Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W095/13291 PCT~S94/12858
CA21 73731
NEURON-GLIA CELL AD~ESION MOLECU~E, NG-CAM,
IN TR~TM~T OF NERVE n~M~
This invention was funded in part by a research
grant from the National Institutes of Health, which
provides to the United States Government certain rights
in this invention.
R~CR~-~OUND OF THE lN V ~:N'l'lON
Field of the Invention
The present invention in the field of neuroscience
and medicine relates to methods for promoting neurite
growth and for promoting the growth and regeneration of
nerves and treating spinal cord injury using neuron-glia
cell adhesion molecule (Ng)-CAM or a functional
derivative thereof.
Description of the Backqround Art
It has been estimated that more than 200,000 nerve
repair procedures are performed ~nn~ ly in the United
States. Unfortunately, the outcome o~ current peripheral
nerve repair is generally poor (Arch;h~ld~ S . J. et al .,
1991, J. Comp. Neurol 306:685-696). The limited regrowth
of nerve fibers in the mature nervous system of higher
vertebrates is due largely to an inability of the axons
to elongate in this environment rather to an intrinsic
inability of the neurons to regrow. Strategies to
improve nerve regrowth using permissive guides such as
-- 1 --
W095/1329l PcT~sg~/l2858
l~A~ ~ 7~73 ~
.
peripheral nerve grafts and silicone tubes have
resulted in some, but limited, regrowth (Archibald et
al., supra; LeBeau, J.M. et al., 1988, J. Neurocytol.
17:161-172).
Current strategies to repair peripheral nerve
transection are directed to suturing of the proximal
nerve to the distal stump with microsurgical techniques
so that the regenerating fibers can course through the
degenerating distal stump to reinnervate the original
target. This i5 effective only for simple transection
but not in the majority of cases where gaps are generated
between the proximal fibers and the distal nerve stumps.
When gaps of several millimeters occur, nerve
regeneration is very poor. Attempts have been made to
bridge the gaps using nerve autografts. As stated above,
silicone tubes and collagen-based conduits are as, or
more, effective in promoting regeneration in rodents and
nonhllm~n primates (Archibald et al., supra; Gibson, K.L.
et al., 1989, Microsurgery 10: 126-129). Gibson et al.
discloses the implantation of nerve cuffs or guide tubes
("entubulization") as a method for repair of transected
nerves. In this approach, pro~ l and distal nerve
stumps are introduced into each end of a tube and are
held together by one or two epineurial sutures. The
regenerating axons travel through the lumen of the guide
tube toward the distal stump. Several types of materials
are known to serve as nerve cuffs, including natural
materials including veins and autogenous collagen, and
synthetic substances including lactate polymers,
polygalactin mesh, polyethylene and silicone tubing.
The entubulation techniques open new possibilities
for improving regeneration by incorporating various
materials. Much has recently been learned about proteins
WO 95/~3291 . I e PCT/US94/12858
~A~173~3~
.
normally expressed abundantly, but transiently, during
nerve development which are re-expressed during nerve
regeneration in animals. The present inventor has turned
his attention to the use of such neurally active proteins
in promoting nerve repair and regrowth.
Cell Adhesion Molecules and Na-CAM
Cell-cell adhesion is a primary process that is
critical for embryonic development and for pattern
formation in the nervous system (Edelman, G.M. et al.,
1990, MORPHOREGULATORY MOLECU~ES, John Wiley & Sons, New
York). The ability of neurons to organize into specific
patterns depends on their interactions with other
neurons, with glia, and with the extracellular
environment (Jacobson, M. ,1991, DEVELOPMENTAL
NEUROBIOLOG~, 3rd Edition, Plenum Press, New York). Many
of these interactions between neural cells are mediated
by cell adhesion molecules (CAMs) (Edelman, G.M., 1983,
Science 219:450-457) which fall primarily into two
different families. Members of the immunoglobulin
superfamily (Edelman, G.M., 1987, Immun. Rev. 100:11-45)
contain ;mmllnoglobulin domains and fibronectin type III
repeats and have calcium-independent binding, while
members of the cadherin family share distinct repeated
~o~;nR and have calcium-dependent binding (Takeichi, M.,
1988, Development 102:639-655).
CAM8 such as the neuron-glia CAM, Ng-CAM (Grumet, M.
et al., 1984, J Cell Biol 98:1746-1756) mediate cell-cell
interactions and are expressed early during development
in æpatially and temporally restricted patterns (Edelman,
G.M., 1988, Biochemistry 27:3533-3543; Jessell, T.M.,
1988, Cell 1: 3-13). Certain CAMs are transmembrane
proteins which may participate in the transmission of
signals between cells to modulate cell behavior and
WO95/~3291 PcT~s94/l28~8
CA21 73731
differentiation (Edelman G.M., 1976, Science 192:218-226;
Schuch, U. et al., 1989, Neuron 3:13-20; Bixby, J.L.,
1989, Neuron 3:287-297).
Ng-CAM is a large neuronal CAM of around 200 kDa
that can mediate neuron-neuron and neuron-glia adhesion,
and has been implicated in neuronal migration and the
formation of nerve bundles. The biochemistry and biology
of Ng-CAM is reviewed in Grumet, M., 1992, J. Neurosci.
Res. 31:1-13, which is hereby incorporated by reference
in its entirety. The biochemical properties of Ng-CAM
are described below. Purified Ng-CAM presented as a
substrate for neurons in culture can promote neuritic
fiber extension of about 100 ~m in several hours. Ng-CAM
binds homophilically (to itself) and heterophilically to
several cell surface proteins. It is structurally
related to a human protein called Ll (Reid, R.A. et al.,
1992, J. Mol. Neurosci. 3:127-135), and it can bind to
m~mm~l ian Ll ~Grumet, M. et al., 1986, J. Cell Biol.
106:487-503; Lemmon, V. et al., 1989, Neuron 2:1597-
1603). The binding of certain adhesion molecules,
including Ll, to neurons generates signals such as an
increase in intracellular calcium that have been
associated with promotion of neurite growth (Schuch et
al., supra; Williams, E.J. et al., 1992, J. Cell Biol.
119:883-892). Thus, these proteins which have been
implicated as major mediators of ~O~Al growth (a) are
expressed at high levels at times and locations of nerve
formation during development, (b) decrease their
expression during maturation of the nervous system, (c)
dramatically increase expression following injury to the
nervous system and (d) return to normal levels after
recovery from injury (Daniloff, J.K. et al., 1986b, J.
Cell. Biol. 103:929-945)).
WO 95/13291 PCT/US94112858
CA21 73~31
.
Ng-CAM shares certain properties with the neural
CAM, N-CAM, which was the first CAM isolated from brain
(Hoffman, S. et al., 1982, J. Biol. Chem. 257:7720-7729).
Unlike Ng-C~M which is specific to the nervous system,
N-CAM is found in muscle and other tissues. N-CAM is a
large cell surface glycoprotein which binds
homophilically to N-CAM on other cells (Hoffman, S. et
al., 1983, Proc Natl Acad Sci USA 80:5762-5766) and to
heparin by a different mechanism (Cole, G.J. et al.,
1989, Neuron 2:1157-1165). N-CAM has a role in cell
adhesion (Brackenbury et al., 1977, ~. Biol. Chem.
2~2:6835-6840) retinal layering (Buskirk et al., 1980,
Nature 285:488-489 ), retinotectal mapping (Fraser et
al., 1984, Proc. Natl. Acad. Sci. USA 81:4222-4226) and
neuron-myotube interaction (Rutishauser et al., ~. Cell
Biol. 97:145-152 1983). N-CAM, however, does not play a
major role in adhesion between embryonic neurons and glia
in as much as most glial cells express low levels of N-
CAM (Grumet et al., 1983, Science 222:60-62). Although
both N-CAM and Ng-CAM are involved in neuron-neuron
adhesion and are members of the ;mmllnQglobulin
superfamily, they are distinct molecules (Cunningham,
B.A. et al., 1987, Science 236:799-806; Burgoon, M.P. et
al., 1991, ~. Cell Biol. 112:1017-1029). Chick proteins
isolated independently and nAmeA by others (e.g., G4
(Rathjen, F.G. et al., 1987a, ~. Cell Biol. 104:343-353)
and 8D9 antigen (T-~mmn~ , V. et al., 1986, ~. Neurosci.
6:2987-2994) are probably identical to Ng-CAM (~urgoon et
al., supra).
Following peripheral nerve injury, the amount of N-
CAM and Ng-CAM increased dramatically in the region
surrounding the site of injury, in the pro~ m~ 1 nerve,
and in the dorsal root ganglia while Ng-CAM levels
wo 95/l3291 C A 2 1 7 3 7 3 1 PCT/US9~,l285~
.
decreased moderately in the spinal cord. The levels of
Ng-CAM returned to normal after nerve regeneration. It
was pointed out that such correlations may have practical
applications in studying nerve repair (Daniloff et al.,
5 supra) . Remsen, L . G . et al ., 1990, Exper. Neurol .
110: 268-273, showed that when tubes applied to transected
sciatic nerves contained monoclonal antibodies (mAbs)
specific to N-CAM, functional neuronal recovery was
inhibited, indicating a role for N-CAM in nerve
regeneration. Daniloff, U.S. Patent No. 4,955,892 (11
Sept 1990) discloses use of N-CAM in nerve prostheses for
repair of peripheral nerve damage and restitution of
muscle innervated by the regenerated nerve. This
reference has no disclosure of Ng-CAM in such a
therapeutic setting.
Characterization of Nq-CAM Protein
Monoclonal antibodies specific for Ng-CAM recognize
a major component having a relative MW of 135 kDa and
lesser amounts of two closely spaced components at about
200 kDa in extracts from chick brain (Grumet et al.,
1984a, Proc Natl Acad Sci USA 81:7989-7993). SDS-PAGE
and protein st~;ning of ;mmllnoaffinity purified Ng-CAM
reveals an additional component of about 80 kDa which is
not directly recognized by the anti-Ng-CAM mAbs. Rabbit
antibodies were raised against each of the above three
fractions. Antibodies against the 200 kDa protein
recognized all three components. Antibodies against the
135 kDa protein recognized the 200 kDa but not the 80 kDa
component. Antibodies against the 80 kDa component
recognized the 200 kDa but not the 135 kDa protein
~Grumet et al ., 1984a, supra; Wolff, J.M. et al ., 1987,
Eur. ~. Biochem. 168:551-561). Thus, each of the smaller
components is antigenically related to the larger one but
WOg5~13291 PCT~Sg~/l2858
CA21 7373~
not to each other, indicating possible cleavage of the
200 kDa component to yield the two smaller polypeptides.
Pulse-chase biosynthesis experiments indicated that
Ng-CAM is first synthesized as a 200 kDa species with the
135 kDa appearing later. The 200 kDa and 80 kDa
components were difficult to analyze directly because of
their relative instability even in the presence of
protease inhibitors (Grumet et al ., 1988, J Cell Biol
106:487-503). Nevertheless, peptide mapping showed that
the two smaller components of Ng-CAM share fragmentation
patterns with the larger but not with each other (Wolff
et al., supra). The amino terminal sequences of Ng-CAM
polypeptides are identical for the two components at ~200
kDa (i. e., 210 and 190 kDa) and the 135 kDa component,
and these differ from the amino terminal sequence of the
80 kDa species (Burgoon et al., supra).
The findings regarding the protein structure of
Ng-CAM are summarized in the form of a linear model
(Figure 10) of its components which include amino
terminal sequence similarities, immunological
relationships, and structural relationships among the
various components. Moreover, the sequence of Ng-CAM
cDNA and analysis of its mRNA support this model.
SUMMARY OF THE lNv~NlION
The present inventor has conceived of the use of Ng-
CAM or ~mm~l ian L1 or NILE protein, or functional
derivatives thereof such as peptides representing regions
of the full protein, to promote ne~rite sprouting and
nerve growth. These proteins or derivatives are
therefore useful in promoting nerve regeneration and
WO 95/13291 PCT/US94/12858
~A21 73731
repair, for example, in the treatment of spinal cord
injuries. These cell adhesion molecules may contribute
to nerve regeneration and neuronal repair by: (1)
promoting neuronal adhesion, (2) neutralizing inhibitory
effects of extracellular matrix molecules such as
chondroitin sulfate proteoglycans (e.g., neurocan and 3F8
proteoglycan (PG)), and (3) providing stimulatory signals
to neurons that alter intracellular messengers to
promote neurite growth.
Ng-CAM/L1 may be particularly effective in promoting
nerve regrowth in peripheral nerve injuries as well as in
lesions in the central nervous system (CNS).
The present invention is thus directed to a method
of promoting the regeneration of a nerve in a subject
having peripheral or central nerve damage, comprising
administering to a subject in need of such treatment an
amount of Ng-CAM, or a functional derivative thereof, or
cells expressing Ng-CAM, effective in promoting
regeneration of nerves.
In another embodiment of the above method, Ng-CAM is
~m; n; gtered in combination with an effective amount of
at least one other agent that is capable of promoting
neuronal survival, growth, differentiation or
regeneration. Preferably the other agent is nerve growth
factor, nerve growth factor (NGF), brain-derived
neurotrophic factor (BDNF), ciliary neurotrophic factor
(CNTF), or another neurotrophin including NT-3, NT-4 or
NT-5, or l~m;n;n.
The above methods are useful in treating peripheral
nerve damage a8sociated with physical or surgical trauma,
infarction, bacterial or viral infection, toxin exposure,
degenerative disease or malignant disease that affects
peripheral or central neurons. Such disease further
WO 95/13291 PCT/US94/12858
~, CA~l 73731
include CNS lesions, gliosis, Parkinson's disease,
Alzheimer's disease, neuronal degeneration, and the like.
The present methods are useful for treating any disorder
which induces a gliotic response or inflammation.
In the above methods, Ng-CAM or its functional
derivative may be administered in a form associated with
a solid or semisolid phase support material, preferably
collagen gel.
The present invention is further directed to a
pharmaceutical composition useful in the treatment of
peripheral or central nerve damage, comprising:
(a) an amount of Ng-CAM effective for treating
peripheral nerve damage; and
(b) a pharmaceutically acceptable carrier.
The pharmaceutical composition may also comprise at
least one other agent that is capable of promoting neuron
survival, growth, differentiation or regeneration or a
material such as collagen gel which serves as a substrate
for nerve growth.
The present invention also provides a method of
promoting regeneration of a injured or severed nerve,
comprising exposing an injured or severed nerve to a
concentration of Ng-CAM that is effective in promoting
the regeneration of neurons. This method may be carried
out in vitro or in vivo.
Also provided is a method for promoting the
regeneration of a severed nerve in a subject, comprising
surgically entubulating the nerve in an entubulation
device which contains an amount of Ng-CAM effective in
promoting the regeneration. The device may further
contain at least one other agent that is capable of
promoting neuron survival, growth, differentiation or
regeneration.
g
W095/13291 PCT~S94/12858
CA21 73731 1--
In another embodiment, the present invention is
directed to a method for promoting neuronal survival or
neurite growth by neutralizing or overcoming the
inhibitory effect of a chondroitin sulfate proteoglycan,
preferably neurocan, on the survival or growth, which
method comprises contacting a nerve fiber inhibited in
its survival or growth by a chondroitin sulfate
proteoglycan with an amount of Ng-CAM effective in
neutralizing or overcoming the inhibitory effect, thereby
promoting neuronal survival or neurite growth.
The present invention is further directed to a
method of diagnosing a neuronal disorder associated with
an abnormal level of a substance which binds to Ng-CAM in
a subject, comprising:
(a) measuring the level of the Ng-CAM-binding substance
in a sample from the subject; and
(b) comparing the levels of the substance measured in
step (a) with the level of the substance in an
analogous sample from a normal individual or a
standard level of the substance,
thereby detecting an abnormality in the level of the Ng-
CAM-binding substance in the subject, the abnormality
being diagnostic of the neuronal disorder. The Ng-CAM-
binding substance is preferably a cho~oitin sulfate
proteoglycan, such as the 3F8 proteoglycan.
Also provided is a method for identifying a compound
or agent which binds to Ng-CAM or to a functional
derivative thereof, comprising:
(a) exposing said compound or agent to Ng-CAM or a
functional derivative thereof, preferably bound to a
solid phase carrier or support;
(b) measuring the binding of the compound or agent to
the Ng-CAM or functional derivative.
-- 10 --
Wo95/~3291 PcT~s94/12858
CA21 73731
The present invention also provides a method of
diagnosing a neuronal disorder, such as a brain tumor,
associated with an abnormal level of Ng-CAM in a subject,
comprising:
(a) measuring the level of Ng-CAM in a sample from the
subject; and
(b) comparing the levels of Ng-CAM measured in step (a)
with the level of Ng-CAM in an analogous sample from
a normal individual or a standard level,
thereby detecting an abnormality in the level of Ng-CAM
in the subject, the abnormality being diagnostic of the
neuronal disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure l is a graph showing binding of neurocan and
aggrecan to CAMs and extracellular matrix proteins.
Wells of removable 96-well plates were coated with
unlabeled proteins (l. 25 ~g/ml) and incubated with ~
labeled proteins (~160,000 cpm). The fraction of the
input bound to the different substrates is given as a
percentage. Nonspecific binding to BSA was also
determined, and specific binding (percent bound) was
calculated as total minus nonspecific binding. Speci~ic
activities in the experiments shown here were 2.5 to 2.9
x lo18 cpm/mole. All values are means of duplicate
determinations i SEM.
Figure 2 is a graph showing binding of neurocan to
CAMs and collagen I under hypotonic and isotonic
conditions. Labeled proteoglycans were applied at an
average of 160,000 cpm per well. All values are means of
duplicate determinations + SEM.
WO 95113291 C A 2 1 7 3 7 3 1 PCT/US94/128!;8
Figure 3 shows saturation curves (panels A and B)
and Scatchard plot analysis (panels C and D) of l25I-
labeled 7-d neurocan (Panels A and C) and neurocan-C
(panels B and D) binding to Ng-CAM. Binding values
represent specific binding (total cpm bound minus cpm
bound to BSA). Neurocan was tested at 0.5-50 ng/well (6.8
x 10l8 cpm/mole), and neurocan-C at 0.2-37 ng/well (4.8 x
10l8 cpm/mole). Bars in the saturation curves represent
the SEM of duplicate determinations.
Figure 4 is a series of graphs showing saturation
curves (panels A and B) and Scatchard analysis (panels C
and D) of I~I-labeled neurocan (panels A and C) and
neurocan-C (panels B and D) binding to N-CAM. Binding
values represent specific binding (total cpm bound - cpm
bound to BSA). Neurocan was tested at 0.4-40 ng/well and
neurocan-C at 1-60 ng/well. Bars in saturation curves
represent the SEM of duplicate determinations.
Figure 5 is a graph showing the binding of I~I-
labeled neurocan-C to Ng-CAM in the presence of other
soluble molecules. Wells coated with Ng-CAM were
incubated with I~I-labeled neurocan-C (160,000 cpm/well)
in the presence of unlabeled neurocan-C 0),
aggrecan (-), chon~roitin sulfate (0), chondroitin
sulfate disaccharides (~), and fibronectin (0) at the
concentrations indicated. Specific binding of neurocan-C
to Ng-CAM in the absence of soluble molecules corresponds
to 0~ inhi~ition; background binding to BSA corresponds
to 100~ inhibition. Values are the means of duplicate
determinations and the SEM is less than 5~ of the mean
value.
Figure 6 is a set of graphs showing effects of
chon~roitinase and heat treatment on the binding of
neurocan to Ng-CAM (panel A) and to N-QM (Panel B).
- 12 -
WO 95/~3291 C A 2 1 7 3 7 3 1 PCT/US94112858
.
"native" - Native proteoglycans; "control" -
proteoglycans incubated at 37C for 2 h in chondroitinase
buffer; "ch-ase" - chondroitinase-treated proteoglycans;
"heat" - proteoglycans incubated at 95C for 15 min;
"ch-ase+heat" - chondroitinase and heat-treated
proteoglycans. ~I-labeled neurocan were used at an
average of 55,000 cpm/well. All values are means of
duplicate determinations i SEM.
Figure 7 is a series of micrographs showing
imm~noperoxidase staining of 7-d postnatal rat brain
cerebellum with antibodies to neurocan, Ng-CAM, and N-
CAM. Panel A: mAb 2C2, which recognizes a cytoplasmic
region that is highly conserved between avian Ng-CAM and
mAmm~lian NILE/L1; Panel B: mAb lDl specific for
neurocan; Panel C: mAb 5B8 specific for N-CAM. Bar = 50
~m.
Figure 8 is a series of micrographs showing effects
of neurocan on neurite growth on Ng-CAM and anti-Ng-CAM
mAbs. Substrates were incubated first with 50 ~g/ml Ng-
CAM (Panels A and B) or 3 ~g/ml mAb anti Ng-CAM IgG
(Panels C and D), followed by incubation with either 30
~g BSA (A and C) or neurocan (B and D). Brain cells from
9-d chick embryos were added to the substrates, fixed
after 2 days in culture and photographed under phase
microscopy. Bar = 50 ~m.
Figure 9 is a series of graphs showing effects of
neurocan on neurite outgrowth. Brain cells from 9-d
chick embryos were plated on substrates coated with 50
~g/ml Ng-CAM (Panel A) or 3 ~g/ml mAb anti Ng-CAM IgG
(Panel B), and either 30 ~g BSA or 11 ~g neurocan.
After 2 days in culture, cells were fixed and, for each
bound cell, the length of the longest neurite was
determined. The histograms show the percentage of
WO95/t329l PCT~s9~/l2858
~ A 2 ~
neurites having the designated lengths. The following
were the values of mean neurite lengths in ~M ~ SEM:
Ng-CAM minus neurocan: 19.3 i 1.8 (n = 148);
Ng-CAM plus neurocan: 6.7 + 1.1 (n - 66);
anti Ng-CAM minus neurocan: 19.4 + 1.7 (n c 89);
anti Ng-CAM plus neurocan: 6.3 i 0.9 (n = 152).
Figure 10 shows a model of Ng-CAM protein and domain
structure. (a) Linear representation of Ng-CAM
components aligned to indicate relationships between
them. The 135 kDa component, which is the predominant
form found in the chick, and the 80 kDa component are
derived from the 200 kDa by proteolysis. The general
locations of N-linked carbohydrates (CHO), phosphorylated
amino acids (P), and covalently bound fatty acids
(swiggled line) are indicated. (b) The six
immunoglobulin domains (each ~100 amino acids) are
represented by loops near the amino terminus (NH2) and
the fibronectin type III repeats (each ~100 amino acids)
are represented by the rectangles. The two last repeats
(shown as open rectangles) have much lower identities to
fibronectin type III repeats. The transmembrane domain
(23 amino acids) is indicated by the vertical stippled
bar, and the cytoplasmic region (113 amino acids) is near
the carboxy terminus (COOH). The location of the amino
terminus of the 80 kDa component is indicated by an
arrow.
D~SCRIPTION OF T~E ~K~KK~'~ EMBODIMENTS
Ng-CAM/L1 binds homophilically to other Ng-CAM
molecules and heterophilically to several cell surface
proteins that are found in the nervous system. The
- 14 -
WO 95/13291 PCT/US94/12858
~ CA21 73731
binding of certain adhesion molecules (including Ll) to
neurons generates intracellular signals (such as increase
in calcium) associated with promotion of neurite growth.
The present inventor recognized that the ability of Ng-
CAM/L1 to provide signals that promote neurite growth as
well as to serve directly as favorable substrates for
neuronal adhesion and migration, make it an excellent
candidate for use in (1) improving nerve regeneration or
promoting nerve survival, (2) treatment of peripheral
nerve injury and spinal cord injury and (3) stimulation
of growth of endogenous, implanted or transplanted CNS
tissue.
Because Ng-CAM normally reappears during
regeneration in a delayed manner (Daniloff et al. 1986,
supra), introduction of Ng-CAM or a derivative thereof
soon after injury is particularly important for
accelerating the rate and the extent of recovery.
Cell repulsion or avoidance is a process that occurs
during normal neural development and may be important for
regeneration. Nerves will not grow into regions
myelinated by oligodendrocytes; certain proteins are
believed to be responsible for this repulsion.
~hon~roitin sulfate proteoglycans have repulsive effects
on neuronal adhesion and fiber growth. The present
inventor and his collaborators have recently shown that
two cho~oitin sulfate proteoglycans from brain,
neurocan and 3F8 (Rauch, U. et al ., 1991 , J. Biol . Chem.
266:14785-14801) inhibit Ng-CAM function and bind to Ng-
CAM with high affinity (Grumet, M. et al., 1993, J.
Cell. Biol 120:815-824; Friedlander, D.R. et al., 1993,
J. Neurosci . 19: 626a). These proteoglycans may therefore
inhibit nerve regrowth and neuronal cell division.
According to the present invention, Ng-CAM is useful to
WO 9Stl329 1 PCTIUS94/12858
~A21 ~3731 ~
neutralize or counteract their inhibitory properties on
neurons.
In particular, the present inventor and his
coworkers have found that neurocan and 3F8 proteoglycan,
chondroitin sulfate proteoglycans of brain, in soluble
form, bind with high affinity to Ng-CAM. Neurocan-
mediated inhibition of neuronal adhesion was related to
blockage of binding to substrate-bound Ng-CAM in in vitro
assays. Longer term assays showed that neurocan
inhibited neurite outgrowth on Ng-CAM substrates under
conditions similar to those that inhibited neuronal
adhesions. Moreover, Ng-CAM reversed the inhibition by
substrate-bound neurocan and stimulated both neuronal
adhersion and neurite growth.
On the basis of these observations, the present
inventor conceived of the use of Ng-CAM to promote neural
recovery from injury, such as spinal cord injury, by
neutralizing the inhibitory action of proteins such as
neurocan on the repair process.
The ability of Ng-CAM/Ll to provide signals that
favor neurite growth as well as to serve directly as
favorable substrates for adhesion and migration, make it
an excellent candidate for use to improve nerve
regeneration. Although these molecules normally reappear
during regeneration, they do so in a delayed manner in
the peripheral nervous system. Therefore, their
introduction soon after injury may be particularly
important for accelerating the rate and the extent of the
recovery.
- 16 -
W095/1329l PcT~s94/l2858
CA21 73731
Ng-CAM/L1 PROTEINS, PEPTIDES AND THEIR FUNCTIONAL
DERIVATIVES
It will be understood that the Ng-CAM/Ll protein
useful in the methods and compositions of the present
invention can be biochemically purified from a cell or
tissue source. For preparation of naturally occurring
Ng-CAM, tissues such as brain, especially of human
origin, are preferred.
Alternatively, because the gene encoding Ng-CAM or
L1 can be isolated or synthesized, the polypeptide can be
synthesized substantially free of other proteins or
glycoproteins of m~mm~lian origin in a prokaryotic
organism or in a non-m~mm~lian eukaryotic organism, if
desired.
Alternatively, methods are well known for the
synthesis of polypeptides of desired sequence on solid
phase supports and their subsequent separation from the
support.
In a further embodiment, the invention provides
"functional derivatives" of Ng-CAM/~1. By "functional
derivative" is meant a "fragment,~ variant,~ "analog,"
or "chemical derivative" of the Ng-CAM/L1. A functional
derivative retains at least a portion of the function of
Ng-CAM, such as binding to Ng-CAM, binding to neurocan,
binding to a specific anti-Ng-CAM antibody, or
stimulation of neurite growth, which permits its utility
in accordance with the present invention.
A "fragment" of Ng-CAM/L1 protein refers to any
subset of the molecule, that is, a shorter peptide.
A "variant" of Ng-CAM/~1 refers to a molecule sub-
stantially similar to either the entire protein or a
fragment thereof. Variant peptides may be conveniently
prepared by direct chemical synthesis of the variant
- peptide, using methods well- known in the art.
- 17 -
WO95/13291 PCT~S9~/128~8
C A 2 1 7 37 3 1 ~
Alternatively, amino acid sequence variants of the
protein or peptide can be prepared by mutations in the
DNA which encodes the synthesized peptide. Such variants
include, for example, deletions from, or insertions or
substitutions of, residues within the amino acid
sequence. Any combination of deletion, insertion, and
substitution may also be made to arrive at the final
construct, provided that the final construct possesses
the desired functional activity. Obviously, the
mutations that will be made in the DNA encoding the
variant peptide must not alter the reading frame and
preferably will not create complementary regions that
could produce secondary mRNA structure (see European
Patent Publication No. EP 75,444).
At the genetic level, these variants ordinarily are
prepared by site-directed mutagenesis (as exemplified by
Adelman et al., DNA 2:183 (1983)) of nucleotides in the
DNA encoding the Ng-CAM/L1 protein or a peptide fragment
thereof, thereby producing DNA encoding the variant, and
thereafter expressing the DNA in recombinant cell culture
(see below). The variants typically exhibit the same
qualitative biological activity as the nonvariant
peptide.
A preferred group of variants of Ng-CAM/~1 are those
in which at least one amino acid residue in the protein
or in a peptide fragment thereof, and preferably, only
one, has been removed and a different residue inserted in
its place. For a detailed description of protein
chemistry and structure, see Schulz, G.E. et al.,
PRINCIPLES OF PROTEIN STRUCTURE, Springer-Verlag, New
York, 1978, and Creighton, T.E., PROTEINS: STRUCTURE
AND MO~ECULAR PROPERTIES, W.E. Freeman & Co., San
Francisco, 1983, which are hereby incorporated by
- 18 -
wosstl329l PCT~S94/12858
CA21 73731
reference. The types of substitutions which may be made
in the protein or peptide molecule of the present
invention may be based on analysis of the fre~uencies of
amino acid changes between a homologous protein of
different species, such as those presented in Table 1-2
of Schulz et al. (6upra) and Figure 3-9 of Creighton
( supra) ~ Base on such an analysis, conservative
substitutions are defined herein as exchanges within one
of the following five groups:O l. Small aliphatic, nonpolar or slightly polar
residues: Ala, Ser, Thr (Pro, Gly);
2. Polar, negatively charged residues and their amides:
Asp, Asn, Glu, Gln;
3. Polar, positively charged residues:
His, Arg, Lys;
4. ~arge aliphatic, nonpolar residues:
Met, Leu, Ile, Val (Cys); and
5. Large aromatic residues: Phe, Tyr, Trp.
The three amino acid residues in parentheses above
have special roles in protein architecture. Gly is the
only residue lacking any side chain and thus imparts
flexibility to the chain. Pro, because of its unusual
geometry, tightly constrains the chain. Cys can
participate in disulfide bond formation which is
important in protein folding. Note the Schulz et al.
would merge Groups 1 and 2, above. Note also that Tyr,
because of its hydrogen bonding potential, has some
kinship with Ser, Thr, etc. Substantial changes in
functional or ;mmllnological properties are made by
selecting substitutions that are less conservative, such
as between, rather than within, the above five groups,
which will differ more significantly in their effect on
maintaining (a) the structure of the peptide backbone in
-- 19 --
WO 95/~3291 PCT/US91/12858
~A21 73731
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. Examples of such substitutions are
(a) substitution of gly and/or pro by another amino acid
or deletion or insertion of gly or pro; (b) substitution
of a hydrophilic residue, e.g., ser or thr, for (or by) a
hydrophobic residue, e.g., leu, ile, phe, val or ala;
(c) substitution of a cys residue for (or by) any other
residue; (d) substitution of a residue having an
electropositive side chain, e.g., lys, arg or his, for
(or by) a residue having an electronegative charge, e.g.,
glu or aspi or (e) substitution of a residue having a
bulky side chain, e.g., phe, for (or by) a residue not
having such a side chain, e.g., gly.
Most deletions and insertions, and substitutions
according to the present invention are those which do not
produce radical changes in the characteristics of the
protein or peptide 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
which are described in more detail below. For example,
a change in the ;mmllnological character of the protein
peptide molecule, such as binding to a given antibody, is
measured by a competitive type ~mmllnoassay. Biological
activity i8 screened in an appropriate bioassay, as
described below.
Modifications of such peptide properties as redox or
thermal stability, hydrophobicity, susceptibility to
proteolytic degradation or the tendency to aggregate with
- 20 -
WO95/l3291 PcT~s94/l2858
~ CA2~7373~
carriers or into multimers are assayed by methods well
known to the ordinarily skilled artisan.
An "analog" of Ng-CAM/L1 refers to a non-natural
molecule substantially similar to either the entire
molecule or a fragment thereof.
A "chemical derivative" of Ng-CAM/Ll contains addi-
tional chemical moieties not normally a part of the
peptide. Covalent modifications of the peptide 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
an organic derivatizing agent that is capable of reacting
with selected side chA; n~ or terminal residues.
Additionally, modified amino acids or chemical
derivatives of amino acids of Ng-CAM/L1 or fragments
thereof, according to the present invention may be
provided, which polypeptides contain additional chemical
moieties or modified amino acids not normally a part of
the protein. Covalent modifications of the peptide are
thus included within the scope of the present invention.
The following examples of chemical derivatives are
pr~ ided by way of illustration and not by way of
limitation.
Aromatic amino acids may be replaced with D- or
L-naphthylalanine, D- or L-phenylglycine, D- or
L-2-thienylalanine, D- or L-l-, 2-, 3- or ~-pyrenyl-
alanine, D- or L-3-thienylalanine, D- or L-(2-pyridi-
nyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- or
~-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenyl-
glycine, D-(trifluoromethyl)-phenylglycine, D-(tri-
fluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D-
or L-p-biphenylphenylalanine, D- or L-p-methoxy-
biphenylphenylalanine, D- or L-2-indole(alkyl)alanine,
- 21 -
wosslt32sl PCT~Sg~/12858
~A21 73731
and D- or L-alkylalanine where alkyl may be substituted
or unsubstituted methyl, ethyl, propyl, hexyl, butyl,
pentyl, iso-propyl, iso-butyl, sec-isotyl, iso-pentyl,
non-acidic amino acids, of C1-C20.
Acidic amino acids can be substituted with non-
carboxylate amino acids while maintaining a negative
charge, and derivatives or analogs thereof, such as the
non-limiting examples of (phosphono)-alanine, glycine,
leucine, isoleucine, threonine, or serine; or sulfated
(for example, -SO3H) threonine, serine, tyrosine.
Other substitutions may include unnatural
hydroxylated amino acids may made by combining "alkyl"
(as defined and exemplified herein) with any natural
amino acid. Basic amino acids may be substituted with
alkyl groups at any position of the naturally occurring
amino acids lysine, arginine, ornithine, citrulline, or
(guanidino)-acetic acid, or other (guanidino)alkyl-acetic
acids, where "alkyl" is define as above. Nitrile
derivatives (e.g., cont~;n;ng the CN-moiety in place of
COOH) may also be substituted for asparagine or
glutamine, and methionine sulfoxide may be substituted
for methionine. Methods of preparation of such peptide
derivatives are well known to one skilled in the art.
In addition, any amide linkage in any of neurocan
polypeptides can be replaced by a ketomethylene moiety,
e.g., (-C(=O)-CH2-) for (-(C=O)-NH-). Such derivatives
are expected to have the property of increased stability
to degradation by enzymes, and therefore possess
advantages for the formulation of compounds which may
have increased in vivo half lives, as administered by
oral, intravenous, intramuscular, intraperitoneal,
topical, rectal, intraocular, or other routes.
WO 95/13291 PCT/US94/12858
~ 7~
In addition, any amino acid representing a component
of the said peptides can be replaced by the same amino
acid but of the opposite chirality. Thus, any amino acid
naturally occurring in the L-configuration (which may
also be referred to as the R or S, depending upon the
structure of the chemical entity) may be replaced with an
amino acid of the same chemical structural type, but of
the opposite chirality, generally referred to as the D-
amino acid but which can additionally be referred to as
the R- or the S-, depending upon its composition and
chemical configuration. Such derivatives have the
property of greatly increased stability to degradation by
enzymes, and therefore are advantageous in the
formulation of compounds which may have longer in ~ivo
half lives, when administered by various routes.
Additional amino acid modifications of amino acids
of a Ng-CAM/L1/NILE protein or peptide according to the
present invention may include the following.
Cysteinyl residues most commonly are reacted with
alpha-haloacetates (and corresponding amines), such as
chloroacetic acid or chloroacetamide, to give
carboxymethyl or carboxyamidomethyl derivatives.
Cysteinyl residues also are derivatized by reaction with
bromotrifluoroacetone, alpha-bromo- beta-(5-
imidozoyl)propionic acid, chloroacetyl phosphate, N-
alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-
pyridyl disulfide, p-chloromercuribenzoate, 2-
chloromercuri-4- nitrophenol, or chloro-7-nitrobenzo-2-
oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with
diethylprocarbonate at pH 5.5-7.0 because this agent is
relatively specific for the histidyl side chain. Para-
bromophenacyl bromide also is useful; the reaction is
- 23 -
W095/1329l PcT~s94/128~8
CA21 73731 ~
preferably performed in 0.1 M sodium cacodylate at pH
6Ø
Lysinyl and amino terminal residues are reacted with
succinic or other carboxylic acid anhydrides.
Derivatization with these agents has the effect of
reversing the charge of the lysinyl residues. Other
suitable reagents for derivatizing alpha-amino-containing
residues include imidoesters such as methyl
picolinimidate; pyridoxal phosphate; pyridoxal;
chloroborohydride; trinitrobenzenesulfonic acid; O-
methylisourea; 2,4 pentanedione; and trans~m;n~e-
catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one
or several conventional reagents, among them
phenylglyoxal, 2,3- butanedione, 1,2-cyclohexanedione,
and ninhydrin. Derivatization of arginine residues
requires that the reaction be performed in alkaline
conditions because of the high p~ of the guanidine
functional group. Furthermore, these reagents may react
with the groups of lysine as well as the arginine
epsilon-amino group.
The specific modification of tyrosyl residues Per se
has been studied extensively, with particular interest in
introducing spectral labels into tyrosyl residues by
2~ reaction with aromatic diazonium compounds or
tetranitromethane. Most commonly, N-acetylimidizol and
tetranitromethane are used to form O-acetyl tyrosyl
species and 3-nitro derivatives, respectively.
Carboxyl side groups (aspartyl or glutamyl) are
selectively modified by reaction with carbodiimides (R'-
N-C-N-R') such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)
carbodiimide or 1- ethyl-3-(4-azonia-4,4-dimethylpentyl)
carbodiimide. Furthermore, aspartyl and glutamyl
- 24 -
WOgS/~3291 PcT~S94/12858
CA21 73731
residues are converted to asparaginyl and glutaminyl
residues by reaction with ammonium ions.
Glutaminyl and asparaginyl residues are frequently
deamidated to the corresponding glutamyl and aspartyl
residues. Alternatively, these residues are deamidated
under mildly acidic conditions. Either form of these
residues falls within the scope of this invention.
Derivatization with bifunctional agents is useful
for cross-linking the peptide to a water-insoluble
support matrix or to other macromolecular carriers.
Commonly used cross-linking agents include, e.g., 1,1-
bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-
hydroxysuccinimide esters, for example, esters with 4-
azidosalicylic acid, homobifunctional imidoesters, in-
cluding disuccinimidyl esters such as 3,3'-
dithiobis(succinimidyl-propionate), and bifunctional
maleimides such as bis-N-maleimido-1,8-octane.
Derivatizing agents such as methyl-3-[(p-
azidophenyl)dithio]propioimidate yield photoactivatable
intermediates that are capable of forming crosslinks in
the presence of light. Alternatively, reactive water-
insoluble matrices such as cyanogen bromide-activated
carbohydrates and the reactive substrates described in
U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128;
4,247,642; 4,229,537; and 4,330,440 are employed for
protein immobilization.
Other modifications include hydroxylation of proline
and lysine, phosphorylation of hydroxyl groups of seryl
or threonyl residues, methylation of the alpha-amino
groups of lysine, arginine, and histidine side ChA; nR
(Creighton, 5upra) ~ acetylation of the N-terminal amine,
and, in some instances, amidation of the C rermina
carboxyl groups.
- 25 -
-
wog5/~32s1 PCT~S94/12858
CA21 73731
Such derivatized moieties may improve the
solubility, absorption, biological half life, and the
like. The moieties may alternatively eliminate or
attenuate any undesirable side effect of the protein and
the like. Moieties capable of mediating such effects are
disclosed, for example, in Remington~s Pharmaceutical
Sciences, 16th ed., Mack Publishing Co., Easton, PA
(1980)
PRODUCTION OF CHICKEN NG-CAM AND HUMAN Ll FUSION
PROTEINS THAT PROMOTE NEURITE GROWTH
Fusion proteins representing different polypeptide
regions in Ng-CAM or Ll are used to identify regions of
Ng-CAM and human Ll that have the desired functional
activity (binding, stimulating neurite growth, etc.).
When combined with the PCR, it is thus possible and
expedient to express in bacteria nearly any selected
region of the protein.
To facilitate unidirectional subcloning of the PCR
products, sense and antisense oligonucleotides have been
designed to include BamHl recognition sequences at the 5'
end and EcoRl recognition sequences at the 3' end,
respectively; appropriately digested PCR products are
then be ligated directly into a vector (such as the
pGEX-2T vector).
Use of this methodology allows construction of
vectors and purification of several fusion proteins in
less than one month.
The pGEX vector is preferred chosen because the
glutathione-S-transferase (GST) fusion proteins can be
purified rapidly by binding to glutathione-agarose beads.
In addition, because cDN~s are cloned into pGEX-2T, the
portion of the fusion protein representing the GST can be
cleaved with thrombin and the engineered polypeptide can
- 26 -
WO95/l3291 PCT~Ss4/l2858
~A2~ 73731
generally be recovered free of the GST protein which can
be removed using glutathione-agarose beads (Ausubel,
F.M., et al., l990, CURRENT PROTOCOLS IN ~OLECULAR
BIOLOGY, John Wiley ~ Sons, New York.
Ng-CAM or fusion proteins thereof may also be
expressed in insect cells using baculovirus expression
system. Production of Ng-CAM or functional derivatives
thereof, including fusion proteins, in insects can be
achieved, for example, by infecting the insect host with
a baculovirus engineered to express Ng-CAM by methods
known to those of skill. Thus, in one embodiment,
sequences encoding Ng-CAM may be operably linked to the
regulatory regions of the viral polyhedrin protein
(Jasny, 1987, Science 238 :1653). Infected with the
recombinant baculovirus, cultured insect cells, or the
live insects themselves, can produce the Ng-CAM or
functional derivative protein in amounts as great as 20
to 50~ of total protein production. When live insects
are to be used, caterpillars are presently preferred
hosts for large scale production according to the
invention.
Fragments of Ng-CAM are purified by conventional
affinity chromatography using monoclonal antibodies that
recognize the appropriate regions of Ng-CAM.
2~ Several constructs have been purified from existing
cDNA clones (Burgoon et al., supra) that represent
different portions of the Ng-CAM ectodomain. The
purified fusion proteins have been tested for binding to
Ng-CAM, and for their ability to promote growth of
neurites in culture. The results suggest that regions in
the first two or three Ig-like domain mediate homophilic
Ng-CAM binding and can promote neurite growth (Figure
10) .
- 27 -
WO95/13291 PCT~S94/12858
CA21 73731
In addition, other regions such as in the fifth and
sixth Ig-like domains do not bind to Ng-CAM but
nevertheless promote neurite growth. These regions may
be involved in Ng-CAM binding to neurocan. The combined
results suggest that different Ig-like domains in Ng-CAM
may have different functions that are mediated by binding
to different ligands.
Given the se~uence similarities in the Ig-like
domains between chicken Ng-CAM and human Ll (about 55~
identity and ~65~ similarity when conservative amino acid
substitutions are considered), it is likely that
comparable regions in the molecules serve similar
functions. To determine which Ig-like domains in Ll
mediate neuronal binding and promote neurite growth,
constructs representing different domains are prepared in
pGEX. The choice of the regions to be expressed is
initially guided by the functional properties of the
existing Ng-CAM fusion proteins, where, for example, the
first three Ig-like domains have been found to promote
neurite growth. The fusion proteins will be expressed
and characterized using established assays described
herein, with neurons and Ll protein obtained from rats.
Inasmuch as rat Ll is ~92~ identical in amino acid
sequence to human Ll, it is a suitable model for the
h~ n protein has not yet been isolated in sufficient
quantitie8 for these studies.
The mAbs specific for the most highly conserved
regions in Ng-CAM can be used to purify Ll protein from
rat brain; studies confirmed that rat Ll so isolated
binds to Ng-CAM.
- 28 -
W095/1329l PCT~S94/l2858
~A21 73731
ASSAYS FOR PROTEINS AND PEPTIDES HAVING Ng-CAM-LIKE
ACTIVITY
To characterize functions of dif~erent regions in
Ng-CAM and Ll, as well as peptides derived therefrom and
other functional derivatives thereof, different assays
for molecular binding, cell adhesion and neurite growth
are used. These assays may be used routinely to analyze
the biological functions of derivatives of Ng-CAM, such
as peptide fragments. The combined use of these assays
allows analysis of molecular mechanisms of binding as
well as neurite growth.
CovasPhere Assays:
Ng-CAM or fusion proteins thereof are covalently
coupled to fluorescent beads and tested (1) for self-
aggregation (measured using a Coulter Counter), and(2) for coaggregation with differently colored
Covaspheres (observed by fluorescence microscopy and
measured using a fluorescence activated cell sorter)
and/or (3) for binding to cells expressing ligands for
Ng-CAM ((Grumet et al ., 1988 , supra; Grumet et al .,
1993, supra; Kuhn, T.B. et al., J. Cell Biol. 115:1113-
1126).
Radioligand Bindinq Assays
Proteins or peptides are labeled with I~I and tested
2~ for binding to unlabeled proteins adsorbed to microwells
of microplates (such as Immulon plate, Dynatech Labs).
Such assays indicated that both I~I-labeled Ng-CAM and
l~I-labeled neurocan bound to Ng-CAM with high affinity.
Furthermore, cells may be transfected with DNA encoding a
portion of the Ng-CAM molecule, representing about 60~ of
the extracellular region; such cells expressing this
portion of Ng-CAM also bind ~ proteoglycan.
GravitY Cell Adhesion Assa~
- 29 -
WO95/13291 PCT~S91/12858
3 ~ ~ 1
Proteins are adsorbed to 35 mm polystyrene petri
dishes in 1 ~l drops by incubation in a humid chamber;
the unadsorbed proteins are removed and the rem~; n; ng
surface of the dishes is blocked with bovine serum
albumin (BSA). Cells numbering from about 2 - 5 x 106 are
incubated on the dishes for 1 h, the dishes are washed to
remove unbound cells, and the binding of cells to
different proteins is visualized by phase contrast
microscopy. The number of cells bound is measured as
described by Friedlander, D.R. et al. (1988), ~. Cell
Biol. 107:2329-2340; Grumet et al., 1993, supra.
Neurite Growth Assay
Petri dishes are coated with proteins as described
above for the gravity cell adhesion assay. Dissociated
primary neurons from brain are prepared for culture by
light trypsinization with EDTA as described by Grumet et
al., 1988, supra, and cultured in defined medium (DMEM
plus ITS+).
Specific parameters of neurite growth which are
recorded include the percentage of neurons with neurites,
mean number of neurites per cell, and mean neurite length
(Hoffman, S. et al., 1986, J. Cell Biol . 103:145-158;
Rogers, S.L. et al., 1983, Dev. Biol. 98:212-220). As an
estimate of neurite length, the distance between the cell
body and the tip of the most distant neurite, the
"neurite reach", is determined with an eyepiece
micrometer.
cDNAs encoding human L1 (Reid et al., supra) were
obta;ne~ from Dr. John-Hemperly. Both chicken and
m~mm~lian cells are tested for binding. Previous studies
indicated that chicken Ng-CAM could bind to rodent L1.
The assays described above are used to functionally
characterize engineered proteins representing regions of
- 30 -
WO95/13291 PcT~ss4/l2858
~ 7:~ ~3 ~
Ll. If, as expected, regions homologous to domains of
Ng-CAM have similar activities, an analysis of their
conserved sequences may provide additional clues for
selecting particular amino acid sequences that are
critical for binding in Ng-CAM and L1.
RELATIONSHIP BETWEEN BINDING DOMAINS IN AVIAN NG-CAM
AND MAMMALIAN Ll: IDENTIFICATION OF ACTIVE PEPTIDES
The mutual binding of avian Ng-CAM and m~mm~lian L1
indicate that the amino acid sequences involved are
conserved. Comparisons between amino acid sequences of
Ng-CAM with Ll have revealed certain highly conserved
regions. For example, although the overall amino acid
identity between Ng-CAM and L1 in the first three Ig-like
domains is only about 55~ (somewhat higher if one allows
for conservative substitutions), there are blocks of
invariant sequence as opposed to those that are highly
variable.
Using the assays described above, individual Ig-like
~om~;nR and other peptide sequences will be ~x~m-ned for
Ng-CAM-like function, such as induction o~ aR neurite
growth. This will define the amino acid sequences that
mediate specific functions and will allow selection of
peptides for therapeutic uses.
Thus, peptides of about 10-20 residues are
synthesized and tested directly for:
(a) binding to L1;
(b) binding to neurocan;
(c) activity in promoting neuronal adhesion;
(d) activity in promoting neurite growth.
- 31 -
W O 95/13291 PCT~US94/12858
C~21 ~3~
HETEROPHILIC BINDING OF Ng-CAM/L1:
INTERACTIONS WITH BRAIN PRQTEOGLYCANS
In addition to homophilic binding, recent studies
indicate that several molecules including neurocan and
3F8 proteoglycan may be heterophilic ligands for
Ng-CAM/L1. Both neurocan, a chondroitin sulfate
proteoglycan that is secreted by neurons, and 3F8
proteoglycan that is associated with astroglial cells,
bind to Ng-CAM and inhibit homophilic Ng-CAM binding as
well as neuronal adhesion and neurite growth to Ng-CAM in
culture.
Rat neurocan and a second chondroitin sulfate
proteoglycan, 3F8 (Rauch, U. et al., 1991, supra), were
obtained from Dr. Richard Margolis, New York University.
Ng-CAM/L1 binding sites for these proteoglycans will
be identified for the preparation of peptides useful as
potent neutralizers of inhibition of neuronal adhesion to
Ng-CAM. Recent experiments by the present inventor and
his colleagues have shown that I~I-labeled neurocan and
3F8 proteoglycan bound to rat L1 and not to other
adhesion proteins such as fibronectin and myelin
associated glycoprotein. Both proteoglycans bound to
Ng-CAM with high affinity (~=1.3 x 10-9 for neurocan, and
~=10-1 for 3F8 proteoglycan). The proteoglycans also
bound to cells transfected to express about 60~ of the
extracellular portion of Ng-CAM.
The proteoglycans may act simply by blocking
homophilic binding sites on cell adhesion molecules such
as Ng-CAM. Alternatively, they may provide "inhibitory"
signals to cells by binding to cell surface receptors.
Both possibilities are of potential importance to neural
regeneration because Ng-CAM polypeptides may directly
promote neurite growth while at the same time they also
WOg5/l3291 PcT~ss4/12858
CA~ 7 7~73 ~
may neutralize "inhibitory" signals that may be generated
by certain proteoglycans.
The radioligand and Covasphere assays described
above will be used to test the binding of neurocan and
3F8 to fusion proteins, peptides and functional
derivatives thereof, representing different regions of
Ng-CAM/Ll. Neurocan inhibits homophilic binding between
purified Ng-CAM on Covasphere beads, suggesting that
neurocan binds at or near the homophilic binding site, or
alters the conformation of Ng-CAM by binding to a
different site. The use of radioligand binding assays
will indicate which domains in Ng-CAM/Ll are involved in
homophilic binding and heterophilic binding to neurocan
and 3F8 proteoglycans.
Peptides of varying lengths derived from Ng-CAM/Ll
that bind to the proteoglycans with high affinity are
useful for neutralizing the inhibitory effects of the
proteoglycans on neuronal adhesion and neurite growth.
Such peptides may be expressed in bacteria, or, more
preferably, by a baculovirus expression system in insect
cells. Such peptides are tested for their ability to
bind to proteoglycans; peptides that can neutralize the
inhibitory effects of the proteoglycans on neurite growth
are useful as therapeutic agents.
THERAPEUTIC APPLICATIONS OF Nq-CAM/Ll
The present invention provides for methods of
treatment of a neuronal disorder, preferably a motor
neuron disorder, in particular peripheral nerve damage or
transection, which methods comprise administering to a
subject in need of such treatmen~ an effective amount of
Ng-CAM/Ll or a functional derivative thereof, that
- 33 -
W095/1329l PCT~S94/128~8
CA21 73731 ~
supports the survival, growth of the neurons and
regeneration of the damaged nerve.
The disorders that may be treated according to this
invention include, but are not limited to, disorders such
as physical or surgical trauma, infarction, infection,
toxin exposure, degenerative disease or malignant disease
that affects peripheral or central neurons as well as any
other components of the nervous system.
Effective doses of Ng-CAM/Ll/NILE for therapeutic
uses discussed above may be determined using methods
known to one skilled in the art. Effective doses may be
determined, preferably in vitro, in order to identify the
optimal dose range using various of the methods described
herein. In a preferred embodiment, an aqueous solution
of a Ng-CAM/Ll/NILE protein or peptide is administered by
subcutaneous injection. Each dose may range from about
0.1 ~g to about 100 ~g/kg body weight, or more
preferably, from about 1 ~g to 50 ~g/kg body weight.
The dosing schedule may vary from once a week to daily
depending on a number of clinical factors, including the
type of disease, severity of disease, and the subject's
sensitivity to the protein. Nonlimiting examples of
dosing schedules are 3 ~g/kg ~m; n; stered twice a week,
three times a week or daily; a dose of 7 ~g/kg twice a
week, three times a week or daily; a dose of 10 ~g/kg
twice a week, three times a week or daily; or a dose of
30 ~g/kg twice a week, three times a week or daily. In
the case of more severe disease, it may be preferable to
administer doses such as those described above by
alternate routes, including intravenously or
intrathecally. Continuous infusion may also be
appropriate.
- 34 -
Wo95~13291 PCT~S94/12858
C A 2 1 73 73 1
Ng-CAM/Ll/NILE may also be administered via a
cellular, solid or semi-solid implant to achieve blood
levels of the protein or peptide similar to those
attained by subcutaneous ~mi n i stration. A cellular
implant may comprise cells naturally producing, or
genetically altered to produce Ng-CAM/Ll/NILE which
secrete the protein or peptide in vivo in a subject
following inoculation.
Ng-CAM/Ll/NILE or a functional derivative may also
be administered in combination with an effective amount
of at least one other agent that is, itself, capable of
promoting neuron survival, growth, or regeneration.
The Ng-CAM/Ll/NILE may be administered in any
pharmaceutically acceptable carrier. The administration
route may be any mode of administration known in the art,
including but not limited to intravenously,
intrathecally, subcutaneously, or intracranially by
injection into involved tissue, intraarterially, orally,
or via an implanted device. Preferably, the Ng-CAM/Ll is
added in combination with a nerve entubulation device or
a gel, such as a collagen gel, to promote nerve regrowth.
The entubulation techniques, described above, for
promoting nerve regeneration, can be combined with the
use of proteins and peptides as described herein to
promote nerve regeneration. Given their ability to
promote neurite growth by various mechanisms, Ng-
CAM/hl/NILE and functional derivatives thereof, are
particularly useful for nerve regeneration when
incorporated in synthetic entubulation devices.
For use in entubulation devices, Ng-CAM/Ll/NILE or a
functional derivative thereof in a dose ranging from
about O.Ol ~g/ml to about 2 mg/ml, more preferably, from
about l ~g/ml to lO0 ~g/ml.
- 35 -
WO 95/13291 PCTIUS94/12858
C A2 1 7 3731 ~
In addition to the utility of Ng-CAM/L1 in the
promotion of peripheral nerve regeneration, its mode of
action indicates additional utilities. Ng-CAM/L1/NILE
can act as a substrate for neurite growth, in the
generation growth promoting signals in neurons, and as an
agent capable neutralizing inhibitory effects of brain
proteoglycans.
Thus, Ng-CAM/L1/NILE or functional derivatives
thereof may also be used to treat other neural disorders
which would benefit from increasing neurite sprouting and
growth. In this regard, previous studies have shown that
Ng-CAM can promote the formation and growth of complex
growth cones which are a key hallmark of development and
a vital aspect of regeneration. Introduction of
polypeptides or peptides from Ng-CAM/L1 that promote
growth may facilitate nerve regrowth in injuries to other
neural regions such as the spinal cord and the brain
where the potential for surgery is even more limited than
in the peripheral nervous system.
Although direct introduction of polypeptides into
the CNS is difficult and is limited by the blood brain
barrier, penetration from the circulation does occur at
sites of injury where the barrier is broken. Major
efforts are currently under way to develop technologies
to deliver proteins and polypeptides into the CNS. The
present inventor's laboratory has found that Ng-CAM
promotes outgrowth from explants of vertebrate nervous
tissues, including brain, spinal cord and dorsal root
ganglia. Thus, the presen~ invention also includes the
use of Ng-CAM or a functional derivative thereof to
promote the growth and innervation by implanted neural
tissues in a m~mm~l .
- 36 -
wo 9j,~3291 C A 2 1 7 3 7 3 1 PcT/rJsg4/l2858
In another embodiment, the Ng-CAM/L1/NILE or
functional derivative can be impregnated into an
implantable delivery device such as a cellulose bridge or
sling prosthesis. Preferably, such a device is covered
with glia, as described by Silver, J. et al., 1983,
Science 220:1067-1069 (1983), which reference is hereby
incorporated by reference in its entirety. Thus, such a
form of neuronal or axonal engineering can be used to
rebuild a major nerve tract.
The present invention also provides pharmaceutical
compositions comprising an amount of Ng-CAM/~l or a
functional derivative thereof effective to promote neuron
growth or nerve regeneration, and effective to treat a
disease associated with nerve damage or dysfunction, in a
pharmaceutically acceptable carrier. Also provided is a
pharmaceutical composition comprising an effective amount
of Ng-CAM/~1 together with one or more additional
neurotrophic factors in a pharmaceutically acceptable
carrier.
Having now generally described the invention, the
same will be more readily understood through reference to
the following examples which are provided by way of
illustration, and are not intended to be limiting of the
present invention, unless specified.
Wo95/13291 PCT~s9~/12858
CA21 73731 ~
EXAMPLE I
MATERIALS AND METHODS
Proteins and Antibodies
Ng-CAM and N-CAM were purified from 14-d embryonic
chicken brains by immunoaffinity chromatography using
mAbs lOF6 and 3G2 that specifically recognize Ng-CAM, and
mAb anti-N-CAM No. 1, respectively. NILE/Ll was purified
from 7-day postnatal rat brain using a combination of two
other anti-Ng-CAM mAbs, 2C2 and l9H3, that recognize the
cytoplasmic region of Ng-CAM, which is highly conserved
between chicken Ng-CAM, mouse Ll, and rat NILE. The
protein purified from detergent extracts of rat brain
membranes contains on SDS/PAGE two major components at Mr
of 200 kDa and 140 kDa and small amounts of a component
at 80 kDa. Polyclonal antibodies against human Ll
(kindly provided by Dr. John Hemperly) recognized the 200
kDa and 140 kDa species on ;mml~noblots, confirming that
it is NILE/Ll.
The 5B8 mAb (obtained from the Developmental Studies
Hybridoma Bank) was used to purify N-CAM from 7-day
postnatal rat brain; this antibody recognizes
cytoplasmic regions of N-CAM. When the rat N-CAM was
resolved on SDS/PAGE and stained with Coomassie Blue, the
characteristic heterodisperse pattern of polysialylated
N-CAM was observed.
Neurocan was isolated and analyzed as described
previously (Rauch et al. ~ supra; Grumet et al~ ~ 1993,
5upra). Brains of 7-day or 2- to 3-month-old Sprague-
Dawley rats were extracted with PBS, and proteoglycans
were purified by ion e~ch~nge chromatography and gel
filtration (Kiang et al., 1981). Neurocan was purified
by ;mmllnoaffinity chromatography using the lDl mAb (Rauch
- 38 -
WO 951~3291 PCT/US94/12858
CA21 73731
et al., supra). Rat chondrosarcoma chondroitin sulfate
proteoglycan (aggrecan) was isolated by CsCl density
gradient centrifugation. For studies of the core
proteins, proteoglycans were digested for 45-60 min at
37C with protease-free chondroitinase ABC (Seikagaku
America Inc., Rockville, MD) in 100 mM Tris-HCl buffer
(pH 8.0 at 37C) containing 30 mM sodium acetate, and
completeness of digestion was confirmed by SDS-PAGE.
Myelin associated glycoprotein (a recombinant form
including the ectodomain (Pedraza, L. et al ., 1990, J.
Cell Biol . lll: 2651-2661) and epidermal growth factor
receptor were kind gifts from Drs. J.L Salzer and J.
Schlessinger, respectively.
Commercial reagents included l~m; n; n, type I and IV
collagens (Collaborative Research), fibronectin (New York
Blood Bank, NY), and bovine serum albumin (BSA) (ICN
Biomedical, Lisle, IL). Sturgeon notochord chondroitin
sulfate, consisting of 80~ chondroitin 4-sulfate and 20
chondroitin 6-sulfate, was obtained from Seikagaku
America Inc.
MAbs against chicken Ng-CAM were prepared as
previously described (Grumet M et al., 1984, ~. Cell
Biol. 98:1746-1756). A mAb specific for the lD1
proteoglycan has been described previously (Rauch et al.,
supra). The Ig was precipitated from ascites fluid with
~mmo~;um sulfate and further purified on DE-52 columns.
Radioliqand Bindinq Assay
Proteoglycans were labeled to a specific activity of
2.~-6 x 10l8 cpm/mole with I~I by the
lactoperoxidase/glucose oxidase method using Enzymobeads
(Biorad). Typically, 50 ~g in of protein were labeled
per reaction. Free iodine was removed by gel filtration
with a PD-10 column (Pharmacia, Piscataway, NJ). Binding
WO 9S/13291 PCTIUS9~/128S8
CA21 73731
assays were performed essentially as described by Zisch,
A.H. et al., 1992, ,J. Cell Biol. 119:203-213). One to 30
~g of soluble proteins in binding buffer (16 mM Tris, pH
7.2; 50 mM NaCl; 2 mM CaCl2; 2 mM MgCl2; 0.02 NaN3) were
adsorbed to removable Immulon-2 wells (Dynatech,
Chantilly, VA) by overnight incubation at room
temperature. Unbound proteins were removed with three
washes in binding buffer containing 0.02~ Tween- 20, and
the wells were blocked by incubation with heat treated
BSA/PBB, 1 mg/ml. Wells were then emptied and 50 ~l/well
of labeled proteins or mixtures of labeled and unlabeled
proteins in PBB, 1 mg/ml, were incubated for 2 h at room
temperature. Unbound proteoglycan was removed by four
washes with TBS (50 mM Tris pH 7.2; 150 mM NaCl; 0.02~
Tween-20). Radioactivity bound to wells was determined
with a gamma counter. Scatchard plots were generated and
the Kd was determined using the MacIntosh version of the
Ligand program (Munson et al ., 1980, Anal. Biochem.
107: 220-239).
Cells
Dissociated cells were prepared essentially as
described before (Brackenbury, R. et al., Proc. Natl.
Acad. Sci. USA 78:387-391). In brief, 9-d chick embryo
brains were treated with trypsin/EDTA (GIBCO, Grand
Island, NY) followed by trituration in DME (GIBCO)
containing 10 ~ fetal calf serum and S0 ,ug/ml DNase I
(Worthington, Freehold, NJ). The cells were washed twice
with ITS+ (Collaborative Research, Bedford, M~)/DME and
once by centrifugation through a 3.5~ BSA/PBS step
gradient.
Sllhstrates
Substrates for cell adhesion and neurite growth
assays consisted of a circular array (~1 cm diameter) of
-- 40 --
wossll329l PcT~s94/12858
~ ~,A21 73731
8 to 12 small circular regions that were coated with
adsorbed proteins. Coated regions were prepared by
incubating l to 3.5 ~l droplets of protein solutions in a
humidified chamber for 30 min (Friedlander et al ., 1988,
supra). After removing the droplets by suction, the
dishes were washed 3 times with PBS and blocked with l~
BSA. Coating solutions included both single proteins and
mixtures of proteins. For double coats, blocking
solution was applied only once, following the second
coating. In selected experiments, one of the protein
solutions included 1-5 ~g/ml rhodamine-labeled BSA as a
marker to identify coated regions, which helped in
determining the location of boundaries between different
substrata.
For quantitative determination of protein binding to
plastic, radiolabeled proteins were used to coat dishes
following the same procedures used for the cellular
assays. After the final wash, the dishes were dried,
their walls removed with pliers, and the bottom of the
dishes were exposed to a PhosphorImager screen (Molecular
Dynamics, Sunnyvale, CA) to determine the relative
amounts of radioactivity in the central region of each
spot by using interactive software (ImageQuant, Molecular
Dynamics). Absolute values of bound protein were
obtained by comparing the relative values with the total
radioactivity adsorbed to a similar set of spots that
were dried completely without prior washing. For
measuring total radioactivity, this method yielded
results similar to those obtained with a gamma counter.
For measuring surface density of adsorbed proteins,
however, the PhosphorImager method is more reliable
because it avoids uncertainties that may be caused by
differences in spot size and boundary effects.
WO95/13291 pcT~ss~ll28s8
.
~21 73731
Cell adhesion assays
250 ~1 of DME/ITS+ containing 2 to 6 x 105 cells were
deposited in the central region of 35 mm polystyrene
dishes that had been coated with proteins. Following
incubation for 80 min at 37C, unattached cells were
removed by washing with PBS and the remaining cells were
fixed with 3.5~ formalin. Attached cells were counted
under a microscope at 200X magnification.
Neuri~e arQwth
105 brain cells were incubated for 2 days under the
same conditions used for cell adhesion assays and were
fixed with formalin. Neurite length was defined as the
distance between the furthest removed neurite tip and the
cell body. Quantitation was done under phase contrast
microscopy with the help of a measuring eyepiece.
Immunocytochemistry
The ;mmunocytochemical localization of neurocan,
NILE/L1, and N-CAM was performed on sagittal Vibratome
sections of 7-d rat cerebellum using the lD1, 2C2, and
5B8 m~bs~ respectively. Rats were perfusion-fixed with
picric acid-paraformaldehyde-glutaraldehyde, and sections
were stained with peroxidase-conjugated second antibody
as described previously (Rauch et al., supra).
~nalytical Methods
Proteins were resolved on SDS/PAGE and were either
transferred to nitrocellulose and ;mmunohlotted with
antibodies (Grumet et al ., 1984, supra; Towbin et al .,
1979, Proc. Natl. Acad. Sci. USA 76:4350-4354) or stained
with Coomassie Blue. Radiolabeled proteins were detected
by autoradiography, and protein concentrations were
determined using either the Lowry (for neurocan) or the
Bradford protein assays (Bio-Rad Laboratories, Ri~hmo~,
- 42 -
WO 95/13291 PCT/US94112858
~ CA2 1 73731
CA); concentration of aggrecan was determined
gravitometrically.
EXAMPLE II
BINDING OF NEUROCAN TO Na-CAM AN3 N-CAM
Binding of neurocan to various cell surface proteins
including neural CAMs and ECM proteins was determined
using a radioligand binding assay (Figure 1). Both rat
neurocan and neurocan-C bound to chicken Ng-CAM and to
its presumed rat homologue NILE/Ll (Grumet, 1992, supra;
Sonderegger and Rathjen, 1992, ~. Cell Biol. 119:1387-
1394). Neurocan also bound to N-CAMs from chicken and
rat. These results suggest that the proteoglycan binding
domains in Ng-CAM and N-CAM have been conserved during
the evolution of avian and m~mm~l ian species. Neurocan
bound to a lesser extent to collagen I but not to myelin-
associated glycoprotein (MAG), collagen IV, or EGF-
receptor.
Under our standard assay conditions (wells coated
with proteins at a concentration of 1.25 ~g/ml) neurocan
did not bind to lAm;n;n but some binding (9~ of total
counts) was detected when a 10 ~g/ml o~ l~m; n; n was used.
The fraction of neurocan that bound specifically to Ng-
CAM was consistently ~20-25~, with a signal to background
ratio as high as 65:1. The percent of neurocan bound to
N-CAM was usually lower and varied to a greater extent in
different experiments ranging from -8-15~ bound with a
signal to background ratio of 18-20:1. By comparison,
~ labeled aggrecan, a chon~roitin sulfate proteoglycan
from chondrosarcoma, bound very weakly to Ng-CAM and N-
CAM (Figure 1), suggesting that the interactions between
neurocan and these neural CAMs are related to structural
domains in neurocan.
- 43 -
WO95/13291 PcT~S94/12858
~21 73731 ~
The radioligand binding assays described above were
performed in hypotonic buffer containing 50mM NaCl, which
yielded higher signal to background ratios a buffer with
150 mM NaCl. At physiological salt concentration,
binding of neurocan to Ng-CAM and N-CAM was ~;m;n;shed by
35-60~, and binding to collagen I was reduced to baseline
levels (Figure 2). These results suggest that, in vivo,
neurocan would not bind to collagen I but it could bind
to the neural CAMs. Because only limited amounts of
proteoglycans were isolated from rat brain, it was
decided to perform saturation and inhibition experiments
(see below) using the hypotonic conditions.
To analyze further the binding of neurocan to Ng-CAM
and N-CAM, saturation experiments were performed with
increasing amounts of labeled proteoglycans. The binding
of 7-d neurocan and neurocan-C to both Ng-CAM (Figure 3)
and N-CAM (Figure 4) was saturable, indicating the
presence of a limited number of neurocan binding sites on
Ng-CAM and N-CAM. Scatchard analysis of these data
yielded linear plots for the binding of both forms of
neurocan to Ng-CAM and N-CAM, indicating a single class
of binding sites for 7-d neurocan and neurocan-C. In all
four cases, the dissociation constants derived from the
plots were quite similar (ranging from 0.21 to 0.42 nM)
indicating high affinity b;n~;ng that is comparable to
specific receptor binding.
The specificity of binding of neurocan to these
neural CAMs was also investigated in competition
experiments (Figure 5).. Substrate-bound CAMs and labeled
ligands were first incubated with increasing
concentrations of unlabeled competitors including
neurocan, aggrecan, chondroitin sulfate, chondroitin
sulfate disaccharides (resulting from chondroitinase
- 44 -
WO95/13291 PCT~S94/12858
~ CA21 7373~
treatment of the proteoglycans) and fibronectin, and then
binding of ~ neurocan was measured. In these
competition experiments, binding of 12sI-neurocan-C to Ng-
CAM was inhibited by soluble neurocan-C in a
concentration-dependent manner. When the labeled
neurocan was added at a concentration of O.l ~g/ml (0.7
nm), 50~ inhibition of binding was obtained with
unlabeled neurocan-C at a concentration between O.l and
0.2 ~g/ml (0.7 - l.4 nM). Half-maximal inhibition was
therefore obtained with a 2:l-l:l molar ratio of
unlabe~ed to labeled ligand, in general agreement with
the ~ obtained from the Scatchard analysis.
Significant concentration-dependent inhibition of
neurocan binding to Ng-CAM and N-CAM was also produced by
free chondroitin sulfate chains (Figure 5). In contrast,
sulfated disaccharides were not effective, implying that
intact chondroitin sulfate chA;n.~ are required to inhibit
proteoglycan binding to CAMs. In the same assay,
fibronectin was ineffective in competing for binding of
neurocan. Using the same set of soluble proteins as
competitors, similar patterns of inhibition were observed
for binding of neurocan-C to N-CAM and for binding of 7-d
neurocan to Ng-CAM and to N-CAM. The results suggest
that chon~roitin sulfate, which constitutes ~20~ by
weight of neurocan, is important in the binding of
neurocan to the neural CAMs. By comparison, equivalent
amounts of chon~roitin sulfate in the form of free ChA; n.
were less effective competitors of binding, suggesting
that structural features in neurocan in addition to
chondroitin sulfate itself are involved in the binding of
neurocan to neural CAMs. The combined results
suggest that chondroitin sulfate ~hAin~ may bind weakly
to Ng-CAM and N-CAM, and may increase the binding
- 45 -
WO95/t3291 PCT~S9~/l2~58
CA21 73731
affinity for CAMs when present on neurocan, but are not
sufficient by themselves to support high affinity
binding.
To analyze more directly the role of chondroitin
sulfate ch~; nR in interactions of neurocan with neural
CAMs, the binding of native and chondroitinase-treated
neurocan to Ng-CAM and N-CAM were compared (Figure 6).
Chondroitinase treatment reduced binding of 7-d neurocan
and neurocan-C to Ng-CAM by ~70~, and to N-CAM by ~80~.
Heat treated proteoglycans retained the ability to bind
to the CAMs at levels that were somewhat lower than those
of controls. However, chondroitinase treatment followed
by heat treatment further reduced the levels of binding
nearly to background. These results are consistent with
idea that chondroitin sulfate ch~;ns on neurocan are
involved in binding to Ng-CAM and N-CAM. Nevertheless,
the data also indicate that the core protein of neurocan
retains specific binding for Ng-CAM and N-CAM even in the
absence of cho~roitin sulfate ch~in.~ which allow for
higher levels of binding.
EXAMPLE III
COLOCALIZATION OF NEUROCAN, Ng-CAM, AND N-CAM
IN DEVELOPING BRAIN
The biological significance of interactions between
neurocan and neuronal CAMs would be supported by finding
these molecules appearing in at least some of the same
locations at certain times during development. The
ability to isolate by immunoaffinity chromatography
significant amounts of neurocan (using mAb lDl) as well
as NILE/Ll (using anti-Ng-CAM mAbs 2C2 and l9H3) and N-
CAM (using mAb 5B8) from 7-d postnatal rat brain is
evidence that all these proteins are present at this
- 46 -
WO95/13291 PcT~ss4/12858
~ CQ21 73731
stage of development (see Materials and Methods). To
determine in more detail the histological localization of
these proteins, specific mAbs were used for
immunoperoxidase staining of sections of early postnatal
rat cerebellum. The staining with mAbs against these
three molecules was similar insofar as it was strongest
in the molecular layer and in the deeper layers of the
cerebellum including the fiber tracts (Figure 7). This
general staining pattern was also observed in 4-d and l0-
d postnatal brain, and is characteristic of severalneural cell adhesion molecules, but differs considerably
from the patterns obtained using mAbs against unrelated
proteins such as glial fibrillary acidic protein or
calbindin.
A more detailed analysis of the staining patterns
with the antibodies against neurocan, Ng-CAM and N-CAM
revealed subtle differences between them. No staining
was detected in the external granule cell layer except
for a low level of reactivity with anti-N-CAM. Whereas
all three antibodies stained long processes in the fiber
tracts, antibodies against neurocan also stained strongly
in a region surrounding the fiber tracts. In addition,
antibodies against neurocan stained weakly in the
Purkinje cell layer (Figure 7, panel b).
25 ~ St~;n;ng experiments in developing rat spinal cord
and brain also indicated that Ng-CAM and the
proteoglycans nuerocan and 3F8 proteoglycan are co-
expressed during development
EXAMPLE IV
EFFECTS OF NEUROCAN ON NEURONAL ADHESION TO Nq-CAM
Given the results described above indicating that
neurocan binds with high affinity to Ng-CAM and to N-CAM,
- 47 -
-
W095/13291 PcT~S94/12858
CA21 73731
and that these molecules are present in partially
overlapping patterns during development, studies were
performed ex~m; n; ng how neurocan inhibits neuronal
adhesion to Ng-CAM in culture. Previous studies from
the present inventor's laboratory showed that neurons do
not bind to substrates prepared using mixtures containing
equal amounts of neurocan and Ng-CAM (Grumet et
al.,1993a). In those studies, the two proteins were
mixed in solution and then adsorbed simultaneously to the
substrates. In the present studies, two different
proteins were sequentially adsorbed, and the substrate
was then tested for binding of neurons. To help
interpret the results, the amounts of protein adsorbed to
the substrate for the various combinations of protein
concentrations and coating orders were determined (see
Materials and Methods, above). The results are shown in
Table I, below.
When individual proteins were adsorbed to the
substrates, neurons bound strongly to Ng-CAM, weakly to
N-CAM, negligibly to BSA, and not at all to neurocan;
because of the weak levels of neuronal binding to N-CAM,
it was omitted from the double coating experiments
described below. As expected, neurons bound to
substrates that were coated first (protein 1) with Ng-CAM
and then (protein 2) with BSA (Table I). Neurons also
bound to substrates that were first coated with BSA and
then with Ng-CAM. Even when relatively high
concentrations of BSA were adsorbed first, a second
adsorption with Ng-CAM was able to support binding of
neurons. When substrates were coated first with Ng-CAM
and then with high concentrations of neurocan-C, neuronal
binding was inhibited (Table I). The degree of
inhibition was not closely correlated with the density of
- 48 -
Wo95/13291 PcT~S94/12858
CA21 73731
neurocan that bound to the substrate which did not vary
significantly for coating solutions in the ll - lO0 ~g/ml
range. Rather, it was more closely related to the
concentration of soluble neurocan that was uqed to
produce the second coat.
These observations suggest that the amount of
substrate-bound neurocan per se is not the critical
factor in causing inhibition. In support of this
interpretation, changing the order of the adsorptions, so
that the Ng-CAM was added after neurocan, allowed for
high levels of neuronal binding (Table I). The promotion
of neuronal adhesion by Ng-CAM was observed at all
concentrations tested. Qualitatively similar results
were obtained for both 7-d neurocan and neurocan-C,
however, 7-d neurocan was a slightly more potent
inhibitor.
Because neurocan could produce these effects not
only by blocking sites on substrate-bound Ng-CAM but also
by interacting directly with cell surface ligands
including Ng-CAM, it was important to determine whether
neurocan had direct effects on cells. To explore this
possibility, experiments were done to test the effect of
neurocan on the binding of neurons to substrates coated
with mAbs against Ng-CAM, which promoted neuronal
adhesion (Table II) but did not bind to neurocan.
Substrates first coated with BSA and then with anti-Ng-
CAM Ig were able to support neuronal attachment, and the
degree of attachment increased with the density of the
adsorbed antibody. When substrates were coated first
with anti-Ng-CAM Ig and then with neurocan, neuronal
adhesion was inhibited. The inhibition was most apparent
at lower densities of bound anti-Ng-CAM Ig. In addition,
substrates coated first with neurocan and then with anti-
- 49 -
WO9S/13291 PCT~S94/12858
CA21 73731 ~
Ng-CAM Ig did not support neuronal adhesion even though
Ng-CAM was present at densities that supported
significant levels of neuronal adhesion. For example,
neurons bound to Ng-CAM (0.25 ng/ml) when it was coated
following BSA, but no b;~; ng was detected even when
greater amounts of Ng-CAM (0.42 ng/ml) were obtained on a
first coat of neurocan (Table II). Because neurocan does
not bind to anti-Ng-CAM, these results suggest that the
inhibition was mediated by direct interaction of neurocan
with the cell surface.
- 50 -
W O 95ll3291 PCTrUS94/12858
~ CA21 73731
TABLE I
ADHESION OF CELLS TO SUBSTRATES COATED WITH NBUROCAN AND Nq-CAM
~rotein 1Conc 1 Density 1Protein 2 Conc 2 Den3ity 2 Attached cells
~9~m~ nq/mm2 uq/ml nq/m*cells/mm2
Ng-CAM 50 2.69 BSA 100 ND359 + 6
2.69 33 ND375 + 9
17 0.75 100 ND287 + 74
17 0.75 11 ND410 + 52
BSA 100 ND Ng-CAN 50 2.16268 + 33
33 ND 50 2.54407 i 11
100 ND 17 0.62270 + 44
11 ND 17 0.74439 + 15
Ng-CAM 50 2.69 neurocan-C 100 1.15 28 i 13
2.69 33 1.18163 + 15
17 0.75 100 1.0213 + 17
17 0.75 11 1.15476 + 69
neurocan100 1.11 Ng-CAM 50 1.39351 + 35
33 1.15 50 1.05455 + 26
100 1.11 17 0.31456 + 50
11 0.96 17 0.41533 + 98
Ng-CAM 50 2.69 7d n~-.oc~, 100 1.56 -6 + 3
2.69 33 1.2269 + 2
17 0.75 100 1.31-8 + 2
17 0.75 11 1.00126 + 61
7d - oc~. 100 1.31 Ng-CAM 50 1.31 285 + 26
33 1.20 50 1.26440 i 2
100 1.31 17 0.41 428 i 61
11 1.01 17 0.50520 + 26
Substrates were incubated se~-~nt;~lly with soluble protein 1 and protein 2 at the
indicated c~ncentrations. The resulting surface densities of adsorbed protein
(densities 1 and 2) were determined in parallel experiments using the same
substrate, proteins, cQnr~nt~ations and liquid volume as in the cell adhesion
assays. To det~- 'nf density 1, duplicate samples of different c~n~entrations of
labelled protein 1 (Ng-CAM, neurocan-C and 7-d neurocan) were spotted on the
substrate, and the substrate-bound radioactivity was measured as described in
Materials and Methods (sets of identical values of density 1 appear in the table,
e.g. 2.69 ng/mm2 for Ng-CAM in lines 1, 2, etc., because they were based on the same
experi t~l point. Data not included in the table indicated that the amount ofbound radioactivity of protein 1 was not lowered by applying a second protein coat
with unlabelled protein). To determine density 2, duplicate spots of l~nl ~h~l 1 ed
protein 1 (BSA, Ng-CAM, ne~Lo~ C and 7-d neurocan) were prepared first, then
labelled protein 2 (Ng-CAM, neurocan~C and 7-d neurocan) was added, and the bound
radioactivity was measured. In the parallel cell adhesion assays with ~nl Ah~l led
proteins, ~;~so~i~ted brain cells from 9 d chick embryos were added to substrates,
and the numbers of attached cells after an 80 min incubation period were obtained as
indicated in Materials and Methods. Ni '_L~ of attached cells were obtained by
subtracting the nl '- of cells adhering to the BSA-coated backy u~-d (20 _ 3) from
the total numbers. Data represent averages (n = 2) + mean deviations. ND = not
determined.
WO95/13291 PCTrUS94/12858
~A21 73731 ~
TABLE II
A & esion of Cellæ to Substrates Coated with Neurocan and anti-Nq-CAM
Protein 1 Conc 1 Density 1 Protein 2 Conc 2 Density 2 Attached cells
uq/ml nq/mm2 uq/ml nq/m* cell~/mm2
anti-Ng-CAM 30 1.50 BSA 33 ND 522 ~ 32
0.73 33 ND 273 + 53
BSA 33 ND anti-Ng-CAM 30 0.55 292 + 73
33 ND 10 0.25 208 i 52
anti-Ng-CAM 30 1.50 neurocan 33 0.41 373 i
0.73 33 0.76 -13 i
neurocan 33 1.20 anti-Ng-CAM 30 0.42 -12 + O
33 1.20 10 0.17 -13 + 0
See Table I for methods. Numbers of attached cells were obtained by subtracting the
number of cells adhering to the BSA-coated background (5 i 2) from the total
numbers. Data represent averages (n = 2) + mean deviations, where shown.
ND ~ not determined.
EX~MP~E ~
EFFECTS OF NEUROCAN ON NEURITE OUTGROWTH
A critical aspect of neuronal development is the
growth of processes. Studies were therefore done to
explore the effects of neurocan on neurite growth in
culture using double-coated substrateæ prepared
essentially as described above for the neuronal adhesion
experiments. A major difference in these experiments was
that the non-adherent cells were not ~el--o~d by washing
after 80 min of incubation and many cells eventually
adhered to substrates even in the presence of neurocan.
On substrates coated first with either Ng-CAM or
anti-Ng-CAM Ig and then with BSA, neurons extended
numerous processes (Figure 7, panels a and c,
respectively). When the second coating was performed
using neurocan, neurite extension was dramatically
~;m; n; shed on substrates coated with either Ng-CAM or
WO95/13291 PcT~ss4/12858
CA21 73731
anti-Ng-CAM Ig (Figure 7, panels b and d). In
quantitative experiments, the neurite length histograms
for both Ng-CAM and anti-Ng-CAM substrates (Figure 8)
showed a significant fraction of neurites longer than 20
~m and gradually fewer neurites of increasing length. In
contrast, neurons grown on neurocan plus either Ng-CAM or
anti-Ng-CAM had most neurites in the ~ - 20 ~m range with
very low levels of longer neurites. The average length
of neurites growing on Ng-CAM substrates was l9.3 + l.8
in the absence of neurocan and 6.7 + l.l in the presence
of neurocan. The respective values for anti-Ng-CAM
substrates were very similar, l9.4 i l.7 ~m in the
absence and 6.3 i 0.9 in the presence of neurocan. The
combined results indicate that neurocan is a potent
inhibitor of neurite growth both on proteins to which it
can bind ( e . g., Ng-CAM) and to which it does not bind
( e . g., anti-Ng-CAM Ig).
DISCUSSION OF EXAMP~ES I-V
The major observations of the studies presented
above are that
(a) neurocan binds with high affinity to Ng-CAM and N-
CAM, two of the most prevalent neural CAMs that play
key roles in cell adhesion, neuronal migration, and
axonal growth during development;
(b) these three molecules are coexpressed during
critical stages of cerebellar histogenesis; and
(c) neurocan inhibits neuronal adhesion and neurite
growth.
R; n~; ~ of Neurocan to Neural CAM8
Of the CAMs and ECM molecules tested for binding of
rat neurocan, chicken Ng-CAM exhibited the strongest
- 53 -
WO95/13291 PCT~S94112858
CA21 7373~ ~
binding, followed by rat NILE/Ll which is presumed to be
its m~mm~l ian homologue (Grumet, 1992, supra; Sonderegger
et al., supra). Neurocan also bound strongly to chicken
and rat N-CAM. The Kd of binding to Ng-CAM and N-CAM
obtained by Scatchard analysis was of high af~inity (-0.3
nM), while collagen I and l~- n l n showed much lower
levels of binding. The potential biological importance
of these lower affinity interactions may be more relevant
in the peripheral nervous system, where these ECM
proteins are more abundant than in developing brain.
The observation that the ~ o~ neurocan-C binding to
Ng-CAM and N-CAM is comparable to that obtained using the
full-length proteoglycan, suggests that neurocan-C (which
represents the C-terminal half of neurocan) contains at
least one binding site for these neural CAMs. This
region of neurocan contains a number of motifs that have
been implicated in binding of other proteins including
two EGF-like repeats, a lectin-like domain, and a
complement regulatory protein-like se~uence (Rauch et
al., J. Biol . Chem. 267:19536-19547) . Neurocan-C
contains a single 32 kDa chondroitin 4-sulfate chain that
is linked at serine-944, whereas three additional
potential cho~roitin sulfate attachment sites (only two
of which are utilized) are present in the N-terminal
portion of neurocan. The observation that the b; n~; n~
affinities of neurocan for Ng-CAM and N-CAM are quite
similar, raises the possibility that there may be a
binding site that is shared by these two CAMs which are
comprised of multiple Ig and fibronectin type III domains
as well as immunologically similar N-linked carbohydrates
epitopes (Burgoon et al.m supra; Grumet, M et al ., 1984,
Proc. Natl . Acad. Sci . USA 81:267-271).
- 54 -
WO 95/~3291 PCT/US94/12858
.
~ e A 2 1 73 73 1
Several of the experiments above (Figures 5 and 6)
suggested a role for chondroitin sulfate in the function
of neurocan. Little is known regarding the mechanism of
inhibition by these chondroitin sulfate containing
molecules. However, the present findings suggest that
Ng-CAM and N-CAM may be specific neuronal receptors for
neurocan. In addition, proteoglycans that have
previously been identified in non-neural tissues may also
be found in the brain. For example, aggrecan, which was
first identified in cartilage, was found by the present
inventor and his collaborators to be in rat brain; the
primary structure of its core protein has regions that
are homologous to those in neurocan (Rauch et al., 1992,
supra) .
Glycosaminoglycans usually occur in tissues only in
the form of proteoglycans, although some proteins occur
in both glycanated and non-glycanated forms ("part time"
proteoglycans), e . g ., the amyloid ~ precursor protein,
chromogranin A, invariant chain, lymphocyte homing
receptor (Margolis, R.K. et al., 1993, Experientia
49:429-446; Ruoslahti, 1989, J. Biol. Chem. 264:13369-
13372). While there is no evidence that neurocan occurs
in brain without its chondroitin sulfate rh~; n.~, the
present results indicate that such a molecule would bind
to Ng-CAM and N-CAM.
Colocalization of Neural CAM8 with Neurocan
The ;mmllnolocalization experiments demonstrate that
neurocan, Ng-CAM/L1/NILE, and N-CAM are prevalent during
brain development and colocalize extensively at least in
the cerebellum. Inasmuch as the anti-N-CAM mAb used
above recognizes the cytoplasmic region of N-CAM, it
revealed only the larger N-CAM species that parallels the
expression pattern of Ng-CAM/Ll/NILE in developing
- 55 -
WO95/~3291 PCT/US94/12858
CA21 7373~ ~
cerebella (Pollerberg, E.G. et al., 1985, J. Cell Biol.
101:1921-1929). Developing cerebellar neurons express
Ng-CAM/L1/NILE and N-CAM in vitro, and recent in situ
hybridization histochemistry studies by the present
inventor's collaborators indicate that mRNA for neurocan
is synthesized by granule cells in rat cerebella. These
results suggest that granule cells may be major
contributors to the high levels of neurocan that appear
in the molecular layer of the developing cerebellum.
These observations, together with the effects of
mixtures of neurocan and Ng-CAM on cells, raise the
possibility of at least two opposing but not exclusive
modes of action that may occur in vivo:
(1) Binding of neurocan to Ng-CAM, and other cell
surface proteins including N-CAM, results in inhibition
of neuronal adhesion and axonal migration consistent with
the hypothesis that proteoglycans act as barriers against
neuronal penetration (Perris, R. et al., 1991,
Development 111:583-599; Oakley, R.A. et al., 1991, Dev.
Biol. 147:187-206; Snow, D.M. et al., 1990, E~p. Neurol.
109:111-130; Pindzola, R.R. et al., 1993, Dev. Biol.
156:34-48; Cole, G.J. et al., 1991, Neuron 7:1007-1018.
This could account for the observation that the parallel
processes of developing granule cells do not enter the
molecular layer in the developing cerebellum (Jacobson,
1991, supra; Grumet, M. et al., 1993a, J. Cell Biol.
120: 815-824) .
(2) Binding of Ng-CAM (and possibly other CAMs such as N-
CAM) to neurocan results in neutralization of the
inhibitory effects of the proteoglycan.
Given that substantial amounts of neurocan can be
extracted simply with PBS from developing brain, it is
likely that this secreted proteoglycan can diffuse
WO 95/13291 C A ~ 1 7 3 7 3 I PCT~S94/12858
.
locally between cells and bind to Ng-CAM and N-CAM.
Whereas these CAMs are found primarily associated with
the plasma membrane, small proportions of extracellular
forms and large proteolytic fragments of these CAMs have
5 been found in extracts prepared from brain tissue. For
example, chicken Ng-CAM as well as m~m~l ian Ll/NILE
have large forms that may be released from neurons in
response to stimuli or as a result of proteolytic
cleavage (Burgoon et al., supra; Richter-~andsberg, C. et
al., 1984, J. Neurochem. 43 : 841-848 ; Sweadner, K.J.,
1983, ~. Neurosci. 3 : 2504 -2517 ; Sadoul, K. et al., 1988,
. Neurochem. ~0 : 510-521 ; Nybroe, O. et al., 1990, Int.
. Dev. Neurosci 8: 273 -281) .
Although the radioligand assay revealed high
affinity binding of radiolabeled neurocan to Ng-CAM,
binding of soluble labeled Ng-CAM to immobilized neurocan
was not found, despite the fact that labeled Ng-CAM binds
to substrate bound Ng-CAM (Grumet, M. et al., 1993b, Cell
Adhesion Commlmic. 1:177-190). It is possible that the
20 ability of neurocan to bind to Ng-CAM depends on its
configuration which could be modified by binding to a
substrate. Attempts to present neurocan in a different
conformation, for example, by using monoclonal antibodies
against neurocan as a linker to the substrate did not
yield significant levels of Ng-CAM binding. In addition,
the present inventor observed that when neurocan was
bound to Covaspheres, it coaggregated with Ng-CAM coated
Covaspheres only when the neurocan had been treated with
chon~roitinase. It is possible that concentrating the
highly charged neurocan either on a substrate or on the
surface of Covaspheres produces a highly charged local
environment that may inhibit ligand binding. This affect
and the fact that Ng-CAM is usually found in a membrane
- 57 _
WO 95/13291 C A ~ 1 7 3 7 3 1 PCT/US9~112858
.
and binds in a highly cooperative manner, may explain the
inability to measure binding of soluble Ng-CAM to
substrate bound neurocan. In any case, the solubility of
neurocan and its binding properties are consistent with
its high level of co-localization with Ng-CAM and N-CAM
in vi vo .
Effects of Neurocan on Neurons
The present results support the notion that neurocan
binding to neurons modulates their behavior. Using a
centrifugation binding assay the present inventor's group
previously found that neurons could bind to substrates
coated with neurocan and that the lD1 antibody
specifically inhibited this binding (Grumet et
al.,1993a, supra) . In the present study, experiments
with cells ~mo~trated that neurocan inhibited neuronal
adhesion and neurite growth, although the mechanism for
this effect was not clear. The "inhibitor" may act
enzymatically to alter CAMs and inactivate them. In this
regard, no change in the mobility of Ng-CAM on gels has
been observed following incubation in physiological
buffers with neurocan (Grumet et al.,1993a). A second
possibility is that the "inhibitor" binds to the cell
surface either to block an adhesion molecule and/or to
generate a signal into the cell. This possibility
appears more likely inasmuch as Fab' fragments of
antibodies against Ng-CAM and N-CAM inhibited the binding
of I~I-neurocan to neurons.
The ability of cells to respond negatively to
neurocan suggests that.its binding to cell surface
molecules such as Ng-CAM and N-CAM may generate signals
that influence the response of the cell. In this regard,
recent studies indicate that binding of other ligands to
both of these CAMs at the ~urface of neurons (Schuch et
WO 95/13291 PCTIUS94/12858
~ CA21 7373~
al., 1989, Neuron 3:13-20; von Bohlen und Halbach, F . et
al., 1992, Eur. J. Neurosci. 4:896-909) and binding of
chondroitin sulfate proteoglycans to growth cones (Snow,
D.M. et al., 1993, ~. Neurobiol. 23:322-336) produced
increases in intracellular levels of calcium, but the
mechanisms of transmembrane signalling mediated by these
interactions are unclear. These observations raise the
possibility that the inhibitory effects of neurocan,
either directly or indirectly, involve its binding to Ng-
CAM and N-CAM on the cell surface.
The cellular assays employed here were designed to
investigate the mechanisms by which neurocan influences
neuronal behavior. When anti-Ng-CAM antibodies were used
as a permissive substrate, neurocan inhibited neuronal
adhesion and neurite growth. Because neurocan does not
bind to anti-Ng-CAM, the results suggest that neurocan
inhibited neuronal adhesion and neurite growth by
interacting directly with the cell surface.
The interpretation of the effects of neurocan on the
adhesion of neurons to Ng-CAM itself is somewhat more
complex due to potential interactions of neurocan with
both substrate-bound Ng-CAM and the neuronal plasma
membrane. When Ng-CAM was used as a permissive
substrate, inhibition occurred only when neurocan was
incubated on the substrate after Ng-CAM had been
adsorbed, and not when neurocan was coated before Ng-CAM.
This occurred even when the amounts of neurocan adsorbed
to the substrate were the same. Thus, the ability of the
mixed substrates to support neuronal adhesion depended
not only on the amounts of the proteins, but also on
their configuration on the substrate. A likely
interpretation, based on the high binding affinity of
soluble neurocan to immobilized Ng-CAM, is that at least
- 59 -
WO95/13291 PCT~S94/12858
CA21 73731 ~
part of the inhibition was due to binding of neurocan to
substrate-bound Ng-CAM. In support of this view, when
higher concentrations of soluble neurocan (which causes
neurocan to bind to a larger proportion of the Ng-CAM,
Figure 3) were incubated with Ng-CAM-bound substrates,
higher levels of inhibition were observed, (Table I).
Analysis of neurite outgrowth also indicated that
neurocan is a potent inhibitor of this critical aspect of
neuronal development. These results are important
because there is not always a direct relationship between
the ability of a particular protein to promote neuronal
adhesion and neurite growth (Lemmon, V. et al., 1992, J.
Neurosci. 12:818-826; Calof, A.L. et al., l99l, ~. Cell.
Biol . 115: 779-794) . The results of the current study
suggest that whether a particular region will promote,
allow, or inhibit cell adhesion and axonal growth will
depend not only on the relative amounts of the CAMs and
proteoglycans but also on the their sequences of
expression and organization during development.
EXAMPLE VI
TRANSFECTION OF NG-CAM INTO MAMMALIAN CELLS
To analyze the function of Ng-CAM and Ll expressed
in m~mm~l ian cells, expression vectors have been
constructed for the transfection of Ng-CAM into m~mm~l ian
cells.
One construct includes the cDNA for most of the
extracellular region of Ng-CAM that is linked to a signal
sequence for phospholipid attachment to the plasma
membranes. Expression of this construct in CHO cells
indicated that Ng-CAM was localized to the plasma
membrane. Experiments are in progress to analyze the
- 60 -
-
WOg5/~3291 PCT~S94/12858
CA21 73731
binding properties of this transfected form of Ng-CAM as
well as mutated forms of this molecule and the human Ll
protein.
The cells transfected to express Ng-CAM bind to Ng-
CAM and to chondroitin sulfate proteoglycans from brain
including 3F8 proteoglycan and neurocan. These results
suggest that this form of the molecule retains at least
some of the activities of the native Ng-CAM protein.
Such cells will also be cotransfected with a
construct encoding phospholipase D at the cell surface.
Such a double transfection should result in a cell line
that has the capability of synthesizing a membrane
anchored glycoprotein that is constitutively released
because of the action of the phospholipase D enzyme at
the surface of the same cell. This provides a useful
system for analyzing functions of the cell adhesion
molecules. In addition, it may provide new technologies
for producing and releasing various membrane proteins
from cells for therapeutic purposes.
~XAMPLE VII
Implantation of Tubes cont~;ning Ng-CAM
in a Sup~ort Matrix into Rats
To analyze the ability of Ng-CAM to promote
regeneration in ~n;m~l S, and as a therapeutic model, Ng-
CAM protein will be incorporated into silicone tubes. As
described previously (LeBeau et al., supra), the proxim~l
and distal stumps of severed rat sciatic nerves are
sutured into the openings of silicone tubes. The insides
of the tubes are filled with a saline solution containing
l-lO0 ~g/ml of Ng-CAM. Albumin serves as a control
protein. At various intervals following the initial
surgery, the ability of the Ng-CAM to promote recovery of
- 61 -
WO 95/1.3291 PCT/US91/128S8
CA~l 73731
neuromuscular function is assessed in the live animals by
measuring evoked muscle action potentials in the
gastrocnemius muscles, as described by Archibald, et al.
( supra) . The nerves are then surgically removed from the
rats, fixed and analyzed by microscopy for nerve
regrowth.
Based on the ability of Ng-CAM to promote the growth
of neurites, it is expected that Ng-CAM will accelerate
the rate of the regeneration process and increase the
maximal level of regeneration and recovery of
neuromuscular function.
EXAMPLE VIII
Implantation of Tissue Guides Impregnated
with Nq-CAM in into Rats
As another method to analyze the ability of Ng-CAM
to promote regeneration in animals, and as a therapeutic
model, Ng-CAM protein is incorporated into collagen based
nerve guides as conduits for peripheral nerve
regeneration. As described by Archibald et al . ( supra),
the prox; m~l and distal stumps of severed rat sciatic
nerves are sutured into the collagen based nerve guides.
The collagen guides are impregnated with a saline
2S solution con~in;ng 1-100 ~g/ml of Ng-CAM or a control
protein (such as albumin). At various intervals
following initial surgery, the ability of the Ng-CAM to
promote recovery of neuromuscular function is assessed in
the live ~n;m~ls by measuring evoked muscle action
potentials in the gastrocnemius muscles, as described by
Arch;h~ld, et al. (supra). The nerves are then
surgically removed from the rats, fixed and analyzed by
microscopy for nerve regrowth (LeBeau et al., supra) .
-- 62 --
WOgS/l3291 PcT~s94/12858
~ CA21 7373~
Based on the ability of Ng-CAM to promote the growth
of neurites, it is expected that Ng-CAM will accelerate
the rate of the regeneration processes and increase the
maximal level of regeneration and recovery of
neuromuscular function.
The references cited above are all incorporated by
reference herein, whether specifically incorporated or
not.
Having now fully described this invention, it will
be appreciated by those skilled in the art that the same
can be performed within a wide range of equivalent
parameters, concentrations, and conditions without
departing from the spirit and scope of the invention and
without undue experimentation.
While this invention has been described in
connection with specific embodiments thereof, it will be
understood that it is capable of further modifications.
This application is intended to cover any variations,
uses, or adaptations of the invention following, in
general, the principles of the invention and including
such departures from the present disclosure as come
within known or customary practice within the art to
which the invention pertains and as may be applied to the
essential features hereinbefore set forth as follows in
the scope of the appended claims.
- 63 -
