
such factors and how to compensate the dosage ranges of the composition
accordingly.
The majority of spinal cord injuries result from damage to the vertebral
column
surrounding the spinal cord. This damage includes fractures, dislocations, or
both.
Much of the damage to the spinal cord is due to secondary phenomena that occur
within
hours following the injury. At this point, the resultant damage may be
reversible;
consequently, a critical factor for recoverable CNS function is the amount of
time that
evolves between injury and the institution of therapy. Most preferably, when
the
nervous system dysfunction is a result of injury, administration of the
composition to
the subject will be as close in time to the time of the injury as possible.
A composition according to the method of the invention can be administered to
a
subject parenterally by injection or by gradual infusion over time. For
example, the
composition can be administered intrathecally or injected directly into the
spinal cord.
The compositions of the present invention can be administered in any manner
which is
calculated to bring the therapeutically effective components to the vicinity
of the
injured axons to be regenerated. Preferably, the composition is injected in a
pharmaceutically acceptable liquid carrier directly to the site.
Alternatively, an implant
bearing components of the composition may be surgically inserted. Such an
implant
may consist of any material, such as nitrocellulose, which can absorb these
components
and slowly release it at site of implantation.
One of the most common method to surmount some of the physical barriers
preventing
drug delivery to the central nervous system has been through the use of pumps.
A
variety of pumps have been designed to deliver drugs from an externally worn
reservoir
through a small tube into the central nervous system.
To be successful, it does not since just to deliver the drug within the
central nervous
system. The drug must be delivered to the intended site of action, at the
required rate of
41
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
administration, and in the proper therapeutic dose. Commercially, the
Medtronic pump
system known for the administration of baclofen intrathecally and the Alzet
osmotic
mini-pump have become acceptable, very useful, and successful means of
delivering
drugs at a controlled rate and dose over extended periods within the central
nervous
system.
Still another technique that has been developed has been disclosed, for
example, in U.S.
Patent No. 5,360,610, which makes use of polymeric microspheres as injectable,
drug-
delivery systems for use to deliver bioactive agents to sites within the CNS.
In
particular, bioactive agents are contained within a compatible biodegradable
polymer
which is then administered to a patient in need of therapy. The compositions
of the
present invention can be prepared in a series of microspheres which can be
implanted at
the site of injury or disorder, thereby allowing a sustained release of the
therapeutically
active components. U.S. Patent Nos. 4,883,666 and 5,601,835 also disclose
polymeric
drug delivery systems for controlled release of any substance to the CNS. A
worker
skilled in the art can easily adapt such polymeric drug delivery systems to
facilitate
administration of the compositions of the present invention to a patient in
need of such
therapy.
Other means of delivery will be apparent to those skilled in this art and are
intended to
be comprehended within the scope of the present invention.
Preparations for parenteral administration are contained in a pharmaceutically
acceptable carrier that is compatible with both the components of the
composition and
the patient. Such carriers include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents include propylene
glycol, polyethylene glycol, metabolizable oils such as olive oil or squalane,
and
injectable organic esters such as ethyl oleate. Aqueous carriers include
water,
alcoholic/acqueous solutions, and emulsions or suspensions, including saline
and
buffered media. Parenteral vehicles include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Preservatives
and other additives may also be present such as, for example, antimicrobials,
anti-
42
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
oxidants, chelating agents, and inert gases and the like. A preferred carrier
is artificial
cerebrospiral fluid.
Kit
The materials for use in the method of the invention are ideally suited for
the
preparation of a kit. Such a kit may comprise a carrier means
compartmentalized to
receive in close confinement one or more container means, such as vials,
tubes, and the
like, each of the container means comprising one of the separate elements to
be used in
the method. For example, one of the container means may comprise a GaIC-
specific
antibody. Alternatively, the antibody and complement may be present in the
same
container. The constituents may be present in liquid or lyophilized form, as
desired.
Needles and/or other equipment that facilitates delivery of the complement and
antibody to the site of damage may include:
a) silastic, Polyethylene, Tygon (Norton Performance Plastics) tubing;
b) subcutaneous pumps, (such as the Medtronic pump system known for the
administration of baclofen intrathecally);
c) spinal needle for direct intraspinal administration, or for short-term
intrathecal administration.
One example of a method using such a kit can be described as, a 14-gauge Tuohy
needle is inserted into the lumbar subarachnoid space. A 5-F catheter is
coaxially
placed with the tip at L 10 and tunneled to the flank (or appropriate
location). This type
of instruction would be understood by one familiar with the technique. Tubing
is
placed intrathecally, and connected to the pump. The pump, containing a finite
folume
of the reagents is placed under the skin this can be refilled in the Doctor's
office, via a
needle inserted into a septa in the pump. Or the Infusaid pump may be used in
the
alternative.
Advantages over Current Methods
The compositions and their uses of the present invention have a number of
advantages
over methods currently available for the regeneration of neuronal growth in
the CNS.
43
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
Interventional therapies, including opiate antagonists, thyrotropin-releasing
hormone,
local cord cooling, dextran infusion, adrenergic blockade, corticosteroids,
and
hyperbaric oxygen, are targeted at reducing secondary inflammatory damage
after a
traumatic injury to the spinal cord in order to prevent the spread of damage
to uninjured
neurons. Unlike the present invention, however, they do not promote
regeneration of
the damaged neurons.
Peripheral nerve transplants and the grafting of donor cells into the CNS are
useful in
that axons can grow into them; however, the axons cannot grow out of them into
the
surrounding CNS due to the inhibitory myelin present. In contrast, the present
invention disrupts the inhibitory myelin to allow regrowth of neurons in the
CNS.
The present invention is described in further detail in the following non-
limiting
examples. It is to be understood that the examples described below are not
meant to
limit the scope of the present invention. It is expected that numerous
variants will be
obvious to the person skilled in the art to which the present invention
pertains, without
any departure from the spirit of the present invention. The appended claims,
properly
construed, form the only limitation upon the scope of the present invention.
EXAMPLE I: REGENERATION OF BRAINSTEM-SPINAL AXONS
The following example illustrates that the transient developmental suppression
of
myelination or the disruption of mature myelin by local intraspinal infusion
of serum
complement proteins along with a complement-fixing, myelin-specific antibody
facilitates brainstem-spinal axonal regeneration after spinal transection in a
mammalian
subj ect.
Materials and Methods:
Surgical Spinal Transection and Transient Immunological Myelin Disruption:
Ten to 12 week old adult female rats (Sprague-Dawley), approximately 200g in
weight,
were anaesthetized with Ketamine/Xylazine (60mg/kg and 7.Smg/kg,
respectively).
After a limited dorsolateral laminectomy at T10, a left-side spinal cord
hemisection
lesion was made with micro-scissors. The extent of the lesion was then
confirmed by
44
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
passing a sharp scalpel through the lesion site three times (Fig. 1A).
Immediately after
the lesion, an intraspinal cannula was implanted at T 11 (n=22 total) and
connected to an
Alzet osmotic pump (14 day) to subsequently deliver a continuous intraspinal
infusion
(@ O.Sp,I/hr) of serum complement (GIBCO BRL, #19195-015, 33% v/v) along with
a
complement-fixing IgG antibody to galactocerebroside (either our own
polyclonal
antibody or Chemicon Intl. Ltd., #AB 142, 25% v/v). Cannulae were held in
place by
means of dental acrylic applied to the vertebral bone. Muscle layers were then
sutured
over the dental acrylic, and the superficial tissue and skin were closed. The
extent of the
hemisection lesion was always confirmed histologically at the end of the 5-
week
treatment and recovery period.
All control animals received an identical hemisection lesion and were then
intraspinally
infused via an osmotic pump, for the same time period, with either vehicle
alone (0.1 M
phosphate buffered saline, PBS, n=5), antibody alone (25% v/v, n=2), or serum
complement alone (33% v/v, n=6). All surgical procedures and subsequent animal
care
protocols were in accordance with Canadian and University of British Columbia
Animal Care Committee guidelines.
Electron Microscopy:
Tissue for ultrastructural analysis was obtained from 10-12 week old adult
female
Sprague-Dawley rats sacrificed 7 days after infusion of serum complement along
with a
complement-fixing IgG antibody to GaIC (see above for details) via an osmotic
pump.
Animals were lethally anaesthetised with Ketamine/Xylazine (120mg/kg and
lSmg/kg,
respectively), then perfused intracardially with 200 ml of O.1M PBS (pH 7.4)
followed
by 100 ml of 4% glutaraldyhyde in O.1M PB, (pH 7.3) and subsequently postfixed
overnight in the same fixative. The infusion site and surrounding cord was cut
into
lmm transverse blocks and processed to preserve rostral-caudal sequence.
Blocks were
washed in O.1M sodium cacodylate buffer (24 hours), postfixed in 2% Os04,
dehydrated through ascending alcohols, and embedded in Spurrs' resin according
to
standard protocols. Tissue blocks from experimental and untreated-control
animals
were processed in parallel. Thin sections (lam) were cut from each block,
stained with
alkaline Toluidine Blue, and examined under a light microscope. For electron
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
microscopic examination, blocks were trimmed then sections were cut at 80-
100nm,
mounted on copper grids, stained with uranyl acetate and lead citrate, and
viewed under
a Ziess EM l OC electron microscope (at 80k~.
Retrograde Neuronal Labeling:
If a retrograde tracer (single label) is injected into the rostral lumbar cord
(1 cm caudal
to the injury site), it should be incorporated and transported back to the
cell bodies of
origin by both intact axons, as well as regenerated projections. Consequently,
it is
essential that the retrograde tracer reliably and extensively label most, if
not all,
descending spinal projection neurons. An equally important parameter is that
the tracer
must be injected in a controlled and reproducible manner at a distance
suWciently
caudal to the spinal injury to prevent any direct difl'usion of the tracer to
the level of the
hemisection injury. The retrograde label that best satisfies all these
conditions is
Fluorogold (Sahibzada, et al., ( 1987) Brain Res. 415:242-256). Fluorescent
dextran
amines, such as RDA, require a recent axonal injury to facilitate axonal
uptake (Heimer
and Zaborszky Neuroanatomical Tract-tracingMethods 2: Recent Progress (New
York: Plenum, 1989)), and are therefore better suited for use in the double
label
retrograde-tracing studies.
Single Label Studies:
Twenty-eight days after the hemisection lesion and, consequently, 14 days
after
completion of the intraspinal infusion of the immunological reagents, each
adult rat was
anaesthetized with Ketamine/Xylazine (60mg/kg and 7.Smg/kg, respectively).
Fluorogold (FG, 100-150n1 total volume, 5% w/v in sterile dH20; Fluorochrome
Inc.
Englewood, CO, USA) was injected (SO-75n1) bilaterally into the middle of the
spinal
tissue at the L1 level, approximately lcm caudal to the lesion site.
The specific effect of the demyelinating protocol on the extent of diffusion
of FG was
also assessed. Rats (n=8) were experimentally treated as described above;
however,
animals were killed at 12, 24, 72, and 120 hours after injection of FG into
the L1 cord.
Eight other rats served as controls, where the pump contained vehicle only,
and were
processed in parallel with the experimentally treated animals. Cryostat
sections (25p,m
thick) were analyzed for the extent of FG diffusion from each injection site
(Fig. 1B).
46
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
There were no significant differences in the extent of visible FG diffusion,
as detected
at the light microscope level, between experimentally treated and control
treated
animals. In all cases, the range of FG diB'usion was 4-6mm (1-1.5 spinal
segments)
from the injection site or at least 1.5 spinal segments caudal to the lesion
site.
Double Label Studies:
At the time of lesion, the hemisection site was packed with gel-foam soaked
with 12%
(w/v in sterile dH20) rhodamine-conjugated dextran amine (RDA, 10,000MW
FluoroRuby, Molecular Probes) for 30 minutes. The gel-foam was then removed,
and
the remaining surgical procedures were completed (as outlined above). After 28
days
survival, all animals were anaesthetized with Ketamine/Xylazine (60mg/kg and
7.Smg/kg, respectively). FG (100-150n1 total volume, 5% w/v in sterile dH20)
was
injected (50-75n1) bilaterally into the spinal parenchyma at the L1 level of
the cord
(n=6).
Analysis of Axonal Regeneration:
Seven days following the injection of the FG tracer into the lumbar cord,
animals were
lethally anaesthetised with Ketamine/Xylazine (120mg/kg and l5mg/kg,
respectively)
and then perfused intracardially with 200m1 of O.1M PBS (pH 7.4) followed by
100 ml
of 4% paraformaldehyde in O.1M PBS, (pH 7.3). The brain and spinal cord were
then
removed and postfixed overnight in the same fixative. Subsequently, each brain
and
spinal cord was cleared of fixative and cryo-preserved by placing the tissue
in a series
of sucrose solutions (15% followed by 21°io). Coronal or parasagital
sections were cut at
25~.m thickness on a cryostat. The brainstem and spinal cord tissue sections
were
examined under a Zeiss Axioskop with a 100W mercury bulb (excitation/emission
wavelengths: FG, 365/420nm; RDA, 546/590nm).
The brainstem-spinal nucleus used to assess the axonal regenerative abilities
of
experimentally treated animals was the Red Nucleus (RN, origin is
contralateral to the
hemisection). Spinal-projecting axons from each RN cross to the opposite side
of the
midbrain and descend throughout the spinal cord within the contralateral
dorsolateral
funiculus. This contralateral spinal projection pathway is known to be a
completely
lateralized tract with the possible exception of 2-5 % of the axons, which may
project to
47
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
the cord via an ipsilateral route (Brown (1974) J. Comp. Neurol. 154:169-188;
Huisman
et al., (1981) Brain Res. 209:217-286; Shieh et al., (1983) J. Comp. Neurol.
214:79-86;
Waldron and Gwyn (1969) J. Comp. Neurol. 137:143-154).
Using a single-blind protocol, the number of retrograde labeled neurons within
the Red
Nucleus (RN) were counted in every other tissue section throughout the nucleus
to
avoid counting the same neuron twice. Only those cells exhibiting a nucleus
and a
neuronal morphology (i.e. mufti-polar in appearance), and that were
specifically labeled
with FG (i.e. not visible using other fluorescent filters; see above)
extending into the
proximal processes, were deemed to be positively labeled spinal-projecting
neurons.
The percentage of regenerating neurons was then determined in comparison to
the
number of labeled neurons within the contralateral (uninjured) control nucleus
within
the same animal.
Results:
Extent of Spinal Cord Demyelination and Myelin Disruption after Immunological
Treatment
Direct intraspinal infusion of 33% heterologous (guinea pig) serum complement
along
with polyclonal antibodies to GaIC (25%) in PBS over 7 days (@ O.Sp,I/hr)
resulted in
extensive demyelination up to 2mm away from the infusion cannula (total
rostrocaudal
distance of 4mm or approximately 1 spinal segment (Fig. 2A). This zone of
demyelination was bounded on either side by a further 8mm or 2 segments of
spinal
cord characterized by disrupted myelin (i.e. myelin that is extensively de-
laminated,
having an unraveled appearance, Fig. 2C). As shown in previous studies
(Keirstead et
al., (1995) J. Neurosci. 15:6963-6974; Keirstead et al., (1992) Proc. Natl.
Acad.
Sci.(USA) 89:11664-11668; Keirstead et al., (1997) Brain. Res. Bull. 44:727-
734),
control infusions of heterologous serum complement alone, myelin-specific
antibody
alone, or PBS alone resulted in only limited non-specific damage immediately
centered
around the cannula site. There was no surrounding zone of demyelination or
myelin
disruption (Fig. 2D).
The immunological demyelination and disruption of myelin within the
experimentally-
48
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
treated adult rat spinal cord is an active process extending throughout the
entire cross-
sectional profile of the cord. Immunological myelin disruption commences
within 1 day
and is associated with an invasion of macrophages or resident microglia and
polymorphonuclear cells (e.g. leukocytes such neutrophils, eosinophils and
basophils).
Many macrophages/microglia contain myelin fragments and complete their
phagocytic
activity within 7 days (Fig. 2B). This pattern of demyelination and myelin
disruption
can be maintained for as long as the serum complement and myelin-specific
antibody
are infused. Recent evidence suggests that after the immunological infusion is
terminated, remyelination begins within 2 weeks (Keirstead and Blakemore
(1997) Glia
(In Press); Dyer, Bourque, and Steeves (unpublished observations)); the new
myelin
originates from differentiating oligodendrocyte progenitors, although invading
Schwann
cells and surviving "mature" oligodendrocytes may also contribute to
remyelination.
Choice of Retrograde Tracer and Its D~usion Distance from the Injection Site
In this study, the major anatomical evidence for axonal regeneration within
the
hemisected and immunologically myelin-suppressed spinal cord of adult rats
depends
on a comparison between the number of retrogradely-labeled neurons within a
homologous pair of brainstem-spinal nuclei. For these comparisons to be valid,
the
candidate brainstem spinal nuclei must have highly unilateral projections that
are
confined to one side of the spinal cord at all levels. A left thoracic
hemisection (Fig.
1A) severed the contralaterally-projecting magnocellular neurons of the right
red
nucleus (RN), but left the projections from the left RN undamaged (as they
project
through the intact right dorsolateral funiculus of the thoracic cord).
In all cases, the Fluorogold label (100-150n1) was injected bilaterally within
the rostral
lumbar cord (lcm or 3 spinal segments caudal to the hemisection injury site).
We
assessed the time course and degree of rostrocaudal diffusion of Fluorogold
within the
lumbar and thoracic spinal cord of normally myelinated (control) animals and
experimentally treated rats (i.e. under demyelinated and myelin disrupted
conditions).
Random 25~m sections of experimental and control-treated spinal cords
(extending
from L2 to T8) were examined under a fluorescent microscope using the highest
49
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
intensity setting of the 100W mercury lamp. Spinal tissue was examined for the
extent
of Fluorogold diffusion at varying survival intervals after injection,
including: l2hr
(n=4), 24hr (n=4), 3d (n=4), and 5d (n=4). The maximum rostral diffusion
distance
observed was 4-6mm (or 1- 1.5 spinal segments) and occurred within a time span
of
24h. The degree of Fluorogold diffusion within the lumbar cord did not change
over the
subsequent time points examined (Fig. 1B).
In summary, no animal (experimental or control) showed any evidence of the
Fluorogold label within the spinal cord at the level of the hemisection lesion
(T10);
thus, by this criteria, no animals had to be excluded from this study. The
available
evidence indicates that the retrograde label was restricted to labeling intact
and
regenerating brainstem-spinal neurons having axonal projections caudal to the
T10
injury site.
Evidence for Brainstem-spinal Axonal Regeneration by Retrograde Neuronal
Labeling
28 animals (12 experimental (9 retrogradely single-labeled, 3 double-labeled)
and 16
control (13 retrogradely single-labeled, 3 double-labeled)) were subjected to
a left-side
lateral hemisection of the T10 spinal cord. Immediately after hemisection, an
infusion
cannula (connected to a 14d osmotic pump) was inserted directly into the
spinal cord 4-
5 mm (1 spinal segment) caudal to the injury site. The osmotic pump contained
one of a
number of 3 different control solutions or the experimental treatment (i.e.
PBS vehicle
alone, serum complement alone, anti-galactocerebroside antibody alone, or
serum
complement with anti-GaIC antibodies, respectively). Animals were then allowed
to
recover for 28 days before the Fluorogold was injected into the rostral
lumbar, lcm (i.e.
3 spinal segments) caudal to the lesion site. After a further 7 days survival,
each animal
was killed, and the brain and spinal cord were removed for examination and
analysis
(see Materials and Methods for criteria used to determine a labeled neuron).
The extent of the hemisection lesion was assessed in every animal. In all but
one
experimental and one control-treated animal, the left thoracic spinal cord was
hemisected (Fig. 1A). Most importantly, the region ofthe rubrospinal tract
(dorsolateral
funiculus) was severed. The right side white matter tracts were always
remained intact
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
and undamaged; usually the gray matter of the contralateral side was also
undamaged.
Comparing "blind" counts of the number of labeled neurons within each RN (Fig.
3A-
B, Table 1), the data indicated that 31.8% ~ 13.38% (n=9, range 10-50%) of the
injured
magnocellular RN neurons had regenerated a sufficient distance into the caudal
lumbar
cord to incorporate and retrogradely transport the Fluorogold (Fig. 4). In
contrast,
control treated animals, receiving either the PBS vehicle alone, GaIC antibody
alone, or
serum complement alone, did not exhibit a significant amount of RN labeling:
1.49% ~
0.84%, (Fig. 3C-D; Fig. 4, n=13, range 0-3, Table 1). The labeling of some
neurons
within the injured right RN nucleus may represent the small number of RN that
do not
project to the opposite side of the midbrain and descend within the
ipsilateral
(uninjured) cord (Shieh et al., (1983) J. Comp. Neurol. 214:79-86). No
retrograde-
labeling of cells was observed within the parvocellular region of the RN. This
was
expected since this RN region predominantly projects only as far as the
cervical region
of the cord.
Double retrograde labeling of the injured and myelin-suppressed rubrospinal
tract was
also qualitatively assessed (Fig. 3E and F). Large numbers of RDA-positive
(first label)
magnocellular RN neurons were observed after direct labeling of the lesion
site at the
time of hemisection injury to the thoracic spinal cord. After intraspinal
myelin-
suppression and subsequent injection of Fluorogold caudal to the lesion site,
a small
overlapping population of FG-positive neurons was observed (i.e. some neurons
were
labeled with both RDA and FG). Cells labeled exclusively by the first or the
second
tracer were also present in every brainstem analysed. The low number of double
labeled
brainstem-spinal neurons may in part be due to the failure of a severed axon
to take up
RDA prior to the sealing of the cut end, i.e. must be freshly injured (Heimer
and
Zaborszky Neuroanatomical tract-tracing methods 2: Recent Progress (New York:
Plenum, 1989)). Also the population of rubrospinal neurons that do not cross
the
brainstem will also appear as FG-positive cells in the "injured" nucleus. Due
to the
small number of animals that were assessed, we did not attempt to quantify
these
results. Nevertheless, they probably represent an under-estimate of the axonal
regeneration facilitated by immunological demyelination and myelin disruption,
but
51
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
definitely not an over-estimate of the degree of brainstem-spinal regeneration
after
myelin suppression.
As compared with prior art using spinal transection (Keirstead et al., ( 1995)
.I.
S Neurosci. 15:6963-6974; Keirstead et al., (1992) Proc. Natl. Acad. Sci.
(USA) 89:11664-
11668), the present invention is demonstrated using a hemisection model for
this study
so that each animal could serve as its own internal control (i.e. axonal
regeneration from
injured brainstem-spinal projections could be readily compared to the
uninjured
contralateral homologue). In addition, the present invention strove to
minimize the
degree of cyst cavity formation that often occurs with larger spinal lesions,
as well as
the amount of animal discomfort over the relatively long recovery periods
required for
this study.
Examinations for any functional or behavioral differences during the 5 week
recovery
period after experimental treatment indicated no notable differences in
locomotor
patterns between injured animals and uninjured control animals (i.e. all
animals walked
and all animals were comparable with respect to basic reflex functions). These
observations were true regardless of the treatment infused intraspinally after
a
hemisection injury (e.g. PBS alone, GaIC antibody alone, serum complement
alone, or
serum complement plus GaIC antibody).
These findings indicate that the immunological suppression of myelin
(demyelination
and myelin disruption) facilitate anatomical regeneration of brainstem-spinal
axons
within the injured adult rat spinal cord.
EXAMPLE II: EFFECTS OF REMOVAL OF A SINGLE COMPLEMENT
PROTEIN ON IMMUNOLOGICAL DEMYELINATION
Materials and Methods:
Surgical Spinal Transection and Transient Immunological Myelin Disruption:
Ten to 12 week old adult female rats (Sprague-Dawley), approximately 200g in
weight,
were anaesthetized with Ketamine/Xylazine (60mg/kg and 7.Smg/kg,
respectively). A
52
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
limited dorsolateral laminectomy was performed at T 10, and connected to an
Alzet
osmotic pump (14 day) to subsequently deliver a continuous intraspinal
infusion (@
0.5~,1/hr) of C3-depleted serum complement (Sigma 58788, 33% v/v) along with a
complement-fixing IgG antibody to galactocerebroside (either our own
polyclonal
antibody or Chemicon Intl. Ltd., #AB142, 25% v/v). Cannulae were held in place
by
means of dental acrylic applied to the vertebral bone. Muscle layers were then
sutured
over the dental acrylic, and the superficial tissue and skin were closed.
All control animals were intraspinally infused via an osmotic pump, for the
same time
period, with whole human serum complement (Sigma S1764, 33% v/v) along with a
complement-fixing IgG antibody to galactocerebroside (either our own
polyclonal
antibody or Chemicon Intl. Ltd., #AB142, 25% v/v). All surgical procedures and
subsequent animal care protocols were in accordance with Canadian and
University of
British Columbia Animal Care Committee guidelines.
Electron microscopy was performed as described in Example I.
Results:
As seen in Figure 5, the removal of the C3 component of complement results in
a lack
of myelin-removal. This indicates that this protein has a fundamental role in
either (i)
opsonization, or (ii) the propagation of the cascade to the lytic membrane
attack
complex (MAC), the final lytic pathway complex.
EXAMPLE HI: REGENERATION OF CHRONICALLY INJURED NEURONS
Materials and Methods:
11 animals (6 experimental and 5 control ) were subjected to a left-side
lateral
hemisection ofthe T10 spinal cord as follows: 10 to 12 week old adult female
rats
(Sprague-Dawley), approximately 2008 in weight, were anaesthetized with
Ketamine/Xylazine (60mg/kg and 7.Smg/kg, respectively). After a limited
dorsolateral
laminectomy at T10, a left-side spinal cord hemisection lesion was made with
micro-
scissors. The extent of the lesion was then confirmed by passing a sharp
scalpel through
the lesion site three times.
53
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
One month (5 animals) or 2 months (6 animals) after hemisection, an infusion
cannula
(connected to a 14d osmotic pump) was inserted directly into the spinal cord 4-
5 mm (1
spinal segment) caudal to the injury site. Cannulae were held in place by
means of
dental acrylic applied to the vertebral bone. Muscle layers were then sutured
over the
dental acrylic, and the superficial tissue and skin were closed. The osmotic
pump
delivered a continuous intraspinal infusion (0.5~,1/hr) of guinea-pig serum
complement
(33% v/v) along with a complement-fixing IgG antibody to galactocerebroside
(either
our own polyclonal antibody or Chemicon Intl. Ltd., #AB 142, 0.25 mg/mL).
All control animals received an identical hemisection lesion and were then
intraspinally
infused via an osmotic pump for the same time period with whole guinea-pig
serum
complement (33% v/v) alone.
Animals were then allowed to recover for 28 days before Fluorogold was
injected into
the rostral lumbar, lcm (i.e. 3 spinal segments) caudal to the lesion site, as
described in
Example I. After a further 7 days survival, each animal was killed, and the
brain and
spinal cord were removed for examination and analysis as described in Example
I.
The extent of the hemisection lesion was confirmed histologically at the end
of both the
S-week treatment and the recovery period. All surgical procedures and
subsequent
animal care protocols were in accordance with Canadian and University of
British
Columbia Animal Care Committee guidelines.
Results:
The extent of the hemisection lesion was assessed in every animal. In all
animals the
region of the vestibulospinal tract was severed. The right side white matter
tracts always
remained intact and undamaged while the gray matter of the contralateral side
usually
remained undamaged.
Comparing "blind" counts of the number of labeled neurons within each LVe
(Fig. 6),
the data indicated that in the 1 month chronically injured animals, 31.5% ~ 5%
(n=3) of
54
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
the injured lateral vestibulospinal neurons had regenerated a sufficient
distance into the
caudal lumbar cord to incorporate and retrogradely transport the Fluorogold.
In contrast,
control treated animals, receiving serum complement alone, did not exhibit a
significant
amount of LVe labeling: 3.6% t 2.7%, (n=2). Of those animals in which
treatment was
delayed for 2 months before treatment commenced, 26.8% ~ 13% (n=3) of the
injured
lateral vestibulospinal neurons had regenerated a sufficient distance into the
caudal
lumbar cord to incorporate and retrogradely transport the Fluorogold. In
contrast,
control treated animals, receiving serum complement alone, did not exhibit a
significant
amount of LVe labeling: 5.4% ~ 1.8%, (n=2). These results indicate that the
compositions of the present invention are useful for promoting regrowth,
repair, and
regeneration of chronically injured neurons in the CNS of a mammalian subject.
EXAMPLE IV: SURGICAL SPINAL TRANSECTION AND TRANSIENT
IMMUNOLOGICAL MYELIN DISRUPTION
Ten to 12 week old adult female rats (Sprague-Dawley), approximately 200g in
weight,
were anaesthetized with Ketamine/Xylazine (60mg/kg, 7.Smg/kg respectively).
After a
limited laminectomy at T10, a left-side spinal cord hemisection lesion was
made with
micro-scissors and the extent of the lesion was then confirmed by passing a
sharp
scalpel through the lesion site (Fig. 7). Immediately after the lesion, an
intraspinal
cannula was implanted at T 11 (n=22 total) and connected to an Alzet osmotic
pump ( 14
day) to subsequently deliver a continuous intraspinal infusion (@ O.Sp,I/hr)
of serum
complement (GIBCO BRL, #19195-015, 33% v/v) along with a complement-fixing IgG
antibody to galactocerebroside (either our own polyclonal antibody or Chemicon
Intl.
Ltd., #AB 142, 25% v/v). Cannulae were held in place by means of dental
acrylic
applied to the vertebral bone. Muscle layers were then sutured over the dental
acrylic,
and the superficial tissue and skin closed. The extent of the hemisection
lesion was
always confirmed histologically at the end of the 5-week treatment and
recovery period.
All control animals received an identical hemisection lesion and were then
intraspinally
infused via an osmotic pump, for the same time period, with either vehicle
alone (0.1 M
phosphate buffered saline, PBS, n=5), antibody alone (25% v/v, n=2), or serum
complement alone (33% v/v, n=6). All surgical procedures and subsequent animal
care
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
protocols were in accordance with Canadian and UBC Animal Care Committee
guidelines.
Electron Microscopy:
Tissue for ultrastructural analysis was obtained from 10-12 week old adult
female
Sprague-Dawley rats sacrificed 7 days after infusion of serum complement along
with a
complement-fixing IgG antibody to GaIC (see above for details) via an osmotic
pump.
Animals were lethally anaesthetised with Ketamine/Xylazine (120mg/kg, lSmg/kg
respectively), then perfused intracardially with 200 ml of O.1M PBS (pH 7.4)
followed
by 100 ml of 4% glutaraldyhyde in O.1M PB, (pH 7.3) and subsequently postfixed
overnight in the same fixative. The infusion site and surrounding cord was cut
into
1mm transverse blocks and processed to preserve rostral-caudal sequence.
Blocks were
washed in O.1M sodium cacodylate buffer (24 hours), post fixed in 2% Os04,
dehydrated through ascending alcohols and embedded in Spurrs' resin according
to
standard protocols. Tissue blocks from experimental and untreated-control
animals
were processed in parallel. Thin sections (l~,m) were cut from each block,
stained with
alkaline Toluidine Blue and examined under a light microscope. For electron
microscopic examination blocks were trimmed and sections cut at 80-100nm,
mounted
on copper grids, stained with uranyl acetate and lead citrate and viewed under
a Ziess
EM lOC electron microscope (at 80kV).
Retrograde Neuronal Labeling:
1. Single label Studies:
Twenty-eight days after the hemisection lesion and consequently 14 days after
completion of the intraspinal infusion of the immunological reagents, each
adult rat was
anaesthetized with Ketamine/Xylazine (60mg/kg, 7.Smg/kg respectively).
Fluorogold
(FG, 100-150n1 total volume, 5% w/v in sterile dH20; Fluorochrome Inc.
Englewood,
CO, USA) was injected (50-75n1) bilaterally into the middle of the spinal
tissue at the
L1 level, approximately lcm caudal to the lesion site (Fig. 7).
2. Double Label Studies:
At the time of lesion, the hemisection site was packed with gel-foam soaked
with 12%
(w/v in sterile dH20) rhodamine-conjugated dextran amine (RDA, 10,000MW
56
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
FluoroRuby, Molecular Probes) for 30 minutes. The gel-foam was then removed
and
the remaining surgical procedures were completed (as outlined above). After 28
days
survival, all animals were anaesthetized with Ketamine/Xylazine (60mg/kg,
7.Smg/kg
respectively) and FG (100-150n1 total volume, 5% w/v in sterile dH20) was
injected
(SO-75n1) bilaterally into the spinal parenchyma at the L1 level of the cord
(n=6).
Analysis of Regeneration:
Seven days following the injection of the FG tracer into the lumbar cord,
animals were
lethally anaesthetised with Ketamine/Xylazine (120mg/kg, l5mg/kg respectively)
and
then perfused intracardially with 200 ml of O.1M PBS (pH 7.4) followed by 100
ml of
4% paraformaldehyde in O.1M PBS, (pH 7.3). The brain and spinal cord were then
removed and postfixed overnight in the same fixative. Subsequently, each brain
and
spinal cord was cleared of fixative and cryo-preserved by placing the tissue
in a series
of sucrose solutions (15% followed by 21%). Coronal or parasagital sections
were cut at
25p,m thickness on a cryostat. The brainstem and spinal cord tissue sections
were
examined under a Zeiss Axioskop with a 100W mercury bulb (excitation/emission
wavelength at: FG, 365/420nm; RDA, 546/590nm; fluorescein, 490/515nm)
The two brainstem-spinal nuclei used to assess the axonal regenerative
abilities of
experimentally treated animals were the Red Nucleus (RN) (origin is
contralateral to the
hemisection) and the Lateral Vestibular (LVe) Nucleus (origin is ipsilateral
to the
hemisection). Spinal-projecting axons from each RN cross to the opposite side
of the
midbrain and descend throughout the spinal cord within the contralateral
dorsolateral
funiculus. This contralateral spinal projection pathway is known to be a
completely
lateralized tract with the possible exception of 2-5 % of the axons which may
project to
the cord via an ipsilateral route (Waldron and Gwyn 1969; Brown, 1974; Huisman
et
al., 1981; Shieh et al., 1983). The LVe tract projects from the dorsolateral
pontine
hindbrain, maintaining an exclusive ipsilateral course throughout the
brainstem and the
ventrolateral white matter of the spinal cord (Zemlan et al., 1979; Shamboul,
1980).
Using a single-blind protocol, the number of retrograde labeled neurons within
the Red
Nucleus (RN) (contralateral to the hemisection) and the Lateral Vestibular
(LVe)
57
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
Nucleus (ipsilateral to the hemisection) were counted in every other tissue
section
(throughout these brainstem nuclei) to avoid counting the same neuron twice.
Only
those cells exhibiting a nucleus, a neuronal morphology (i.e. mufti-polar in
appearance)
and specifically labeled with FG (i.e. not visible using other fluorescent
filters; see
above) extending into the proximal processes, were deemed to be positively
labeled
spinal-projecting neurons. The percentage of regenerating neurons for each
brainstem-
spinal projection was then determined in comparison to the number of labeled
neurons
within the contralateral (uninjured) control nucleus within the same animal.
Extent of Spinal Cord Demyelination and Myelin Disruption after Immunological
Treatment
Direct intraspinal infusion over 7 days (@ 0.5~,1/hr) of 33% heterologous
(guinea pig)
serum complement along with polyclonal antibodies to GaIC (25%) in PBS
resulted in
extensive demyelination up to 2mm away from the infusion cannula (total
rostrocaudal
distance of 4mm or approximately 1 spinal segment (Fig. 2A). This zone of
demyelination was bounded on either side by a further 8mm or 2 segments of
spinal
cord characterized by disrupted myelin (i.e. myelin that is extensively de-
laminated,
having an unraveled appearance, Fig. 2B). As shown in previous studies
(Keirstead et
al., 1992, 1995), control infusions of heterologous serum complement alone,
myelin-
specific antibody alone, or PBS alone resulted in only limited non-specific
damage
immediately centered around the cannula site. There was no surrounding zone of
demyelination or myelin disruption (Fig. 2C).
The immunological demyelination and disruption of myelin within the
experimentally-
treated adult rat spinal cord was an active process extending throughout the
entire cross-
sectional profile of the cord. Immunological myelin disruption commenced
within 1 day
and was associated with an invasion of macrophages or resident microglia and
polymorphonuclear cells (e.g. leukocytes such neutrophils, eosinophils and
basophils).
Many macrophages/microglia contained myelin fragments and completed their
phagocytic activity within 7 days (Fig. 2D). This pattern of demyelination and
myelin
disruption could be maintained for as long as the serum complement and myelin-
specific antibody were infused. Recent evidence suggests that after the
immunological
58
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
infusion is terminated remyelination begins within 2 weeks (Keirstead and
Blakemore,
1997; Dyer, Bourque and Steeves unpublished observations) and the new myelin
originates from differentiating oligodendrocyte progenitors, although invading
Schwann
cells and surviving "mature" oligodendrocytes may also contribute to
remyelination.
Choice of Retrograde Tracer and Its D~sion Distance from the Injection Site
In this study, the major anatomical evidence for axonal regeneration within
the
hemisected and immunologically myelin-suppressed spinal cord of adult rats
depends
on a comparison between the number of retrogradely-labeled neurons within a
homologous pair of brainstem-spinal nuclei. For these comparisons to be valid,
the
candidate brainstem spinal nuclei must have highly unilateral projections that
are
confined to one side of the spinal cord at all levels. As summarized in Fig.
7A, a left
thoracic hemisection severed the contralaterally-projecting magnocellular
neurons of
the right red nucleus (RN), but left the projections from the left RN
undamaged (as they
project through the intact right dorsolateral funiculus of the thoracic cord).
Likewise, a
left thoracic hemisection severed the ipsilateral projecting neurons of the
left lateral
vestibulospinal nucleus (LVe), but left the axons from the right LVe nucleus
undamaged (as they also project through the intact right side of the thoracic
cord via the
ventrolateral white matter).
If a retrograde tracer (single label) is injected into the rostral lumbar cord
(I cm caudal
to the injury site), it should be incorporated and transported back to the
cell bodies of
origin by both intact axons, as well as regenerated projections. Consequently,
it is
essential that the retrograde tracer reliably and extensively label most, if
not all,
descending spinal projection neurons. An equally important parameter is the
tracer must
be injected in a controlled and reproducible manner at a distance sufficiently
caudal to
the spinal injury to prevent any direct diffusion of the tracer to the level
of the
hemisection injury. The retrograde label that best satisfied all these
conditions was
Fluorogold (Sahibzada, et al., 1987). Fluorescent dextran amines, such as RDA,
require
a recent axonal injury to facilitate axonal uptake (c.f. Heimer and Zaborszky,
1989), and
were therefore better suited for use in the double label retrograde-tracing
studies (see
description below).
59
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
In all cases, the Fluorogold label (100-150n1) was injected bilaterally within
the rostral
lumbar cord (1 cm or 2-3 spinal segments caudal to the hemisection injury
site, Fig. 7).
We assessed the time course and degree of rostrocaudal diffusion of Fluorogold
within
the lumbar and thoracic spinal cord of normally myelinated (control) animals
and
experimentally treated rats (i.e. under demyelinated and myelin disrupted
conditions).
Random 25~m sections of experimental and control-treated spinal cords
(extending
from L2 to T8) were examined under a fluorescent microscope using the highest
intensity setting of the 100W mercury lamp. Spinal tissue was examined for the
extent
of Fluorgold diffusion at varying survival intervals after injection,
including: l2hr
(n=6), 24hr (n=6), 3d (n=6), Sd (n=6) and 7d (n=22). The maximum rostral
diffusion
distance observed was 4-6 mm (or 1- 1.5 spinal segments) and occurred within a
time
span of 24h. The degree of Fluorogold diffusion within the lumbar cord did not
change
over the subsequent time points examined (Fig. 7).
Evidence for Brainstem-spinal Axonal Regeneration by Retrograde Neuronal
Labeling
In brief, 28 animals; 12 experimental (9 retrogradely single-labeled, 3 double-
labeled)
and 16 control (13 retrogradely single-labeled, 3 double-labeled) were
subjected to a
left-side lateral hemisection of the T10 spinal cord. Immediately after
hemisection, an
infusion cannula (connected to a 14d osmotic pump) was inserted directly into
the
spinal cord 4-5 mm (1 spinal segment) caudal to the injury site. The osmotic
pump
contained one of a number of 3 different control solutions or the experimental
treatment
(i.e. PBS vehicle alone, serum complement alone, anti-galactocerebroside
antibody
alone, or serum complement with anti-GaIC antibodies, respectively). Animals
were
then allowed to recover for 28 days before the Fluorogold was injected into
the rostral
lumbar, lcm (i.e. at least 2 spinal segments) caudal to the lesion site. After
a further 7
days survival, each animal was killed and the brain and spinal cord were
removed for
examination and analysis (see above for criteria used to determine a labeled
neuron).
The extent of the hemisection lesion was assessed in every animal. In all but
one
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
experimental and one control-treated animal, the left thoracic spinal cord was
hemisected (Fig. 7). Most importantly, the regions of the rubrospinal tract
(dorsolateral
funiculus) and the lateral vestibulospinal tract (ventrolateral funiculus)
were severed.
The right side white matter tracts were always remained intact and undamaged
and
usually the gray matter of the uninjured side was also undamaged.
As discussed above, the 2 pairs of brainstem-spinal nuclei examined for
evidence of
retrograde labeling (after spinal cord hemisection and immunological myelin
suppression) were the RN and the LVe. These brainstem-spinal nuclei were
chosen for
their unilateral projection patterns within the thoracic and lumbar cord,
enabling
comparisons to be made between the retrograde-labeling within an injured
nucleus and
the uninjured contralateral homologue. Comparing "blind" counts of the number
of
labeled neurons within each RN (Fig. 3A-B), the data indicated that 31.8% t
4.7%
(n=8, range 10-50%) of the injured magnocellular RN neurons had regenerated a
sufficient distance into the caudal lumbar cord to incorporate and
retrogradely transport
the Fluorogold (Fig. 9). In contrast, control treated animals, receiving
either the PBS
vehicle alone, GaIC antibody alone, or serum complement alone did not exhibit
a
significant amount of RN labeling; 1.49% ~ 0.23%, (Fig. 3C-D; Fig. 9, n=13,
range 0-
3). The labeling of some neurons within the injured right RN nucleus may
represent the
small number of RN that do not project to the opposite side of the midbrain
and descend
within the ipsilateral (uninjured) cord (Shieh et al., 1983). No retrograde-
labeling of
cells was observed within the parvocellular region of the RN.
Retrograde-labeling of regenerating LVe neurons was also observed, but only
after
experimental demyelination and disruption of spinal cord myelin (Fig. 8). In 8
experimental animals, the mean percentage of regenerating LVe labeling, in
comparison
to the uninjured contralateral control nucleus, was 41.8% ~ 3.1% (n=8, range
33-49%).
In control-treated animals (see above) the percent LVe labeling was 2.24% ~
0.55%
(Fig. S, n=13, range 0-6).
Double retrograde labeling of the injured and myelin-suppressed rubrospinal
tract was
also qualitatively assessed (Fig. 9E and F). Large numbers of RDA-positive
(first label)
61
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
magnocellular RN neurons were observed after direct labeling of the lesion
site at the
time of hemisection injury to the thoracic spinal cord. After intraspinal
myelin-
suppression and subsequent injection of Fluorogold caudal to the lesion site
(see above
for details) a small overlapping population ofFG-positive neurons was observed
(i.e.
some neurons were labeled with both RDA and FG). Cells labeled exclusively by
the
first or the second tracer were also present in every brainstem analysed.
Examinations for any functional or behavioral differences during the 5 week
recovery
period after experimental treatment indicated no notable differences in
locomotor
patterns between injured animals and uninjured control animals (i.e. all
animals walked
and all animals were comparable with respect to basic reflex functions). These
occurred
regardless ofthe treatment infused intraspinally after a hemisection injury
(e.g. PBS
alone, GaIC antibody alone, serum complement alone, or serum complement plus
GaIC
antibody). Thus, subtle differences were very difficult to observe or quantify
and
'gross' motor patterns were essentially the same.
As compared with prior art using spinal transection (Keirstead et al., 1992,
1995), the
present invention is demonstrated using a hemisection model so that each
animal could
serve as its own internal control (i.e. axonal regeneration from injured
brainstem-spinal
projections could be readily compared to the uninjured contralateral
homologue). In
addition, the present invention strove to minimize the degree of cyst cavity
formation
that often occurs with larger spinal lesions, as well as the amount of animal
discomfort
over the relatively long recovery periods required.
The present invention also illustrates that the demyelination produced by the
intraspinal
infusion of serum complement and a myelin-specific antibody (e.g. GaIC)
produced a
rapid and active demyelination over 1-2 segments of the cord with myelin
disruption
extending a further 2 segments, either side of the infusion site. Resident
microglia
and/or invading macrophages were observed to contain myelin debris. The
immunological suppression of spinal cord myelin surrounding the thoracic
hemisection
facilitated significant axonal regeneration by 2 unilaterally projecting
brainstem-spinal
pathways, the rubrospinal and lateral vestibulospinal (RN and LVe,
respectively) tracts.
62
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
Control treated animals (hemisection injury plus local intraspinal infusion of
PBS alone,
GaIC antibody alone, or serum complement alone) showed little or no retrograde
labeling within the injured RN or Lve.
EXAMPLE V: EFFECTS OF REMOVAL OF SINGLE COMPLEMENT
PROTEINS ON IMMUNOLOGICAL DEMYELINATION
In order to demonstrate the relative necessity of the different complement
proteins in
the composition, we assessed the effects of serum infusions deficient in a
particular
complement protein (eg. C3, C4, C6, and Factor B). We evaluated spinal treated
tissue
ultrastructurally using a transmission electron microscope and phenotypically
by
indirect immunofluorescence.
Materials and Methods:
Surgical Spinal Transection and Transient Immunological Myelin Disruption:
1 S Ten to 12 week old adult female rats (Sprague-Dawley), approximately 200g
in weight,
were anaesthetized with Ketamine/Xylazine (60mg/kg and 7.Smg/kg,
respectively). A
limited dorsolateral laminectomy was performed at T10, and connected to an
Alzet
osmotic pump (14 day) to subsequently deliver a continuous intraspinal
infusion (@
O.Sp,I/hr) of depleted serum complement (Sigma S8788, 33% v/v) along with a
complement-fixing IgG antibody to galactocerebroside (either our own
polyclonal
antibody or Chemicon Intl. Ltd., #AB 142, 25% v/v). Cannulae were held in
place by
means of dental acrylic applied to the vertebral bone. Muscle layers were then
sutured
over the dental acrylic, and the superficial tissue and skin were closed.
All control animals were intraspinally infused via an osmotic pump, for the
same time
period, with whole human serum complement (Sigma S 1764, 33% v/v) along with a
complement-fixing IgG antibody to galactocerebroside (either our own
polyclonal
antibody or Chemicon Intl. Ltd., #AB 142, 25% v/v). All surgical procedures
and
subsequent animal care protocols were in accordance with Canadian and
University of
British Columbia Animal Care Committee guidelines.
63
SUBSTITUTE SHEET (RULE 26)
CA 02371592 2001-10-25
WO 00/64473 PCT/CA00/00440
Electron microscopy was performed as described in Example I.
Results:
The results are demonstrated in Figures 12 and 13.
Upon removal of the C3 protein, common to both the classical and alternative
pathways
(see Figure 11), or the C4 protein, a classical pathway protein, normal myelin
was
observed. Using serum deficient in Factor B, an alternative pathway protein,
demyelination was observed; in addition, large numbers of macrophages
containing
myelin debris were present in these regions. These results suggest that the
classical
serum complement pathway has a fundamental role in this immunological
protocol.
Upon removal of C6, a Membrane Attack Complex protein, a less extensive patchy
distribution of naked axons was observed, once again, accompanied by many
macrophages, suggesting that some of the demyelination may be occurring
through
macrophage mediated events involving complement-derived anaphylatoxins and
membrane receptors.
64
SUBSTITUTE SHEET (RULE 26)
