Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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NEURON AND NEURAL TUMOR GROWTH REGULATORY SYSTEM,
ANTIBODIES THERETO AND USES THEREOF
BACKGROUND
Following trauma in the adult central nervous system (CNS) of m~mm~l~, injured neurons do not
regenerate their transected axons. An important barrier to regeneration is the axon growth
inhibitory activity that is present in CNS myelin and that is also associated with the plasma
membrane of oligodendrocytes, the cells that synthesize myelin in the CNS (see Schwab, et al.,
Ann. Rev. Neurosci., 16, 565-595, 1993 for review). The growth inhibitory properties of CNS
myelin have been demonstrated in a number of different laboratories by a wide variety of
techniques, including plating neurons on myelin substrates or cryostat sections of white matter,
and observations of axon contact with mature oligodendrocytes (Schwab et al., 1993). Therefore,
it is well documented that adult neurons cannot extend neurites over CNS myelin in vitro.
It has also been well documented that removing myelin in vivo improves the success of
regenerative growth over the native terrain of the CNS. Regeneration occurs after irradiation of
newborn rats, a procedure that kills oligodendrocytes and prevents the appearance of myelin
proteins (Savio and Schwab, Neurobiology, 87, 4130-4133, 1990). After such a procedure in rats
and combined with a corticospinal trait lesion, some corticospinal axons regrow long distances
beyond the lesions. Also, in a chick model of spinal cord repair, the onset of myelination
correlates with a loss of its regenerative ability of cut axons (Keirstead, et al., Proc. Nat. Acad.
Sci. (USA), 89, 11664-11668, 1992). The removal of myelin with anti-galactocerebroside and
complement in the embryonic chick spinal cord extends the permissive period for axonal
regeneration. These experiments demonstrate a good correlation between myelination and the
failure of axons to regenerate in the CNS.
Until recently the identity of specific proteins important for the inhibitory activity remained
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elusive, although they have been sought since 1988 (Schwab et al., 1993). One component of
the myelin-derived inhibitors as myelin-associated glycoprotein (MAG) has been identified
(McKerracher et al., Neuron, 13, 229-246 and 805-811, 1994). This finding was at first
surprising because MAG does not have the biochemical properties or distribution of the
myelin-derived inhibitor reported by Schwab et al., (1993).
There have been some expectations of the properties of the non-MAG inhibitor in myelin, based
on the work of Martin Schwab (reviewed in detail by Schwab et al., 1993). It was reported to be
attributed to two different proteins of 35 kDa and 250 KDa. Myelin- derived growth inhibitory
activity was also reported to be a property of CNS myelin but not PNS myelin. It has since been
determined that PNS has inhibitory activity, but the inhibitory activity is masked by l~ninin
(David et al., 42, 594-602, 1995).
Schwab has sought to determine the identity of the myelin-derived inhibitors of neurite
outgrowth, and his fintlin~ have been extensively reviewed (Schwab et al., 1993). Schwab
determined a possible molecular weight of the growth inhibitory proteins in the following way.
Myelin proteins were separated by SDS PAGE under den~ ring conditions, the gel was cut into
slices and proteins were eluted from the slices and inserted into liposomes. The liposomes were
tested for inhibitory activity. Regions of the gel corresponding to 250 kDa and 35 kDa were
identified as most inhibitory, and heat destroyed the inhibitory activity. The loss of activity with
heat suggested that the activity was due to a protein that required native conformation. Why this
putative protein retains biological activity after the den~hlring conditions of SDS-PAGE remain
a mystery. The evidence to claim the 250 kDa and 35 kDa proteins as the major myelin
inhibitors is weak.
The evidence for the 250 kDa and 35 kDa proteins as myelin-derived inhibitors comes mainly
from the work of Schwab with their IN-l antibody. Schwab raised monoclonal antibodies to the
inhibitory proteins eluted from gels and cloned one monoclonal antibody, called IN-l, which is a
low-affinity IgM. It has been used to characterize the myelin-derived inhibition. The antibody is
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reported to bind to the 35 kDa and 250 kDa proteins, but the Western blots indicate that it lacks
specificity and that many additional bands are also recognized (Caroni and Schwab, Neuron, 1,
85-96, 1988). The immunoprecipitation data presented in the same publication was given in
tabular form rather than by showing the gels, as a rigorous analysis requires, and these data
cannot be easily evaluated. However, application of the antibody to various in vitro preparations
has been shown to partially block the inhibitory properties of myelin. Also, the application of this
antibody in vivo allows a small number of corticospinal axons to elongate long distances after
CNS injury (Schnell and Schwab, Nature, 343, 269-272, 1990; Schnell et al., Nature, 367, 170-
173, 1994). Moreover, raphe spinal serotonergic neurons also regenerate, and there is
improvement in some aspects of locomotor function (Bregman et al., Nature, 378, 498, 1995).
Therefore, the evidence to date suggests that blocking the myelin-derived inhibitors of neurite
outgrowth will be an important component of any therapeutic strategy to improve regeneration in
the adult CNS. Because the proteins identified by the antibodies have not been identified, the
components of myelin that block axon growth, in addition to MAG, remain unknown. It has
been noted that both MAG and the new inhibitor arretin, that is described herein, appear to be
acidic proteins. Therefore, to date, the identity of the non-MAG inhibitory components of myelin
remain unknown, and the proteins that the IN-l antibody recognizes remain uncharacterized.
While the findings of MAG as an inhibitor of neurite outgrowth were surprising, other
laboratories have now subst~nti~te~l our in vitro documentation that MAG is an important
myelin-derived inhibitor of neurite growth (Mukhopadhay et al., Neuron, 13, 757-767, 1994;
Schafer et al., Neuron, In press, 1996; DeBellard, Mol. Cell Neurosci., 7, 7616-7628, 1996). The
contribution of MAG has also been examined in vivo, and the results indicate that other growth
inhibitory proteins in myelin exist (Li et al., J. Neurosci. Res., In press, 1996). In these studies it
has been shown that some differences occur in axon extension after lesions in MAG null mutant
mice, a finding that differs from that reported for a similar study of a different line of
MAG-deficient mice (Bartsch et al., Eur. J. Neurosci., 7, 907-916, 1995; Bartsch et al., Neuron,
15, 1375-1381, 1995 ). In both cases, however, the results from the studies of MAG knock out
mice injured in the CNS are less dramatic than reported with treatment with the IN-l antibody
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(Bartsch et al., 1995 - see below), suggesting the non-MAG inhibitors that remain in CNS
myelin form an important barrier to regeneration; indeed their expression in the absence of MAG
expression may have been upregulated during CNS development.
Data has suggested that MAG may not be acting alone. To date, the presence of another protein
had not been shown nor were its properties known. The present invention has, for the first time,
demonstrated the presence and properties of such a protein.
Tenascins
Four members of the tenascin family have been identified and characterized: tenascin-C,
tenascin-R, tenascin-X and tenascin-Y (Bristow et a/., Cell Biol., I22, 265-278, 1993; Erickson,
H.P., J. Cell Biol., I20, I079-1081, 1993). Tenascin-X and tenascin-Y are not prominent in the
nervous system. Tenascin-C is important in the development of the nervous system and it is the
best characterized member of this protein family. It is generated by alternative splicing (Weller et
a/., J. Cell Biol., I 12, 355-362, 1991; Sriramarao and Bourdon, Nucl. Acids Res., 2I, 347-362,
1993) and the variants are expressed both in the nervous system and in several non-neural
tissues. Tenascin-C has been suggested to be involved in neuron-glia adhesive and migratory
events and to promote axon outgrowth after injury of peripheral nerves.
Tenascin-R (TN-R), has a modular structure similar to TN-C, previously designated Jl-160/180
and janusin in rodents, or restriction in chicken (Pesheva et al., J. Cell Biol., I09, 1765-1778,
1989; Fuss et al., J. Neurosci. Res., 29, 299-307, 1991, and J. Cell Biol., I20, 1237-1249, 1993).
Tenascin-R is predomin~ntly expressed by oligodendrocytes during the onset and early phases of
myelin formation and remains detectable in myelin-forming oligodendrocytes in the adult, and is
also expressed by neurons (Pesheva et al., 1989, Fuss et al., 1993). Tenascin-R has been shown
to be involved in promotion of neurite outgrowth and morphological polarization of
differentiating neurons when presented as a uniform substrate (Lochter and Schachner, J.
Neurosci., I3, 3986-4000, 1993; Lochter et a/., Eur. J. Neurosci., 6, 597-606, 1994). When
offered as a sharp substrate boundary with a neurite outgrowth conducive molecule, tenascin-R is
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repellent for growth cone advance (Taylor et al., J. Neurosci. Res., 35, 347-362, 1993; Pesheva et
al., 1993).
Tenacins are not thought to be an important component of the myelin-derived inhibitory activity
because they lack the specific myelin distribution, they are not restricted to the CNS, and their
molecular weight differs from the p-~ul--ptive proteins identified by Schwab. However, studies
have indicated that both tenascin R and tenascin C are minor inhibitory components of
octlyglucoside extracts of myelin. The data indicate that growth inhibitory proteins from the
CNS matrix may become associated with isolated myelin fragments.
Chondroitin Sulfate Proteoglycans (CSPGs)
Proteoglycans (PGs) are proteins that are found predomin~ntly on the cell surface and in the
extracellular matrix; they are covalently bound to complex carbohydrates called
glycosaminoglycans. Glycosarninoglycans (GAGs) are polymers of disaccharide repeats, which
are mostly highly sulphated and negatively charged. The main glycosaminoglycans in PGs are
chondroitin sulfate, d~rm~t~n sulfate, heparan sulfate, and keratan sulfate. (Ruoslahti, E., Ann.
Rev. Cell Biol., 4, 229-255, 1988). The number of GAG chains can vary from one to over one
hundred.
Proteoglycans are known to be important for the development and regeneration of the nervous
system, but they have not been considered to be myelin proteins or form part of the growth
inhibitory activity of myelin. Moreover, proteoglycans have not been reported to be recognized
by the IN-l antibody or to form a major growth inhibitory component of white matter regions of
the CNS.
Chondroitin sulfate proteoglycans (CSPGs) constitute the major population of PGs in the CNS.
The different patterns of localization and developmental expression of CSPGs throughout the
nervous system implicate them in diverse roles in development and in regeneration. After injuries
in the adult CNS, CSPGs are thought to be important in the formation of the glial scar. They
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have been implicated as both positive and negative modulators of axonal growth. Recent
observations indicate that DSD-l-PG, a neural chondroitin sulfate proteoglycan, promotes
neurite outgrowth of embryonic day 14 mesencephalic and embryonic day 18 hippocampal
neurons from rat (Faissner et al., J. Neurochem., 54, 1004-1015, 1994). However, NG2, an
integral membrane CSPGs expressed on the surface of glial progenitor cells, inhibits neurite
growth. The NG2 proteoglycan also inhibits neurite growth after digestion with chondroitinase
ABC, indicating that the inhibitory activity is a property of the core protein and not the
covalently attached chondroitin sulfate glycosaminoglycan chains (Dou and Levine, J. Neurosci.,
14, 7616-7628, 1994), but for many other types of CSPGs the inhibitory activity resides in the
glycosaminoglycan. Chondroitin sulfate proteoglycan immunoreactivity is increased after
cerebral cortical (McKeon et al., J. Neurosci., 11, 3398-3411, 1991), spinal (Pindzola et al.,
Dev. Biol., 156, 34-48, 1993) and optic nerve lesions (Brittis et al., Science, 255, 733-736,
1992). In vitro studies indicate that CSPG immunoreactivity on astrocytes increases when they
are plated on monolayers of leptomeningeal cells (Ness and David, Glia, In press, 1997). Similar
increases in CSPG immunoreactivity have been reported on Schwann cells co-cultured with
astrocytes ((~Thimik~r and Eng, Glia, 14, 145-152, 1995). This highly sulfated proteoglycan
which is a potent inhibitor of neurite growth in vitro (Snow et al., Neurol, 109, 111 - 130, 1990),
has been shown to be involved in the differentiation of developing retinal ganglion cells, and by
acting as an inhibitory substrate serves to a~pLopl;ately guide ganglion cell axons toward the
optic disc (Brittis and Silver, Proc. Nat. Acad. Sci. USA., 19, 7539-7542, 1992). McKeon et al.,
J. Neurosci., 11, 3398-3411, 1991) have reported that astrocytes harvested from the site of
cerebral cortical lesions express increased amounts of CSPG, which reduces neurite growth on
these cells in vitro. The expression of CSPG on the surface of a subset of cultured astrocytes has
also been shown to correlate with their reduced capacity to support neurite growth (Meiners et
al., J. Neurosci., 15, 8096-8108, 1995). The collapse of the growth cone is an important response
of the growing exon to inhibitory cues in the el~vilo~ lent. Collapse of the lamellipodium is
sometimes followed by retraction of the neurite (Kapfhammer and Raper, J. Neurosci., 7, 201-
212, 1987; Raper and Grunewald, Exp. Neurol., 109, 70-74, 1990; Bandtlow et al., J. Neurosci.,
10, 3837-3848, 1990). Many previously characterized inhibitory molecules found in the
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developing nervous system have been shown to cause growth cone collapse in vitro (Davies et
al., Neuron, 4, 11-20, 1990; Stahl et al., Neuron, 5, 735-743, 1990; Bandtlow et al., 1990;
Keynes et al., Ann. N.Y. Acad. Sci. 633, 562, 1991; Luo et al., Cell, 75, 217-227, 1993). Such
collapsing activity has been observed previously in the adult chicken brain and shown to bind to
PNA, and be associated with glycoproteins with molecular weights of 48 and 55 kDa (Keynes et
al., 1991). Others, such as the 33 kDa inhibitor in the developing chicken tectum also binds to
PNA (Stahl et al, 1990). Because proteoglycans are a very heterogenous class of proteins with
diverse biological activities it is essential that individual, identified proteins be considered.
Relevant to the present invention are the proteoglycans phosphocan and versican, because the
protein of the present invention, arretin, has common immunological eptitopes with these
1 5 proteins.
Phosphacan.
Phosphacan is a proteoglycan in brain recognized by the 3F8 antibody (Maurel et al., Proc. Nat.
Acad. Sci. USA, 91, 2512-2516, 1994), and by the 6B4 antibody (Maeda et al., Neurosci., 67,
23-35, 1995). Phosphacan is a splice variant of a receptor-type protein tyrosine phosphatase,
although phosphacan itself lacks the phosphatase domains. It is a protein with an apparent
molecular weight of approximately 500 kDa, having a core glycoprotein of approximately 400
kDa. The HNK-l monoclonal antibody recognizes a 3-sulphated carbohydrate epitope, and this
epitope is strongly represented in phosphacan from 7-day brain, but not in adult brain (Rauch et
al., J. Biol. Chem., 266, 14785-14801, 1991). In development phosphacan is immunostained on
radial glia and on neurons (Maeda et al., 1995) and generally it is expressed in both white matter
and grey matter regions (Meyer-Puttlitz, et al., J. Comp. Neurol. 366, 44-54, 1996). and
therefore, unlike the myelin inhibitors, it is not localized only to white matter areas. It appears to
be synthesized only by astroglia (Engel et al, 1996).
Versican.
Versican, a CSPG originally isolated from fibroblasts, also called PG-M, has an apparent
molecular weight of approximately 900 kDa, with a core protein of approximately300 to 400
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kDa (Braunewell et al., Eur. J. Neurosci., 7, 792-804, 1995; Naso et al, 1994). Versican belongs
to a family of aggregating CSPGs; other members of the family include the cartilage-derived
aggrecan, and two PGs expressed in the nenous system, neurocan and brevican
(Dours-Zimmermann and Zimmerermann, J. Biol. Chem., 269, 32992-32998, 1994). Versican is
widely distributed in adult human tissues, associated with connective tissue of various organs, in
certain muscle tissues, epithelia, and in central and peripheral nenous tissues. Four versican
isoforms are known (Vo, Vl, V2, V3), derived by alternative splicing. They vary in calculated
mass from approximately 370 kDa (Vo) to approximately 72 kDa (V3). It has been suggested
that the association of versican expression with cell migration and proliferation in vivo and its
a&esion inhibitory properties in vitro point to pathological processes such as tumorigenesis and
metastasis (Bode-Lesniewska et al., Histol. & Cyto., 44, 303-312, 1996; Naso et al,. J. Biol.
Chem., 269, 32999-33008, 1994).
Other CSPGs related to versican are brevican (Mr approximately 145 kDa) and neurocan (Mr >
300 kDa). Neither of these is known to be expressed by oligodendrocytes and are therefore not
expected to be present in CNS myelin (Engel et al., J. Comp. Neurol. 366, 34-43, 1996; Yamada
et al., J. Biol. Chem., 269, 10119-10126, 1994).
Another CSPG family member that is not related to either versican or phosphacan, is NG2.
Although it is expressed by 02A progenitor cells in the developing rat nenous system, it has no
apparent homology to arretin-relevant GSPG's, and has an Mr approximately 400-800 kDa with
a core protein of approximately 300 kDa (Nishiyama et al., J. Cell Biol., 114, 359-371, 1991).
Neuroblastoma
Neuroblastoma arises from neuroectoderm and contains anaplastic sympathetic ganglion cells
(reviewed in Pinkel and Howarth, 1985, In: Medical Oncology, Calabrese, P., Rosenberg, S. A.,
and Schein, P. S., eds., MacMillan, N.Y., pp. 1226-1257). One interesting aspect of
neuroblastoma is that it has one of the highest rates of spontaneous regression among human
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tumors (Everson, 1964, Ann. N.Y. Acad. Sci. 114:721-735) and a correlation exists between such
regression and maturation of benign ganglioneuroma (Bolande, 1977, Am. J. Dis. Child.
122: 12- 14). Neuroblastoma cells have been found to retain the capacity for morphological
maturation in culture. The tumors may occur anywhere along the sympathetic chain, with 50% of
such tumors origin:~ting in the adrenal medulla.
Neuroblastoma affects predomin~ntly preschool aged children and is the most common
extracranial solid tumor in chil&ood, constituting 6.5% of pediatric neoplasms. One half are less
than two years of age upon diagnosis. Metastases are evident in 60% of the patients at
presentation usually involving the bones, bone marrow, liver, or skin. The presenting symptoms
may be related to the primary tumor (spinal coral compression, abdominal mass), metastatic
tumor (bone pain) or metabolic effects of substances such as catecholamines or vasoactive
polypeptides secreted by the tumor (e.g. hypertension, diarrhea). Experimental evidence indicates
that an altered response to NGF is associated with neuroblastoma (Sonnenfeld and Ishii, 1982, J.
Neurosci. Res. 8:375-391). NGF stimulated neurite outgrowth in one-half of the neuroblastoma
cell lines tested; the other half was insensitive. However, NGF neither reduced the growth rate
nor enhanced survival in any neuroblastoma cell line.Present therapies for neuroblastoma involve
surgery and/or chemotherapy. Radiation therapy is used for incomplete tumor responses to
chemotherapy. There is a 70- 100% survival rate in individuals with localized tumors, but only a
20% survival rate in those with metastatic disease even with multiagent chemotherapy. It appears
that patients less than one year have a better prognosis (70%) than older children.
This background information is provided for the purpose of making known information believed
by the applicant to be of possible relevance to the present invention. No admission is necessarily
intended, nor should be construed, that any of the preceding information constitutes prior art
against the present invention. Publications referred to throughout the specification are hereby
incorporated by reference in their entireties in this application.
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5 SUMMARY OF THE INVENTION
The present invention relates to a neuron and neural tumor growth regulatory system, antibodies
directed against the components of this system and diagnostic, therapeutic, and research uses for
each of these aspects. The concept of a system is used to denote the functional relationship
10 between the genes (for the regulatory factors and the receptors), their encoded protein-regulatory
factors which regulate neuron growth (particularly neurite growth), and the receptors which are
activated by the protein. The functional relationship allows one to use one component to identify
and determine another. For example, having identified the protein component (factor or
receptor), one can use techniques well known in the art to identify the gene.
In accordance with the present invention, a protein has now been identified, hereinafter referred
to as arretin, as one of the molecular components involved in contact-mediated growth inhibition
on myelin. This protein has an apparent molecular weight of approximately 70 kDa, but it could
be derived from a molecular complex. Given the purified protein, procedures for obtaining the
20 other parts of the system are well known to those skilled in the art to purify the other components
to the system. For example, the protein can be used in very standard techniques to obtain the
amino acid sequence which can be used to obtain probes for nucleic acid sequences encoding
arretin. Alternatively, arretin protein may be tagged for use as a reporter to detect receptors of
arretin, which are then sequenced and used to obtain probes for the nucleic acid sequences
25 encoding arretin receptors. Moreover, the production of antibodies to each of these components
is also standard procedure.
The present invention further relates to arretin receptors and fragments thereof as well as the
nucleic acid sequences coding for such arretin receptors and fragments, and their therapeutic and
30 diagnostic uses. Substances which function as either agonists or antagonists to arretin receptors
are also envisioned and within the scope of the present invention.
The present invention further relates to the nucleic acid sequences coding for arretin and its
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S receptors, in addition to their therapeutic and diagnostic uses.
In accordance with another aspect of the present invention, there is provided the use of arretin for
the regulation of growth of neurons and neural tumors.
In a further aspect of the present invention, there is provided a method for inhibiting growth of
neural tumors, comprising the steps of introducing into the growth environment of the neurons a
growth inhibiting amount of arretin, fragments thereof, or an arretin agonist.
In yet a further aspect of the present invention, arretin can be used to design small molecules to
block neurite outgrowth and neural tumor growth. These small molecules will be useful to block
growth in situations involving aberrant sprouting, epilepsy, or metastasis.
A further embodiment involves a method of suppressing the inhibition of neuron growth,
comprising the steps of delivering to the nerve growth ~llvhulllllent, antibodies directed against
arretin in an amount effective to reverse said inhibition.
In another aspect of the present invention arretin can be used to design antagonist agents that
suppress the arretin-neuronal growth regulatory system. These antagonist agents can be used to
promote axon regrowth and recovery from trauma or neurodegenerative disease.
In accordance with another aspect of the present invention, there is provided an assay method
useful to identify arretin antagonist agents that ~iUppl ess inhibition of neuron growth, comprising
the steps of:
a) culturing neurons on a growth permissive substrate that incorporates a growth-inhibiting
amount of arretin; and
b) exposing the cultured neurons of step a) to a candidate arretin antagonist agent in an
amount and for a period sufficient prospectively to permit growth of the neurons;
thereby identifying as arretin antagonists the candidates of step b) which elicit neurite outgrowth
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5 from the cultured neurons of step a).
In yet another aspect of the present invention, there is provided an assay method useful for
screening for compounds that stimulate cell adhesion and neurite growth, comprising the steps
of:
10 a) coating a growth permissive substrate with a growth-inhibiting amount of arretin; and
b) adding a test compound and neuronal cells to the arretin-coated substrate;
c) washing to remove unattached cells;
d) measuring the viable cells attached to the substrate,
thereby identifying the cell a&esion candidates of step b) which elicit neurite outgrowth from
15 the cultured neurons of step a).
In accordance with another aspect of the present invention, there is provided a method to
suppress the inhibition of neurons, comprising the steps of delivering, to the nerve growth
environment, an antagonist for arretin or its receptor in an amount effective to reverse said
20 inhibition.
In another embodiment, the nucleic acids encoding arretin and/or its receptor can be used in
antisense techniques and therapies.
25 Arretin inhibits neurite outgrowth in nerve cells and neuroblastoma cells. Such inhibitory
protein comprises a 70,000 dalton molecular weight protein, aggregates, and analogs,
derivatives, and fragments thereof. Arretin and its related proteins proteins may be used in the
treatment of patients with malignant tumors which include but are not limited to melanoma and
nerve tissue tumors (e.g., glioma, or neuroblastoma). The present invention also relates to
30 antagonists of arretin, including, but not limited to, antibodies. Such antibodies can be used to
neutralize the neurite growth inhibitory factors for regenerative repair after trauma, degeneration,
or infl~mm~tion. In a further specific embodiment, monoclonal antibody may be used to
promote regeneration of nerve fibers over long distances following spinal cord damage.
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Various other objects and advantages of the present invention will become apparent from the
detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Analysis of growth inhibition after separation of myelin proteins by DEAE anion
exchange chromatography.
A. Western blots of column fractions probed with anti-MAG antibody.
B. Neurite growth inhibition and protein profile present in the column fraction shown in A.
Figure 2. Identification of 70 kDa components in DEAE chromatographic fractions from CNS
myelin as chondroitin sulphate proteoglycans. Myelin extracts (lane 1), DEAE chromatographic
fractions 10, 25, and 32 (lanes 2, 3, and 4) were subjected to SDS-PAGE (6-16% acrylamide
gradient) under reducing conditions, and detected by silver staining (A) and Western blots with
anti-MAG (B), anti-TN-C (C), anti-TN-R (D), and anti-CS 473 antibodies (E). The position and
molecular weight in kDa of marker proteins is indicated.
Figure 3. Western blot analysis of PNA affinity purification of the 70 kDa CSPGs from DEAE
chromatographic fractions 20-34. A. Pooled DEAE chromatographic fractions 20-34 (lane 1),
fractions 2 and 6 (lanes 2 and 3) of Hepes buffer wash, fractions 2 and 6 (lanes 4 and 5) of high
salt buffer wash, and fractions 2, 4, 6, and 8 (lanes 6, 7, 8, and 9) were subjected to SDS-PAGE
(6-16% acrylamide gradient) under reducing conditions, and detected by Western blots with
anti-CS 473 antibody. B. Pooled DEAE chromatographic fractions 20-34 (lane 1), flow-through
of PNA affinity column (lane 2), fraction 2 (lane 3) and pooled eluate (lane 4) were subjected to
SDS-PAGE (6- 16% acrylamide gradient) under reducing conditions, and detected by Western
blots with anti-MAG antibodies. The position and molecular weight in kDa of marker proteins is
indicated.
Figure 4. Identification of the 70 kDa components as phosphacan and versican-related molecules.
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5 A and B. Western blot analysis with 3F8 polyclonal anti-phosphacan (A) and with polyclonal
antibodies against recombinant versican (B). Fractions 20, 22, 24, 26, 28, 30, 32, and 34 (lanes
1-8) from DEAE chromatophy were subjected to SDS-PAGE (6-16% acrylamide gradient) under
reducing conditions. C, D, and E. Western blot analysis with 473 anti-CS antibody (C), 3F8
polyclonal anti-phosphacan (D) and polyclonal anti-recombinant versican (E). Myelin extracts
(lane 1), pooled DEAE chromatographic fractions 20-34 (lane 2), pooled flow-through from the
PNA affinity column (lane 3), and pooled eluates from PNA affinity column (lane 4) were
subjected to SDS-PAGE (6-16% acrylamide gradient) as in A and B. The position and molecular
weight in kDa of marker proteins is indicated.
Figure 5. Analysis of the 70 kDa CSPGs after chondroitinase ABC treatment. Pooled eluates
from the PNA affinity column (lane 1) and chondroitinase ABC treated pooled eluates from PNA
affinity column (lane 2) were subjected to SDS-PAGE (6-16% acrylamide gradient) under
reducing conditions and detected by amido black staining (A) and by Western blots with
polyclonal anti-phosphacan 3F8 (B). A bands at 28 kDa (in A. lane 1) is PNA (artifactually
eluted). Two bands above 72 kDa (in A. lane 2) are chondrontinase ABC. The position and
molecular weight in kDa of marker proteins is indicated.
Figure 6. Determin:~tion of cell-type expression of the 70 kDa CSPGs. Total membrane proteins
(100 ~ g) from brain (lane 1), myelin (lane 2), oligodendrocytes (lane 3), astrocytes (lane 4),
cerebellar neurons (lane 5), hippocampal neurons (lane 6), NG 108-15 cells (lane 7), and L-cells
(lane 8) were subjected to SDS-PAGE (6-16% acrylamide gradient) under reducing conditions
and detected by Western blots with polyclonal anti-phosphacan 3F8. The position and molecular
weight in kDa of marker proteins is indicated.
Figure 7. Inhibitory effects of the 70 kDa CSPGs on neurite outgrowth from cerebellar neurons.
Cerebellar neurons were plated as single cell suspensions on the 70 kDa CSPGs (arretin) and
other substrates applied to PORN-treated nitrocellulose substrates. Cells were m~int~ined for 24
h before fixation and staining with toluidine blue. Error bars indicate standard deviation. Coating
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concentrations were about 50 nM (1:25 dilution) and 10 nM (1:125 dilution) for arretin and
denatured arretin (DN) and 10 nM for l~minin. Bars represent percent neurons with neurites
(mean + SD).
Figure 8. Inhibitory effects of the 70 kDa CSPGs on neurite outgrowth from hippocampal
neurons. Hippocampal neurons were plated as single cell suspensions on the 70 kDa CSPGs
(arretin) and other substrates applied to PORN-treated tissue culture plastic. Cells were
maintained for 24 h before fixation and staining with toluidine blue. Error bars indicate standard
deviation. Coating concentrations were about 50 nM (1:25 dilution) and 10 nM (1:125 dilution)
for arretin and denatured arretin (DN) and 10 nM for l~minin Bars represent percent neurons
with neurites (mean + SD).
Figure 9. inhibitory effects of the 70 kDa CSPGs on neurite outgrowth from NG108-15 cells.
NG108 cells were plated as single cell suspensions on the 70 kDa CSPGs (arretin; inhib.p) and
other substrates applied to PLL-treated tissue culture plastic. Cells were m~int~ined for 24 h
before fixation and staining with toluidine blue. Coating concentrations were about 50 nM (1 :5
dilution) for arretin (inhib.p) and denature arretin (denat. inhib.p) and 10 nM for l~minin (LM).
Bars represent neurons with neurites (% growth). PLL= polylysine.
Figure 10. SDS-PAGE showing purification of arretin. The polypeptide was visualized by dyes
after gel electrophoresis. Lane 1 shows arretin purified by peanut agglutinin (PNA) affinity
chromatography. Two bands at approximately 70kDa are visible. A band at 28 kDa was
identified as a peanut agglutinin cont~min~nt. Lane 2 shows pooled fractions from a DEAE
chromatographic column that were applied to the PNA column for further purification of the
arretin bands. Lane 3 shows myelin starting material from which arretin was extracted. Lane 4
shows molecular weight markers.
Figure 11. Two-dimensional gel electrophoresis separation of arretin obtained from PNA
column chromatography. Polypeptides were separated in the first dimension by isoelectric
CA 02221391 1997-11-17
5 focusing followed by SDS-PAGE separation according to size in the second dimension. Spots
1,2, and 3 at approximately 70 kDa are separated from each other by size and charge. The spot at
28 kDa is peanut agglutinin, verified by Western blotting (not shown).
Figure 12. Anti-arretin antibody 1 8D2 neutralizes neurite outgrowth inhibition and cell body
repulsion by arretin on NG 108-15 cells. Picture A demonstrates cells growing normally on a
substrate of arretin-polylysine overlaid with anti-arretin 1 8D2. Picture B shows cell growth is
inhibited on a substrate of arretin-polylysine treated with control antiserum.
Figure 13. Western blot showing that culture supernatant from monoclonal antibody 1 8D2
15 recognizes the approximately 70 kDa arretin component. Lane 1 (arros) shows partially purified
arretin. Lane 2 shows myelin. Lane 3 shows octylglucoside/salt extract of myelin.
Figure 14. Growth cone collapse by arretin. A. Collapsed growth cones (arrows) after addition
of arretin. B. Growth cones treated with DMEM as a control remain spread. Explants of P2 rat
20 dorsal root ganglion neurons were plated on laminin can cultured overnight to allow neurite
extension. Arretin purified by lectin chromatography (A) or conkol medium (B) was added to
the cultures. The cultures were fixed with paraformaldehyde 30 min. later and viewed by phase
contrast microcopy. The numbers of collapsed growth cones were counted. Arretin caused
significantly more growth cone collapse than the PBS or DMEM controls.
DETAILED DESCRIPTION OF THE INVENTION
For the purpose of the present invention the following terms are defined below.
30 The term, neurite growth regulatory factor, refers to either arretin or its receptor.
"Agonist" refers to a pharmaceutical agent having biological activity of inhibiting the neurite
outgrowth of neurons cultured on a permissive substrate or inhibiting the regeneration of
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CA 02221391 1997-11-17
damaged neurons. It would be desirable to inhibit neuron growth in cases of epilepsy,
neuroblastoma, and neuromas, a disease state in a m:~mmAl which includes neurite outgrowth or
other neural growth of an abnormal sort which causes pain at the end of an amputated limb.
Antagonists which may be used in accordance with the present invention include without
limitation a arretin fragment, an analog of arretin of the arretin fragment, a derivative of either
arretin, the arretin fragment or said analog, an anti-idiotypic arretin antibody or a binding
fragment thereof, arretin ectodomain and a pharmaceutical agent.
"Antagonist" refers to a ph~rm~ceutical agent which in accordance with the present invention
which inhibits at least on biological activity normally associate with arretin, that is blocking or
suppressing the inhibition of neuron growth. Antagonists which may be used in accordance with
the present invention include without limitation a arretin antibody or a binding fragment of said
antibody, a arretin fragment, a derivative of arretin or of a arretin fragment, an analog of arretin
or of a arretin fragment or of said derivative, and a pharmaceutical agent, and is further
characterized by the property of ~u~plessillg arretin-mediated inhibition of neurite outgrowth.
An arretin antagonist is therefore, a chemical compound possessing the ability to alter the
biological activity of the neuronal receptor for arretin such that growth of neurons or their axons
is suppressed. The agonist or antagonist of arretin in accordance with the present invention is not
limited to arretin or its derivatives, but also includes the therapeutic application of all agents,
referred herein as pharmaceutical agents, which alter the biological activity of the neuronal
receptor for arretin such that growth of neurons or their axon is suppressed. The receptor can be
identified with know technologies by those skilled in the art (Mason, (1994) Curr. Biol., 4:1158-
1161) and its association with arretin or fragments thereof can be determined. The neuronal
receptor for arretin may or may not be the same as cell surface molecules that recognize and bind
arretin in an adhesion assay (Kelm et al., (1994) Curr. Biol., 4:965-972). Once the active arretin-
recognition domain of the receptor(s) is/are known, a~plopl;ate peptides or their analogs can be
designed and prepared to serve as agonist or antagonist of the arretin-receptor interaction.
CA 02221391 1997-11-17
5 The term "effective amount" or "growth-inhibiting amount" refers to the amount of
pharmaceutical agent required to produce a desired agonist or antagonist effect of the arretin
biological activity. The precise effective amount will vary with the nature of pharmaceutical
agent used and may be determined by one or ordinary skill in the art with only routine
experimentation.
As used herein, the terms "arretin biological activity" refers to cellular events triggered by
arretin, being of either biochemical or biophysical nature. The following list is provided, without
limitation, which discloses some of the known activities associated with contact-mediated
growth inhibition of neurite outgrowth, adhesion to neuronal cells, and promotion of neurite out
15 growth from new born dorsal root ganglion neurons.
Use of the phrase "substantially pure" or "isolated" in the present specification and claims as a
modifier of DNA, RNA, polypeptides or proteins means that the DNA, RNA, polypeptides or
proteins so designated have been separated from their in vivo cellular environment. As a result
20 of this separation and purification, the substantially pure DNAs, RNAs, polypeptides and
proteins are useful in ways that the non-separated, impure DNAs, RNAs, polypeptides or
proteins are not.
As used herein, the term "biologically active", or reference to the biological activity of arretin or,
25 or polypeptide fragment thereof, refers to a polypeptide that is able to produce one of the
functional characteristics exhibited by arretin or its receptors described herein. In one
embodiment, biologically active proteins are those that demonstrate inhibitory growth activities
central nervous system neurons. Such activity may be assayed by any method known to those of
skill in the art.
Based on the present evidence that arretin is a growth inhibitory protein in myelin, the means
exist to identify agents and therapies that suppress arretin-mediated inhibition of nerve growth.
Further, one can exploit the growth inhibiting properties of arretin, or arretin agonists, to
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CA 02221391 1997-11-17
5 suppress undesired nerve growth. Without the critical finding that arretin has growth inhibitory
properties, these strategies would not be developed.
The description of the present invention comprising a neuron and neural tumor growth regulatory
system can be divided into the following sections solely for the purpose of description: (1)
10 isolation, purification and characterization of arretin; (2) production of arretin-related derivatives,
analogs, and peptides; (3) arretin antagonists and assay methods to identify arretin antagonists;
(4) characterization of arretin receptors; (5) molecular cloning of genes or gene fragments
encoding arretin and its receptors; (6) generation of arretin related derivatives, analogs, and
peptides; (7) production of antibodies against the components of the arretin growth regulatory
15 system, (ie. arretin, its receptors, and the nucleic acid sequences coding for these proteins); (8)
the diagnostic, therapeutic and research uses for each of these components and the antibodies
directed thereto.
1. Isolation, Purif cation, and Characterization of Arretin
The present invention relates to CNS myelin associated inhibitory proteins of neurite growth and
receptors of CNS myelin associated inhibitory proteins of neurite growth. The CNS myelin
associated inhibitory proteins of the invention may be isolated by first isolating myelin and
subsequent purification therefrom. Isolation procedures which may be employed are described
25 more fully in the sections which follow. Alternatively, the CNS myelin associated inhibitory
proteins may be obtained from a recombinant expression system. Procedures for the isolation
and purification of receptors for the CNS myelin associated inhibitory proteins are described
below.
30 Isolation and Purification of Arretin Proteins
Arretin proteins can be isolated from the CNS myelin of higher vertebrates including, but not
limited to, birds or m~mm~ (both human and nonhllm~n such as bovine, rat, porcine, chick,
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CA 02221391 1997-11-17
5 etc.). Myelin can be obtained from the optic nerve or from central nervous system tissue that
includes but is not limited to spinal cords or brain stems. The tissue may be homogenized using
procedures described in the art (Colman et al., 1982, J. Cell Biol. 95:598-608). The myelin
fraction can be isolated subsequently also using procedures described (Colman et al., 1982,
supra).
In one embodiment of the invention, the CNS myelin associated inhibitory proteins can be
solubilized in detergent (for e.g., see McKerracher et al., 1994). The solubilized proteins can
subsequently be purified by various procedures known in the art, including but not limited to
chromatography (e.g., ion exchange, affinity, and sizing chromatography), centrifugation,
15 electrophoretic procedures, differential solubility, or by any other standard technique for the
purification of proteins. In one aspect, the solubilized proteins can be subjected to one or two-
dimensional electrophoresis, followed by elution from the gel. Gel-eluted proteins can be further
purified and/or used to generate antibodies.
20 Alternatively, the CNS myelin associated inhibitory proteins may be isolated and purified using
immunological procedures. For example, in one embodiment of the invention, the proteins can
first be solubilized using detergent. The proteins may then be isolated by immunoprecipitation
with antibodies. Alternatively, the CNS myelin associated inhibitory proteins may be isolated
using immunoaffinity chromatography in which the proteins are applied to an antibody column
25 in
solubilized form.
2. Production of Arretin-Related D.;,.~uli~, Analogs, and Peptides
30 The production and use of derivatives, analogs, and peptides related to arretin are also
envisioned, and within the scope of the present invention and include molecules antagonistic to
neurite growth regulatory factors (for example, and not by way of limitation, anti-idiotype
antibodies). Such derivatives, analogs, or peptides which have the desired inhibitory activity can
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CA 02221391 1997-11-17
5 be used, for example, in the treatment of neuroblastoma. Derivatives, analogs, or peptides related
to a neurite growth regulatory factor can be tested for the desired activity by assays for
nonpermissive substrate effects or for growth cone collapse.
The neurite growth regulatory factor-related derivatives, analogs, and peptides of the invention
10 can be produced by various methods known in the art. The manipulations which result in their
production can occur at the gene or protein level. For example, a cloned neurite growth
regulatory factor gene can be modified by any of numerous strategies known in the art (Maniati~,
et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.). A given neurite growth regulatory factor sequence can be cleaved at
15 appl~.pliate sites with restriction endonuclease(s), subjected to enzymatic modifications if
desired, isolated, and ligated in vitro. In the production of a gene encoding a derivative,
analogue, or peptide related to a neurite growth regulatory factor, care should be taken to ensure
that the modified gene remains within the same translational reading frame as the neurite growth
regulatory factor, uninterrupted by translational stop signals, in the gene region where the desired
20 neurite growth regulatory factor-specific activity is encoded.
Additionally, a given neurite growth regulatory factor gene can be mutated in vitro or in vivo, to
create and/or destroy translation, initiation, and/or tçrmin:~tion sequences, or to create variations
in coding regions and/or forrn new restriction endonuclease sites or destroy preexisting ones, to
25 facilitate further in vitro modification. Any technique for mutagenesis known in the art can be
used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, et al., 1978, J.
Biol. Chem. 253:6551), use of TAB® linkers (Pharmacia), etc.
30 3. Arretin Antagonists and Assay Methods to Identify Arretin Antagonists
In one embodiment suitable as arretin antagonist candidates are developed comprising fragments,
analogs and derivatives of arretin. Such candidates may interfere with arretin-mediated growth
CA 02221391 1997-11-17
5 inhibition as competitive but non-functional mimics of endogenous arretin. From the amino acid
sequence of arretin and from the cloned DNA coding for it, it will be appreciated that arretin
fragments can be produced either by peptide synthesis or by recombinant DNA expression of
either a truncated domain of arretin, or of intact arretin could be prepared using standard
recominant procedures, that can then be digested enzymically in either a random or a site-
10 selective manner. Analogs of arretin or arretin fragments can be generated also by recombinantDNA techniques or by peptide synthesis, and will incorporate one or more, e.g. 1-5, L- or D-
amino acid substitutions. Derivatives of arretin, arretin fragments and arretin analogs can be
generated by chemical reaction of the parent substance to incorporate the desired derivatizing
group, such as N-t~rmin~l, C-terminal and intra-residue modifying groups that have the effect of
15 m~kin~; or stabilizing the substance or target amino acids within it.
In specific embodiments of the invention, candidate arretin antagonists include those that are
derived from a detf~rmin~tion of the functionally active region(s) of arretin. The antibodies
mentioned above and any others to be prepared against epitopes in arretin, when found to be
20 function-blocking in in vitro assays, can be used to map the active regions of the polypeptide as
has been reported for other proteins (for example, see Fahrig et al., (1993) Europ., J. Neurosci.,
5: 1118- 1126; Tropak et al., (1994) J. Neurochem., 62: 854-862). Thus, it can be determined
which regions of arretin are recognized by neuronal receptors and/or are involved in inhibition of
neurite outgrowth. When those are known, synthetic peptides can be prepared to be assayed as
25 candidate antagonists of the arretin effect. Derivatives of these can be prepared, including those
with selected amino acid substitutions to provide desirable properties to enhance their
effectiveness as antagonists of the arretin candidate functional regions of arretin can also be
determined by the preparation of altered forms of the arretin domains using recombinant DNA
technologies to produce deletion or insertion mutants that can be expressed in various cell types
30 as chim~ric proteins that contain the Fc portion of immunoglobulin G (Kelm et al., (1994) Curr.
Biol., 4: 965-972). Alternatively, candidate mutant forms of arretin can be expressed on cell
surfaces by transfection of various cultured cell types. All of the above forms of arretin, and
forms that may be generated by technologies not limited to the above, can be tested for the
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CA 02221391 1997-11-17
presence of functional regions that inhibit or ~;ul)l)LeS~ neurite outgrowth, and can be used to
design and prepare peptides to serve as antagonists.
In accordance with an aspect of the invention, the arretin antagonist is formulated as a
pharmaceutical composition which contains the arretin antagonist in an amount effective to
suppress arretin-mediated inhibition of nerve growth, in combination with a suitable
pharmaceutical carrier. Such compositions are useful, in accordance with another aspect of the
invention, to suppress arretin-inhibited nerve growth in patients diagnosed with a variety of
neurological disorder, conditions and ailments of the PNS and the CNS where treatment to
increase neurite extension, growth, or regeneration is desired, e.g., in patients with nervous
system damage. Patients suffering from traumatic disorders (including but not limited to spinal
cord injuries, spinal cord lesions, surgical nerve lesions or other CNS pathway lesions) damage
secondary to infarction, infection, exposure to toxic agents, malignancy, paraneoplastic
syndromes, or patients with various types of degenerative disorders of the central nervous system
(Cutler, (1987) In: Scientific American Medicines, vol. 2, Scientific American Inc., N.Y., pp. 11-
1-11-13) can be treated with such arretin antagonists. Examples of such disorders include but are
not limited to Strokes, Alzheimer's disease, Down's syndrome, Creutzfeldt-Jacob disease, kuru,
Gerstman-Straussler syndrome, scrapie, tr~n~mi.csible mink encephalopathy, Huntington's
disease, Riley-Day f~mili~l dysautonomia, multiple system atrophy, amylotrophic lateral
sclerosis or Lou Gehrig's disease, progressive supranuclear palsy, Parkinson's disease and the
like. The arretin antagonists may be used to promote the regeneration of CNS pathways, fiber
systems and tracts. Administration of antibodies directed to an epitope of arretin, or the binding
portion thereof, or cells secreting such antibodies can also be used to inhibit arretin function in
patients. In a particular embodiment of the invention, the arretin antagonist is used to promote
the regeneration of nerve fibers over long distances following spinal cord damage.
In another embodiment, the invention provides an assay method adapted to identify arretin
antagonists, that is agents that block or suppress the growth-inhibiting action of arretin. In its
most convenient form, the assay is a tissue culture assay that measures neurite out-growth as a
-23 -
CA 02221391 1997-11-17
convenient end-point, and accordingly uses nerve cells that extend neurites when grown on a
permissive substrate. Nerve cells suitable in this regard include neuroblastoma cells of the
NG108 lineage, such as NG108-15, as well as other neuronal cell lines such as PC12 cells
(American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 USA, ATCC
accession NO. CRL 1721), human neuroblastoma cells, and primary cultures of CNS or PNS
neurons taken from embryonic, postnatal or adult animals. The nerve cells, for instance about
103 cells-microwell or equivalent, are cultured on a growth permissive substrate, such as
polylysine or l~minin, that is over-layed with a growth-inhibiting amount of arretin. The arretin
incorporated in the culture is suitably myelin-extracted arretin, although forms of arretin other
than endogenous forms can be used provided they exhibit the arretin property of inhibiting
neuron growth when added to a substrate that is otherwise growth permissive.
In this assay, candidate arretin antagonists, i.e., compounds that block the growth-inhibiting
effect of arretin, are added to the arretin-cont~ining tissue culture preferably in amount sufficient
to neutralize the arretin growth-inhibiting activity, that is between 1.5 and 15,ug of arretin
antagonists per well co~ g a density of 1000 NG108-15 cells/well cultured for 24 hr. in
Dulbecco's minim~l essential medium. After culturing for a period sufficient for neurite
outgrowth, e.g. 3-7 days, the culture is evaluated for neurite outgrowth, and arretin antagonists
are thereby revealed as those candidates which elicit neurite outgrowth. Desirably, candidates
selected as arretin antagonists are those which elicit neurite outgrowth to a statistically
significant extent compared to neurons plated on arretin alone.
Screening for compounds that stimulate cell adhesion and neurite growth on arretin-coated
substrates.
Arretin not only prevents neurite growth but also reduces the adhesion of cells to the substrate.
Since cell adhesion is technically far easier to assay quantitatively than neurite growth, cell
adhesion can be used as a first screen for high-through-put screening of a large number of
compounds. This can be done using the MTT [3 {4-5-dimethylthiazol-2-yl]-2,5-
-24-
CA 02221391 1997-11-17
S diphenyltertrazolium bromide) assay. MTT is taken up by live cells and converted by the
mitochondria into a blue substrate that can be quantified by a densitometer. For this assey, 96-
well plates are coated with arretin. After washing wells the add chemical compounds can be
added to the well for 1-2 hours or along with neuronal cells such as NG108-15 cells. After 2-4
hours or overnight incubation with the cells, the cultures are washed to remove unattached cells.
MTT is then added to the cells at a concentration of 0.5mg/ml in culture medium. Incubate for 4
hours at 370C in a 5% CO2 incubator. Wash once with PBS and add acid isopropanol(lOOul/well), and mix with a pipette. After 5 minlltes the plates are read with ELISA reader at
550nm.
Other assay tests that could be used include without limitation the following: 1) The growth
cone collapse assay that is used to assess growth inhibitory activity of collapsin (Raper, J.A.,
and Kapfhammer, J.P., (1990) Neuron, 2:21-29; Luo et al., (1993) Cell, 75:217-227) and of
various other inhibitory molecules (Igarashi, M. et al., (1993) Science, 259:77-79) whereby the
test substance is added to the culture medium and a loss of elaborate growth cone morphology is
scored. 2) The use of patterned substrates to assess substrate plerelellce (Walter, J. et al., (1987)
Development, 101:909-913; Stahl et al., (1990) Neuron, 5:735-743) or avoidance oftest
substrates (Ethell, D.W. et al., (1993) Dev. Brain Res., 72:1-8). 3) The expression of
recombinant proteins on a heterologous cell surface, and the transfected cells are used in co-
culture experiments. The ability of the neurons to extend neurites on the transfected cells is
assessed (Mukhopadhyay et al., (1994) Neuron, 13:757-767). 4) The use of sections of tissue,
such as sections of CNS white matter, to assess molecules that may modulate growth inhibition
(Carbonetto et al., (1987) J. Neuroscience, 7:610-620; Savlo, T. and Schwab, M.E., (1989) J.
Neurosci., 9: 1126- 1133). 5) Neurite retraction assays whereby test substrates are applied to
differentiated neural cells for their ability to induce or inhibit the retraction of previously
extended neurites (Jalnink et al., (1994) J. Cell Bio., 126:801-810; Sudan, H.S. et al., (1992)
Neuron, 8:363-375; Smalheiser, N. (1993) J. Neurochem., 61 :340-342). 6) The repulsion of
cell-cell interactions by cell aggregation assays (Kelm, S. et al., (1994) Current Biology, 4:965-
972; Brady-Kainay, S. et al., (1993) J. Cell Biol., 4:961-972). 7) The use of nitrocellulose to
CA 02221391 1997-11-17
5 prepare substrates for growth assays to assess the ability of neural cells to extend neurites on the
test substrate (Laganeur, C. and Lemmon, V., (1987)PNAS, 84:7753-7757; Dou, C-L and Levine,
J.M., (1994) J. Neuroscience, 14:7616-7628).
Useful arretin antagonists include antibodies to arretin and the binding fragments of those
10 antibodies. Antibodies which are either monoclonal or polyclonal can be produced which
recognize arretin and its various epitopes using now routine procedures. For the raising of
antibody, various host animals can be immunized by injection with arretin or fragment thereof,
including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase
the immunological response, depending on the host species, and including but not limited to
15 Freund's (complete and incomplete), mineral gels such as alllminum hydroxide, surface active
substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet hemocyanins, dinmitrophenol, and potentially useful human adjuvants such as BCG
(Bacille Calmette-Guerin).
20 4. Isolation and Purif cation of Receptors for Arretin
Receptors for arretin can be isolated from cells whose attachment, spreading, growth and/or
motility is inhibited by arretin. Such cells include but are not limited to fibroblasts and neurons.
In a prt;r~ ;d embodiment, neurons are used as the source for isolation and purification of the
25 receptors.
In one embodiment, receptors to arretin may be isolated by affinity chromatography of neuronal
plasma membrane fractions, in which a myelin associated inhibitory protein or peptide fragment
thereof is immobilized to a solid support. Alternatively, receptor cDNA may be isolated by
30 expression cloning using purified arretin as a ligand for the selection of receptor-expressing
clones.
Alternatively, arretin protein may be tagged for use as a reporter to detect receptors of arretin,
-26 -
CA 02221391 1997-11-17
5 using techniques that are well known in the art. There are many different types of tags that may
be employed such as flourescence radioactive tags.
5. Molecular Cloning of Genes or Gene Fr~ en~ Encod;~tgArretin and Its Receptors
Any m~mm~ n cell can potentially serve as the nucleic acid source for the molecular cloning of
the genes encoding arretin or its receptors. The DNA may be obtained by standard procedures
known in the art from cloned DNA (e.g., a DNA "library"), by chemical synthesis, by cDNA
cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired
15 m:~mm~lian cell. (See, for example, Maniatis et al., 1982, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985,
DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U. K., Vol. I, II.) Clones derived
from genomic DNA may contain regulatory and intron DNA regions, in addition to coding
regions; clones derived from cDNA will contain only exon sequences. Whatever the source, a
20 given neurite growth regulatory factor gene should be molecularly cloned into a suitable vector
for propagation of the gene.
In the molecular cloning of a neurite growth regulatory factor gene from genomic DNA, DNA
fragments are generated, some of which will encode the desired neurite growth regulatory factor
25 gene. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively,
one may use DNAse in the presence of m~ng~nese to fragment the DNA, or the DNA can be
physically sheared, as for example, by sonication. The linear DNA fragments can then be
separated according to size by standard techniques, including but not limited to, agarose and
polyacrylamide gel electrophoresis and column chromatography.
Once the DNA fragments are generated, identification of the specific DNA fragment co~ g a
neurite growth regulatory factor gene may be accomplished in a number of ways. For example, if
an amount of a neurite growth regulatory factor gene or its specific RNA, or a fragment thereof,
-27-
CA 02221391 1997-11-17
5 is available and can be purified and labeled, the generated DNA fragments may be screened by
nucleic acid hybridization to the labeled probe (Benton and Davis, 1977, Science 196:180;
Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961-3965). For example, in a
preferred embodiment, a portion of a neurite growth regulatory factor amino acid sequence can
be
10 used to deduce the DNA sequence, which DNA sequence can then be synthesized as an
oligonucleotide for use as a hybridization probe. Alternatively, if a purified neurite growth
regulatory factor probe is unavailable, nucleic acid fractions enriched in neurite growth
regulatory factor may be used as a probe, as an initial selection procedure. It is also possible to
identify an appropl ;ate neurite growth regulatory factor-encoding fragment by restriction enzyme
15 digestion(s) and comparison of fragment sizes with those expected according to a known
restriction map if such is available. Further selection on the basis of the properties of the gene, or
the physical, chemical, or immunological properties of its expressed product, as described above,
can be employed after the initial selection.
20 A neurite growth regulatory factor gene can also be identified by mRNA selection using nucleic
acid hybridization followed by in vitro translation or translation in Xenopus oocytes. In an
example of the latter procedure, oocytes are injected with total or size fractionated CNS mRNA
populations, and the membrane-associated translation products are screened in a functional assay
(3T3 cell spreading). Preadsorption of the RNA with complementary DNA (cDNA) pools
25 leading to the absence of expressed inhibitory factors indicates the presence of the desired
cDNA. Reduction of pool size will finally lead to isolation of a single cDNA clone. In an
alternative procedure, DNA fragments can be used to isolate complementary mRNAs by
hybridization. Such DNA fragments may represent available, purified neurite growth regulatory
factor DNA, or DNA that has been enriched for neurite growth regulatory factor sequences.
30 Immunoprecipitation analysis or functional assays of the in vitro translation products of the
isolated mRNAs identifies the mRNA and, therefore, the cDNA fragrnents that contain neurite
growth regulatory factor sequences. An example of such a functional assay involves an assay for
nonpermissiveness in which the effect of the various translation products on the spreading of 3T3
CA 02221391 1997-11-17
5 cells on a polylysine coated tissue culture dish is observed. In addition, specific mRNAs may be
selected by adsorption of polysomes isolated from cells to immobilized antibodies specifically
directed against a neurite growth regulatory factor protein. A radiolabeled neurite growth
regulatory factor cDNA can be synthesized using the selected mRNA (from the adsorbed
polysomes) as a template. The radiolabeled mRNA or cDNA may then be used as a probe to
10 identify the neurite growth regulatory factor DNA fragments from among other genomic DNA
fragments. Alternatives to isolating the neurite growth regulatory factor genomic DNA include,
but are not limited to, chemically synthesizing the gene sequence itself from a known sequence
or making cDNA to the mRNA which encodes the neurite growth regulatory factor gene. Other
methods are possible and within the scope of the invention. The identified and isolated gene or
15 cDNA can then be inserted into an a~propliate cloning vector. A large number of vector-host
systems known in the art may be used. Possible vectors include, but are not limited to, cosmids,
plasmids or modified viruses, but the vector system must be compatible with the host cell used.
Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or
plasmids such as pBR322 or pUC plasmid derivatives. Recombinant molecules can be20 introduced into host cells via transformation, transfection, infection, electroporation, etc.
In an alternative embodiment, the neurite growth regulatory factor gene may be identified and
isolated after insertion into a suitable cloning vector, in a "shot gun" approach. Enrichment for a
given neurite growth regulatory factor gene, for example, by size fractionation or subtraction of
25 cDNA specific to low neurite growth regulatory factor producers, can be done before insertion
into the cloning vector. In another embodiment, DNA may be inserted into an ~ression vector
system, and the recombinant ~ s~ion vector colll~hlillg a neurite growth regulatory factor gene
may then be detected by functional assays for the neurite growth regulatory factor protein.
30 The neurite growth regulatory factor gene is inserted into a cloning vector which can be used to
transform, transfect, or infect al)plopliate host cells so that many copies of the gene sequences
are generated. This can be accomplished by ligating the DNA fragment into a cloning vector
which has complementary cohesive termini. However, if the complementary restriction sites used
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CA 02221391 1997-11-17
5 to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may
be enzymatically modified. Alternatively, any site desired may be produced by ligating
nucleotide
sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific
chemically synthesized oligonucleotides encoding restriction endonuclease recognition
10 sequences. In an alternative method, the cleaved vector and neurite growth regulatory factor gene
may be modified by homopolymeric tailing. Identification of the cloned neurite growth
regulatory factor gene can be accomplished in a number of ways based on the properties of the
DNA itself, or alternatively, on the physical, immunological, or functional properties of its
encoded protein. For example, the DNA itself may be detected by plaque or colony nucleic acid
hybridization to labeled probes (Benton, W. and Davis, R., 1977, Science 196: 180; Grunstein, M.
and Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Alternatively, the presence of a
neurite growth regulatory factor gene may be detected by assays based on properties of its
expressed product. For example, cDNA clones, or DNA clones which hybrid-select the proper
mRNAs, can be selected which produce a protein that inhibits in vitro neurite outgrowth. If an
20 antibody to a neurite growth regulatory factor is available, a neurite growth regulatory factor
protein may be identified by binding of labeled antibody to the putatively neurite growth
regulatory factor-synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay)-type
procedure. In specific embodiments, transformation of host cells with recombinant DNA
molecules that incorporate an isolated neurite growth regulatory factor gene, cDNA, or
25 synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene
may be obtained in large quantities by growing kansformants, isolating the recombinant DNA
molecules from the transformants and, when necessary, retrieving the inserted gene from the
isolated recombinant DNA. If the ultimate goal is to insert the gene into virus expression vectors
such as vaccinia virus or adenovirus, the recombinant DNA molecule that incorporates a neurite
30 growth regulatory factor gene can be modified so that the gene is flanked by virus sequences that
allow for genetic recombination in cells infected with the virus so that the gene can be inserted
into the viral genome. After the neurite growth regulatory factor DNA-collt~ ;llg clone has been
identified, grown, and harvested, its DNA insert may be characterized as described herein. When
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CA 02221391 1997-11-17
5 the genetic structure of a neurite growth regulatory factor gene is known, it is possible to
manipulate the structure for optimal use in the present invention. For example, promoter DNA
may be ligated 5' of a neurite growth regulatory factor coding sequence, in addition to or
replacement of the native promoter to provide for increased ~2~L lession of the protein. Many
manipulations are possible, and within the scope of the present invention.
Expression of the Cloned Neurite Growth Regulatory Factor Genes.
The nucleotide sequence coding for a neurite growth regulatory factor protein or a portion
thereof, can be inserted into an applopliate expression vector, i.e., a vector which contains the
15 necessary elements for the transcription and translation of the inserted protein-coding sequence.
The necessary transcriptional and translation signals can also be supplied by the native neurite
growth regulatory factor gene and/or its fl~nking regions. A variety of host-vector systems may
be utilized to express the protein-coding sequence. These include but are not limited to
m~mm~liAn cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell
20 systems infected with virus (e.g., baculovirus); microorg~ni~m~ such as yeast co~ g yeast
vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. The
~lession elements of these vectors vary in their strengths and specificities. Depending on the
host-vector system utilized, any one of a number of suitable transcription and translation
elements may be used.Any of the methods previously described for the insertion of DNA
25 fragments into a vector may be used to construct ~ les~ion vectors co~ g a chimeric gene
consisting of a~pr~pliate transcriptional/translational control signals and the protein coding
sequences. These methods may include in vitro recombinant DNA and synthetic techniques and
in vivo recombinations (genetic recombination).
30 Expression vectors cont~ining neurite growth regulatory factor gene inserts can be identified by
three general approaches: (a) DNA-DNA hybridization, (b) presence or absence of "marker" gene
functions, and (c) expression of inserted sequences. In the first approach, the presence of a
foreign gene inserted in an expression vector can be detected by DNA-DNA hybridization using
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CA 02221391 1997-11-17
5 probes comprising sequences that are homologous to an inserted neurite growth regulatory factor
gene. In the second approach, the recombinant vector/host system can be identified and selected
based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase
activity, resistance to antibiotics, transformation phenotype, occlusion body formation in
baculovirus, etc.) caused by the insertion of foreign genes in the vector. For example, if a given
10 neurite growth regulatory factor gene is inserted within the marker gene sequence of the vector,
recombinants cont~ining the neurite growth regulatory factor insert can be identified by the
absence of the marker gene function. In the third approach, recombinant expression vectors can
be identified by assaying the foreign gene product expressed by the recombinant. Such assays
can be based on the physical, immunological, or functional properties of a given neurite growth
15 regulatory factor gene product.
Once a particular recombinant DNA molecule is identified and isolated, several methods known
in the art may be used to propagate it. Once a suitable host system and growth conditions are
established, recombinant expression vectors can be propagated and prepared in quantity. As
20 previously explained, the expression vectors which can be used include, but are not limited to,
the following vectors or their derivatives: human or animal viruses such as vaccinia virus or
adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,
lambda), and plasmid and cosmid DNA vectors, to name but a few.
25 In addition, a host cell strain may be chosen which modulates the ~ es~ion of the inserted
sequences, or modifies and processes the gene product in the specific fashion desired. Expression
from certain promoters can be elevated in the presence of certain inducers; thus, expression of
the genetically engineered neurite growth regulatory factor protein may be controlled.
Furthermore, different host cells have characteristic and specific mech~nism~ for the translational
30 and post-translational processing and modification (e.g., glycosylation, cleavage) of proteins.
Appropriate cell lines or host systems can be chosen to ensure the desired modification and
processing of the foreign protein expressed. For example, ~ ession in a bacterial system can be
used to produce an unglycosylated core protein product. Expression in yeast will produce a
CA 02221391 1997-11-17
5 glycosylated product. Expression in m~mm~ n (e.g. COS) cells can be used to ensure "native"
glycosylation of the heterologous neurite growth regulatory factor protein. Furthermore, different
vector/host expression systems may effect processing reactions such as proteolytic cleavages to
different extents.
10 Identification and Purification of the Expressed Gene Product
Once a recombinant which expresses a given neurite growth regulatory factor gene is identified,
the gene product can be purified and analyzed as described above. The amino acid sequence of
arretin and its receptor protein can be deduced from the nucleotide sequence of the cloned gene,
15 allowing the protein, or a fragment thereof, to be synthesized by standard chemical methods
known in the art (e.g., see Hunkapiller, et al., 1984, Nature 310:105-111). In particular
embodiments of the present invention, such neurite growth regulatory factor proteins, whether
produced by recombinant DNA techniques or by chemical synthetic methods, include but are not
limited to those col-t~ il-g altered sequences in which functionally equivalent amino acid
20 residues are substituted for residues within the sequence resulting in a silent change. For
example, one or more amino acid residues within the sequence can be substituted by another
amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent
alteration. Substitutes for an amino acid within the sequence may be selected from other
members of the class to which the amno acid belongs. For example, the nonpolar (hydrophobic)
25 amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and
methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine,
and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic
acid. Also included within the scope of the invention are neurite growth regulatory factor
30 proteins which are differentially modified during or after translation, e.g., by glycosylation,
proteolytic cleavage, etc.
Characterization of the Neurite, Growth Regulatory Factor Genes
CA 02221391 1997-11-17
The structure of a given neurite growth regulatory factor gene can be analyzed by various
methods known in the art.
The cloned DNA or cDNA corresponding to a given neurite growth regulatory factor gene can be
analyzed by methods including but not limited to Southern hybridization (Southern, 1975, J.
Mol. Biol. 98:503-517), Northern hybridization (Alwine, et al., 1977, Proc. Natl. Acad. Sci.
U.S.A. 74:5350-5354; Wahl, et al., 1987, Meth. Enzymol. 152:572-581), restriction
endonuclease mapping (M~ni~t~ et al., 1982, Molecular loning, A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), and DNA sequence analysis. DNA
sequence analysis can be performed by any techniques known in the art including but not limited
to the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy
method (Sanger, et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467), or use of an
automated DNA sequenator (e.g., Applied Biosystems, Foster City, Calif.).
6. Production of Antibodies Against the Components of the Arretin Growth Regulatory
System
Antibodies can be produced which recognize neurite growth regulatory factors or related
proteins. Such antibodies can be polyclonal or monoclonal.Various procedures known in the art
may be used for the production of polyclonal antibodies to epitopes of a given neurite growth
25 regulatory factor. For the production of antibody, various host anim~l.c can be immunized by
injection with a neurite growth regulatory factor protein, or a synthetic protein, or fragment
thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to
increase the immunological response, depending on the host species, and including but not
limited to Freund's (complete and incomplete), mineral gels such as alul~ lulll hydroxide,
30 surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. A monoclonal antibody to
an epitope of a neurite growth regulatory factor can be prepared by using any technique which
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CA 02221391 1997-11-17
5 provides for the production of antibody molecules by continuous cell lines in culture. These
include but are not limited to the hybridoma technique originally described by Kohler and
Milstein (1975, Nature 256:495-497), and the more recent human B cell hybridoma technique
(Kozbor et al., 1983, Immunology Today 4:72) and EBV-hybridoma technique (Cole et al., 1985,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In a particular
10 embodiment, the procedure described . may be used to obtain mouse monoclonal antibodies
which recognize arretin and its receptors.
The monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric
human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be
15 made by any of numerous techniques known in the art (.RTMq., Teng et al., 1983, Proc. Natl.
Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al.,
1982, Meth. Enzymol. 92:3-16). Chimeric antibody molecules may be prepared col-t~il-il-g a
mouse antigen-binding domain with human constant regions (Morrison et al., 1984, Proc. Natl.
Acad. Sci. U.S.A. 81 :6851, Takeda et al., 1985, Nature 314:452). A molecular clone of an
antibody to a neurite growth regulatory factor epitope can be prepared by known techniques.
Recombinant DNA methodology (see e.g., Maniatis et al., 1982, Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) may be used to
construct nucleic acid sequences which encode a monoclonal antibody olecule, or antigen
binding region thereof.
A monoclonal antibody to an epitope of arretin can be prepared by using any technique which
provides for the production of antibody molecules by continuous cell lines in culture. These
include but are not limited to the hybridoma technique originally described by Koler and
Milstein ((1975) Nature, 256:495-497), and the more recent human B cell hybridoma technique
(Kozbor et al., (1983) Immunology Today, 4:72) and EBV-hybridoma technique (Cole et al.,
(1985) In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp 77-96). In a
particular embodiment, the procedure described by Nobile-Orazio et al. ((1984) Neurology,
34:1336-1342) may be used to obtain antibodies which recognize recombinant Arretin (for
CA 02221391 1997-11-17
example of techniques, see Attia S. et al., (1993) J. Neurochem., 61: 718-726).
The monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric
human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be
made by any of numerous techniques known in the art (e.g. Tan et al., (1983) Proc. Natl. Acad.
Sci. U.S.A., 80: 7308-7312; Kozbor et al., (1983) Immunology Today, 4: 72-79; Olsson et al.,
(1982) Meth. Enzymol., 92: 3-16). Chimeric antibody molecules may be prepared cont:~ining a
mouse antigen-binding domain with human contact regions (Morrision et al., (1984) Proc. Natl.
Acad. Sci. U.S.A., 81: 6851; Takeda et al., (1985) Nature, 314: 452).
A molecular clone of an antibody to a Arretin epitope can be prepared by known techniques.
Recombinant DNA methodology may be used to construct nucleic acid sequences which encode
a monoclonal antibody molecule, or antigen binding region thereof (see e.g., Maniatis et al.,
(1982) In Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.).
For use, arretin antibody molecules may be purified by known techniques, such asimmunoabsorption or immunoaffinity chromatography, chromotographic methods such as HPLC
(high performance liquid chromatography), or a combination thereof, etc.
Arretin antibody fragments which contain the idiotype of the molecule can be generated by
known techniques. For example, such fragments include but are not limited to: the F (ab')2
fragment which can be produced by pepsin digestion of the antibody molecule; the Fab,
fragments which cen be generated by reducing the disulfide bridges of the F (ab')2 fragment, and
the two Fab or Fab fragments which can be generated by treating the antibody molecule with
papain and a reducing agent.
Monoclonal antibodies known to react with human arretin may be tested for their usefulness to
serve as arretin antagonists (Nobile-Orazio et al., (1984) Neurology, 34: 1336-1342; Doberson et
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CA 02221391 1997-11-17
al., (1985)Neurochem. Res., 10: 499-513).
Antibody molecules may be purified by known techniques, e.g., immunoabsorption or
immunoaffinity chromatography, chromatographic methods such as HPLC (high performance
liquid chromatography), or a combination thereof, etc.Antibody fragments which contain the
10 idiotype of the molecule can be generated by known techniques. For example, such fragments
include but are not limited to: the F(ab')<sub>2</sub> fragment which can be produced by pepsin
digestion of the antibody molecule; the Fab, fragments which can be generated by reducing the
disulfide bridges of the F(ab')<sub>2</sub> fragment, and the 2 Fab or Fab fragments which can be
generated by treating the antibody molecule with papain and a reducing agent.
7. Diagnostic, Therapeutic and Research Uses for each of these Components and the
Antibodies Directed Thereto
Arretin, its receptors, analogs, derivatives, and subsequences thereof, and anti-inhibitory protein
20 antibodies or peptides have uses in diagnostics. Such molecules can be used in assays such as
immunoassays to detect, prognose, diagnose, or monitor various conditions, diseases, and
disorders affecting neurite growth extension, invasiveness, and regeneration. In one embodiment
of the invention, these molecules may be used for the diagnosis of malignancies. Alternatively,
the CNS myelin associated inhibitory proteins, analogs, derivatives, and subsequences thereof
25 and antibodies thereto may be used to monitor therapies for diseases and conditions which
ltim~tely result in nerve damage; such diseases and conditions include but are not limited to
CNS trauma, (e.g. spinal cord injuries), infarction, infection, malignancy, exposure to toxic
agents, nutritional deficiency, paraneoplastic syndromes, and degenerative nerve diseases
(including but not limited to Alzheimer's disease, Parkinson's disease, Huntington's Chorea,
30 amyotrophic lateral sclerosis, progressive supra-nuclear palsy, and other dementias). In a specific
embodiment, such molecules may be used to detect an increase in neurite outgrowth as an
indicator of CNS fiber regeneration. For example, in specific embodiments, the absence of the
CNS myelin associated inhibitory proteins in a patient sample co~ lg CNS myelin can be a
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CA 02221391 1997-11-17
diagnostic marker for the presence of a malignancy, including but not limited to glioblastoma,
neuroblastoma, and melanoma, or a condition involving nerve growth, invasiveness, or
regeneration in a patient. In a particular embodiment, the absence of the inhibitory proteins can
be detected by means of an immunoassay in which the lack of any binding to anti-inhibitory
protein antibodies is observed. The immunoassays which can be used include but are not limited
to competitive and non-competitive assay systems using techniques such as radioimmunoassays,
ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, precipitation
reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A
immunoassays, immunoelectrophoresis assays, and immunohistochemistry on tissue sections, to
name but a few.
In accordance with another aspect of the invention, arretin and related compounds that retain the
arretin property of inhibiting neurone growth (herein referred to as arretin agonists) are used
therapeutically to treat conditions in which ~upples~ion of undesirable neuronal growth is
desired. These include for example the treatment of tumors of nerve tissue and of conditions
resulting from uncontrolled nerve sprouting such as is associated with epilepsy and in the spinal
cord after nerve injury. In one embodiment patients with neuroblastoma, and particularly with
neuropathies associated with circulating arretin antibody, can be treated with arretin or arretin
agonist.
Useful for nerve growth suppression are pharrnaceutical compositions that contain, in an amount
effective to suppress nerve growth, either arretin or a arretin agonist in combination with an
acceptable carrier. Arretin can be obtained either by extraction from myelin as described above
or, more practically, by recombinant DNA ~ s~ion of Arretin-encoding DNA, for example, in
the manner reported for MAG by Attia S., et al., J. Neurochem.,61, 718-726, 1993. Useful
arretin agonists are those compounds which, when added to the permissive substrate described
above, suppress the growth of neuronal cells. Particularly useful Arretin agonists are those
compounds which cause a statistically significant reduction in the number of neuronal cells that
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CA 02221391 1997-11-17
extend neurites, relative to control cells not exposed to the agonist. Candidate Arretin agonists
include fragments of Arretin that incorporate the ectodomain, including the ectodomainper se
and other N- and/or C-terminally truncated fragments of Arretin or the ectodomain, as well as
analogs thereof in which amino acids, e.g. from 1 to 10 residues, are substituted, particularly
conservatively, and derivatives of Arretin or Arretin fragments in which the N- and/or C-t~ rrnin~l
10 residues are derivatized by chemical stabilizing groups. Such Arretin agonists can also include
anti-idiotypes of Arretin antibodies and their binding fragments.
In specific embodiments of the invention, candidate Arretin agonists include specific regions of
the Arretin molecule, and analogs or derivatives of these. These can be identified by using the
same technologies described above for identification of Arretin regions that serve as inhibitors of
neurite outgrowth.
The Arretin related derivatives, analogs, and fragments of the invention can be produced by
various methods known in the art. The manipulations which result in their production can occur
at the gene or protein level. For example, Arretin-encoding DNA can be modified by any of
numerous strategies known in the art (Maniatis et al., Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982), such as by cleavage at
al)propliate sites with restriction endonuclease(s), subjected to enzymatic modifications if
desired, isolated, and ligated in-vitro.
Additionally, the Arretin-encoding gene can be mutated in-vitro or in-vivo for instance in the
manner applied fro production of the ectodomain, to create and/or destroy translation, initiation,
and/or terrnin~tion sequences, or to create variations in coding regions and/or form new
restriction endonuclease sites or destroy preexisting ones, to facilitate further in-vitro
30 modification. Any technique for mutagenesis known in the art cab be used, including but not
limited to, in-vitro site directed mutagenesis (Hutchinson, et al., J. Biol. Chem., 253,6551,
1978), use of TABTM linkers (Pharmacia), etc.
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CA 02221391 1997-11-17
5 For delivery of Arretin, Arretin agonist or Arretin antagonist, various known delivery systems
can be used, such as encapsulation in liposmes or semipermeable membranes, ~ les~,ion in
suitably transformed or transfection glial cells, oligodendroglial cells, fibroblasts, etc. according
to the procedure known to those skilled in the are (Lindvall et al., Curr. Opinion Neurobiol., _,
752-757, 1994). Linkage to ligands such as antibodies can be used to target delivery to myelin
10 and to other therapeutically relevant sites in-vivo. Methods of introduction include, but are not
limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and
intranasal routes, and transfusion into ventricles or a site of operation (e.g. for spinal cord
lesions) or tumor removal. Likewise, cells secreting Arretin antagonist activity, for example, and
not by way of limitation, hybridoma cells encapsulated in a suitable biological membrane may be
15 implanted in a patient so as to provide a continuous source of Arretin inhibitor.
In another specific embodiment, ligands which bind to arretin or its receptors can be used in
im~gin~ techniques. For example, small peptides (e.g., inhibitory protein receptor fragments)
which bind to the inhibitory proteins, and which are able to penetrate through the blood-brain
20 barrier, when labeled ~p~opliately, can be used for im~ging techniques such as PET (positron
emission tomography) diagnosis or scintigraphy detection, under conditions noninvasive to the
patient.
Neurite growth inhibitory factor genes, DNA, cDNA, and RNA, and related nucleic acid
25 sequences and subsequences, including complementary sequences, can also be used in
hybridization assays. The neurite growth inhibitory factor nucleic acid sequences, or
subsequences thereof comprising about at least 15 nucleotides, can be used as hybridization
probes. Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions,
disorders, or disease states associated with changes in neurite growth inhibitory factor expression
30 as described supra. For example, total RNA in myelin, e.g., on biopsy tissue sections, from a
patient can beassayed for the presence of neurite growth inhibitory factor mRNA, where the
amount of neurite growth inhibitory factor mRNA is indicative of the level of inhibition of
neurite outgrowth activity in a given patient.
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CA 02221391 1997-11-17
Therapeutic Uses of Arretin
CNS myelin associated inhibitory proteins of the present invention can be therapeutically useful
in the treatment of patients with malignant tumors including, but not limited to melanoma or
tumors of nerve tissue (e.g. neuroblastoma). In one embodiment, patients with neuroblastoma can
be treated with arretin or analogs, derivatives, or subsequences thereof, and the human functional
equivalents thereof, which are inhibitors of neurite extension.
In an alternative embodiment, derivatives, analogs, or subsequences of CNS myelin inhibitory
proteins which inhibit the native inhibitory protein function can be used in regimens where an
increase in neurite extension, growth, or regeneration is desired, e.g., in patients with nervous
system damage. Patients suffering from traumatic disorders (including but not limited to spinal
cord injuries, spinal cord lesions, or other CNS pathway lesions), surgical nerve lesions, damage
secondary to infarction, infection, exposure to toxic agents, malignancy, paraneoplastic
syndromes, or patients with various types of degenerative disorders of the central nervous system
(Cutler, 1987, In: Scientific American Medicines v. 2, Scientific American Inc., N.Y., pp.
11-1-11-13) can be treated with such inhibitory protein antagonists. Examples of such disorders
include but are not limited to Alzheimer's Disease, Parkinsons' Disease, Huntington's Chorea,
amyotrophic lateral sclerosis, progressive supranuclear palsy and other dementias. Such
antagonists may be used to promote the regeneration of CNS pathways, fiber systems and tracts.
Administration of antibodies directed to an epitope of, (or the binding portion thereof, or cells
secreting such as antibodies) can also be used to inhibit arretin protein function in patients. In a
particular embodiment of the invention, antibodies directed to arretin may be used to promote the
regeneration of nerve fibers over long distances following spinal cord damage.
Various delivery systems are known and can be used for delivery of arretin, related molecules, or
antibodies thereto, e.g., encapsulation in liposomes or semipermeable membranes, ~ es~ion by
bacteria, etc. Linkage to ligands such as antibodies can be used to target myelin associated
protein-related molecules to therapeutically desirable sites in vivo. Methods of introduction
-41 -
CA 02221391 1997-11-17
5 include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, oral, and intranasal routes, and infusion into ventricles or a site of operation (e.g.
for spinal cord lesions) or tumor removal. Likewise, cells secreting CNS myelin inhibitory
protein antagonist activity, for example, and not by way of limitation, hybridoma cells,
encapsulated in a suitable biological membrane may be implanted in a patient so as to provide a
10 continuous source of anti-CNS myelin inhibiting protein antibodies.
In addition, any method which results in decreased synthesis of arretin or its receptors may be
used to (limini~h their biological function. For example, and not by way of limitation, agents
toxic to the cells which synthesize arretin and/or its receptors (e.g. oligodendrocytes) may be
15 used to decrease the concentration of inhibitory proteins to promote regeneration of neurons.
Arretin Receptors
Arretin receptors as well as analogs, derivatives, and subsequences thereof, and anti-receptor
20 antibodies have uses in diagnostics. These molecules of the invention can be used in assays such
as immunoassays or binding assays to detect, prognose, diagnose, or monitor various conditions,
diseases, and disorders affecting neurite growth, extension, invasion, and regeneration. For
example, it is possible that a lower level of ~ ession of these receptors may be detected in
various disorders associated with enhanced neurite sprouting and plasticity or regeneration such
25 as those involving nerve damage, infarction, degenerative nerve diseases, or malignancies. The
CNS myelin associated inhibitory protein receptors, analogs, derivatives, and subsequences
thereof may also be used to monitor therapies for diseases and disorders which ultimately result
in nerve damage, which include but are not limited to CNS trauma (e.g. spinal cord injuries),
stroke, degenerative nerve diseases, and for malignancies.
The assays which can be used include but are not limited to those described above.
Arretin receptor genes and related nucleic acid sequences and subsequences, including
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CA 02221391 1997-11-17
5 complementary sequences, can also be used in hybridization assays, to detect, prognose,
diagnose, or monitor conditions, disorders, or disease states associated with changes in neurite
growth inhibitory factor receptor expression.
Arretin Receptors
Arretin receptors or fragments thereof, and antibodies thereto, can be therapeutically useful in the
treatment of patients with nervous system damage including but not limited to that resulting from
CNS trauma (e.g., spinal cord injuries), infarction, or degenerative disorders of the central
nervous system which include but are not limited to Alzheimer's disease, Parkinson's disease,
15 Huntington's Chorea, amyotrophic lateral sclerosis, orprogressive ~ul)l~luclearpalsy. For
example, in one embodiment, arretin receptors, or subsequences or analogs thereof which contain
the inhibitory protein binding site, can be ~(lmini~tered to a patient to "compete out" binding of
the inhibitory proteins to their natural receptor, and to thus promote nerve growth or regeneration
in the patient. In an alternative embodiment, antibodies to the inhibitory protein receptor (or the
20 binding portion thereof or cells secreting antibodies binding to the receptor) can be ~(lministered
to a patient in order to prevent receptor function and thus promote nerve growth or regeneration
in the patient. Patients in whom such a therapy may be desired include but are not limited to
those with nerve damage, stroke, or degenerative disorders of the central nervous system as
described supra.
Various delivery systems are known and can be used for delivery of arretin receptors, related
molecules, or antibodies thereto, e.g., encapsulation in liposomes, expression by bacteria, etc.
Linkage to ligands such as antibodies can be used to target arretin-related molecules to
therapeutically desirable sites in vivo. Methods of introduction include but are not limited to
30 intr~rl( rm~l, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intranasal routes,
and infusion into ventricles or a site of tumor removal.
The present invention is directed to genes and their encoded proteins which regulate neurite
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CA 02221391 1997-11-17
5 growth and the diagnostic and therapeutic uses of such proteins. The proteins of the present
invention (arretin and its receptors) include proteins associated with central nervous system
myelin with highly nonpermissive substrate properties, termed herein neurite growth inhibitory
factors.
10 The present invention is also directed to antibodies to and peptide fragments and derivatives of
the neurite growth inhibitory proteins and their therapeutic and diagnostic uses. These antibodies
or peptides can be used in the treatment of nerve damage resulting from, e.g., trauma (e.g., spinal
cord injuries), stroke, degenerative disorders of the central nervous system, etc. In particular,
antibodies to arretin proteins may be used to promote regeneration of nerve fibers. In a specific
15 embodiment of the invention, monoclonal antibodies directed to arretin and/or its receptors may
be used to promote the regeneration of nerve fibers over long distances following spinal cord
damage.
The present invention is described in further detail in the following non-limiting examples. It is
20 to be understood that the examples described below are not meant to limit the scope of the
present invention. It is expected that numerous variants will be obvious to the person skilled in
the art to which the present invention pertains, without any departure from the spirit of the
present invention. The appended claims, properly construed, form the only limitation upon the
scope of the present invention.
EXAMPLES
Example I: Isolation and characterization of a novel neurite growth inhibitory
30 molecule from m~mm~ n central nervous system myelin
Animals.
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ICR mice and Wistar rat embryos were obtained from the animal facilities at Charles River.
Materials.
The following lectins were purchased from Sigma: Maclura pomifera (osage orange), Arachis
hypogaea (PNA), Ulex europaeus (gorse), Phaseolus vulgaris PHA-L (red kidney bean), Triticum
vulgaris (wheat germ), and Concanavalin A (jack bean). T, ~tninjn from EHS sarcoma,
Poly-L-o.l.ill,ine (PORN), Poly-L-lysine (PLL), Chondroitinase ABC (chondroitin ABC lyase,
E.C. 4.2.2.4. from Proteus vulgaris, protease-free), heparinase and PNA agrose beads were also
purchased from Sigma. Horseradish peroxidase (HRP)- conjugated secondary antibodies to
rabbit, rat or mouse IgG and IgM were purchased from Amersham and Jackson Labs.
Antibodies.
Monoclonal antibody 473-HD is a mouse IgM against a chondroitin sulphate epitope on mouse
brain proteoglycans (Faissner et al., J. Cell Biol., 126, 783-799, 1994). Rabbit polyclonal
anti-versican antibodies were generated against recombinantly expressed human versican fusion
proteins. We used monoclonal anti-L2 antibody (412) from rat (Kruse et al., Nature, 316, 146-
148, 1985) and polyclonal antibody 3F8 against phosphacan (Engel et al., J. Comp. Neurol., 366,
34-43, 1996; Meyer-Puttlitz et al., J. Comp. Neurol., 366, 44-54, 1996).
Multiple neurite growth inhibitory activities are present in extracts of CNS myelin after DEAE
chromatography. We have previously shown that two peaks of neurite growth inhibitory activity
are present in fractions of myelin extracts following DEAE chromatography (McKerracher et al.,
Neuron, 13, 805-811, 1994). The largest of these peaks is associated with the earlier fractions
eluted off the DEAE column by a 0.2 to 2 M gradient. A substantial proportion of the inhibitory
activity in this peak is associated with myelin-associated glycoprotein (MAG). The inhibitory
activity in column fractions was assayed by an in vitro bioassay using a neuronal cell line
(NG108-15). These results suggest that molecule(s) other than MAG also contribute to the
inhibitory activity associated with CNS myelin (Fig. 1).
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Identification of a 70k Da protein associated with CNS myelin
In addition to MAG and the NI35/250 inhibitory molecules associated with myelin
(McKerracher,
et al., 1994; Mukhopadhyay et al., Neuron, 13, 757-767, 1994; Schwab et al., Ann. Rev.
Neurosci., 16, 565-595, 1993), three extracellular matrix, molecules namely, tenascin-C (TN-C),
tenascin-R (TN-R) and chondroitin sulfate proteoglycans (CSPGs) that are distributed in many
CNS and non-CNS tissues are also known to have neurite growth inhibitory activity (Schachner
et al., 1994). We therefore investigated which of these inhibitory molecules are found in the two
inhibitory peaks obtained after DEAE chromatography of CNS myelin extracts. DEAE column
chromatographic fractions that contained the first (fractions 10) and second (fraction 26)
inhibitory peaks were subjected to SDS-PAGE on a 6-16% polyacrylamide gradient gel under
reducing conditions. These gels were either silver stained (Fig. 2A) or Western blotted with
anti-MAG, TN-C, TN-R, and a monoclonal antibody against chondroitin sulfate (mAb 473) (Fig.
2B-E). The silver stained gels (2A) showed any bands. Anti-MAG antibody recognizes a 100
kDa band that is highly enriched in fractionlO but is much weaker in fractions 26 and 32 (Fig
2B). The intensity of the 200 and 220 kDa bands labelled with anti-TN-C was similar to that of
the MAG antibody, i.e., enriched in fraction 10 (Fig. 2C). However, the 160 and 180 kDa bands
recognized by the anti-TN-R antibody were present only in the total myelin extract and in
fraction 10 (Fig. 2D). Interestingly, the anti-CS mAb 473 recognized 70 kDa band and a slightly
small minor band in fractions 26 and 32 but not in the octylglucoside extract of myelin and/or in
fraction 10. This shows that these components can only be detected immunochemically after
substantial enrichment during the purification steps. These experiments show that MAG, TN-C
and TN-R may contribute to the inhibitory effects of the first peak, and that MAG, TN-C and the
70 kDa bands may contribute to the second inhibitory peak. Western blots of samples of brain
membranes probed with mab 412 that recognizes the HNK-l epitope indicates that this
carbohydrate epitope is not found in the 70 kDa components (data not shown).
Enzymatic hydrolysis with chondroitinase ABC and heparinase
Proteins were treated with chondroitinase ABC (0.02 U/ml) in 50 mM Tris-acetate (pH 8.0) for
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CA 02221391 1997-11-17
2.5 h at 37~c in the presence of protease inhibitors (5 mM benzamidine, 1 mM iodoacetamide and
5 mM p-tosyl-L-lysine chloromethyl ketone, sodium salt). Heparinase digestion was done
according to the m~nllf~cturer's instructions.
Purification of Arretin.
Preparation of myelin extracts and their fractionation by DEAE chromatography have been
described (McKerracher et al., 1994; see Fig. l). For further purification by lectin affinity
chromatography, PNA-conjugated agarose beads (1.2 ml) were used. DEAE chromatographic
fractions number 20 to 34 (2 ml each) were pooled (about 30 ml), diluted with 3 volume of H2O,
and loaded on the PNA-agarose colurnn. The flow-through was reloaded three times, and the
column was subsequently washed with 12 ml Hepes buffer (pH7.5, 0.08% Sodium azide, 10 mM
Hepes, 0.15 mM NaCl, 0.1 mM Ca2+, and 0.01 mM Mn2+), followed by 12 ml of a high salt
buffer (pH7.5, 2 M NaCl, and 20 mM Triethanolamine). The column was eluted with 20 ml of
elution buffer (2 M NaCl, 20 mM Trithanolamine, pH7.5, and 0.5 M D-galactose). Appropriately
pooled fractions were dialysed against 1000 ml of H2O at 40~C, lyophilised, and dissolved in 1
ml of H2O, such that the final concentration was about 0.16 M NaCl, 1.6 mM Trithanolamine,
pH7.5, and 0.04 M D-galactose. Samples were aliquoted, and stored at -70~c. The protein profile
was determined by SDS-PAGE on gradient gels (6 to 16% polyacrylamide) (T ~rnmli, U.K.,
Nature, 277, 680-685, 1970), by two-dimensional electrophorens and by Western blots (Towbin
et al., Proc. Nat, Acad. Sci. USA., 76, 4350-4354, 1979). Protein concentrations were estimated
according to Bradford (1976).
Reactivity of Arretin with lectins.
Proteins tranferred to membranes were blocked with 2% bovine serum albumin (BAS) in TBS
buffer (20 mM Tris-HCl, 500 mM NaCl, pH7.5) for 1 h, and incubated separately with 6g/ml of
different HRP- or biotin-conjugated lectins for 2 h. The membranes were washed with TTBS (20
Mm Tris-HCl, 500 mM NaCl, 0.05% Tween-20, pH7.5) for lh and complexes were detected by
ECL (DU PONT) or the AP-ABC (VECTOR) Kit according to the m~llf;~cturer's instructions.
As positive controls for lectin binding, several sugars, including galactose, glucose, glucosamine,
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CA 02221391 1997-11-17
5 galactosamine, fucose, and mannose (at 20 mg/ml), were applied as spots on nitrocellulose.
Purification by Lectin affinity chromatography
To further purify the 70 kDa components from DEAE fractions cont~ining the
second inhibitory peak, we screened the ability of the components to bind the following
10 lectins: Maclura pomifera (osage orange), Arachis hypogaea (PNA), Ulex europaeus uea I (gorse
or furze). Phaseolus vulgaris (PHA-L), Triticum vulgaris (wheatgerm agglutinin) and
Concanavalin A (Con-A). Nitrocellulose membranes electro blotted with pooled DEAE fractions
20 to26 after protein separation by SDS-PAGE were probed with the various lectins. All the
lectins except Con-A bound only to the 70 kDa bands (not shown).
We next tested whether the 70 kDa components could be purified by binding
to lectin. For this, PNA-conjugated agarose beads were chosen. Fractions 20 to 26 obtained
from DEAE column chromatography of bovine CNS myelin extracts were pooled and incubated
with PNA-conjugated beads in an Eppendorf tube. After washing the beads, the proteins
20 bound to the PNA-beads were separated by SDS-PAGE, electrophoretically blotted onto
nitrocellulose membrane and probed with anti-MAG, TN-C and the 473 antibodies. As expected
only the 70 kDa bands were recognized by the mAb 473. No labeling was observed with the
other two antibodies, indicating that PNA lectin can be used to separate the 80 kDa molecule
from MAG and TN-C (not shown).
A two-step purification of the 70 kDa components was therefore attempted. Octylglucoside
extracts of bovine CNS myelin were passed though a DEAE column, and the material eluted by a
NaCl gradient, and fractions 20-34 were pooled. The pooled fractions were then subjected to
PNA-affinity chromatography. The material eluted from the PNA column was separated on a
30 SDS-PAGE gradient gel (6-16% acrylamide) under reducing conditions. The gels were then
stained with silver, or Western blotted and probed with anti-MAG, TN-C and 473 antibodies. A
70 kDa doublet was seen after Amido black staining (Fig. 5A). This major band was recognized
only by the 473 anti-CS antibody (Fig. 3A), but not by anti-MAG (Fig. 3B) or anti-TN-C
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CA 02221391 1997-11-17
5 antibodies (not shown). The minor component just below the major band was not visible in this
preparation.
The 70 kDa components are novel phosphocan-versican-related molecules. We further
investigated whether the 70 kDa bands purified from CNS myelin shared epitopes with other
10 known CSPGs. On Western blots of the DEAE chromatographic fractions the 70 kDa bands
also reacted with polyclonal antibodies against phosphacan and recombinant versican (Fig. 4A
and B). Both these antibodies plus the 473 anti-CS recognized the 70 kDa PNA affinity purified
polypeptides (Fig. 4C, D, E). After chondroitinase ABC treatment, the major 70 kDa proteins
were found to have an apparent Mr of 50 kDa (Fig. 5A) which did not react with the anti-CS
15 mAb 473 (not shown), but did react with anti-phosphacan (Fig. 5B) and anti-versican. Since
native phosphacan has a molecular weight of 500-600 kDa (core protein 400 kDa), and versican
is a very large proteoglycan with a molecular weight of 900 kDa (core protein 400 kDa), the 70
kDa components that we have isolated from CNS myelin are likely to be novel proteins. We call
these proteins arretin (collectively). The 2 bands may represent 2 isoforms, or the smaller
20 component may be an altered version of the larger, due to degradation.
The 70 kDa proteins inhibit neurite growth. The present invention involved a test that examined
effects of the 70 kDa myelin-derived proteins in mod~ tin~ neurite growth from rat
hippocampal and cerebellar granule celleurons. The 70 kDa proteins inhibited neurite growth
25 from neonatal rat cerebellar and hippocampal neurons (Figs. 7 and 8), as well as from cultured
NG108-15 cells (Fig. 9). This inhibitory activity was lost after heat denaturation. These result
indicate that novel myelin-associated 70 kDa proteins are inhibitors of neurite growth, and are
likely to be largely responsible for the activity associated with the second inhibitory peak in
fractions obtained after DEAE separation of CNS myelin extracts. The present invention
30 comprises these new inhibitors collectively termed as arretin.
Assays for repulsion of growth cones and cell bodies.
Tissue culture dishes (Becton Dickinson) with 24 wells were coated with methanol-solubilized
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CA 02221391 1997-11-17
5 nitrocellulose according to Lagenaur and Lemmon (1987) and air-dried in a sterile hood. For
assays addressing the effect of arretin on growth cones, nitrocellulose and poly-L-lysine (PLL
0.01%) coated dishes were used as described (Xiao et al., Neurosci., 8, 766-782, 1996). The
dishes were washed three times with PBS and dried in a sterile hood. Different test proteins
(arretin, denatured (80~c for 30 min) arretin, TN-R, and l~rninin), each at concentrations of 2 nM,
10nM, and 50nM, were applied in duplicate as 2.5 111 single spots to the dishes and incubated
overnight at 37~c in a humidified atmosphere.
Determin~tion of subskate coating efficiency was been described by Xiao et al., 1996. Before
plating the NG108 cells or cerebellar neurons, the dishes were washed with Ca 2+_ and Mg2+-free
Hanks' balanced salt solution (CMF-HBSS). Explants were prepared from cerebella of 6 to
7-day-old mice and m~int~ined in a chemically defined medium (Fischer et al., J. Neurosci., 6,
605-612, 1986; Fischer, G., Neurosci. Lett., 28, 325-329, 1982). Explants were allowed to grow
neurites for 72 h and then fixed with glutaraldehyde in PBS at a final concentration of 2.5%.
After fixation, cultures were stained with 0.5% toluidine blue (Sigma) in 2.5% sodium carbonate,
washed five times with water and air dried. All experiments were performed at least three times.
Assay for neurite outgrowth. Hippocampal neurons from 18- to 1 9-day-old rat embryos were
prepared as described (Keilhauer et al., Nature, 316, 728-730, 1985; Lochter et a/., J. Cell Biol.,
1 13, 1159-1171, 1991; Dorries et a/., 1995 ?). For the assays on neurite outgrowth, hippocampal
neurons were m~int~ined in chemically defined medium (Rousselet et al., Ann. Rev. Cell Biol.,
129, 495-504, 1988; Lochter and Schachner, J. Neurosci., 13, 3986-4000, 1993; Xiao et a/.,
1996).
Briefly, 96-well plates (Nunc) were pretreated with 5g/ml poly-L-ornithine (PORN) for 1 to 2
hours at 37~c, washed twice with water and air-dried. Proteins at concentrations of 2 nM, 10 nM,
and 50 nM were coated on the dried surfaces overnight at 37~c in a humidified atmosphere.
Determin~tion of substrate coating efficiency as described Xiao et al., 1996. The plates were
washed three times with CMF-HBSS and hippocampal neurons prepared from 18- to l9-day-old
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CA 02221391 1997-11-17
rat embryos (Keilhauer et al., 1985; Lochter et al., 1991; Dorries et al., 1995) were plated at a
density of 3,000 cells per well in 100~11 a chemically defined medium (Rousselet et al., 1988;
Lochter and Schachner, 1993; Xiao et al., 1996). After 12 h, cells were fixed without a
preceding washing step by gentle addition of 25% glutaraldehyde to a final concentration of
2.5%. After fixation, cultures were stained with toluidine blue and morphological parameters
were quantified with an IBAS image analysis system. For morphometric analysis, only cells
without contact with other cells were evaluated. Neurites were defined as those processes with a
length of at least one cell body diameter. The total neurite length per cell was determined by
analysing 50 cells in each of two wells. To determine the number of cells with neurites, 100
neurons in each of two wells were counted per experiment. Raw data from at least three
independent experiments were analyzed by ANOVA and by the Newman-Keuls test with P <
0.05 and P < 0.01 being considered significant or highly significant, respectively. All graphs
comprise data derived from at least three independent experiments.
From the foregoing description, one skilled in the art can easily ascertain the essential
characteristics of this invention, and without departing from the spirit and scope thereof, can
make various changes and modifications to the invention to adapt it to various usages and
conditions. Such changes and modifications are properly, equitably, and intended to be within
the full range of equivalence of the following claims.
