Note: Descriptions are shown in the official language in which they were submitted.
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CNS NEURITE OU'rGROWTH MODULATORS, AND COMPOSITIONS,
CELLS AND METHODS EMBODYING AND USING SAME
BACKGROUND OF THE INVENTION
Field of the Invention
5 This invention relates generally to the modulation of neural growth in the central
,, nervous system, and more particularly to methods and associated agents, constructs
and compositions for improving CNS neural growth. Specifically, the invention
relates to the use of cellular adhesion molecules, and preferably neural cell
a&esion molecules such as Ll, to foster and improve such neural growth.
DescriPtion of the Related Art
The ability of neurons to extend neurites is of prime importance in establishingneuronal connections during development. It is also required during regc.l~.alion to
re-establish connections destroyed as a result of a lesion.
Neurites elongate profusely during development both in the central and peripheral
15 nervous systems of all animal species (Cajal (1928) Degeneration and regeneration
in nervous system~ Oxford University Press. London). This phenomenon pertains
to axons and dendrites. However, in adults. axonal and dendritic regrowth in thecentral nervous system is i.l-,lea illgly lost with evolutionary progression.
In the peripheral nervous system, after infliction of a lesion, axons of all vertebrate
20 species are able to regrow (Cajal (1928); Martini (1994) J. Neurocytol. 23:1-28).
However, in m~mm~l~, neurite regrowth following damage is limited to neuritic
sprouting. Regrowth of neuronal processes is, however, possible in lower
vertebrate species (Stuerrner et al. (1992) J. Neuro~iol. 23:537-550). In contrast, in
the central nervous system, most. if not all neurons of both higher and lower
25 vertebrate adults possess the potential for neurite regrowth (Aguayo (1985) "Axonal
regeneration from injured neurons in the adult mammalian central nervous svstem."
In: Synaptic Plasticitv (Cotman. C.~., ed.) ~ew York. The Guilford Press, pp.
457-484-)
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Glial cells are the decisive determin~nt~ for controlling axon regrowth. M~mm~ nglial cells are generally permissive for neurite outgrowth in the central nervous
system during development (Silver et al. (1982) J. Comp. lVeurol. 210:10-29; Miller
et al. (1985) Del,elop. Biol. 111:35-41; Pollerberg et al. (1985) J. Cell. Biol.101:1921-1929) and in the adult peripheral nervous system (Fawcett et al. (1990)Annu. Rev. Neurosci 13:43-60). Thus, upon infliction of a lesion, glial cells of the
adult m~mm~ n pelil,he.al nervous system can revert to some extent to their
earlier neurite outgrowth-promoting potential, allowing them to foster regell~.ation
(Kalderon (1988) J. Neurosci Res. 21:501-512; Kliot et al. "lnr~-ce~l regeneration of
dorsal root fibres into the adult m~mm~ n spinal cord," In: Current Issues in
Neural Regeneration, New York, pp. 311-328; Carlstedt et al. (1989) Brain Res.
Bull. 22:93-102). Glial cells of the central nervous system of some lower
vertebrates remain permissive for neurite regrowth in adulthood (Stuermer et al.(1992) J. .~eurobiol. 23:537-550). In contrast, glial cells of the central nervous
system of adult m~rnmzll~ are not conducive to neurite regrowth following lesions.
Several recognition molecules which act as molecular cues underlying promotion
and/or inhibition of neurite gro~th have been identified (Martini (1996). Among
the neurite outgrowth promoting recognition molecules, the neural cell adhesion
molecule Ll plavs a prominent role in me~ tin~ neurite outgrowth (S~h~rhner
(1990) Seminars in the Neurosciences 2:497-507). Ll-dependent neurite outgrowth
is m~ t~cl by homophilic interaction. Ll enh~n~es neurite outgrowth on L1
cs~ g neurites and Schwann cells, and Ll transfected fibroblasts (Bixby et al.
(1982) Proc. Nat'l Acad Sci. U.S.A. 84:2555-2559; Chang et al. (1987) J: Cell.
Biol. 104:355-36~; Lagenaur et al. (1987) Proc. hatl. Acad. Sci USA 84:7753-
7757; Seilheimer et al. (1988) J. Cell. Biol. 107:341-351; Kadmon et al. (199Oa) J.
Cell. Biol. 110: 193-208; Williarns et al. (1992) J. Cell. Biol. 119:883-892).
Expression of L 1 is enhanced dramatically after cutting or crushing peripheral
nerves of adult mice (Nieke et al. (1985) Di.~erentiation 30:1~1-151; Mar~ini et al.
(1994a) Glia 10:70-74). Within two days Ll accumulates at sites of contact
between neurons and Schwann cells being concentrated mainly at the cell surface
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WO 96t329S9
of Schwann cells but not neurons (~Iartini et al. (1994a)). Furthermore, the
homophilic bindinP ability of Ll is enhanced by molecular association with the
neural cell adhesion molecule N-CAM. allowing binding to occur through
homophilic ~Csict~nce (Kadmon et al. (199Oa); Kadmon et al. (199Ob) J. Cell Biol.
110:209-218 and 110:193-208; Horstkorte et al. (1993) J. Cell. Biol. 121:1409-
1421). Besides its neurite outgrowth promoting ~lo~,Lies~ L1 also participates in
cell adhesion (Rathjen et al. (1984) El~fBO J. 3:1-10; Kadmon et al. (1990b) J.
Cell. Biol. 1 10:209-218; Appel et al. (1993) J. Neurosci., 13 :4764-4775), granule
cell mi,~ration (Lindner et al. (1983) Na-ure 305:4~7-430) and myelination of axons
10 (Wood et al. (1990) J. Neurosci 10:3635-3645).
Ll consists of six immunoglobulin-like domains and five fibronectin type III
homologous repeats. Ll acts as a signal tr~n~ cer, with the recognition process
being a first step in a complex series of events leading to changes in steady state
levels of intracellular m~s.~en~ers. The latter include inositol phosphates, Ca2+, pH
15 and cyclic nucleotides (Schuch et al. (1990) Neuron 3:13-20; von Bohlen und
Hallbach et al. (199'7) Eur. J. Neurosci. 4:896-909: Doherty et al. (1992) Curr.Opin. Ne2~robiol. 2:593-601) as well as changes in the activities of protein kinases
such as protein kinase C and pp60C-s~c (Schuch et al. (1990) Neuron 3:13-20; Atashi
et al. (1992) Neuron 8:831-842). Ll is also associated with a casein type II kinase
20 and another unidentified kinase which phosphorvlates Ll (Sadoul et al. (1989) J.
Neurochem 328:251-254). L1-mediated neurite outgrowth is sensitive to the
blockage of L type Ca'' channels and to pertussis toxin. These fin~ling~ indicate
the il.lpolL~-ce of both Ca~' and G proteins in L1-mediated neurite outgrowth
(Williarns et al. (199'7) J. Cell. Biol. 119:883-89'7). Ll is also present on
25 proliferating, imm~hlre astrocytes in culture and neurite outgrowth is promoted on
these cells far better than on differenti~t~1 L1 imm~n-neg~tive astrocytes (Saad et
al. (1991) J. Cell. Biol. 115:473-484). ~n vivo, however, astrocytes have been
found to express L 1 at any of the developmental stages e~;~min.-~1 from embryonic
day 13 until adulthood (Bartsch et al. (1989) J.Comp. Neurol 284:451-462: and
30 unpublished data).
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Given the capabilitv of Ll to promote neurite outgrowth. it is pertinent to
investi~te whether astrocytic exyre~ion of Ll and other members of the
immunoglobulin s-lptlr~lily to which Ll belongs. may overcome potentially
inhibitory molecular cues reported to be present on glial cells and myelin in the
adult central nervous system (S~h~rhner et al., Perspectil,es in Developm. .,
Nez~robiol. in Press; Schwab et al. (1993) Ann. Re~ eurosci. 16:~65-595). This is
of particular relevance to the development of effecti~,e strategies for the l,c~ t
of debilitation caused by the malformation of or injury to neural tissues of theCNS, and it is toward such objectives that the present invention is directed.
SUMMARY OF THE rNVENTION
In accordance with the present invention, an agent and corresponding methods aredisclosed for the modulation of neural growth and particularly, such growth as can
be promoted in the co~ lent of the central nervous system (CNS), and
specifically~ in myelinated nerve tissue. The agents of the present invention are
15 notable in their ability to promote such neural _rowth in an environment that has
been traditionallv viewed as inhibitory to the growth promoting stimulus of known
neurite outgrou,th factors. Specifically, this inhibitory environment includes
inhibitory molecular cues which are present on glial cells and myelin the central
nervous system.
20 The agents of the present invention are broadly selected from a group of celladhesion molecules, and more preferably neural cell adhesion molecules. Most
preferably. the agents of the present invention are selected from the group of
molecules belonging to the immunoglobulin superfamily, and particularly to thosemembers that mediate Ca'~-independent neuronal cell adhesion, of which L1, N-
:~ CAM and myelin-associated glycoprotein are particular members. Other cell
adhesion molecules which may also influence CNS neural gro~th include l~minin~
fibronectin~ N-cadherin, BSP-2 (mouse N-CAM)~ D-2, ~2~-lA6-Al, Ll-CAM,
NILE, Nr-CArvI. TAG- 1 (axonin- 1), Ng-CAM and F3/F 1 1.
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In a further aspect of the present invention~ the agents of the invention belong to a
new family referred to herein as the LI family of neural recognition molecules.
Tnis fannily includes Ll, NgCAM, neurofascin, Drosophila neuroglian, zebrafish
L l . l and Ll.2. and others. This group of agents all demonstrate the Ig-liKe
S domains and FN-like repeats that are characteristic of Ll, and in this connection,
exhibit a remarkable colinearity, a high degree of N-glycosidically linked
carbohydrates, which include the HNK-1 carbohydrate structure, and a pattern of
protein fr~ment~ comprising a major 185 kD band and smaller bands of 165 and
125 kD.
10 The agents of the present invention also include fragments of cell adhesion
molecules and cognate molecules, congeners and mimics thereof which modulate
neurite growth in the CNS. In particular. the agents include molecules w'nich
contain structural motifs characteristic of extracellular matrix molecules, in
particular the fibronectin type III homologous repeats and immunoglobulin-like
15 domains. Preferably, these .L~u~iluldl motifs include those structurally similar to
fibronectin type III homologous repeats 1-2. and immnnoglobulin-like domains I-II,
III-IV and V-VI.
The invention extends to methods of promoting and enhancing neural regeneration
in vivo, and to the corresponding genetic constructs. such as plasmids. vectors~20 transgenes, and the like, and to pharm~rentical compositions, all of which may be
used to accomplish the objectives of such methods. More specifically, the agentsof the present invention may be prel,~ed as vectors or plasmids, and introduced
into neural cells located at a site in the CNS where regeneration is needed, forexample, by gene therapy techni~ues, to cause the c;~uie~.~.ion of an agent of the
25 present invention and to thereby promote the re~uisite neural growth. Anotherstrategy contemplates the formulation of one or more of the appropriate agents in a
composition that may likewise be directly delivered to a CNS site~ as by parenteral
~lmini~;tration. As certain of the agents. such as Ll, have demonstrated homophilic
binding, the ~lmini~tration of such a composition may serve the purpose of
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inhibiting rather than promoting neural growth. This effect may be desirable in
particular instances where unwanted or uncontrolled growth may occur or is
occurring, and therefore the invention extends to this use as well.
Correspondingly, the capability of the agents to engage in homophilic binding
5 renders antagonists to the agents. including antibodies thereto, capable of acting as
agonis~s, and thereby participating in the promotion of neural growth and
regeneration. Thus. the invention extends to the ~re~.~dlion of a~op~iate
constructs and compositions CO~ g the antibodies to the agents, for the
th~ uLic purposes set forth herein. Also, and as demonstrated later on herein,
10 antibodies to Ll, for example. may serve as part of a drug discovery assay or the
like, to identify further agents that may possess activity and utility both ~ gnostic
and therapeutic, in accordance with the present invention. Particularly, and as
illustrated later on herein with reference to the isolation and characterization of
CHLl, an L1 analog, antibodies such as polyclonal antibodies, may be used to
15 identify further members of the Ll CAM family. and the invention accordingly
extends to such CAM members as are isolated by use of such antibodies
The in~ention also covers diagnostic applications. where for example, it is desirable
to assess the potential for or actual development of CNS neural growth by the
detection and measurement of the presence. amount or activity of one or more of
20 the agents of the invention. Likewise, and as described hereinafter, the invention
also extends to assays, including drug discovery assays, that capitalize on the
activity of the agents of the present invention in the modulation of CNS neural
growth. For example, prospective dru_s may be tested for CNS neural growth
modulation by means of an assay cont~ining an agent of the invention, or a cell
25 line or culture developed in conjunction herewith mav serve as the assay system.
Briefly, the present invention also features transgenic mouse lines expressing a t
neural adhesion molecule in differentiated ~strocvtes an'd glial cells, and cells ~nd
tissues derived therefrom. In particular. the neural adhesion molecule is Ll. The
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astroglial Ll t~ plession çnh~nres neurite outgrowth on central nervous system
tissue derived from these transgenic mice.
Also as discussed~ the invention features methods for enhancing neuronal ouL,lowLl
of CNS neurons, for enhancing memor,v and for increasing synaptic efficacy, as
o 5 measured by stabilization of long term potentiation, and other similarly useful
methods. Also featured are methods of testing drugs and other manipulations
which modulate the effects of the neural adhesion molecule, and assay systems
suitable for such methods.
Accordingly, it is a principal object of the present invention to provide a tr~ncgenic
m~mm~l the glial cells of which express an exogenous neural adhesion molecule.
A funher object of the invention is to provide a cell culture cont~ining the glial
cells of the transgenic m~mm~l
Yet another object of the invention is to provide a cell culture system co.,l~;"il-p
lesioned or unlesioned optic nerves or other pans of the nervous system of the
transgenic ms~mm~l
Still a further object of the invention is to provide a method for enhancing neuronal
outgrowth of CNS neurons, which includes culturing the neurons on the glial cellculture system.
A further object of the invention is to provide a method for enhancing neuronal
outgrowth of CNS neurons, which includes culturing the neurons on the optic nerve
or other parts of the nervous system placed in the cell culture system.
A still further object of the invention is to provide a method for enh~ncin~ -
neuronal outgrowth of CNS neurons. which includes the secretion of neural
adhesion molecule bv implanted cells.
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Another object of the invention is to provide a method for enhancing memory,
which includes ~1mini.ctering to the brain of a m~mm~l in need of memory
enhancemPnt an amount of the cells of the glial cell culture system effective toenhance the memory of the m~mm~l .
5 Yet another object of the invention is to provide a method for enhancing memory,
including ~lminictPring to the brain of a m~mm~l in need of memory enhancement,
an arnount of the cells of the optic nerve or other parts of the nervous system
placed in the cell culture system effective to Pnh~nce the memory of the m~mm~l
A still further object of the invention is to provide a method for enhancing
10 memory, including delivering to the glial cells of the brain of a m~mm~l in need of
such memory enhancement. a vector which allows for the e;~ sion of a neural
adhesion molecule in the glial cells.
A further object of the invention is to provide a method for enhancing memory,
which includes the secretion of neural adhesion molecule by implanted cells.
15 Another object of the invention is to provide a method for increasing synaptlc
efficacy in the CNS of a m~mm~l in need of such an increase, including
~lmini.~tering to the brain of the m~mm~l, an amount of the cells of the glial cell
culture system effective to increase synaptic efficacy in the brain of the m~mm~l
Yet a further object of the invention is to provide a method for increasing synaptic
20 efficacy in the CNS of a m~mm~l in need of such an increase, including
~lmini~tPring to the brain of the m~mm~l, an arnount of the cells of the optic nerve
or other parts of the nervous system placed in the cell culture system effective to
increase synaptic efficacy in the brain of the mzlmm~
A still further obiect is to provide a method for increasing synaptic efficacy in the
25 CNS of a m~mm~l in need of such an increase which includes delivering to the
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glial cells of the brain of a m~mm~l in need of such enhancement, a vector whichallows for the expression of a neural adhesion molecule in the glial cells.
A further object of the invention is to provide a method for increasing synapticefficacy, which includes the secretion of neural adhesion molecule by implanted
5 cells.
Another object of the invention is to provide a method of testing the ability of a
drug or other entity to modulate the activity of a neural adhesion molecule, which
includes adding CNS neurons to the glial cell culture system; adding the drug under
test to the cell culture system; measuring the neuronal outgrowth of the CNS
lO neurons; and correlating a difference in the level of neuronal outgrowth of cells in
the presence of the drug relative to a control culture to which no drug is added to
the ability of the drug to modulate the activity of the neural adhesion molecule.
Another object of the invention is to provide a method of testing the ability of a
drug or other entity to modulate the activity of a neural adhesion molecule which
15 includes adding CNS neurons to the optic nerve or other parts of the nervous
system cell culture system; adding the drug under test to the cell culture system;
measuring the neuronal outgrowth of the CNS neurons; and correlating a difference
in the level of neuronal outgrowth of cells in the presence of the drug relative to a
control culture to which no drug is added to the ability of the drug to modulate the
20 activity of the neural a&esion molecule.
Yet another object of the invention is to provide an assay system for screening
drugs and other agents for ability to modulate the production of a neural adhesion
molecule, which includes the ~lial cell culture system; and CNS neurons added tothe cell culture system.
2~ A further object of the invention is to provide an assay system for screening drugs
and other agents for ability to modulate the production of a neural adhesion
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molecule7 which includes culturing the ~llal cell culture system inoculated with a
drug or agent; adding CNS neurons to the cell culture system, and ex~minin~
neuronal outgrowth to determine the effect of the drug thereon.
Yet another object of the invention is to provide an assay system for screening
5 drugs and other agents for ability to modulate the production of a neural a&esion
molecule, which includes culturing the optic nerve or other parts of the nervoussystem in the cell culture system inoculated with a drug or agent; adding CNS
neurons to the cell culture system; and e~c~mining neuronal outgrowth to determine
the effect of the drug thereon.
10 Another object of the invention is to provide an assay system for scrcenillg drugs
and other agents for ability to modulate the production of a neural adhesion
molecule, which includes inoculating a culture of CNS neurons with a drug or
agent; adding a soluble neural adhesion molecule: and e~c~mininE~ neuronal
outgrowth to determine the effect of the drug thereon.
15 Other objects and advantages will become appalent to those skilled in the art from
a review of the ensuing detailed description taken with reference to the following
illustrative drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 depicts the map of the GFAP-LI chimeric transgene. A 4.05 kb mouse
20 Ll cDNA was inserted into exon 1 of a modified GFAP gene using Not I linkers.In this construct, the Ll cDNA is preceded 5' by an SV40 late gene splice (V) and
followed 3' by an SV40 polyadenylation signal (pA). The locations of the L1
ATG and the polyadenylation signal are indicated. Exons are shown as boxes.
FIGURE 2 depicts a Northern blot analysis of brain RNA from different trans~enic25 lines. 10 ~Lg of total RNA of whole adult brain was loaded in each lane and
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Il
probed with mouse Ll cDNA. Exposure time was 3 days. Lanes 1-3, brains from
different transgenic offspring (lane 1: line 3426; lane 2: line 3427; lane 3: line
3418; lane 4. brain from non-l-dllsg~-~ic control). Note that the level of transgenic
Ll mRNA (arrow) is different in the three transgenic lines, with levels being
5 highest in line 3476~ intermeAi~te in line 3427 and lowest in line 3418. The
position of 28S and 18S rRNA is shown on the right margin.
FIGURE 3 depicts the loc~li7~tion of Ll mRNA in adult unlesioned (A, C and E)
and lesioned (15 days after the lesion. B and D) optic nerves from non-transgenic
(A, B and E) and transgenic mice (C and D) of line 3426 by in situ hybridization.
10 In wild type ~nim~lc, Ll mRNA is detectable only in neuronal cells of the retina
but not in the glial cells of the optic nerve (A and B). In transgenic ~nim~lc, cells
cont~ining Ll transcripts are visible in the optic nerve (C and D). The density of
L1 positive cells is highest in the unmvelinated proximal part of the nerve. Thedensity of L 1 mRNA positive cells in the nerve is slightly increased after a lesion
15 (compare C and D). In the optic nerve, the distribution of cells ~x~lc;,:,hlg Ll (C
and D) is similar to that of cells e~.es,illg GFAP (E). Scale bar in E: 100 ~m (for
A to E).
FIGURE 4 depicts the double immunofluorescence microscopic localization of Ll
(A and B) and GFAP (C) in unlesioned (A and C) and lesioned (28 days after the
20 lesion, B) optic nerves from adult transgenic (line 3426, A and B) and wild type
(C) ~nim~ls. Ll immnnoreactivity in optic nerves from transgenic ~nim~lc is
significantly increased after a lesion (compare A and B). The pattern of Ll
im~nunoreactivity in lesioned transgenic nerves is similar to the pattern of GFAP
immlm~st~ining in unlesioned wild type nerves. Ll positive unmyelinated retinal
25 cell ganglion axons are present in unlesioned wild type nerve (A). Scale bar in
C:S0 ~m (For A to C).
-
FIGURE S depicts the double immunofluorescence microscopic localization of Ll
(A and D) and GFAP (B and E) in cultured astrocvtes from transgenic ~nim~ls of
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lZ
line 3426 (A, B and C) and wild type ~nim~lc (D. E and F). Note that only the
cells from L~ sge.lic ~nim~lc express Ll. whereas astrocytes from wild type
Qnim~lc are Ll negative. Scale bar in F:30 ~m (for A to F).
FIGUE~E 6 shows (A) Western blot analysis of lesioned (15 days after the lesion)5 and unlesioned optic nerves from transgenic and wild type ~nim~l.c '~5 ~lg of total
protein of lesioned (lanes 1, 3 and 5) or unlesioned nerves (lanes 2, 4, and 6) was
loaded and (letected using affinity purified polyclonal antibodies against Ll.
Protein extracts were made from mice of transgenic lines 3426 (lanes 1 and 2),
3427 (lanes 3 and 4) and from wild type ~nim~lc (lanes 5 and 6). There is an
10 increase in Ll e~ ;,sion in transgenic ~nim~lc compared to non-Ll~sg~ ic
controls. Following optic nerve lesion, an up-regulation of Ll occurred in
transgenic ~nim~lc, whereas the amount of Ll in wild type ~nim~l.c de~,.cased.
Al~alent molecular weights (in kD) are shown on the left margin.
FIGURE 7 depicts examples of neurite outgrowth from mouse cerebellar neurons
15 cultured on cryostat sections of optic nerves from wild type (A and B) and
transgenic (C and D) ~nim~lc (line 3426). A and C l~resen~ unlesioned optic
nerves, B and D le~ sent lesioned optic nerves. Scale bar in D:50 ~m (for A to
D).
FIGURE 8 depicts and COI~ ;S neurite lengths of cerebellar neurons mzlint~in~d
20 on cryostat sections of unlesioned (c) and lesioned (1) optic nerves (28 days after
the lesion) from wild type (WT) and L~ Sgc;niC ~nim~lc (lines 3426, 3427 and
3418). Note that the length of neurites on sections from transgenic ~nim~lc is
greater than on sections from wild type ~nim~lc In transgenic lines neurites arealways longer on lesioned than on unlesioned nerves, whereas neurite lengths on
25 unlesioned and lesioned nerves of wild type ~nim~l.c do not show a significant
difference. Note that the neurite length correlates positively with the levels of L 1
expression in different transgenic lines (see also Western blot data). Mean values +
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13
standard error of the mean from one r._~les~ntaLive experiment (out of 12) are
shown.
FIGURE 9 is a graph measuring neurite lengths of cerebellar neurons m~int~inçCl
on cryostat sections of unlesioned (c) and lesioned (1) optic nerves (28 days after
S the lesion) from wild type (WT) and transgenic ~nim~l.c without and after pre-inr~lh~tion of sections with affinity purified polyclonal antibodies against Ll (anti
L1) and mouse liver membranes (anti liver). Neurite lengths on nerves without
pre-incubation with any antibody were taken as 100% and neunte lengths on
sections of the same nerves obtained after antibody tr~tment were expressed in
10 relation to this value. A significant reduction (60%) of neurite length by Llantibodies was found on cryostat sections from transgenic ~nim~l~ Numbers on
the top ~c~lesel~t the total number of nerves measured for each value. Mean values
i standard error of the mean are from at least four independent e.s~ ents carried
out in duplicate.
15 FIGURE 10 depicts and CU~ JdleS neurite lengths of mouse cerebellar (A) or chick
DRG (B) neurons on astrocytic monolayers prepared from wild type (WT) and
transgenic ~nim~l~ (line 3426) in the absence of antibodies and after pre-incubation
of sections with affinity purified polyclonal antibodies against L2 (anti Ll) and
mouse liver membranes (anti liver). The neurite length on astrocytes without pre-
20 incubation with any antibody was taken as 100% and the neurite lengths onastrocyte monolayers obtained after antibody treatment are ex~l~ ssed in relation tothis value. A significant reduction (about 40%) of neurite length is only visible on
transgenic astrocytes after preincubation of the monolayers with L 1 antibodies.Mean values + standard deviation are from at least 100 neurons from two
25 independent ~ .hllents carried out in quadruplicate. * indicates means that were
significantly different (p~ 0.0~, Mann-Whitney U test) from the control (wild type
- or transgenic astrocytes without any antibody treatment).
FIGURE 11 demonstrates the in vivo regrowth of axons in the optic nerve (0-2000
~m). 6-8 week old GFAP-Ll transgenic mice and wild type mice were crushed
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14
intraorbitally and. after 14 days, traced with a fluorescein-labeled biotin ester to
mark retinal ganglion cell axons by anterograde labelin . Each point ~ cse
one animal.
FIGURE 12 depicts in vivo leg.owLh of axons in the optic nerve (0-800 ~m). 6-8
5 week old GFAP-L 1 transgenic mice and wild type mice were crushed intraorbitally
and, after 14 days, traced with a fluorescein-labeled biotin ester to mark retinal
ganglion cell axons by anterograde labeling. Each point represents one animal.
FIGURE 13 shows the effect of the injection of chicken L1 antibodies into the
IMHV on percent avoidance (retention of memory) on a one-trial passive avoidance10 task. Each point .e~lese.lLs a group of birds who received injections of L1
antibodies (anti-Ll) (closed circles) or saline (open squares) at the time relative to
training indicated. All ~nim~l~ were tested at 24 hours post-kaining (*, p<0.05
between saline and antibody groups, %').
FIGURE 14 comprises two graphs depicting the effect of injections of Ig I-IV and15 FN fr~gment~ at -30 minlltes and +5.5 hours on retention of memory for passive
avoidance task. All ~nim~lc were tested at 24 hours post-training. The number of~nim~l~ in each group is shown in the histograms (*p<0.05; **p<0.005).
FIGURE 15 comprises a series of graphs showing the influence of antibodies
against L1 (anti-Ll) on LTP in pyramidal neurons in the CAl region of rat
20 hippocampal slices. a, Averaged (n=4) EPSP's recorded before and 50 minl-tes
after (arrow) TBS at the control site not injected with antibodies. b, Averaged
(n=4) EPSPs recorded before and 50 min. after TBS (arrow) in the presence of
rabbit polyclonal antibodies against mouse L1 (Rathjen et al. (1984)). c, Time-
course of the EPSP initial slope before and after TBS in the presence of Ll
25 antibodies (IgG fraction, 6.2 mg/ml O) or polyclonal antibodies to the
immllnoglobulin-like domains I-VI recombinantly expressed in CHO cells (Hynes
(1992) Cell. 69:11-''5) (antiserum cont~inin(J. approximately 1 mg/ml of specific
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antibodies. ~) and the following controls: (I) Control LTP (no antibodies. O), (2)
in the presence of the IgG fraction of the polyclonal antibodies to mouse liver
membranes (3.5 m~/ml,--), which react strongly with rat hippocampal slices
(Lindner et al. (1983) Nature 305:4'~7-430), (3) in the presence of rabbit non-
5 immune serum, and (4) in the presence of Ll antibodies without induction of LTPby TBS (6.~ mg/ml. O; see also e, f). Results are ~ e.,~ed as means + S.E.M. ofthe EPSP initial slope i percent of the baseline values recorded during the '70 min.
before TBS (n=~) slices from at least 3 ~nim~l~; values for LTP's in the ple~,nce
of L1 antibodies differ from the control LTP at p<0.001 for the antibodies against
10 L1. and at p<0.01 for the antibodies to the immunoglobulin-like domains I-VI). d,
Concentration-depenc~enre of the reduction in LTP by the IgG fraction of
polyclonal antibodies against Ll (6.~ mg/ml; O): 2 mg/mL ~; 0.6 mg/ml, ~;
0.06 mg/ml, ~1; p<0.0001). As a control, the results from polyclonal antibodies
against liver membranes are shown (3.5 mg/ml, ~). e, Averaged (n=4) EPSP's
15 recorded before and 60 min. after (arrow) the application of polyclonal antibodies
against L1 in the ~bs~nce of TBS. f. Averaged (n=4) intracellular excitatory
postsynaptic currents (EPSP) recorded before and 30 min. after (arrow) the
application of polyclonal antibodies against L1 in the absence of TBS.
FIGURE 16 demonstrates the influence of the immunoglobulin-like domains I-VI~
20 polyclonal NCAM antibodies and oligomannosidic glycopeptides on LTP. a, time-course of the EPSP initial slope before and after TBS in the presence of the
imml-noglobulin (Ig)-like domains I-VI (216 ~g/ml; 3.2 mM; in 20 mM Tris/HCl
pH 7.6 O; p<0.01) and the fibronectin (FN) type III homologous repeats I-V (225
~lg/ml; 3.8 mM; in 70 mMTris/HCl pH 7.6, ~) of L1, comparedto control LTP
25 (20 mM Tris/HCl, pH 7.6, O). b, Time-course of the EPSP initial slope before and
after TBS in the presence of antibodies to NCAM (IgG fraction, 3.9 mg/ml, ~), anantiserum against axonin-1 (--), and the following controls: (1) a non-imml-ne
rabbit serum ( ~), ( ) an IgG fraction of non-immune rabbit serum (3.0 mg/ml. ~ ).
and (3) in the presence of NCAM antibodies (3.9 mg/ml. ~1; p<0.06) without
30 induction of LTP by TBS. c, time-course of the EPSP initial slope before and after
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TBS in the presence of oligomannosidic gl~,copeptides (O). control gl~cope~lides(--) derived from asialofetuin (both at 100 ~M). and in the absence of
glycu~e~tides (O). Results are expressed as means + S.E.M. of the EPSP initial
slope in percent of the baseline values recorded during the 20 min. before TBS
5 (n=5 or 6 slices from at least 3 ~nim~
FIGURE 17 graphically depicts the influence of L I antibodies and oligomannosidic
carbohydrates on previously established LTP and on ~fNDA receptor-merlis~tecl
synaptic tr~n~mic~ion. a, Time-course of the EPSP initial slope before and afterTBS in the presence of L1 antibodies applied either throughout the experiment (6.2
10 mg/ml; O) or starting 10 minllSes after TBS (6.2 mg/ml; ~). b, time-course of the
EPSP initial slope before and after TBS in the presence of oligomannosidic
carbohydrates applied either throughout the experiment (100 ,uM;O) or starting 20
minllt~s after TBS (100 ~lM;--). c, Averaged (n~-4) NMDA receptor-dependent
EPSP's recorded in the presence of CNQX (30 ~lM) before and after 30 minl-tes
1~ (arrow) application of L1 antibodies. d, Averaged (n=4) NMDA receptor-dependent
EPSP's recorded in the presence of CNQX (10 ~lM) before and after (arrow) 60
minllt~s of application of oligomannosidic carbohydrates. Results in a and b areexpressed as means + S.E.M. of the EPSP initial slope in percent of the baselinevalues recorded during the 20 min. before TBS (n=5 slices from at least 3 ~nim~lc).
20 FIGURE 18 depicts the nucleotide sequence of the 4.43 kb cDNA insert of clone pX#2 and ~le~ ceA amino acid sequence of mouse CHL1. The longest open
reading frame (bp 296 to bp 3922) contains 1209 amino acids terrnin~ting with a
TGA termination codon. The two hydrophobic regions representing the signal
peptide (amino acids 1-24) and the tr~n~m~mhrane region (1082-1104) are
25 underlined by a bar. Two arrows indicate the ~' and 3- ends of clone 311 isolated
from the ~gtl 1 library. Potential sites of asparagine-linked glycosylation (Hubbard
and Ivatt, 1981) are marked below the amino acid sequence with filled diamonds.
The irnmunoglobulin (Ig)-like domains are numbered with roman numerals from I
to VI below the conserved tryptophan. The characteristic cysteines are indicated by
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circles. The FN-like repeats are nurnbered Fl to F5 and the characteristic
tnptophans (mi~sing in Fl; F2; W 732. F3; W 830, F4: W 936, F5; W 1053) and
t~Tosines/phenylalanines (Fl: Y 682. F'7: Y 781, F3: F888, F4: Y 989, and mie~inE
in F5) are boxed. A bracket highlight~ the RGD and DGEA sequences (amino acid
5 residues 185-187 and 555-558, respectively). Untr~n~l~te~l sequences are shown- numbered in italics. The sequence data are available from EMBL/Genebank/DDBJ
under accession number X94310.
FIGURE 19 depicts the domain structure, codin~ region of the bacterially ~ ;,sedprotein fragment, and hydrophobicity plot of mouse CHLI
10 (a) The diagram sketches the structural features clecl~ce~l from the primary
sequence of CHLl. -Numbers refer to the amino acid sequence starting at the
translation start site. Ig-like domains I to Vl are re~.resellLed by half circles (amino
acid numbers refer to the cysteines forming the disulfide bridges). FN-like repeats
I to 5 are symbolized by boxes (amino acid numbers refer to the domain
15 boundaries). The potential sites for N-glycosylation are indicated by filled circles.
Signal peptide and tr~n~mPmbrane region are denoted by etched boxes. (b) The barindicates the position of the cDNA encoding the recombinant protein produced in
E. coli (c) The hvdrophobicity plot (Kyte and Doolittle, 1982) of the ~led~lceclarnino acid sequence shows the characteristic features of an integral membrane
20 protein with the putative hydrophobic signal sequence and transmembrane domain
(*). Positive values in-lic~te hydrophobicity. Numbering of the abscissa refers to
arnino acid position.
FIGURE 20 shows the ~lignmPnt of the intracellular domains of molecules of the
L1 family. The sequences of the intracellular domains starting with the first amino
25 acid residue after the putative tr~n~memhrane regions are aligned for mouse CHLl,
mouse L1, chicken Nr-CAM, chicken Ng-CAM, chicken neurofascin, Drosophila
neuroglia~ and zebrafish L1.2. The numbers refer to the amino acid positions of
CHLI and gray boxes indicate gaps introduced in the CHLI sequence. Identical
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amino acids occurrinC in the majority of sequences are marked by black boxes.
The three brackets (I. II and III) refer to highly conserved stretches.
FIGURE '21 is a Northern blot analysis of. CHL1 and L1 mRNA in different tissuesof mouse and rat
S (a) Poly (A) RNA (2,1g) from brain minus cerebellum (lanes 1,5), spinal cord
(lanes 3,7). and dorsal root ganglia (lanes 4, 8) and total RNA (10 ~lg) from
cerebellurn (lanes 2.8) of nine-dy-old mice were hybridized with CHLl (lanes 1 to
4) or Ll (lanes S to 8) riboprobes. Poly (A)* RNA (1 ~Lg) from kidney (lane 9),
spleen (lane 10), liver (lane 11), and thymus (lane 12) and poly (A)* RNA (0.5~g)
10 from intestine (lane 13) and lung (lane 14) of nine-day-old mice were hybridized
with the CHLl riboprobe.
(a) Total RNA (20 ~g) of NGF induced (lane 1) and non-inclucecl (lane 2) PC12
cells, COS-l cells (lane 3). and total RNA (30 ~Lg) of cerebellum of nine- (lane 4)
and six- (lane 5) day-old rats were hybridized with the CHLl riboprobe. RNA
15 markers are indicated at the left margins.
FIGURE ''2 demonstrates the specificity of polyclonal antibodies against CHLl and
t:x~ie~ion of CHLl in different tissues.
(a) Western blot analysis of brain derived immunopurified Ll (lane 1 (2 ~lg)), N-
CAM (lane 2 (2 llg)). MAG (lane 3 (2 ,ug)), and recombinant anion exchange
20 chromatography purified CHLl protein fragment (lane 4 (0.1 llg)) using CHLl
antibodies. (b) Western blot analysis of soluble (S) and insoluble (M) fractions of
detergent lysates of crude membranes from brain (lane 1). liver (lane 2), lung (lane
3), kidney (lane 4), and int~stinto (lane 5) of nine-day-old mice. The numbers at
the lef~ (b) refer to the molecular masses of CHLl immunl~-reactive bands of brain
25 (lane 1) and liver (lane 2).
Molecular mass standards are indicated in kD at the left (a) and right (b) margin~.
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FIGURE 73 shows the detection of CHLI on transiently transfected COS-I cells
~Ionolayer cultures of CHLl-transfected (a) and mock-transfected (c) COS-l cellswere irmnunostained ~,vith polyclonal antibodies a~ainst CHLI. (b,d) co.l~a~onding
phase contrast micro raphs for (a.c). res~e~;Li~ely. Bar in d = 30 llm for a to d.
- 5 FIGURE 24 depicts the locali_ation of CHLl and Ll mRNA in sections of mouse
retina, optic nerve. and cerebellar cortex by in situ hybridi_ation analysis. In the
retina of 7-day-old mice, Ll mRNA is detect~hle in ganglion cells located in theganglion cell layer (1 in a) and in amacrine and horizontal cells located in the inner
nuclear layer (2 in 1). Other cells types in the retina or glial cells in the optic
nerve do not contain ~letect~hle levels of Ll transcripts (a). CHLl rnRNA is weekly
detectable in ~anglion cells and in a few cells located at the inner (i.e. vitread)
margin of the inner nuclear layer (b). Glial cells located in pro~imal (i.e. retina-
near) regions of the optic nerve are strongly labeled by the CHLI ~nti~ton~e cRNA
probe whereas glia cells located in more distal re~ions are only weekly labeled (b).
In the cerebellar cortex of two-week-old mice, Ll ~ à~ a are ~1etect~hle in
stellate and basket cells in the molecular layer (mol) and in Golgi and granule cells
in the internal granular layer (Igl:d). CHLl transcripts are distributed in a similar
pattern, with the only exception that hardly any labeling is visible in thinner part of
the molecular layer (b). Sections hybridized with a CHL1 sense cRNA probe are
not labeled (for a 7-day-old retina and optic nerve. see c).
Bar in c = 100 ~lm for a-c: bar in e = 150 ~lmm for d and e.
FIGURE 25 illustrates the irnmunofluoresc~nc~e microscopic loc~1i7~tion of CHLl
in cultures of astrocytes.
Double-immunolabeling of cultured mouse astrocytes was performed with
polyclonal antibodies to CHLl (a,d) and monoclonal antibodies to GFAP (b,e), (c
and f) are the corresponding phase contrast micrographs for (a,b and d,e),
respectively. Bars in c and F = 20 ~m for a-c and d-f respectively.
FIGURE 26 is a Western blot analysis of deglycosylated CHLl
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Soluble (S) and insoluble (M) fractions of detergent Iysates of crude membranes
from brain of seven-day-old mice were incubated with N-glycosidase F (N). O-
glycosidase (O), both enzymes (N+O). or ~ ithout enzyme (-) and reacted with
antibodies against CHL1 in Western blots. Molecular mass standards are indicatedin kD at the right margin. The molecular masses of the glycosylated and *
deglycosylated CHL 1 protein components are indicated in kD in the box below.
FIGURE 27 shows the presence of the MNK-l carbohydrate in CHLl
imrnunoprecipitates from brain tissue. CHLl was imrnuno~leci~i~ated from
d~Le-ge.ll Iysates of whole brain tissue of nine-day-old mouse brain using CHLl
10 antibodies. Brain lysate (lane 1) and immunoprecipitates (lanes 2,3) were resolved
by SDS-PAGE, blotted, and incubated with monoclonal antibody 312 against the
HNK-l epitope (lanes 1,2) or CHLl antibodies (lane 3). Molecular mass standards
are indicated in kD at the right margin.
FIGURE 28: NEURITE OUTGROWTH OF HIPPOCAMPAL NEURONS IN
15 COCULTURES WITH L929-TRANSFECTANTS
Hippocampal neurons derived from rats of embryonal day 18 were cultured in
subconfluent monolayers of L929-transfectants or parental L929 cells. After 11-12
h of coculture the cells were fixed and labeled with monoclonal antibody412
(recognizing the HNK-1 carbohydrate epitope) or a polyclonal antibody against
20 NCAM. For measurement of the total neurite length only the longest neurite per
each branch was deterrnined due to the highly branched character of the neurons in
these cultures.
(A) Neurite outgro~th is promoted bv CHL 1 and inhibitable by antibodies.
Neurons were cocultured with CHLl-transfectants (CHLl) or parental L929 cells
25 (L929) with (+AB) or without polyclonal antibodies against recombinant CHLl
(500 ~Lg/ml of purified IgG, added 45 min after plating) and on Ll-transfectants.
The mean total neurite length of 4 - 5 independent experiments is shown. Error
bars are standard error of the mean.
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(B) Different CHL 1 lines promote neurite outgrowth better than L 1. Neurons
were cultured on two different CHL1-transfectants (CHL1 line 1, CHL1 line 2)
w~th slightly different e~ e~ion levels, parental L979 cells (L979) and Ll
transfectants (L1). Total neurite length is given as percent of L979 cells as a
5 control (ctr). Error bars are standard error of the mean.
- (C) Neurite outgrowth promotion affects all length classes of neurites. Cumulative
frequency distribution plot of the total neurite length of hippocampal neurons
cocultured with CHLl-tran~re~ (CHL1 line 1 and 2) and parental L929 cells
(L929) with (+AB) or without antibody tr~atm~rlt as ~iven in (A). The ~elcent~ge10 of neurons with neurites longer than or equal to a certain length ~ (vertical axis)
was plotted as a function of neurite length x (horizontal axis). Values are from one
cpresçl-t~l;ve experiment.
FIGURE 29: NEURITE OUTGROWTH OF SMALL CEREBELLAR NEURONS
IN COCULTURE WITH L929-TRANSFECTANTS
1~ Cerebellar neurons derived from 6-7 day old mice were cultured for 20 h on
CHL1-transfectants (CHLl), CHL1-transfected non-~ ressi..g L979 cells (Mock),
parental L929, or L1-transfectants (L1). The st~inin~ of the cells was performed as
already described in Figure 7.
(A) CHL1 promotes neurite outgrowth of small cerebellar neurons. The mean of
20 total neurite length of three c;x~elilllents is shown. Error bars are standard error of
the mean.
(B) CHL1 promotes neurite outgrowth also of small cerebellar neurons better tha
L1. The total neurite length is given as percent of L929 cells as a control (ctr).
Error bars are standard error of the mean.
25 (C) Increase of neurite outgrowth of cerebellar neurons by CHL1 affects all size
classes of neurites. Cllm~ tive frequency of distribution plot of the total neurite
length of the percentage of neurons with neurites longer than or equal to a certain
length x (vertical axis) was plotted as a function of neurite length ~ (horizontal
axis). Values from one reples~,L~l;ve experiment are shown.
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FIGURE 30: NEURITE OUTGROWTH OF HIPPOCAMPAL NEURONS
TREATED WITH SOLUBLE CHL1
Hippocampal neurons were cultured on poly-L-lysine coated coverslips for 12 h
with addition of SUpe~lldkll~t:~ (40 llg/ml of total protein) of crude membrane
5 prc~dtions of CHLl-tran~recL~t~ (CHLl), parental L929 cells (L9~9), or L1-
transfectants (L1). Staining and measurement of neurite length was performed as
already described (Figure 7).
(A) Soluble CHLl from L929 transfectants promotes outgrowth of the longest and
the surn of all neurites per cell. Absolute length of longest neurite nad total neurite
10 length are shown. Values are means of three independent experiments. Error bars
are standard error of the mean.
(B) Soluble CHL1 promotes a slight increase of neurite number. Total neurite
length in percent of the neurite length of hippocampal neurons treated with
supernatants derived from parental L929 cells (ctr) are plotted. Values are means
15 of three independent e.~ ents. Error bars are standard error of the mean.
(C) Also soluble CHL l affects neurite outgrowths of all length classes of neurites.
C--m~ ti-e frequency distribution plots of the total neurite length from one
repr~ose~t~tive experiment are shown.
FIGURE 31: QUANTITATIVE AGGREGATION ANALYSIS AND STABILITY
20 OF CHLI- AND L1-PROTEIN IN L9'~9 TRANSFECTANTS.
(A) Quan~ e analysis of aggregation of S2 cell transfectants. To detect
aggregation CHLl- (CHL1) (ctr) and Ll- (L1) transfected cells were cultured (at
densities of about 3X106 cells/ml) for 18 h in culture medium with (+ind) or
without (-ind) induction of transgene expression bv CuS04. Particle number was
25 counted in a hemacytometer at the beginning and at the end of the incubation. The
~ .cenlage of aggregation was calculated by the index (l-N/NO)x100. N18 and
NO represent the particle numbers at the end or the beginning of the incubation
period, respectively. Values are the means of at least four independent
experiments. Error bars are standard deviations.
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(B) Kinetics of a~gregation of L929-transfectants. CHL1-transfected (CHLl),
CHLl-L~ re~;led non-e~iessing (Mock), parental L929 (L929)~ and Ll-transfected
(L1) cells had been detached from tissue culture by treatment with low
concentration of trypsin-EDTA, washed and incubated at 37~C in polystyrene tubes.
5 An aliquot of each sample was withdrawn every 30 min and the particle nurnber
- was counted in a hemacytometer. The results are e,~lessed as described in (A).
Values shown are the means of at least three independent ~ hllents. Bars are
standard deviations.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
OF THE INVENTION
More particularly, the present invention relates to the use of certain agents
identified herein as "CNS neural growth modulators" (CNGMs), and particularly toa class o:E neural cell adhesion molecules as defined herein~ to promote neuriteoutgrowth in the central nervous system (CNS). In general, neurons in the adult
15 central nervous system have been considered in~?p~ble of regrowth, due to
inhibitory molecular cues present on glial cells. The agents and methods of the
present invention can be used to overcome this inhibition and promote CNS neurite
ouL~lo~vlh~
The agents of the invention include and may be selected from any cell adhesion
20 molecule which is capable of mo~ ting or promoting CNS neurite ouL~lowLh, andparticularly to molecules belonging to the imml-noglobulin su~c.r~llily. More
particularly, the molecules are selected from the members of the immunoglobulin
sul,c;lr~llily which mediate Ca~l-independent neuronal cell adhesion, including Ll,
N-CAM and myelin-associated glycoprotein. The invention also contemplates
2~ fr~gmPn~ of these molecules, and analogs, cognates, congeners and mimics of
these molecules which have neurite-promoting activity. Particularly preferable
structural motifs for these fr~gmPnt~ and analogs include domains similar to thefibronectin type III homologous repeats (particularly repeats 1-2) and
imrnunoglobulin-like domains (particularly domains I-II. III-IV and V-VI).
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As the agents of the invention. and particularly, the members of the L 1 CAM
family, e.xhibit homophilic bindin<J. both the agents and their antagonists, andparticularlv. their antibodies. may serve as agonists with respect to the .ece~Lol for J
the agents~ and may thus be employed in both diagnostic and therapeutic
applications in the sarne manner and for the same purpose as the agents themselves.
Thus, Ll acts as a receptor, and its antibody may be emploved as an agonist, to
promote neurite outgrowth as set forth herein, to assist in neural regeneration
particularly in the CNS. This capability is further demonstrated in the ability of
the antibodies to Ll to serve in a method for the identification of further members
10 of the L1 CAM family of neural recognition molecules, that will serve as agents
herein, and the invention accordingly extends to the molecules that are id~ntifierl
isolated and characterized by means of such antibodies. As such, therefore, the
class of materials identified as CNS neural growth modulators hereinbelow, is
considered to include the antibodies to CAMs such as Ll and its analogs, such as1~ CHLl. described later on herein, arnong its nurnbers.
The present invention relates in one aspect to the ectopic expression of CNS neural
growth modulators (CNGMs) or neural cell adhesion molecules on differ~nti~t~-l
astrocytes in vivo. These molecules have been found to enhance neurite outgrowthon monolayer cultures of such astrocytes and cryostat sections of unlesioned and20 lesioned adult mouse optic nerves. and also in vivo, in optic nerve crush
experiments in transgenic ~nim~lc The increased neurite outgrowth-promoting
capacity is proportional to the level of ectopic CNGM ex~l~ aaion. This is
demonstrated bv coll.~isons of the distinct transgenic lines of the invention,
which express different basal levels of transgenic-encoded CNGM, and by
2~ correlations following increased CNGM e~iea-aion after a lesion of the optic nerve.
It should be appreciated that although optic nerves. both lesioned and unlesioned,
are suitable for use with the present invention, that any part of the nervous system
can likewise be used. including portions of the brain and spinal cord.
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Neurite outgro-vth is dependent on the levels of CNGM e~cpression by astrocytes,demonslld~ g the specific effect exerted by CNG~I in promoting neurite ouL~l.,wLh
in the transgenic animal. Inhibition of neurite outgrowth bv polyclonal CNGM
antibodies, but not by antibodies to mouse liver membranes~ filrther supports this
5 specificity. in particular, since both antibodies react well with the cell surfaces of
neurons and astrocytes of L~ sgellic zlnim~lc
In a ~.ef~ d embodiment, the CNGM is L1. Ll's biolo,~ical effects can be
inhibited by L1 antibodies, which indicates that Ll is homophihcally active in atrans configuration at the cell surface of ~ sgellic astrocytes. Furthermore, Ll10 species-specific antibodies that do not react with chicken dorsal root ganglion
neurons inhibit neurite outgrowth of this neuronal cell type on transgenic
astrocytes. These fincling~ unequivocally identifv L1 as a trans-acting active
molecule and show that ectopic ~x~les~ion of Ll by glial cells that normally lack
Ll t;,~ ion significantly enh~n~es neurite outgrowth in vitro.
15 The transgene-mediated ~nh~nce ~-e.-t of neurite outgrowth on glial cells that do not
normally express L1 in YiVo in~lic~tec that glial cells of the adult m~mm~ n
central nervous system can be made more conducive to neurite outgrowth. The
loss of neurite outgrowth-promoting glia-derived molecules with maturation (Smith
et al. (1986) J. Comp. Neurol. 251:23-43; Smith et al. (1990) DeY. Biol. 138:377-
20 390) th-,lefole appears to be colll~ellsated for by cA~ s~ion of a recognition
molecule that is normally highly t;~ ;,sed by glial cells in the adult m~mm~ n
peripheral nervous system (Niecke et al. (1985); Bixby et al. (1988) J. Cell. Biol.
107:353-36''; Seilheimer et al. (1988) J. Cell. Biol. 107:341-351).
The phenotype of adult astrocytes from the present transgenic lines may be
25 modified towards the more Schwann cell-related capacity of lee~u.es~ g Ll after
infliction of a lesion. An increase in L1 t:.~res~ion by Schwann cells is likelymediated by neurotrophins upregulated after damage by autocrine mech~ni~m~
(Seilheimer et al. (1987) EMBO J. 6: 1611 - 1616) . Similarly. L l expression by
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astrocytes in culture can be upregulated by TGF-13 and NGF (Saad et al. (1991)).By generating mice with a GFAP-Ll transgene, the inability of mature astrocytes to
respond to neural injury is overcome with an upregulation of the neurite outgrowth
promoting molecule L1. The ~ s~ion of Ll may be particularly beneficial for
5 neurite outgrowth in myelinated tracts of the central nervous system which
norrnally contain several molecules that are neurite outgrowth inhibiting (Sçh~ hn~r
et al., Perspectives in Deve~opm. Neurobiol. in Press, Schwab et al. (1995) Ann.Rev. Neurosci. 16:56~-595).
The present invention demonstrates that the inhibitory action of astroglial and
10 oligodendroglial cells may be overcome, at least in part, by the neurite outgrowth
promoting ~ lup~.Lies of the agents defined herein. and as particularly illustrated by
the activity of ectopically ~ sed Ll. Expression of Ll by astrocytes seems also
to compensate for inhibitory effects exerted by oligodendrocytes. Permissive andnon-perrnissive molecular cues therefore may not have to be localized on the same
15 cell type for neurite outgrowth to occur. Tn.~te~rl such molecular cues might be
partitioned among different cell types. The cellular and molecular manipulation of
L1 and other neurite outgrowth promoting molecules may therefore allow
enhancement of the regenerative capacity of the adult m~mm~ n central nçrvous
system following injury or ~ e~e.
20 As indicated earlier, the present invention extends to the promotion of neural
growth in the CNS. including such growth as is desired to regenerate structures lost
due to injury or illness, as well as those structures and tissues exhibiting incomplete
or imm~ re formation. The agents of the invention also exhibit a neuroprotectiveor neuropreservative effect as illustrated later on herein. and for example, could be
25 ~Amini~tered to inhibit or counteract neural degeneration or loss of variable etiology.
The invention accordingly extends to constructs and compositions cont~ining or
delivering the agents of present invention, whether by the promotion of the
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'~7
e,~ie;.sion of certain agents via gene therapy or the like, or by the exogenous
sl~irninictratiOn of the agents where ~p~ ,iate and beneficial, in pharm~reutical
compositions to treat injured or ~ice~ceA CNS structures. In this latter connection,
it is contemplated that certain of the agents are able to exert a gro~th promoting
5 effect when so ~lminictered, although it is recognized that members of the
,;,elllly ic~etltifiefl group, such as L1 and N-CAM appear to bind homophilicallv
and may therefore prove more beneficial ~vhen delivered bv means of c:x~les~ion.The invention is intPnclefl to extend to both routes and protocols where feasible.
It should also be appreciated that the present invention relates to the use of CNGM-
10 secreting cells for the modulation of neural outgrowth, regeneration. and neural
survival in the CNS. As such, certain soluble CNGMs and fragments thereof, and
cognate molecules thereof are also within the invention.
Therefore, if appearing herein, the following terms shall have the definitions set out
below.
15 The terms "agent", "CNS neural growth modulator", "CNGM", "neural recognitionmolecule", "recognition factor"~ "recognition factor protein(s)", "neural adhesion
molecule", and any variants not specifically listed. may be used herein
interchangeably, and as used throughout the present application and claims refer to
protein~eous material including single or multiple proteins, and extends to those
20 proteins having the amino acid sequence previously described and the profile of
activities set forth herein and in the Claims. The foregoing terms also include
active fr~gmPntc of such proteins, cognates, congeners. mimics and analogs,
including small molecules that behave similarly to said agents.
Accordingly, proteins displaying substantially equivalent or altered activity are
- 2~ likewise contemplated. These modifications mav be deliberate. for example. such
as modifications obtained through site-directed mutagenesis. or mav be accidental,
such as those obtained through mutations in hosts that are producers of the comple~c
CA 02218599 1997-10-16
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28
or its named subunits. Also, the terms "CNS neural _rowth modulator", "CNGM",
"neural recognition factor", "recognition factor", "recognition factor protein(s)", and
"neural adhesion molecule" are int~n~led to include within their scope proteins
specifically recited herein as ~vell as all substantially homologous analogs and5 allelic variations.
The amino acid residues described herein are preferred to be in the "L" isomericform. However, residues in the "D" isomeric form can be substituted for any L-
arnino acid residue, as long as the desired functional ~iOpl,l Ly of immlmoglobulin-
10 bindin~ is retained by the polypeptide. NH refers to the free amino group presentat the ~-unino terminus of a polypeptide. COOH refers to the free carboxy group
present at the carbo~v terrninus of a polypeptide. In keeping with standard
polvpeptide nomenclature, J. Biol. Chem., 243:35~2-59 (1969), abbreviations for
arnino acid residues are shown in the following Table of Correspondence:
TABLE OF CORRESPONDENCE
SYMBOL AMINO ACID
1-Letter 3-Letter
Y Tyr tyrosine
G Gly glycine
F Phe phenyl~l~nine
M Met methionine
A Ala alanine
S Ser serine
Ile isoleucine
L Leu leucine
T Thr threonine
V Val valine
P Pro proline
K Lys lysine
H His histidine
CA 02218~99 1997-10-16
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29
Q Gln glnt~rnine
E Glu glutamic acid
W Trp tryptophan
R Arg arginine
D Asp aspartic acid
N Asn asparagine
C Cys cysteine
It should be noted that all amino-acid residue sequences are ~ c;sented herein by
formulae whose left and right orientation is in the conventional direction of amino-
10 terminus to carboxy-terminus. Furtherrnore, it should be noted that a dash at the
beginning or end of an amino acid residue sequence indicates a peptide bond to afurther sequence of one or more amino-acid residues. The above Table is
present~cl to correlate the three-letter and one-letter notations which may appear
alternately herein.
15 A "replicon" is any genetic element (e.g., plasmid~ ch~omosome, virus) that
functions as an autonomous unit of DNA replication in vivo; i.e., capable of
replication under its own control.
A "vector" is a replicon, such as a plasmid, phage or cosmid, to which another
DNA segment may be ~tt~rh~-l so as to bring about the replication of the ~ ch~1
20 segmellt.
A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (?~ nine~
guanine, thymine, or cytosine) in its either single stranded forrn, or a double-stranded heli~. This term refers only to the primary and secondary structure of the
molecule~ and does not limit it to any particular tertiary forms. Thus, this term
~5 includes double-stranded DNA fo~md~ inter aZia, in linear DNA molecules (e.g.,
restriction fragments). viruses, plasmids~ and chromosomes. In discussing the
structure of particular double-stranded DNA molecules~ sequences may be described
CA 02218~99 1997-10-16
PC~IUS96J05434
W~ 961329S9
herein according to the normal convention of giving onlv the sequence in the 5' to
3' direction along the nontranscribed strand of DNA (i.e.. the strand having a
sequence homologous to the mRNA).
An "origin of replication" refers to those DNA sequences that participate in DNA
5 synth.o~ic
A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed
and tr~n~l~t~l into a polypeptide in vivo when placed under the control of
~I.ro~l;ate regulatory sequences. The boundaries of the coding sequence are
deterrnined by a start codon at the 5' (amino) terminus and a translation stop codon
10 at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to,
prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequehces
from eukaryotic (e.g.. m~mm~ n) DNA, and even synthetic DNA sequences. A
polyadenylation signal and L~ls~ ion termination sequence will usually be
located 3' to the coding sequence.
15 Transcriptional and translational control sequences are DNA regulatory sequences,
such as prornoters, enh~nrers, polyadenylation signals, terminators, and the like,
that provide for the e~S~res~ion of a coding sequence in a host cell.
A "prornoter sequence" is a DNA regulatory region capable of binding RNA
polymerase in a cell and initi~ting Ll~lsc.i~Lion of a downstream (3' direction)20 coding sequence. For purposes of defining the present invention, the promotersequence is bounded at its 3' terminus by the transcription initiation site and
extends u~sLrealll (5' direction) to include the minimum number of bases or
elements neces~ry to initiate transcription at levels detectable above background.
Within the promoter sequence will be found a transcription initiation site
25 (conveniently defined by mapping with nuclease Sl), as ~vell as protein binding
domains (con~ert~ sequences) responsible for the binding of RNA polymerase. fEukaryotic promoters will often. but not always. contain "TATA" boxes and "CAT"
CA 02218~99 1997-10-16
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31
bo.Yes. Prokarvotic promoters contain Shine-Dal~arno sequences in addition to the
-10 and -3~ concPn~llc sequences.
An "t~ cssion control sequence" is a D~A sequence that controls and regulates
the ~ seli~Lion and translation of another DNA sequence. A coding sequence is
- ~ "under the control" of transcriptional and translational control sequences in a cell
when RNA polymerase transcribes the coding sequence into mRNA, which is then
tr~n~l~tec~ into the protein encoded by the coding sequence.
A "signal sequence" can be included before the coding sequence. This sequence
encodes a signal peptide, N-terminal to the polypeptide, that co"""-."ic~tPs to the
10 host cell to direct the polypeptide to the cell surface or secrete the polypeptide into
the media and this signal peptide is clipped off by the host cell before the protein
leaves the cell. Signal sequences can be found associated with a variety of proteins
native to prokaryotes and eukaryotes.
The term "oligonucleotide", as used herein in referring to probes, is defined as a
15 molecule comprised of two or more ribonucleotides, preferably more than three.
Its exact size will depend upon many factors which. in turn. depend upon the
ultimate function and use of the oligonucleotide.
The term ll~lhl~tl" as used herein refers to an oligonucleotide, whether occurring
naturally as in a purified restriction digest or produced synthetically, which is
20 capable of acting as a point of initiation of synthesis when placed under conditions
in which synthesis of a primer extension product, which is complementary to a
nucleic acid strand, is in~ ce~ i.e., in the presence of nucleotides and an inducing
agent such as a DNA polymerase and at a suitable telllpeldlu~e and pH. The
primer rnay be either single-stranded or double-stranded and must be sufficiently
25 long to prime the synthesis of the desired extensic!n product in the presence of the
inducing agent. The exact length of the primer will depend upon many factors,
including temperature. source of primer and use of the method. For example. for
CA 02218~99 1997-10-16
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3'~
diagnostic applications~ depending on the complexity of the target sequence, theoligomlcleotide primer typically contains 15-25 or more nucleotides, although itmay contain fewer nucleotides.
The primers herein are selected to be "subst~nti~ " complernent~ry to different
S strands of a particular target DNA sequence. This means that the primers must be
sufficiently complementary to hybridize with their respective strands. Therefore,
the prirner sequence need not reflect the exact sequence of the template. For
exarnple, a non-complement~ry nucleotide fragment may be ~tt~rhe~l to the ;' endof the primer, with the rem~in~ier of the primer sequence being complem~nt~ry to10 the strand. Alternatively, non-complementary bases or lon~er sequences can beinte,a~ ed into the primer, provided that the primer sequence has sufficient
complementarity with the sequence of the strand to hybridize therewith and thereby
form the template for the synthesis of the extension product.
As used herein, the terms "restriction endonucleases" and "restriction enzymes"
15 refer to bacterial enzymes, each of which cut double-stranded DNA at or near a
specific nucleotide sequence.
A cell has been "transformed" by exogenous or heterologous DNA when such DNA
has been introduced inside the cell. The transforming DNA may or may not be
integrated (covalently linked) into chromosomal DNA m~kinE up the genome of
20 the cell. In prokaryotes, yeast, and m~mm~ n cells for example, the transforming
DNA may be m~int~ined on an episomal element such as a plasmid. With respect
to eukaryotic cells, a stably transforrned cell is one in which the transforrning DNA
has become integrated into a chromosome so that it is inherited by rl~llghter cells
through chromosome replication. This stability is demonstrated by the ability of25 the eukaryotic cell to establish cell lines or clones comprised of a population of
daughter cells cont~inin~ the transforming DNA. A "clone" is a population of cells
derived from a sinPle cell or common ancestor by mitosis. A "cell line" is a clone
of a primary cell that is capable of stable growth in vitro for many generations.
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33
Two DNA sequences are "subst~nti~lly homologous" when at least about 75%
(preferablv at least about 80%, and most preferably at least about 90 or 95%) ofthe nucleotides match over the defined length of the DNA sequences. Sequences
~, that are subst~nti~lly homologous can be identified by col.l~ing the sequences
5 using standard software available in se~uence data banks, or in a Southern
hybridization ~uelh-lent under, for example, stringent conditions as defined forthat particular system. Defining appropriate hybridization conditions is within the
skill of the art. See. e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra;
Nucleic Acid Hybridization, supra.
10 A "heterologous" region of the DNA construct is an itlPntifi~hle segm~nt of DNA
within a larger DNA molecule that is not found in association with the larger
molecule in nature. Thus, when the heterologous region encodes a m~mm~ n
gene, the gene will usually be fl~nkPd by DNA that does not flank the m~mm~ n
genomic DNA in the genome of the source orp~nicm Another example of a
15 heterologous coding sequence is a construct where the coding sequence itself is not
found in nature (e.g.. a cDNA where the genomic coding sequence contains introns,
or synthetic sequences having codons different than the native gene). Allelic
variations or naturally-occurring mutational events do not give rise to a
heterologous region of DNA as defined herein.
20 An "antibody" is any immllnoglobulin~ including antibodies and fr~gm~ntc thereof,
that binds a specific epitope. The term encomp~c~çs polyclonal, monoclonal, and
chimeric antibodies, the last mentioned described in further detail in U.S. Patent
Nos. 4,816,397 and 4.816,567.
An "antibody combining site" is that structural portion of an antibody molecule
25 comprised of heavy and light chain variable and hypervariable regions that
specifically binds antigen.
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34
The phrase "antibody molecule" in its various grammatical forms as used herein
contemplates both an intact immllnoglobulin molecule and an immunologically
active portion of an imml-noglobulin molecule.
Exemplary antibody molecules are intact immlmoglobulin molecules, subst~nti~lly
5 intact immunoglobulin molecules and those portions of an irnmunoglobulin
molecule that contain the ~a~dLo~ue~ including those portions known in the art as
Fab, Fab', F(ab'), and F(v), which portions are preferred for use in the thc.a~ Lic
methods described herein.
Fab arld F(ab'), portions of antibody molecules are prepared by the proteolytic
10 reaction of papain and pepsin, respectively, on subst~nti~lly intact antibodymolecules by methods that are well-known. See for exarnple, U.S. Patent No.
4,342,~66 to Theofilopolous et al. Fab antibody molecule portions are also well-known and are produced from F(ab'), portions followed by reduction of the
disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and
15 followed by alkylation of the resulting protein melca~ ~ith a reagent such asio~lo~et~mide. An antibody cont~ining intact antibody molecules is preferred
herein.
The phrase "monoclonal antibody" in its various gr~mm~tical forms refers to an
antibody having only one species of antibody combining site capable of
20 immlmnreacting with a particular antigen. A monoclonal antibody thus typically
displays a single binding affinity for any antigen with which it imrnunoreacts. A
monoclonal antibody may therefore contain an antibody molecule having a plurality
of antibody combining sites, each immllnospecific for a different antigen; e.g., a
bispecific (chimeric) monoclonal antibody.
25 The phrase "pharmaceutically acceptable" refers to molecular entities and
compositions that are physiologically tolerable and do not typically produce an
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3~
allergic or similar untoward reaction. such as gastric upset. ~li77in~ss and the like,
when ~-lmini~tPred to a hurnan.
The phrase "therapeutically effective amount" is used herein to mean an amount
sufficient to prevent. and preferably reduce by at least about 30 percent, more
5 preferably by at least 50 percent, most preferably by at least 90 percent, a clinically
significant change in the S phase activity of a target cellular mass, or other feature
of pathology such as for example, elevated blood plci,~ule~ fever or white cell
count as may attend its presence and activity.
A DNA sequence is "operatively linked" to an e.~ ion control sequence when
10 the t~lession control sequence controls and regulates the transcription and
translation of that DNA sequence. The term "operatively linked" includes having
an ~io~liate start signal (e.g., ATG) in front of the DNA sequence to be
c~ ssed and m~ L~ g the correct reading frame to permit ~l. s~ion of the
DNA se~uence under the control of the ~ lession control sequence and production
15 of the desired product encoded by the DNA sequence. If a gene that one desires to
insert into a recombinant DNA molecule does not contain an app-ul,.;ate start
signal, such a start signal can be inserted in front of the gene.
The term "standard hybridization conditions" refers to salt and tell~ dlllre
conditions subss~nti~lly equivalent to Sx SSC and 65~C for both hybridization and
20 wash.
In one aspect. the present invention relates to transgenic ~nim~l~ which express a
CNGM or neural recognition molecule, in particular Ll~ and preferably in
astrocytes. These ~nim~l~ have increased capability for neural outgrowth in the
central nervous system.
25 The invention also includes an assay system for the screening of potential drugs
effective to modulate neural outgro~hth of target m~mm~ n cells by interrupting or
CA 02218~99 1997-10-16
PCT~U~, ~'OS43~1
WO 96/329S9
36
potenti~tin~ the CNGM's neural recognition activity. By "neural recognition
activity" or "neural adhesion activity" is meant anv biological effect which is a
result of the CNGM's binding to another molecule. including intracellular effects
on second messengers. In one inct~nre7 the test drug could be ~minictered either5 to a cellular sample with the ligand that activates the CNS neural growth
modulator, or a transgenic animal e~lc~hlg the CNS neural growth modulator, to
determine its effect upon the binding activity of the modulator to any chemical
sample, or to the test drug, by comparison with a control. Identifying
characteristics of at least one of the present CNS neural growth modulators, in
10 particLlar L1, is its participation in changes in steady state levels of intracellular
mecs~n~ers, including Ca1, pH, and cyclic nucleotides, as well as changes in theactivities of protein kinases such as protein kinase C, pp60'-Sr', a casein type II
kinase and another kinase known to phosphorylate Ll.
The assay system could more h-,~o.Lal-lly be adapted to identify drugs or other
15 entities that are capable of binding to the CNGMs or proteins, either in the
cytoplasm or in the nucleus, thereby inhibiting or potenti~ting transcriptional
activity. Such assay would be useful in the development of drugs that would be
specific to particular cellular activity, such as neural outgrowth or increase in
synaptic efficacy. or that would potentiate such activity, in time or in level of
20 activity. For example, such drugs might be used to modulate neural outgrowth in
response to injury, or to treat other pathologies, as for example, in treating
neurodegenerative tiice~ces such as Parkinson's Disease, ALS, ~lntington's Disease
and Alzheimer's Disease.
In yet a further embodiment, the invention contemplates agonists and antagonists of
2~ the activity of a CNS neural growth modulator. In particular, an agent or molecule
that inhibits the ability of neurons to recognize a CNGM such as L 1 can be used to
block neural outgrowth, where such outgrowth is contraindicated, and as described
earlier, a pharmaceutical composition cont~ining such an agent may be ~rinninictered
directly to the target site. In another embodiment. an agonist can be a peptide
CA 02218~99 1997-10-16
WO 96132959 PCTJU~g''.,!;13
37
having the sequence of a portion of an L1 domain particularly that ~ L~ ~n
fibronectin type III homologoùs repeats 2 and 3. or an antibody to that region.
Either of these molecules may potentially be used where a particular CNGM such
as L1 has the ability to undergo homophilic binding (i.e., Ll can bind to itself, and
5 therefore both antibodies to Ll and fr~gm~ont~ of Ll itself are capable of binding to
Ll)-
One of the diagnostic utilities of the present invention extends to the use of thepresent CNGMs in assays to screen for protein kinase inhibitors. Because the
activity of the CNGMs described herein are phosphorylated, they can and
10 presumably are dephosphorylated by specific phosph~t~ces Blocking of the
specific kinase or phosphatase is t~lele~ole an avenue of pharmacological
intervention that would modulate the activity of these neural recognition proteins.
The present invention like~,vise extends to the development of antibodies against the
15 CNGMs, including naturally raised and recombinantly l,rep~ed antibodies. For
example, the antibodies could be used to screen e~ ion libraries to obtain the
gene or genes that encode the CNGMs. Such antibodies could include both
polyclonal and monoclonal antibodies prepared by known genetic techniques, as
well as bi-specific (chimeric) antibodies, and antibodies including other
20 functionalities suiting them for additional diagnostic use conjunctive with their
- c~p~bility of mo~ ting neural oulg..~Lh
In particular, antibodies against CNS neural growth modulators can be selected and
are included within the scope of the present invention for their particular ability in
binding to the protein. Thus, activity of the neural growth modulators or of the25 specific polypeptides believed to be causally connected thereto may therefore be
followed directly by the assay techniques ~ cl-c.~ed later on. through the use of an
a~o~-iately labeled quantity of the neural growth modulator or antibodies or
analogs thereof.
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wa, 961~29S9
38
Thus, the CNGMs. their analogs. and any antagonists or antibodies that may be
raised thereto, are capable of use in connection with various diagnostic techniques,
including immunoassays, such as a radioimm--nl sic~sly using for example, an
antibody to the CNGM that has been labeled by either radioactive addition,
S reduction with sodium borohydride, or radioiodination.
In an immnnt sl~ssly. a control quantity of the antagonists or antibodies thereto, or
the like may be l~.epalcd and labeled with an enzyme. a specific binding partnerand/or a radioactive element, and may then be introduced into a cellular sample.After the labeled material or its binding partner(s) has had an opportunity to react
10 with sites within the sample, the resulting mass may be exslrnine~l by kr,,own
techniques. which may vary with the nature of the label 5itt~-h.-~ For example,
antibodies against the CNGMs may be selected and a~plopl;ately employed in the
exemplary assay protocol, for the purpose of following protein material as
described above.
1~ In the instance where a radioactive label, such as the isotopes 3H. '~C, 3~P, 35S, 36Cl,
5'Cr, 5~Co, 58Co, 59Fe. 90Y, l25I, '3lI, and '86Re are used, known currently available
counting procedures may be l~tili7P~l In the instance where the label is an enzyme,
detection may be accomplished by any of the presently utilized colorimetric,
spectrophotometric. fluorospectrophotometric, ~llp~unletric or gasometric
2~ techniques known in the art.
The present invention includes an assay system which may be ~,e~ ,d in the form
of a test kit for the q~ ive analysis of the extent of the presence of the neural
growth modulators, or to identify drugs or other agents that may mimic or block
their activit-. The system or test kit may comprise a labeled component prepared25 by one of the radioactive and/or enzymatic techniques discussed herein, coupling a
label to the neural growth modulators. their agonists and/or antagonists, and one or
more additional immunochemical reagents. at least one of which is a free or
immobilized li~and. capable either of binding with the labeled component. its
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WO s6r32sss
39
binding partner. one of the components to be deterrnined or their binding
(S)-
In a further embodiment, the present invention relates to certain thelalJ~ulic
methods which would be based upon the activity of the CNS neural growth
5 modulator(s), its (or their) subunits, or active fragments thereof, or upon agents or
other drugs determined to possess the sarne activity. A first therapeutic method is
associated with the promotion of CNS neural growth resulting from the plese.lce
and ac~ivity of the CNGM, its active fragments, analogs, cognates, congeners or
mimics, and comprises ~-lminict~ring an agent capable of mocl~ ting the
10 production and/or activity of the CNGM, in an arnount effective to promote CNS
development, regrowth or rehabilitation in the host. Conversely, drugs or other
neutralizing binding partners to the CNGM or proteins mav be ~riminictered to
inhibit or prevent undesired neural outgrowth. Also, the modulation of the action
of specific kinases and phosphatases involved in the phosphorylation and
15 dephosphorylation of CNGMs or proteins ~ selll~ a method for mo~ tin, the
activity of the modulator or protein that would concomitantly potentiate therapies
based on CNGM/protein activation.
More specifically, the therapeutic method generallv referred to herein could include
20 the method for the tre~tmPnt of various pathologies or other cellular dysfunctions
and derangements by the ~-iminictration of ph~rm~reutical compositions that may
comprise effective inhibitors or enhancers of the activity of the CNS neural growth
modulator or its subunits, or other e~ually effective drugs developed for in.ct~nre by
a drug screening assay ~repdled and used in accordance with a further aspect of the
25 present invention. For example. drugs or other binding partners to the CNS neural
growth modulator or proteins may be ~lminictered to inhibit or potentiate binding
and second m~ssenger activity.
As mentioned above. the invention extends to the discoverv of a full family of L1
CAMs, and particularly to an analog to Ll known as CHL1. CHL1 comprises an
CA 02218~99 1997-10-16
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WO 96/329S9
N-terrninal signal sequence. six immunoglobulin (Ig)-like domains. and 4.5
fibronectin type III (FN)-like repeats~ a transmembrane domain, and a C-terminal,
most likely intracellular domain of approximately 100 amino acids. CHLl is most
similar in its extracellular domain to chicken Ng-CAM (about 40% amino acid
identity), followed by mouse L1, chicken neurofascin, chicken Nr-CAM, f
Drosophila neuroglian, and zebrafish Ll.l (37 to 28 % arnino acid identity,
c~,Lively), and mouse F3, rat TAG-I. and rat BIG-l (about 27~/o amino acid
identity). The similarity with other members of the Ig superfamily (e.g. N-CAM,
DCC, HLAR, rse) is 16 to 11 %. The intracellular domain is most similar to
10 mouse and chicken Nr-CAM, mouse and rat neurofascin (about 50 % amino acid
identity) followed by chicken neurofascin and Ng-CAM, Drosophila neuroglian,
and zebrafish Ll.l and Ll.2 (about 40 % amino acid identity). Besides the high
overall homology and conserved modular structure arnong previously recognized
members of the Ll family (mouse/human Ll/rat NILE: chicken Ng-CAM;
15 chicken/mouse Nr-CAM; Drosophila neuroglian: zebrafish Ll.l and Ll.2;
chicken/mouse neurofascin/rat ADGP)~ Ll characteristic criteria were i-le~tifiedwith regard to the number of amino acids between positions of conserved amino
acid residues clefining ~lict~nces within and between two ~ ctont Ig-like domains
and FN-like repeats. These show a colinearity in the six Ig-like domains and
20 adjacent four FN-like repeats that is remarkably conserved between Ll and;
molecules cont~ining these modules (~lecign~te~l the Ll family c~csette) including
the GPI linked forms of the F3 subgroup (mouse F3/chicken Fll/human CNTNl;
rat BIG-1/mouse PANG; rat TAG-1/mouse TAX-1/chicken axonin-l). The
colorectal cancer molecule (DCC) previously introduced as an N-CAM like
25 molecule conforms to the Ll farnily c~ccette. Other structural features of CHLI
shared between members of the Ll family are a high degree of N-glucosidically
linked carbohydrates (about 20% of its molecular mass), which include the HNK-l
carbohydrate structure, and a pattern of protein fragmentc comprising a major 185
kD band and smaller fragments of 100 and 125 kD. As for the outer Ll family
30 members, predominant ~ es~ion of CHLI is observed in the nervous system and
at later developmental stages.
CA 02218599 1997-10-16
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41
The following e~camples are presented in order to more fully illustrate the preferred
embo-lim~nt~ of the invention. They should in no way be construed, however, as
limiting the broad scope of the invention.
~ . .
EXAMPLE 1
. S GFAP-Ll transgene and production of transgenic mice
Glial fibrillary acidic protein (GFAP, Eng et al. (1971) Brain Res; 28:351-35~) is
cssed predominately by astrocytes at late stages in the development of the
mouse central nervous system (Landry et al. (1990) J. Neurosci. Res. 25:194-203).
The~e~ore regulatory sequences of the GFAP gene were used to direct the
~ ion of the neural cell adhesion molecule Ll to mature astrocytes of
transgenic mice. The GFAP-L1 transgene (Fig. I) encodes only the neural cell
adhesion molecule Ll since the ATG of the GFAP gene was mllt~tPd and the L1
coding sequence is followed 3' by a translational stop and a polyadenylation signal
(Toggas et al. (1994) Nature 367:188-19,). This construct was used to establish
three different lines of transgenic mice. ~leci~n~t~cl 3418, 3426 and 3427.
The mouse Ll cDNA (Moos et al. (1988) Nature 334:701-703) was inserted into
exon 1 of the murine glial fibrillar,v acidic protein (GFAP) gene modified as
described previously (Toggas et al. (1994) Nature 367:188-193). The 4.05 kb
mouse Ll cDNA cont~ining the entire coding sequence of the protein and 250 3'
non-tr~n~l~t~rl nucleotides was fused with the modified GFAP-L1 transgene.
The 14.5 kb GFAP-Ll kansgene was excised from a modified cloning vector by
digestion with Sfi I, followed by electrophoresis and electroelution from an agarose
gel. Purified DNA was diluted to a final concentration of 2 ~g/ml in T5Eo I (S mM
Tris-HCI, pH 7.4, 0.1 mM EDTA). Approximately 2 pl of diluted DNA were
~5 microinjected into the male pronucleus of fertilized eggs derived from CB6Flfemales (superovulated) mated to C57Bl/6J males. Eggs surviving the
micromanipulation were transferred into oviducts of pseudo-pregnant foster mothers
.
,
CA 02218599 1997-10-16
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42
following describes methods (Hogan et al. (1986) Manipulating A~Iouse ~mbryo,
Cold Springs Harbor Laboratory, New York).
EXAMPLE 2
Southern blot analysis
S Mice were analyzed for the hlLegldLion of the transgene into the mouse genome by
Southern blot analysis of genomic DNA isolated from tail biopsies (Southern
(1975) J. Mol. Biol. 98:503-517). Transgenic founder mice were mated and pups
scle.,llcd in the same manner to establish transgenic lines. Ten ~g ~mples of DNA
were digested with either Bam HI or with Eco RI and Xba I followed by
10 electrophoretic sepaldLion on a 0.7% agarose gel and transfer to Hybond N+
membrane (Amersham) under ~Ik~line conditions. A 3.3 kb Eco RI-fragment of
the Ll cDNA or a ,30 bp ~ind III fragment of SV40 late splice and
polyadenylation site purified from Al.5 plasmid (Maxwell et al. (1989)
Biotechniques 7:276-280) were labelled with ;'~a-CTP by random priming
15 (Boehringer Marmheim) for use as probes. Prehybridization was performed at
65~C for one hour in 5x SSPE, 5:~ Denhardt's solution, 0.5% (w/v) SDS and 0.1
mg/ml sonicated non-homologous DNA. Hybridi_ation was performed overnight.
Final stringency wash conditions for all Southern blots were O.l:c SSPE and 0.1%SDS (w/v) at 65~C.
EXAMPLE 3
Northern blot analysis
.~n~estheti7to-1 adult mice (12-weeks-old) were sacrificed by a lethal dose of
chloralhydrate and brains were removed and immediately frozen in liquid nitrogen.
Total cellular RNA was isolated by pulverizing the tissue in liquid nitrogen. Four
molar guanidinium thiocyanate was added to the pulverized tissue. Isolation of
total RNA was performed as described (Chomczynski et al. (1987) Anal. Biochem.
162:156-159; Pagliusi et al. (1989) AMOG. J. Neurosci. Res. ''2:113-119). RNA
yields were estim~ted from absorbance at 260 nm. Ten ,ug of the RNA were
CA 02218599 1997-10-16
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fractionated on 1% agarose-formaldehyde gels for Northern blot analysis (Thomas
(1980) Proc. Natl. Acad. Sci. US,4 77:201-205).
Randomly primed L 1 cDNA probes were used to simultaneously detect the
endogenous Ll mRNA of 6 kb (Tacke et al. (1987) Neurosci. Lett. 82:89-94) and
= 5 the transgene-derived Ll mRNA of 4.2 ~b. Densitometric analysis of Northern
blots was p~lrolllled on sc~nn~cl images (Arcus scanner, Agfa-Gavaert) of the
original films using the Image Program (NIH, Research Services Branch, NIMH).
Northern blot analysis of total RNA from whole brains of the transgenic ~nim~lc
revealed Ll transcripts of a size (4.2 kb) expected for transgene-derived mRNA
(Fig. 2.). These transcripts are clearly distinct from the endogenous Ll mRNA
which is 6 kb and derived from postmitotic neurons. Densitometric analysis
revealed that the levels of transgene-derived Ll mRNA were 34%, 13% and 8% in
lines 3426, 3427 and 3418, r~ e~;Li~lely, as compared to the levels of endogenous
Ll mRNA (rated 100%).
EXAMPLE 4
~nims~l~
For cultures on cryostat sections. immunocytochemi~try and in situ hybridizationhllents~ control ~nim~lc were taken from stocks of age-m~ h~l normal
C57bl/6J mice or non-transgenic littermates. For isolation of small cerebellar
20 neurons and for l.le~,~udlion of astrocyte cultures six-day-old ICR non-transgenic
pups were used. Dorsal root ganglion (DRG) neurons were ~ ~ed from eight-
day-old chick embryos.
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EXA~IPLE 5
In situ hvbri~li7~tiQn
To verify that astrocytes of transgenic ~nim~l~ e~cpressed Ll in vivo optic nerves
were analvzed by in situ hybridization. The optic nerve was chosen since it
5 contains only glial cells and is free of neuronal cell bodies. Astrocytes in vivo
normally lack ~ c~ion of Ll at any developmental stage (unpublished data).
For detection of Ll mRNA in cryostat sections of fresh-frozen brain sections,
digoxigenin-labelled cRNA was generated by in vitro transcription (Dorries et al.
(1993) Histochemistry 99:251-262). The sequence encoding the extracellular part
10 of Ll (Moos et al. (1988) was subcloned into the pBluescript KS+ (Stratagene)vector. Anti-sense and sense cRNA probes were gel.e.clLed by transcribing the L1insert after linearization of the reslllting plasmid with X~o I or ~fba I, using the T7
and T3 promoters, respectively. For generation of GFAP cRNA probes, a 1.2 kb
fragment of GFAP cDNA (Lewis et al. (1984) Proc. Natl. Acad Sci. 81:2743-
1~ 7745; kindly provided by Dr. N.J. Cowan) encoding the N-terminus of the protein
was subcloned into the pBluescript KS+ vector. Anti-sense and sense cRNA
probes were generated by transcribing the resulting plasmid, linearized with Eco RI
and X~o I. from the T3 and T7 promoters, respectively. To improve tissue
penetration~ anti-sense and sense probes were sized under ~Ik~line hydrolysis
20 conditions to obtain an average fragment length of about 300 nucleotides. ~n situ
hybridization on sections of optic nerves ~r~a ed from adult (12-weeks-old3
~nim~l~ was performed as described by elsewhere (Dorries et al. (1993); Bartsch et
al., J. Neurosci., in press).
In non-transgenic controls L1 transcripts were detected only in nerve cells of the
2~ retina but not in optic nerve, neither before (Fig. 3A) nor after a lesion (Fig. 3B).
By contrast, Ll mRNA was ~ s~d by glial cells of the optic nerves from
transgenic mice (Fig 3C). L1 mRNA positive cells were detectable in both the
distal myelinated and the proximal un~nvelinated parts of the nerve. The intensity
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of the hybridization signal was higher in the unmyelinated proximal part, when
co~ aled to the myelinated distal part of the nerve.
A si~ilar distribution of positive cells and similar differences in labelling intensity
beL~ n unmyelinated and myelinated regions were observed using a GFAP cRNA
- 5 probe (CO~ Figs. 3C and E). The number of Ll mRNA positive cells in the
optic nerve of transgenic ~nims~le was, however, always significantly lower than the
number of GFAP-positive cells, probably due to the lower sensitivity of the Ll
cRNA~ probe. Altel"alively, cletect~hle levels of L 1 mRNA might be achieved only
in astrocytes with high levels of GFAP ex~,es~ion. Such a threshold effect couldbe due to the design of the GFAP-Ll transgene which contains only 2 kb of GFAP
5' fl~nking sequences. In vitro studies suggest that the region between 2 and 6 kb
~slleanl of the transcriptional start site contains sequence elements ~ngmPntin~e~lession of GFAP-driven fusion genes in C6 cells (Sarid (1991) J. Neurosci.
28:217-228). Finally, the modification of the GFAP exon 1, including the
introduction of the large Ll cDNA, might reduce the stability of the chimeric
mRNA as co",~ cd to GFAP mRNA and alter effects exerted by regulatory GFAP
sequences located U~SIlc~ll and downstrearn of the modified region.
After lesioning the optic nerve, an upregulation of Ll expression was observed in
transgenic (Fig. 3D) but not in nontransgenic (Fig. 3B) optic nerves. The numberof cells which e~iessed Ll and the h~telisilv of the Ll hybridization signal were
similar in different individuals of the same transgenic line but varied across
different ~ sg. nic lines. Con.eietent with the results obtained by Northern blot
analysis (see above). Ll mRNA positive cells were most abundant in line 3426
followed by line 3427 and, finally, line 3418. This variability in the level of
transgene ~res~ion in different lines could be related to a number of factors, in
particular, effects caused by the neighboring host chromatin regions fl~nkinE the
dirr~,~"~ transgene integration sites (Proudfoot (1986) .?vature 322:562-565; Reik et
al. (1987) Nature 328:248-251; Sapienze et al. (1987) Na~ure 328:251-254).
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EXAMPLE 6
Antibodies
Production of polyclonal rabbit antibodies against mouse L 1 and purification on an
Ll imm-mcl~ffinity column (Rathjen et al. (1984): Martini et al. (1988) and
5 polyclonal antibodies against mouse liver membrane (Lindner et al. (1983);
Pollerberg et al. (1985) have been described. A mouse monoclonal antibody
against GFAP was purchased (Boehringer Mannheim).
For Western blot analysis, polyclonal and monoclonal antibodies were vi~ li7~o~1 by
horseradish peroxidase conjugated goat anti-mouse or rabbit antibodies (Dianova,10 Hamburg, C:;~,mally). For immunocytochemistry, primary antibodies were ~et~cf.od
using fluorescein isothiocyanate- or tetramethylrhodamine isothiocyanate-conjugated
goat anti-rabbit and goat anti-mouse antibodies (Dianova). Digo~cigenin-labelledcRNA probes for in situ hybridization were vi.cl-~li7P~l by ~lk~line phosphatase-
conjugated Fab fragments to digoxigenin (Boehringer ~nnheim).
EXAMPLE 7
Maintenance of neurons on clyostat sections
To analyze whether optic nerves from transgenic ~nim~l~ are more conducive to
neurite outgrowth than optic nerves from wild type ~nim~ cerebellar neurons
were m~in~in~l on cryostat sections of lesioned and contralateral unlesioned optic
20 nerves (Fig. 7).
Optic nerves of 6 to 16-week-old mice were prepared as described by Bartsch et al.
(1989) J. Comp. l~ieurol. 284:451-462. In brief, lesioned and unlesioned optic
nerves were embedded and frozen in serum-free, hormonally defined mediurn
(Fischer (1986b) Neurosci. Lett. 28:325-329) using liquid nitrogen. Tissue sections
25 (14 ~m thick) were cut longitudinally on a Frigocut 270-cryostat (Jung-Reichardt),
mounted onto poly-L-lysine-coated (Sigma. 0.001~,~o in water) sterile glass
coverslips and air-dried for 2-3 hours in a sterile chamber. After washing the
sections for 5 minutes with medium. Percoll gradient-purified small cerebellar
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neurons (~eilh~lPr et al. (1985) Nature 316:728-730) from six-day-old ICR mice
(6 x 10~ cells in 100 ~1 medium) were applied to each coverslip. Cells were
m:~int~;n.orl in an incl-h~tQr at 37~C with a humidified atmosphere of 5% CO. and
95% air.
5 Neurite outgrowth was also measured in the presence of antibodies. Sections were
pre-incub~te~l with polyclonal L1 antibodies or polvclonal antibodies against mouse
liver membranes (100 ~lg/ml, dialyzed e~lensi~ely against and diluted in culturemedium) for 1 hour at 37~C. After removal of antibodies, sections were washed
carefully with culture medium (5 times, each for 5 min~ltec at room ttl~ ldlLIre)
10 and Percoll gradient purified small cerebellar neurons were added. After 2 days,
cryostat cultures were fixed in 4% paraformaldehyde in PBS for 30 minllt~s at
room telll~ldL-~Ie and the neurite lengths were measured. To avoid "edge effects"
in the measurem~ntc~ we did not evaluate the sections which were situated in theouter rim comprising 20% of the coverslips. Using a semi-~ltom~tic colllpul~l
15 image analysis program (IBAS, Kontron, Zeiss) the lengths of all neurites which
had grown on these sections were measured and the average neurite length per
neuronal cell body calclll~t~cl For each e~ lilllent and optic nerve (lesioned or
unlesioned), the average length of neurites grown on nerves of transgenic ~nim~lc
was related to the corresponding values of control ~nim~lc Twelve independent
20 ~ ~I.c,.hl1ents were performed with lesioned and contralateral unlesioned nerves
using at least two transgenic ~nimz~lc
For trans,~enic ~nim~l~, an increase in neurite length was observed on lesioned
colllp~ed with unlesioned nerves. In contrast, neurite lengths on lesioned and
unlesioned optic nerves of wild type ~nim~l~ were not significantlv different (Fig.
25 8). Neurites of neurons cultured on unlesioned optic nerves from transgenic
~nim~ were con.ci~te~tly longer than neurites of neurons cultured on unlesioned
nerves from ~vild type ~nim~lc A m~Yimal increase in neurite length of about
300% was observed when using sections from line 3426. Similarl~, neurite length
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on lesioned nerves of transgenic lines was increased up to 400% when cor"l)~e~
with lesioned nerves from wild type ~nim~lc
The neurite outgrowth promoting activity of transgenic optic nerves correlated
positively with the level of L1 c~ s~ion (Fig. 8). Unlesioned optic nerves of line
5 3426, which express the highest levels of Ll protein were more potent in
increasing neurite outgrowth than those of lines 3427 and 3418 expressing, by
comparison~ lower levels of L1 (in dec,easillg order). On lesioned optic nerves of
lines 3426 and 3427 (28 days after the lesion), neurite outgrowth was four timeshigher than on lesioned optic nerves of wild type ~nim~ The finding that the
10 increase in neurite outgrowth in lesioned optic nerves was similar for the lines 3426
and 3427 (although line 3426 shows 25% increase in Ll protein e~ e;,~ion after
lesion as compared with line 3427) could indicate that the level of L1 protein in
line 3427 already suffices for maximal induction of neurite outgrowth from smallcerebellar neurons.
15 Pre-incubation of unlesioned optic nerves from wild type ~nim~l~ with polyclonal
antibodies to Ll or mouse liver membranes did not significantly affect neurite
lengths (Fig. 9). In contrast, neurite lengths were reduced by more than 50% when
cryostat sections of unlesioned or lesioned optic nerves from the transgenic line
3426 were pre-incubated with L 1 antibodies (Fig. 9). Antibodies to liver
20 membranes, which strongly bind to optic nerves and small cerebellar neurons (data
not shown), did not show similar inhibitory effects. Inl~ies~ gly, pre-incubation of
lesioned optic nerves from wild type ~nim~lc with Ll antibodies in-lllce-l an
increase in neurite outgrowth compared with lesioned nerves from wild type
~nim~lc without a prior antibody pre-incubation. Antibodies to mouse liver
25 membranes did not show a significant increase under the same conditions,
indicating that addition of cell surface reactive antibodies per se does not disturb
neurite outgrowth.
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EXAMPLE 8
Maintenance of neurons on monolayer cultures of astrocytes
To ~lep~c astrocyte monolayers, fo~ebldil~s from six-day-old mice were cleaned
free of non-neuronal tissue and dissociated as described elsewhere (Schnitzer et al.
5 (1981) J. Neuroimmunol. 1:429-456: Fischer et al. (1982a) Neurosci. Lett. 29:297-
302; Keilh~lçr et al. (1985). Cells were m~ r~l on poly-L-lysine-coated
(Sigma, 0.001% in water) cell culture flasks in BME medium (Gibco) cont~ining
10% horse serurn and 2 mM glllt~mine for 14 to 2 I days. Cont~min~in~
oligodendrocytes and neurons were removed by ~h~king the flasks at every medium
10 change and by subculturing the cells at inteNals of four days. lmmllncst~inin~ for
GFAP after 14 days of m~ tf ~ e showed that more than 90% of the cells were
astrocytes. After 14 days in culture, the cells were trypsinized and m~int~in-orl as
monolayers for five days on poly-L-lysine-coated glass coverslips. Percoll gradient
purified small cerebellar neurons (Schnitzer et al. (1981)) from six-day-old mice
15 and dorsal root ganglion (DRG) (Seilhtoimer et al. (1988) J. Cell. Biol. 107:341-
351) neurons from eight-day-old chick embryos were then added onto the astrocytemonolayers. After 6 hours of co-culture for cerebellar and 12 hours for DRG
neurons, the co-cultures were fixed with 2% paraformaldehyde in PBS and neurite
lengths were analyzed as described in Example 7.
20 Neurite outgrowth from mouse small cerebellar or chick dorsal root ganglion
(DRG) neurons was also studied in monolayer cultures of astrocytes derived from
transgenic (line 3426) or non-transgenic controls (Fig. 10, Table 1).
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TABLE 1
Neurite lengths of cerebellar and dorsal root ganglion (DRG) neurons
maintained on astrocytic monolavers prepared from wild type mice (WT) and
the transgenic line 3426.
Cerebellar DRG
neurons neurons
WT 65 + ~ mm 90 + 10 mm
WT + anti Ll 72 + ~0 mm lOS + 14 mm
WT + anti liver 57 + 24 mrn 107 + 12 mm
0 3426 75 + 41 mm 137 + 10 rnm
3426 + anti Ll 45 + '~5 mIn 88 ~ 8 mrn
3426 + anti liver ~8 + 31 mm 124 + 15 nllll
~eurite lengths on astrocytes without pre-incubation with any antibody or after
c.l~ with polyclonal antibodies against Ll (anti Ll) or antibodies against
15 mouse liver membranes (anti liver) are shown. ~lean values + standard deviation
are from at least 100 neurons from two independent t;~e~ ,ents carried out in
quadruplicate.
Neurite length of cerebellar or DRG neurons on transgenic astrocytes was
approximately 15% or 50% higher. respectively~ when colllp~lcd with neurite
20 length using wild type astrocytes (Table 1). Anti-liver membrane antibodies did
not affect neurite length on astroc,vte monolayers from wild type or transgenic
zlnim~l~ (Fig. 10, Table 1). Pre-incubation of astrocyte monolayers with L1
antibodies did not significantly affect neurite length on cells from wild type
z~nim~l~ In contrast~ it reduced neurite length of cerebellar or DRG neurons grown
25 on cells from transgenic ~nim~l~ by approximately, 40%. It is noteworthy in this
context that the polyclonal antibodies directed against mouse Ll used in this study
do not react with neurons from chicken (Martini et ah, 1994a; data not shown). By
immllnofluorescence analysis it could be sho~n. ho-vever, that these antibodiès
bind as efficiently as Ll antibodies to astrocytes from transgenic ~nim~l~ as well as
30 to mouse small cerebellar neurons (data not sho~n).
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EXAMPLE 9
Immullofluorescence and Aurion-GP immunogold microscopy
Ll and GFAP immunosf~ining of fresh-frozen cross- or longitll-lin~lly sectioned
r optic nerves or astrocytic monolayers of wild type and transgenic ~nim~l~ were
5 pc.Çolllled as described (Bartsch et al. (1989)). For double-labelling, we first
~ incllb?te~l astrocytes as live cells with Ll antibodies (2 ~g/ml in 1% BSA in PBS)
at 4~C for 30 minlltec After permeabilizing the cells with 70% methanol at -20~Cfor 10 minllteC, cells were incubated with GFAP antibody for 30 minnt~s at 4~C.
For quantification of neurite lengths in cryostat culture experiments, the Aurion
10 immuno R-Gent silver enhanced st~ining was used according to the m~nnf~ctl-rer's
instructions (Aurion, Tmmllno Gold Reagents & Accessories Custom Labelling,
Wageningen, The Netherlands) with minor modifications. In brief, cultures were
fixed in 4% paraformaldehyde in PBS for 10 minutes at room telllp~-dlule,
incubated in 50 mM glycine in PBS for 10 mimltes and then treated for 15 min~ltes
15 in blocking buffer (BB, 0.5% BSA in PBS). ARer 3 washes in BB, each for 5
minllt~s, cells were incnh~tPcl with Ll antibodies diluted in BB (2 ~g/ml) for 30
minllteS at room telll~ dlulc. Subsequently, cultures were washed 3 times in BB
each for 5 mimlt-os and secondary antibody diluted 1:20 in BB was added for 1
hour at room te~ -dlule. ARer 3 washes with distilled water, cultures were fixed20 in 2% glutaraldehyde in PBS for 10 mimlt~s at room temperature and washed 3
times with distilled water. A 1:1 mixture of enh~n~er and developer was then
added at room Lt~ laLule. ARer the a~pea,d"ce of the reaction product,
coverslips were washed 3 times with tlictille~l water and embedded in glycerol.
In optic nerves of non-transgenic mice, L1 immunoreactivitv was restricted to
2~ unmyelinated retinal ganglion cell axons (Bartsch et al. (1989)). In unlesioned
optic nerves from transgenic ~nim~lc, weak Ll immunoreactivity was also found inassociation with cell bodies and radially oriented cell processes (Fig. 4A). Theintensity of this Ll immunoreactivity in transgenic optic nerves increased
significantly aRer a lesion (Fig. 4B) and was similar in distribution to the GFAP
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immunoreactivity found in unlesioned (Fig. 4C) or lesioned (not shown) wild typenerves.
Ll expression was additionally analyzed in cultures of astrocytes ~rel,~ed from
forcblaill of six-day-old transgenic ~nim~l~ No L1 immunoreactivity was
5 clçtect~ble on astrocytes from wild type ~nim~lc (Fig. 5D). In contrast, Ll positive
cells were present in cultures from transgenic ~nim~ls (Fig. 5A). As demonstrated
by double-immllnost~ining, the same cells also proved positive for GFAP (Fig. 5Band E) indicating that the cells ~x~les~illg L1 are indeed astrocytes. Since Ll
immunost~ining was perforrned on living cells, it seems likely that in the transgenic
10 ~nimzll~ L1 is also exposed on the cell surface of astrocytes in vivo.
EXAMPLE 10
Western blot analysis
To further qu~llila~e the amount of Ll expression in GFAP-L1 transgenic mice,
detergent extracts of homogenates of unlesioned and lesioned (15 days after the
15 lesion) optic nerves from wild type and ~ sgellic adult mice were analyzed on Western blots (Fig. 6).
Lesioned (15 days after the lesion) and contralateral unlesioned optic nerves from
8-week-old ~nim~l~ were cleaned free of non-neuronal tissues and then frozen in
liquid nitrogen. Care was taken that only myelin~tç~l distal but not Ll
20 immllnoreactive unmyelinated or partly myelin~t~rl proximal regions of the nerves
were used. Nerves were frozen and thawed ten times before sonication with a
Branson B15 sonicator at 4~C for 5 mimltes The tissues were then homogenized
with a Dounce homogenizer in homogenization buffer (1% Triton X-100, 2 M
urea, 5 mM ben7~mic~ine, 0.1 mM iodo~ret~mide 1 mM phenylmeth~nçsulfonyl
25 fluoride, 5 mM Na-p-tosyl-L-lysinechloro-methyl ketone in PBS). Homogenates
were cleared by centrifugation at 16.000g at 4~C for 15 mimltes Supern~t~nt~
were treated with methanol/chloroform to precipitate proteins as described by
Wessel et al. (1984). The protein content was deterrnined in the supernatant
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(Pierce). After SDS-PAGE on 7% slab gels under reducing conditions, proteins
(25 ~Lg) were analyzed by Western blotting using polyclonal Ll antibodies (0.4
~glml). Horseradish peroxidase-conjugated secondary antibody (2 ~g/ml) was
~1etected by the ECL Western blotting detection kit (Amersham). Densitometric
5 analysis of imml-noblots was ~elrolllled on sc~nn~l images (Arcus scanner, Agfa-
Gavaert) of the original films using the Image Program (NIH, Research Services
Branch, NIMH).
Densitometric analysis of the immllnQblots demonstrated that Ll ~ ,s~ion in
unlesioned optic nerves of transgenic ~nim~l~ was about 40% and 13% (lines 3426
10 and 3427, respectively) higher than in unlesioned optic nerves of wild type ~nim~lc
Ll expression in lesioned transgenic nerves was 310% and 200% (lines 3426 and
3477, respectively) higher as colll~,arcd with lesioned nerves of wild type ~nim~lc.
A comparison between lesioned and contralateral unlesioned optic nerves from wild
type ~nim~l~ revealed a decrease in Ll protein ~ le~sion of about 40% on the
15 lesioned side. In contrast, the amount of L1 protein in lesioned nerves of lines
3427 and 3426 increased by approximately 30% when colll~alcd with the
unlesioned contralateral side. The ~,eà~ion level of Ll in line 3426 was
a~loxi,llately 35% and 25% higher than in the line 3427 for unlesioned and
lesioned optic nerves, respectively.
2~ EXAMPLE 11
In vivo r~ of axons in the optic nerve
6-8 week old GFAP-L1 transgenic mice and wild type mice were crushed
intraorbitally and, after 14 days, traced with a fluorescein-labeled biotin ester to
mark retinal ganglion cell axons by anterograde labeling. Results are shown in
25 Figures 11 and 12. Each point ~ es~ s one animal.
E~MPLE 12
Identification of the border between fibronectin type III homologous repeats 2
and 3 of the neural cell adhesion molecule L1 as a neurite Out~rvwll
promoting and signal transducing domain
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To determine the domains of neural cell adhesion molecule Ll involved in neuriteoutgrowth. monoclonal antibodies against Ll were generated and their effects on
neurite outgrowth of small cerebellar neurons in culture investi~terl When the
eleven antibodies were coated as substrate, only antibody 557.B6, which recognizes
an epitope represented by a synthetic peptide comprising amino acids 818 to 832 at
the border between the fibronectin type III homologous repeats 2 and 3, was as
potent as Ll in promoting neurite outgrowth, increasing intracellular levels of Ca~
and sl:imulating the turnover of inositol phosphates. These fintling~ suggest that
neurite outgrowth and changes in these second messengers are correlated. Such a
10 correlation was confirmed by the ability of Ca2'-channel antagonists and pertussis
toxin to inhibit neurite outgrowth on Ll and antibody 557.B6. These observationsindicate for the first time a distinct site on cell surface-bound Ll as a prominent
signal transducing domain through which the recognition events appear to be
funnelled to trigger neurite outgrowth, increase turnover of inositol phosphates and
lS elevate intraceilular levels of Ca'+.
EXAMPLE 13
L2/H[NK-l immunoreactivity in reinnervated peripheral nerve: preferential
expression of previously motor a~on-associated Schwann cells
The carbohydrate epitope L2/HNK-1 (hereafter ~lesign~te~l L2) is t~ essed in the20 adult mouse by myelin~ting Schwann cells of ventral roots and muscle nerves, but
rarely by those of dorsal roots or cutaneous nerves. Since substrate-coated L2
glycolipids promote ouL~lvwLh of cultured motor but not sensory neurons, L2 may
thus influence the yl~rerelllial reinnervation of muscle nerves by regenerating motor
axons in vivo.
2~ Therefore. the influence of regenerating axons on L2 ~ .e~sion by reinnervated
Schwann cells was analyzed by directing motor or sensory axons into the muscle
and cutaneous branches of fe noral nerves of eight-week-old mice. Regenerating
axons from cutaneous branches did not lead to immunocytochemically detectable
L2 e~ c;~ion in muscle or cutaneous nerve branches. Axons regenerating from
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5~
muscle branches led to a weak L2 exl,res~ion by few Schwann cells of the
cutaneous branch, but provoked a strong L2 t;~ s~ion by many Schwann cells of
the muscle branch. Myelin~ting Schwann cells previously associated with motor
axons thus differed from previously sensory axon-associated myelin~tin~ Schwann
5 cells in their ability to express L2 when contacted by motor axons. This
- upregulation of L2 ~ re~ion during critical stages of reinnervation may provide
motor axons regenerating into the ~ ;ate, muscle pathways with an advantage
over those regenerating into the i~ u~liate~ sensory l~lhw~y~.
EXAMPLE 14
10 L1 in consolidation of memory for a passive avoidance task in the chick
Training day-old chicks on a one trial passive avoidance task, in which they learn
to ~u~les~ their ten-l-oncy to peck at a small bright bead if it is coated in the bitter-
tasting methylanthranilate, results in a time-dependant cellular and molecular
c~cc~Ae cnlmin~tin~ in the remodelling of pre- and post-synaptic elements in two15 discrete regions of the forebrain, the intermeAi~t~ medial hyperstriatum ventrale
(IMHV) and Lobus parolfactorius (LOP) (Rose (1991) Trends In lVeurosciences
14:390-397). The r~cc~ involves two distinct waves of glycoprotein synthesis, as
evidenced by enhanced fucose incorporation, occurring in both IMHV and LPO at
varying times following training. Both waves are nececc~ry for long-term (that is.
20 24 hours plus) memory retention for the avoidance tasks, in which ~mnesi~ is
evici~onrecl by chicks, which would otherwise avoid the previously bitter bead,
pecking at a dry bead on test.
Given the role of Ll in meAi~ting cell-cell contact. the present study was
undertaken in order to determine if Ll is amongst the learning-associated
25 glycoproteins participating in either or both waves of glycoprotein synthPsic, and is
necessary for memory formation. If so, antibodies to Ll ~Aminictered at an
a~ .iate time relative to training should prevent the svnaptic remodelling
.~ n~c.oss~ry for long term memory and therefore produce ~mnesi~ for the task.
Similarly, if the extracellular domains of the Ll molecule play a part in the
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56
recognition and adhesion processes which are required for synaptic remodelling and
stabilization, e~ogenously applied extracellular domain fr~sJrn~nt~ which will bind
homophilically to the endogenous molecule might disrupt this process.
Antibodies and Fragments
5 Polyclonal antibodies were plc~ ,d in rabbits by imm~lni7~tion with imml-no-
affinity purified Ll (Ng-CAM, 8D9) following an established immllni7~ti~n
procedure (Rathjen et al. (1984)). L1 was isolated from one-day old chicken brains
using an 8D9 monoclonal antibody (Lagenaur and Lernmon (1987) Proc. Nat'l
Acad. Sci. USA 84:77533-7757) column again using established procedures
10 (Rathjen et al. (1984)). Antibodies were isolated from the serum obtained after the
third immnni7~tion using Protein G Sepharose (Pharmacia LKB) according to the
lr~rtllrer7s instructions. Recombinantly e~lessed fusion proteins in E. coli
repres~ntin~ the si~ immlmoglobulin-like (Ig-I-VI) and five fibronectin type IIIhomologous repeats (FN1-5) were p~ ,d as described by Appel et al (1993).
Sodium dodecyl sul&te polyaclylamide gel electrophoresis (SDS-PAGE)
and immunoblots of chick subcellular fractions
Fifty ~lg of protein from brain homogenate, from crude membranes. from a solublefraction (Burchuladze et al. (1990) Brain Res. 535:131-138) and from postsynaptic
densities (Murakami et al. (1986) J. Neurochem. 46:340-348), all from day-old
20 chicken brains were separated by SDS-PAGE under reducing conditions on a 5-
15% polyacrylamide gradient gel (T ~emmli (1970) Nature 227:146-148),
whereafter they were L~ r~ d to nitrocellulose according to the method of
(Burnette (1981) Anal. Biochem. 112:195-203). After overnight incubation with Llantibodies at a dilution of 1:1,000 in Tris-buffered saline, pH 7.2, cont~inin~; 5%
25 rief~ cl milk powder. immunoreactive bands were detected according to previously
described methods (Scholey et al. (1993) Neurosciences ~:499-509).
Training and testing procedures .
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Day-old Ross chunky chicks of both sexes. h~tchecl in incubators were place in
pairs in small pens, ~ ailled to peck at small (2.5 mm) white beads and then
trained on a larger (4mrn) chrome bead coated with methylanthranilate as described
by Lossner and Rose ((1983) J. Neurosciences 41:1357:1363). Birds which pecked
5 the bitter bead evinced a stereotyped disgust response, .sh~kin~ their heads
vigorously and b~kin~ away from the bead. Twenty four hours following training,
each animal was tested by the ~cs~ on of a dry chrome bead identical to the
one used in training. Retention of passive avoidance learning was indicated in
~nim~l~ avoiding the test bead. In each replication of this protocol, 24-36 chicks
10 were trained and tested. More than 80% of trained, uninjected chicks normallyavoid the bead on test under these conditions, though there is som~tim~s a slight
reduction in avoidance in saline injected birds. By contrast, birds which are trained
on a water-coated bead peck the dry bead avidly on test, and their avoidance score
is rarely above 5-10%. All training and testing was routinely carried out by an
15 ~x~c~ enter blind as to the prior tre~trnent of the zmim~lc
lnje~tions
Ll antibodies? FN1-5 and Ig I-VI fr~gm~ons~ were dialyzed overnight against 0.9%saline and the concentration adjusted to 1 mg/ml for Ll and 250 ~Lg/ml for the
fr~gment~. Chicks received bilateral intracranial injections into the intermediate
20 medial hyperstriatum ventrale (IMHV) of 10 ~11 Ll antibodies per hemisphere;
control ~nim~lc received similar injections of saline. Accurate delivery into the
IMHV was received by the use of a specially ~ ign~l head holder and sleeved
Harnilton syringe (Davis et al. (1982) Pharm. Biochem. Behav. 17:893-896).
Chicks receiving this injection volume of either saline or antibodies prior to
25 training or testing showed no overt behavioral effects, pecking the bead accurately
during training. The large extracellular volume of the brain of the newly h~tch~?d
chick means that injections of this size are well-tolerated, and can be achievedwithout leakage. A previous report has demonstrated (Scholey et al. (1993)) thatthere is a slow diffusion of antibody from the injection site in the hours following
30 injection. The accuracv of placement of the injection was routinely monitored by
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visual inspection of the brains post-mortem. In each replication of the t;~ h~lent.
a balanced group of saline and antibody or fragment-injected chicks were
employed. In the Ll ~ hl.ent, groups of chicks were injected with saline or
antibody at one of eight time points relative to training; 2 hours or ~0 minlltec pre-
5 training, or +1 hour. +3 hours~ +4 hours. +5.5 hours, +8 hours or +12 hours post-
training. On the basis of previous observations, it was predicted that any effects
would be observed in birds injected at either 30 mintltPs prior or 5.5 hours post-
training, and the numbers of replications at these time points were accordingly
greater (N=17, 28, 17, 19, 18, 21, 19 and 18 respectively for antibody injections).
10 Ll fr~m~ntc FN1-5 and lg I-IV were injected at either -30 minnt~s or +5.5 hours
and retention tested at 24 hours. Retention in groups of saline and Ll-antibody or
Ll-fragment-injected chicks was colnpal~ d statistically by X~- Results are shown ~n
Figures 13 and 14.
EXAMPLE 1~
15 Involvement of Ll and NCAM in long term potentiation
Transverse hippocampal slices (400 ~lm) from halothane-~n~sthPtized male Wistar
rats (180-220g) were prepared using standard techniques. Slices were m~int~intodin an interface chamber and initially allowed to recover for 45 min. in a
hyperosmolar (320 mOsm/kg) artificial cerebrospinal fluid (ACSF) at room
20 temperature. The bath temperature was then raised to 30~C and the medium was
changed to a normotonic ACSF (307 mOsm/kg) co.-t~;.,i~-g (in mM): NaCI, 124.0;
KCI, 2.5; MgSO24, 2.0: CaC12, 2.5; KH.P04, 1.25; NaHCO3, 26.0; glucose, 10;
sucrose, 4; bubbled with 95% O./5% CO, (pH 7.4); perfusion rate: 0.75 ml/min.
The Schaffer collateral/commiccllral fibers were stim~ te(l by twisted pl~tinnm-
25 iridium wires (50 ~Lm tii~m~ter) placed in the stratum r~ tllm of the CAl region.Test stimuli consisted in monophasic impulses of 100 ~s duration every 30 seconds
and the stimulus strength was adjusted to obtain 30% of the maximal EPSP
amplitude (maYimal EPSP without superimposed population spike). EPSP s were
recorded from the CA1 stratum r~ tllm bv means of 2 glass micropipettes (2 M
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NaCl, 1-5 MQ) positioned about 300 ,uM apart from the stimt-l~tion electrode on
each side.
.. Af~fer stable rccoldillg for at least 15 minutes, antibodies or protein frt7gmçn~c were
ejected onto the CAI dendritic field in the vicinity (50-75 ~lm) of one lecol~illg
electrode (the one carefully adjusted at 30%) by using a modified microinjectionsystem (Nanoliter injector, WPI) continuously delivering 5 nl every 10 seconds up
to the end of the experiment unless otherwise indicated. A wash-out of the
antibodies with subsequent induction of LTP was not possible for evident reasons,
but it was verified whether LTP could be inrlured within each slice by recordingfrom the second electrode where no antibodies were applied. Although the tip of
the ejection micropipette did not penetrate the slice, a small reduction in the EPSP
amplitude was sometimes observed when the ejection was started. This volume
artifact was independent of the nature of the ejected material. Proteins were
dialyzed against 20 mM PBS at pH 7.4 unless otherwise indicated and
concentrations referred to the pipette concentration.
Twenty min~ltes after initiating the microejection, LTP was in~ ced with a thetaburst stimul~tion (TBS) paradigm concictin~ of three trains spaced by ~ seconds;each train consisted of ten high frequency bursts of 5 pulses at 100 Hz and the
bursts were separated by 200 ms (Reichardt et al. (1991) Annu. Rev heurosci.
14:531-570). Duration of the stim~ tion pulses was doubled during TBS.
Induction of LTP could be totally prevented by perfusion of 10 ~fM D(-)-2-amino-5-phosphonc,l,en~loic acid (D-AP5; Tocris). Whole cell recordings were obtained
from CAl neurons using the "blind" patch clarnp method with an EPC-9 patch
clarnp amplifier. The bath temperature was 30~C. Patch electrodes were pulled
from 1.5 rnm OD borosilicate glass and had recict,.nces between 3 and 8 MS2. Thepipettes were neither fire polished nor coated. The electrodes were routinely filed
f with a solution containing (in mM): potassium gluconate, 12~; KCl, ~; MgCl" l;
CaCl2, 1; N-(l-hydroxyethyl)-piperazine-N'-(2-eth~nes-~lphonic acid) (HEPES), 5;1.2-bis~2-aminophenoxy)ethane-N,N,N'.N'-tetraacetic acid (BAPTA), ~; Na-ATP,
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10 and Na-GTP, 0.3. with pH adjusted to 7.3 using KOH. Series resict~nre was
not compen~tP~l Responses were sampled as an avera~e of three to four signals,
either printed out for visual analysis. or stored on disk for further analysis.
St~tictir~l evaluations were perforrned by analysis of variances with planned
comparisons and contrast analysis; time was considered as a dependent variable .
with one level of repeated measures. Anti-Ll (Rathjen et al. (1984)), anti-Ig I-VI
(Hynes et al. (1992)) and anti-liver membranes antibodies (Linder (et al. (1983))
were produced as previously described. Results are shown in Figure 15.
Ig-like domains I-VI and FN type III homologous repeats I-V of Ll were ex~ressedin bacteria and purified as described (Hynes et al. (1992)). Antibodies to NCAM
and axonin-l were produced as described (Larson et al. (1986) Science 232:985-
988; Bailey et al. (1992) Science 2~6:645-649). Production of oligomannosidic
glycopeptides from ribonuclease B and control gl~/cop~ ides from asialofetuin have
been described (Larson et al. (1986)). Results are shown in Figure 16.
NMDA receptor-mediated EPSP's were isolated by applying 30 ,uM of the non-
NMDA blocker 6-cyano-7-nitro~uinoxaline-2,3-dione (CNQX; Tocris) starting 20
minlltes prior to the application of antibodies or ,glycopeptides. At the end of each
experiment, it was verified that D(-)-2-arnino-5-phosphonopentanoic acid (D-APS;30 ~lM; Tocris) completely ~u~l~ressed these responses. Results are shown in
Figure 17.
EXAMPLE 16
L1 exerts neuropreservative effect
An experiment was perforrned to further elucidate the activity of Ll with CNS
nerve tissue. Specifically. aliquot samples of mouse mesencephalon cells were
plated and cultured on four separate plates having media ~le~ d as follows: the
first control plate was coated with poly-L-lysine alone; a second plate was coated
with poly-L-lysine and Ll; a third control plate was coated with poly-L-lysine and
l~minin; and a fourth plate was coated with poly-L-lysine. I~minin and L1. All
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plates received aliquot amounts of cells and were incubated under identical
conditions. After 7 days. the plates were all stained for the presence of dopamine
and thereafter observed. The plates that were coated with L1 exhibited a growth of
200% to 400% greater than the controls. The plates coated with l~minin e~chibited
5 ~reater neurite outgrowth, but not more cells than those coated ~,ith Ll. The
results demonstrate and suggest that Ll exerts a profound neulopre3el~ative effect,
as cell viability measured by numbers of cells gro~n was dramatically increased
over controls.
EXAMPLE 17
l0 Soluble L1 (L1-Fc) is functionally active and is a potent agent in neuronal
~ur~ival
Soluble Ll was made in COS cells as a recombinant Ll-Fc fusion protein by the
procedure described in Neuron 14:57-66, 1995. The recombinant protein was
purifie,d by Protein A affinity chromatography, and was used either as a substrate
15 coated onto plastic or as a soluble molecule added to the culture medium at
approximately l-l0 ~Lgiml. Neurite outgrowth and survival of mes~n-ephalic
neurons from day 17 rat embryos were examined in culture after 7 days in vitro
m~int~-n~nce. Dopaminergic neurons were recognized by imrnunostaining for
dopamine-~-hydroxylase (DBH) and quantified using IBAS morphometric
20 equipment. Cultures with added soluble Ll-Fc were m~int~inP(l on poly-DL-
ornithine (PORN) and substrate-coated L1-Fc was added on top of previously
coated PORN (under conditions described in Appel et al? J. Neuroscience 13:4764-4775, 1993). NCAM-Fc was used as a control.
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Table - Survival and neurite out~rowth of DBH- neurons after 7 days in ~i~ro
number of neurons~ lenPth of neurites~~
substrate-coated Ll 129 ~ 20 179 + 40
soluble L 1 98 + 7 13 ~ + 27
PORN onlv (control) 14 + q 37 + 9
vlean vaiues are ~'rom at least three mdepenaent e~penments _ SkM
The numbers are from a unit field
~ ~~ The lengths of all neurites (total neurite length) per neuron was determined
(in !lm)
Recognition amonP neural cells is an important prerequisite for the development of
10 a functioning nervous system. Recognition molecules are e.Y~,csscd at the cell
surface, where thev me~ te interaction between nei_hboring cells, like cadherins.
or between the cell surface and the e~ctracellular matri~;. like integrins (Takeichi.
1991; ~-losl~hti~ 1988; Hynes, 1992). The most prominent family of reco~nition
molecules comprises imm~lno~lobulin (Ig)-like domains. The In-like domains
15 reflect a cornmon ancestrv of immunoQlobins and cell adhesion molecules. both of
which are involved in specific reco~nition events (Edelman. 1970!. In the nervous
system the I~ su~c-r~ilv comprises bv now more than two dozen distinct
molecules. Some In-like domain cont~ininE molecules have multiple functions
~,vithin the extracellular domain: Receptors for cvtokines and neurotrophins have
20 hi~h affinitv receptive functions as ~ ell as reco~nition ~lu~c~Lies (T~nn~hill et al..
1995; Pulido et al.. 1992). The three--lim~nsional structure of IP-like domains is
similar to FN-like r~, ~?t,~ et al. 199'': Leah,v et al.. 199'7). which are als~
structural motifs in several e~tracellular matri~ molecules. such as fibronectin.
members of the tenascin famil~. and others (Williams and Barcla~. 1988: Barnn etal.~ 199~: Erickson. 1993) ~'~eural reco~nition molecules of the I~ supefamil~ ha~,-e
characteristic temporal. spatlel. and cell-tvpe specific espression patterns (for
revie~s. see Edelman. 1988: Schachner. 1991. 199~: Rathien and Josseli. 1991:
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Rutiehauser, 1993). Recognition molecules of this farnilv are functionally
overlapping in that all promote cell adhesion and neurite outgrowth. Some
recognition molecules are strongly homophilic, i.e. self binding partners, whereas
others are predomin~ntly heterophilic, i.e. they bind to non-self partners whichoften comprise other members of the Ig s~e~ lily or extracellular matrix
molecules (Briimmen~orf and Rathjen, 199~, 1994). Present knowledge of the
functional ~r~ Lies of the individual Ig-like ~lom~in~ and/or FN-like repeats ofneural recognition molecules in~ te both distinct, and overlapping functional
properties in recognition, neurite outgrowth, and repulsion (Gennarini et al., 1991;
10 Frel et al., 1992; Taylor et al., 1993; Appel et al., 1993, 1995; Pesheva et al., 1993;
Feisenfeld et al., 1994; Hoim et al., 1995).
Among neural recognition molecules of the Ig su~ ~.r~lily, the family of molecules
related to the neural recognition molecule L 1 shows striking similarity in function
and structure. They are potent neurite oul~,.owLh promoters and are ~ essed
15 relatively late during development, mostly at the state when axogenesis occurs.
They are predomin~ntly c~ ed by neurons, although some members of the L1
family are also present on neurite outgrowth promoting glial cells (Martini and
Sch~ ner~ 1986: Bixby et al.l 1988; Seilheimer and Sch~rhner~ 1988).
In the experiments that follow, another member of the Ll family is identified and
20 characterized, that is clesign~t~l a close homolog of Ll (CHLl). It colllahls six Ig-
like domains and FN-like repeats, of which four are highly homologous to the FN-like repeats of other Ll family members. The partial FN-like repeat localizes tothe membrane-adjacent region of the molecule, which is the most variable region
among L1 related molecules. Other features of CHLl shared with members of the
2~ L1 family are its predominant and developmentally late expression in the nervous
system, and its high level of N-glycosylation. including e.~ ea~ion of the HNK-1carbohydrate.
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EXAMPLE 18
MATERIAL AND METHODS
,Anim:~lc
ICR mice and Wistar rats were used for tissue ple~dLions.
Antibodies
Po}yclonal antibodies directed against the recombinantly ex~ulessed e~tracellular part
of CHLI (amino acids 499-1063 (Figures 18 and 19)) and Ll (amino acids 126-
1981 (Appel et al., 1993)) were raised in rabbits as described (Rathjen and
Sçh~hner, 1984). To raise antibodies against CHLl ~00 ~Lg of purified peplitle
was injected into rabbits followed by four additional injections of 100 ~lg in
intervals of three weeks. Ll antibodies were concentrated from serum by
ammonium sulfate precipitation (13.5 mg/ml). Monocional rat antibody 412 was
used to identify the HNK-l carbohydrate epitope (Kruse et al., 1984). Monoclonalantibodies against glial fibrillary acidic protein (GFAP) were obtained from
Boehringer (Mannhelm). The monoclonal antibodies to the Ol antigen(s) has been
described (Sommer and Schachner~ 1981).
Purification of neural adhesion molecules
Ll, N-CAM. and MAG were immllno~ffinity purified from detergent extracts of
crude membrane fractions from adult mouse brain using monoclonal antibody
columns (Rathjen and Sch~f~hn~r7 1984; Falssner et al., 1985; Poltorak et al., 1987).
cDNA libraries and screening
P.~,p~dLion of the ~gtl 1 library derived from poly(A)' RNA of brains from 8-day-
old mice and screening of this library with immunoaffinity purified polyclonal Ll
antibodies were performed as described (Tacke et al., 1987). To obtain longer
cDNA clones. a new DNA library uas constructed: ~NA was purified from brains
of 6 to 14-day-old mice by the ~uanidinium thiocyanate/acid phenol method J
(Chomezynski and Sacchi, 1987). Poly(A)~ RNA was enriched by two subsequent
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passages over an oligo(dT)-cellulose colurnn (Sarnbrook et al.~ 1989). Ei~ht
micrograms of poly(A)+ RNA were used for synthesis of oligo(dT)-primed double-
stranded cDNA using a cDNA synthesis kit (Amersha~n). The cDNA was size-
selected and ligated into the plasmid pXMDl with DraIII-adaptors cont~ining a SalI
S site (KluYen et al.. 1992). For propagation and amplification of the library, E. coli
' strain TOP10 (Invitrogen, Netherlands) was used. For screening, aliquots were
directly plated onto Nylon membranes (BIODYNETM Pall) with a density of about
2x104 bacteria/filter (138 cml). Replica filters were inc~b~ttol1 overnight at 37~C.
Subsequently, the bacteria were lysed (0.5M NaOH; 1.5M NaCl), filters were
10 neutralized (3M NaCl; 0.5M Tris-HCl pH 8.0), washed in a 2xSSC, air-dried, and
baked for 2 hr. at 80~C. The Nylon membranes were prehybridized, hybridized
with a 1 kb fragment (HincII/KpnI) of CHLl (derived from the ~gtl 1 library)
redioiabelled by random-priming (Boehringer Mannheim) according to the
m~nllf~c-turer s protocol, washed under high-stringency conditions at 42~C. and then
15 e~{posed to X-ray film as described else-vhere (Sambrook et al., 1989). Six positive
clones were further characterized by restriction mapping and seql-Pncing according
to standard protocols (Sambrook et al.~ 1989). One clone (pXi~2) cont~inin~ a 4.43
kb CHL1 insert was used for further analysis.
DNA Sequencing and sequence analysis
20 Nucleotide sequences were determined by the dideoxy-chain-termination method
(Sanger et al., 1977) using double-stranded DNA as a template for T7 DNA
polymerase (Pharmacia) and synthetic oligonucleotides as primers. cDNA
sequences were assembled and analyzed with the DNASTAR program (DNASTAR,
Inc., London). Unless otherwise indicated, amino acid sequences were aligned by
25 the Jolun Hein method (Hein, 1990) (gap penalty = 11, gap length penaltv = 5, K
tuple = 2).
Comp~rison of protein sequences
To calculate a similarity index (%) for comparison of the distances between
conserved amino acid residues, several distinct proteins cont~ining SiX Ig-like
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domains and at least four FN-like repeats were aligned at conserved amino acid
residues in the Ig-like domains (~;y~Leil1es which refer to S-S bridges) and in the
FN-like repeats (tryptophan and tyrosine/phenyl~l~nine). The number of amino
acid residues between these conserved positions was determined. This is referredS to as the concPncuc ~ict~n-e The mean value of the ~ict~n~eS~ i.e. the con~er~cl~c
distance and standard deviation (SD) among Ll family members were calc~ t~l
SD values were rounded up to the next integer. The rlict~n(~e for each protein was
compared to the mean ~1ict~n~e and considered as match if the distance value
equaled the mean value + the SD (= con~P~Cllc ~lict~nre). The number of m~tchP~s10 to the l9 conc~Pncllc ~i~t~nt~Pc was calculated for each individual protein (similarity
index = nurnber of m~trl~Ps / 19 x 100). For example: In the CHLl protein 16
distance values match to the consensus ~i~t~n~Pc while three of all criteria did not
match. This leads to a similarity index of 16 / 19 or 84% for CHLl.
EXAMPLE 19
1~ Cell culture and expression of CHLl and L1 in COS-1 cells
The 4.43 kb insert of clone pX#2 was ligated into SalI digested pXMD1 (Kluxen etal., 199'~, Kluxen and Lobbert, 1993). A subfragment (EcoRl (plasmid
polylinker)/Pvull bp 4048) of the mouse L1 cDNA (Moos et al., 1988) was treated
with T4 DNA polymerase and ligated into pXMDl.
20 COS-1 cells were m~int~ined in DMEM (0.1% glucose) supplemented with 10%
(v/v) fetal calf serum at 37~C in a humidified atmosphere with 5% CO2. DEAE
dextran-mediated DNA transfection was performed as described (Kluxen et al.,
1992) with some modifications. Briefly. the cells were seeded at about 10,000
cells/cm'. One day later, after two washes with DMEM (0.45% glucose), the
25 medium was replaced by transfection solution composed of DMEM supplemented
with 10% (v/v) Nu-serum (Becton Dickinson, Switzerland), 0.4 mg/ml (w/v)
DEAE-de?;tran (Pharmacia), 50 ~LM chloroquine. and 1.25 ~g!ml DNA (4 ml per
10 cm dish). The cells were incubated 4 hr at 37~C and 5% CO.. Then the
mediurn was removed and the cells were incubated for '' min in phosphate-buffered
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saline (pH 7.3) cont~ining 10% dimethylsulfoxide (v/v)~ After two washes with
DMEM (0.45% glucose), DMEM supplemented with 10% (v/v) fetal calf serum
and 20 ~lg/ml gentarnycin uas added and the cells were incubated in this medium.24 hr later cells were ~l~t~eh~cl by incubation with 0.01% trypsin and 0.0004%
5 EDTA in Hanks' b,.l~nt~e~l salt solution (HBSS) for 5 min at 37~C, replated for
imml-n~ cytochemi~try at a density of about 20,000 cells/cm- in 2~-well plates
(Falcon) CO.~ P poly-L-lysine coated glass coverslips (11 mm in ni~m~ot~r), and
incubal:ed for an additional 24 hr. For Western blot analysis the cells were replated
on tissue culture dishes and incubated for an additional 48 hr.
10 PC12 cells were m~int~in~cl in DMEM with 10% (v/v) fetal calf serum and 5%
(v/v) horse serum on collagen coated tissue culture dishes. For induction of thecells with nerve growth factor (NGF) the medium was removed from monolayers at
about 50% confluency and replaced with medium of reduced serum content (5%
horse serurn) suppl~m~ont~cl with 100 ng/ml 7s-NGF (Sigma, Switzerland). After
15 two days of incubation the cells were detached by incubation with 0.1% trypsin and
0.04% EDTA, collected and subjected to RNA extraction.
Primary cultures of astrocytes were ~.lepaled according to McCarthv and De Vellis
(1980) with modifications (Guénard et al. 1994) and used for imml-nost~ininP after
one to two weeks in vitro. Primary cultures of oligodendrocytes were prepared as20 described by Laeng et al. (1994) and m~int~in~d in vitro for 12 days.
EXAMPLE 20
Production of antisense RNA
The 4.43 kb insert of clone pXi~2 was ligated into SalI digested pBS II SK.
(Stratagene) followed by deletion of an ApaI (vector)/AvrII (bp 3330 (Figure 18))
25 fr~E~m~nt to obtain the cDNA fragment of CHLI encoding the extracellular part of
the protein (see Figure 18 and 19). A similar construct for L1 was prepared by
ligation of an EcoRI (plasmid polylinker)/EcoNI (bp 3304) fragment of the L1
cDNA (Moos et al.. 1988) treated with T4 DNA ploymerase and ligated into SmaI
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digested pBS II SK-. The plasmids were digested with Xbal and used for synthesisof 32P-labeled ~nti~en~e RNA with T7 RNA polvmerase as described (Melton et al.,1984).
Northern blot analysis
S Poly (A)- mRNA was prepared from different tissues of neonatal and 9-day-old
mice using the OligotexTM Direct mRNA-Method (QIAGEN Inc., Dusseldorf,
Germany) following the m~nllf~f turer s instructions. Poly (A)- mRNA and RNA
marker (RNA ladder~ GIBCO/BRL) were subjected to electrophoresis on a 0.8 %
forrnaldehyde/agarose gel and subsequently transferred to Hybond-N membrane
10 (Amersham) by capillary transfer (Southern, 1975) in 20x SSC. After W
crosclinking (UV-Stratalinker~) 1800, Stratagene, La Jolla, CA), the amount of
RNA transferred and bound to the membrane was controlled by methylene blue
staining (Sambrook et al., 1989). Following prehybridization for 2 hr at 65~C, the
membrane was hybridized overnight using CHLl- and Ll- specific 3~P-labeled
15 ~nticen~e RNA probes in hybridization buffer (5:cSSC, 2.5xDenhart s solution, 50
mM Na2PO.I (pH 6.5). 0.1% SDS, lmM EDTA. ~ ~g/ml salmon sperm DNA, 50%
formamide) at 65~C. The filter was then washed three times at 65~C in O.lxSSC,
0.1% SDS for 1 hr and exposed tO X-ray film.
EXAMPLE 21
20 Expression and purification of recombinant CHL1 protein in E. coli
A 1.7-kb cDNA-fragment of CHL1 (Mscl; bp 1791 (which origin~tçs from the
vector cloning site and the S' end of the ~gtl 1 derived CHLl clone) and BsmAl;
bp 3494) encoding the 6th Ig-like domain (IgVl) and FN-like repeats 1.4.~ (see
Figures 18 and 19b) was subcloned into the unique BamHI restriction site of the
2~ pET-vector (Studier and Moffatt, 1986). The correct sequence of the plasmid was
confirmed by sequencing. E. coli strain BL21 (DE3) was transformed with this
plasmid. E~pression and purification bv anion exchange chromatography of the
recombinant protein ~vere performed according to Appel et al. (1993). SDS-PAGE
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and Coomassie st~ining showed a major band at the e~cpected molecular weight (70kD) which contained at least 80% of the total protein (not shown).
Tissue fractions
Det~rgellt lysales of whole tissue were prepared by homogenization of tissues in 40
S mM Tris-HCl (pH 7.4), 150 mM NaCI, 5 mM EDTA, SmM EGTA, lmM
phenylmethysulfonylfluoride (PMSF), 1% Triton X-100 and m~int~ineci at 4~C for
3 hr under co~ t stirring. The soluble fraction was s~ Led from insoluble
material by centrifugation at 100,000 g.
For l~re~dlion of detergent Iysates of membrane fractions, tissues were
10 homogenized in 1 mM NaHCOz (pH 7.9), 0.2 mM CaC12, 0.2 mM Mgcl~, 1 mM
spermidine. 5 ~lg/ml aprotinin, 10 llg/ml soybean trypsin inhibitor. 1 mM PMSF,
and 0.5 iodoacetamide at 4~C. Membrane and soluble fractions were then
separated and the membrane pellot was resuspended in solubilization buffer (20
mM Tris-HCl (pH 7.9), 0.15 M NaCI. 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-
100, 5 ~Lg/ml Aprotinin, 10 ~Lg/ml soybean trypsin inhibitor, I mM PMSF, and 0.5mM ioclo~et~mide).
Transiently transfected COS-l cells were washed twice with HBSS and incubated
with 1 mM EDTA in HBSS for 10 min at 37~C. The cells were then ~et~ h~l
with a fire polished Pasteur pipette and collected by centrifugation at 200g for 10
20 min at 4~C. The cells were lysed in 20 mM Tris-HCl (pH 7.4), 1~0 mM NaCl, 1
mM EDTA, 1 mM EGTA, 1 mM ioclo~cet~micle, 1 mM PMSF~ and 1% MP-40 and
the supernatant was cleared by centrifugation (13000g). Protein determinations
were performed as described by Bradford (1976).
Western blot analysis
25 Proteins were separated by SDS-PAGE (T.~emmil 1970) on 8% or 10% slab gels
under reducing conditions and transferred to nitrocellulose filters (0.45 ,~Lm~ BA 8~;
Schleicher & Schuell. Dassel. Germany) for immunodetection according to Faissner
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et al. (1985), using CHLl antiserum (diluted 1:500. l:10000 for ECL), Ll
polyclonal antibodies (diluted 1:1000, 1:15000 for ECL). or monoclonal antibody
412 (diluted 1: 1000. 1 :10000 for ECL) and alkaline phosphatase-coupled secondary
anti-rabbit or anti-rat IgG. Bound antibodies were either ~letecte~l by the e~h~n~e~
S chemiluminescence (ECL) method according to the m,.nt-f~rt~lrer's instructionsusing ECL Western blotting detection reagents (Amersham) and ~-ray filrns, or byusing BCIP and NBT as chromogenic substrates.
EXAMPLE 22
Enzyme-linked immunosorbent assay
lO The enzyme-linked immImosorbent assay (ELISA) was performed as described by
HIlem~.nn et al. (1992) with the exception that proteins were coated at
concentrations of 100 ng/ml. CHLl antiserum was used in several dilutions
bet~,veen 1:250 to 1:2Xl06.
Deglycosylation of CHL1
15 Detergent lysates of brain tissue homogenate (200 ~11. 6 mg/ml protein
concentration) from seven-day-old mice were sel,alaled into a soluble fraction and
insoluble material by centrifugation (see Tissue fractions) and incubated with 0.5
units N-glycosidase F or 2.5 units O-glycosidase, or both enzymes at these
concentrations according to the m,.nI~f~cturer s instructions (Boehringer Mannheim,
20 Gerrnany). The Iysates were resolved by SDS-PAGE on 10% gels. The proteins
were transferred to nitrocellulose and incnh~t~ with CHL1 antiserum (1:500
diluted) directed against the recombinant CHL1 protein fragment (see Figure 18).
Immunoprecipitation
The soluble fraction of detergent Iysates of brain tissue homogenate (300 ~1. 5
25 mg/ml protein concentration) from nine-day-old mice (see Tissue fractions) was
incubated ~ith 10 !11. CHL1 antiserum or polyclonal antibodies a~ainst Ll
overnight at 5~C in 5 ml buffer (20 mM Tris-HCI (pH 7.4), 150 mM NaCI. 5 mM
EDTA) cont~ining 1% NP40 and 30 ~11 of G-Sepharose (Pharmacia/LKB). After
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sequentiallv washing the buffers contz~inin~ 0.1% NP40, 0.05% SDS, and then 20
mM Tris-HCl (pH 7.~), the Sepharose beads were boiled for 10 min in 5x sample
buffer (250 mM Tris-HCI (pH 6.6), 10% SDS, 50% glycerol, 0.5% bromophenol
blue, 25% B-mercaptoethanol) and the supernatant was resolved by SDS-PAGE on
5 10% gels. The proteins were transferred to nitrocellulose and ~1~tecte~ with
polyclonal antibodies against Ll. CHLl antiserum, or monoclonal antibody 412 by
Western blot analysis.
Indirect immunofluorescence
For cell surface s~ining (Scl.~ and S~h~rhn~r~ 1981) CHLl-, and mock (vector
10 only)- transfected COS-1 cells plated on coverslips were incubated for 30 min at
room temperature with primary antibody (CHLl antiserurn (1:100 diluted) or Ll
polyclonal antibodies (1:200 diluted)) in DMEM COI-t~;,.;.,g 10% fetal calf serum,
10 mM Hepes (pH 7.3), and 0.0'~% NaN3, and then with secondary antibody. After
innunost~inin~, the cells were fixed with 4% paraforrnaldehyde in phosphate
15 buffered saline (pH 7.3) and mounted in Moviol (Hoechst) cont~ining 2.5%
po~ium iodide. For double-immnnofluorescence st~ining of astrocytes and
oligodendrocoytes, incubation of primary antibodies to cell surface antigens wasperforrned as described for transfected COS-l cells. Subsequently, incubation ofcells with primary antibodies against intercellular antigens was pe~ro~ ed after20 permeabilizing the cells with methanol at -20~C.
EXAMPLE 23
In Situ Hybridization
To generate digoxigenin-labelled ~ntieen~e cRNA probes of equal size from
corresponding parts of CHLl and L1 the same constructs as for Northern blot
25 analysis were used. Sense probes were generated from similar constructs with the
inserts in opposite direction. All the cRNA probes were generated using T7 RNA
polymerase follo~ved by an ~Ik~line treatment to obtain an average fragment length
of 250 nucleotides. In situ hybridization was perforrned as described (Bartsch et
al., 1992; Dorries et al., 1994).
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72
RESULTS AND DISCUSSION
Identification of CHLl cDNA
Screening of a ~gtl I expression library for cDNA clones encoding the cell
adhesion molecule Ll with polyclonal antibodies raised against brain-derived
immlmopurified Ll (Tacke et al. 1987) identified the clone 311. It cont~ined a
partial cDNA homologous to Ll (34.1% according to Lipman and Pearson (198S))
and an open reading frame of 2112 base pairs (bp) coding for 704 amino acids
including the cytoplasmic part. To isolate full length cDNA clones, a DNA
fragment of this clone was used for screening a different cDNA library. Six
independent clones were isolated. Two clones contained 4.2 and 4.4 kb inserts
comprising the entire coding region of a close homolog of Ll (CHL1). The clone
cont~ining the 4.4 kb insert was further investig~tt~A
DNA and deduced amino acid sequences and structural features
The 4.4 kb insert encodes a 5' untr~ncl~tP~I region of 295 bp, an open reading
frame of 3627 bp, and a 3' untr~ncl~t.od region of 518 bp (Figure 18). Although
there is an oligo(A) tract at its 3 terminus, a clear consensus polyadenylation
signal u~sl.c~l- of this sequence is mi~sing. The fl~nking sequences of the AUG
start codon (position 296, Figure 18) do not conform to the optimal con~en~llc
sequence for initiation of translation (Kozak, 1987). However, this AUG is takenas the start codon for translation based on two lines of evidence. It is preceded
Ir~sL,cal~l by stop codons in all three reading frarnes and is followed by a potential
signal sequence with a cleavage site predicted after residues 24 or 25 (scores of
8.65 and 6.40, respectively, according to the algorithm of von Heijne (1986))
(Figure 18).
2~ Translation of the open reading frame yields a protein of 1209 amino acids with a
calculated molecular mass of 134.9 kD and features characteristic of an integralmembrane glycoprotein. The putative extracellular domain is composed of 1081
amino acids. with 18 potential sites for N-glycosylation (Figures 18 and l9a) and
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more than 60 potential O-glycosylation consensus sites (not shown) (Pisano et al.,
1993), followed by a tr~n~membrane domain of 23 amino acids, as judged by
hydro~alhy analysis according to Kyte and Doolittle (1982) (Figure l9c). This
domaim is flanked at its N-terminal end by a polar residue and at its C-terminal end
5 by a basic amino acid, conci.~tent with a stop transfer signal (Figure 18). The
intracellular region is composed of 105 amino acid reci~ es
The extracellular region contains the two major structural motifs of repeated
domains that are characteristic of the Ll farnily: a 685 amino acid stretch withhomology to Ig-like domains and a 472 amino acid stretch with homology to FN-
10 like repeats (Figures 1 and 2a). All of the six Ig-like domains contain the
characteristic pair of cysteine residues located at 47-54 amino acids apart from each
other (Figure 19). A conserved proline (except in the sixth Ig-like domain) at the
end of ~3-strand B in conjunction with a C2-type cluster of conserved amino acids
around the second cysteine residue in each domain (DXGXYXCXAXN) assign the
15 Ig-like domains to the C2-set (Williams and Barclay, 1988). Between the Ig-like
domains and the membrane s~ .;..g region are four domains that are homologous
to the FN-like repeats in fibronectin (Kornblihtt et al., 1985). Each of these
domains of approximately 100 amino acids contains the highly conserved
tryptophan (except for the first FN-like repeat) and tyrosine/phenyl~l~nine residues
20 in the N- and C-terminal regions, respectively. Interestingly, the fifth FN-like
repeat is, in contrast to the other members of the Ll family, only a rllrlim~nt~ry
one-half FN-like repeat (Figure 18). Whether this half FN-like repeat ~ ;,ent~
one of several alternatively spliced forms, one of which contains a full FN-likerepeat, remains to be ~let~rmin~l by other methods than Northern blot analysis. It
25 is noteworthy in this context that no evidence for alternative splicing was found by
restriction analysis of the six independently isolated clones (not shown).
Alternative splicing of the fifth FN-like domain was observed for Nr-
CAM/BRAVO, where cDNA isoforrns were isolated lacking the fifth FN-like
repeat (Grumet et al.. 1991: Kayyem et al.. 1992.) The absence of the fifth FN-
30 like domain in chicken neurofascin (Volkmer et al.. 1992) is most probably also
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due to altemative splicing. since its rat homolog. the ankyrin-binding glycoprotein
(ABGP) (Davis et al.. 1993), contains a fifth FN-like domain. Thus, CHLl adds a
new structural feature to the L1 family; only four and one-half FN-like repeats are
re~i~ed (Figure 19).
5 Another structural feature of CHLl is the presence of an RGD sequence (arnino
acids 185-187) in the second Ig-like domain (Figure 18. This tripeptide has
originally been identified as a cell Qtt~hment site within the tenth type III domain
of fibronectin (Pierschbacher and Rusolahti, 1984) and contributes to il1te~,l;nbinding (for review see Rusolahti and Pierscllb~c~er, 1987). Three ~lim~n~ional
10 structure analysis of FN-like repeats showed that the RGD motif is localized
between the ~3-strands F and G (Main et al., 1992). This motif is also found in
other members of the Ll family. In the third FN-like repeats of chicken Ng-CAM
(Burgoon et al., 1991) and the species homologs chicken neurofascin and rat
ABGP, the RGD sequence is found at the same position, between the J3-strands F
15 and G. RGD motifs are also found in Ll (two in L1 mouse and rat (NILE), and
one in human Ll (Moos et al., 1988; Hlavin and Lemmon, 1991; Prince et al.,
1991). All Ll RGD sequences are found in the sixth Ig-like domain, but in a
different amino acid environment than RGDs in the FN-like modules of fibronectin.
As in Ll, the tripeptide in CHLl is localized on the 13-strand E of the second Ig-
20 like domain. Whether the RGD sequences in these proteins are functionallv activeis currently not known. It is noteworthy in this context that neurite extension
in~ ce~l by TAG-l (Furley et al., 1990), a member of the F3/F11 family
(Briimmen~lorf and Rathjen, 1993, 1994) that contains a RGD motif in the second
FN-like domain depends on B, inte_rin and Ll (Felsenfeld et al.. 199~). This
25 observation raises the possibility of a direct physical interaction between the second
FN-like repeat of TAG-l and 13, integrin.
CHL1 also contains a DGEA sequence (amino acids 555-55~) in the 13-strand C of
the sixth Ig-like domain (Figure 1~). This sequence is not found in other members
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of the Ll family. The DGEA sequence has also been implicated in a~3, h~Le
recognition of type I collagen co"lS ",;I-g this motif (Staatz et al., 1991).
Structural similaritv of CHLl with other recognition molecules of the Ig
superfamily
A comparison of the amino acid sequence of CHLl with the tr~n~l~tecl EMBL gene
sequence ~l~t5lbs~c~ showed that CHLl is 87.2% identical to a 109 amino acid long
stretch and 79.6 identical to a 93 amino acid long stretch previously identified in
human brain (accession number HS2431 and HSXT02610 (Adams et al., 1992,
1993)). Thus, there a~pe~u~ to be a highly conserved CHL1 molecule in human.
10 The sequences of mouse, hllm~n, and rat Ll/NILE, chicken Ng-CAM, chicken Nr-
CAM, zebrafish Ll.l (Tongiorgi et al., 1995), chicken neurofascin/rat ABGP,
Drosophila neuroglian (Bieber et al., 1989), mouse F3/chicken F1/human CNTN1
(C~ i et al.. 1989; ~n~rht, 1988; Briimmen-lorf et al., 1989; Berglund and
~n.~eh~ 1994), rat TAG-1/ chicken axonin-1/ human TAX-1 (Furley et al., 1990;
1~ Hasler et al., 1993; Tsiotra et al., lg93), and rat BIG-l/ mouse PANG (Yoshihara
et al., 1994; Connelly et al., 1994) taken from the tr~n~l~te~ EMBL gene sequence
~l~t~ e were compared with CHL1. The comparison is displayed in Table 1,
below.
CA 02218599 1997-10-16
WO 9613295!~ PCT/US96JO~434
76 ~ C U
O r
~ ~q N
-- S C t) 1:
r U O ~
S~ ~ ~ ~ ..
a) ~ E~ U
E~ l S
~ ~ 3 ;~ O
,~ ~a--O
~
a~ o
~ ~ ~ C
~1) H 1
a 3 Q~
.a ~s ~-1 s
~, --' _ ~ ~ ~ ~ ~ o ~ s
C 0 ~ '
~ ' ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ C8
~,n _ o ~ n
u ~ 13
-- O _~
a
~n o
h ~: h -I h 11
u ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ S ~
a) _l ~ x
~ ~ S ~ l U
a~ cO~U ~ N ~ U 7 ,s~
o _, ~ ,,
a~ n h
~n -~ E ~ ~r ~ ~ ~ ~ ~ N ~ I r ~S U
_~ C U ~-
~ r~ ~o u
r t~ E
r O ~ ~
h ~ a h
C~ U ~ _ _ S
~ O
o ~ ~ a, 3 ~,1
U ~ _ r ~ ~ 5 ~ S
~ -- ~ ~ ~ -- h z~ u
,~ E ~ ~ _ E ~ a) h ) U
m ,~ x ~ s
~ z z ~ ~E-' Q C~~S 5 Z S ~ ~
E~ h E~ O ~ N
CA 02218599 1997-10-16
WO 96/329S9 PCTIUS96/0!;434
77
o ~
s ~ z
s
,. o~ _ o~ ~ 3 ._
_~ _ s -
~ o ~ ~, ~ ~ u .
h
5~ U ~I
C C C ~-
r ~
:~ _ s--
~1 . _~
. ~ ~ ~o
~ . . _ _ ~ _ ~ _ ~ _ ~
. ~
,~, 8 1~ O o _ ~ ~ ,~ S~u
a
- ~ a c
v ~ c
~ ~c a
'S5. ' O a _~
~ ~ . ~ .,. ~ ~ = '' ~ r v ~
3 ~ ~ ~ o
Z ~ _l
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O
c, F
a ~ ~ c '
~ ~ a o
- o ~s
_ ,c 11 ~,
_ ~ s
~ ~ z :E _ ;r 1 S S ~ ~ ~ ~ ~
i~ o 0 r 0
CA 02218599 1997-10-16
PCT/US96/0~;'t3
WO g6/329S9 78
:~, x x
~ g ~ ~ 5 ~' ~ ~
.
~T
-
y ~ _ _ _ _ r. ~ _ O ~ r- ~ .o
C,
j~ ~ 3 s z ~ i y ~ ~ ;
CA 02218599 1997-10-16
WO 961329S9 PC'rJUS96/0~434
79
;. h ~
C ., ~ ~
' V O
C C
,~--a~ C
_ ,c _ ~
- i 5 ~ o~ v. U
~ -- ~ ~8 0
~ ,c ~ o, ~ g C.
~ ~ ~ o '-- '
- 5 5 ~ lovc~
..
-
V . -- ~ i
5 - ~ o ~ 5 ql
,, C .~:~a o
I h C
o
~ ~V"V~
X X ~ u ~ r m ~ r
C ~ ~ ~ C C~
E--1~ . s ~ '
I Z ~ ~ V
Z t-. 11 m s
, <,, O~ O~ O~ ~ O~ O , ~ ~ ~ O
~D ~ S ;~
S
V
" ~ ~ ~. S~ o o o~ ,,0 _ C 14 S _ a C~
~ ., . z m
~ ~ _. _ _ _ _ _ O O ~ ~ a~ 'q~
z ~ I ~a K
S Z -I ~
a ~~ X ~ ~ C
~ ~ ~ _ _ _ _ _ _ ~ ~' '~ ~ ~ ~ O N q ~)
~ ~ .
O ~ _ _
r C.l '
~ ~ ~ 5 .,, :5~ , ~ , , , , ~ q~ _ ~ ~ q
s z
.. ~" .-1 _
3 "Z ~ 8~-- ~
O ~ ~4 ~ q u~
S, .~Sv q)
tn ~o x s _I ~ c
I ~ ~ ~ V ~ ~I
m m
g 3 ~ g ~ c ~ Y c~ ~ ~ x g ~ s~cq ''c~'
o. ~ cr~
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WO 96~32959 80
CHL1 is most similar to chicken Ng-CAM (37% amino acid identitv in the
extracellular domain. Table 1) and mouse Nr-CAM (64% arnino acid identity in theintracellular domain. Table 2). However. the degree of identity, particularly in the
extracellular part, is not sufficient to consider these proteins as species homologs.
5 Recently, a partial cDNA clone of mouse Nr-CAM (Moscoso and Sanes, 1995) was
identified. Mouse Nr-CAM is nearly identical (99%) to chicken NR-CAM (see
Table 2). Therefore. CHL1 is not likely the Nr-CAM homolog in the mouse.
CHL1 is the fourth member of the Ll family in the mouse with Ll, Nr-CAM,
neurofascin (Moscoso and Sanes, 1995), and CHLl, with a highly conserved
10 species homolog in human (Adams et al., 1992, 1993).
Considering the similarity of the intracellular sequences of chicken and mouse Nr-
CAM and of chicken and mouse neurofascin (99% and 87%, respectively, Table 2),
it is highly unlikely that mouse Ll and chicken Ng-CAM are species homologs,
since they show only 01% sequence identity in the intracellular domain (Table 2).
15 Rather, the existence of Ng-CAM as the fifth member of the L1 farnily in the
mouse with a highly conserved intracellular domain is to be expected.
Interestingly, mouse Ll upon heterophillc interaction with chicken Ng-CAM
promotes neurite outgrowth (Lemmon et al.. 1989). suggesting that members of theL1 family may interact with each other.
20 Besides the similarities in the overall structure of L1 family members (Table 1 and
Table 3), the most hi~hly conserved regions can be id~nsifie~l in the cytoplasmic
domain (Figure 20). This striking homology is evident for members of the L1
family in all species so far identified which contain an intracellular domain: L1,
CHL1, Nr-CAM, Ng-CAM, neurofascin, neuroglian, and zebrafish L1.1 and also
25 for the partial sequences of zebrafish L1.2 (Tongiorgi et al., 1995) and mouse Nr-
CAM and neurofascin (Moscoso and Sanes. 1995) (Table 2). Within this region
two stretches, one located close to and partially within the plasma membrane-
sp~nning segment and the other at its C-terminal end (I, III in Figure 20), are
nearly identical. Another amino acid stretch conserved in L1, Ng-CAM, Nr-CAM~
30 neurofascin. Ll.1 and Ll.2 but not in CHL1 (Table 2) contains a RSLE motif (II
in Figure 3) that origin~tes by alternative splicing and is expressed only in neurons
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(Grurnet et al.. 1991: Miura et al., 1991; Volkrner et al., 199'~). Since the
intracellular region is most highly conserved bet~-een these proteins. all members
of the Ll family may use the same signal transduction pathway to activate neurite
extension. It has been demonstrated that the cytoplasmic domains of ABGP, Ll,
5 and Nr-CAM can interact with ankvrin linking cell recognition to the cytoskeletal
scaffold (Davis et al.. 1993, Davis and Bennett, 1994).
E~AMPLE 24
Identil~lcation of structural requirements in the e~tracellular domain to classify
members of the Ll family
10 To further study the general criteria for membership in the Ll family, we
- invçstig~ted the position of highly conserved amino acids in the Ig-like domains
(cysteines ~vhich refer to the S-S bridges) and FN-like repeats (tryptophan,
tyrosine/phenyl~l~nine) for several members of the Ig supc.r~ ily (Table 3).
Molecules cont~ining six Ig-like domains and at least four FN-like repeats (Ll
15 family and the GPI linked F3/FI 1 subgroup (Brummenclorf and Rathjen, 1993))
reveal a very constant number of amino acids separating these conserved amino
acids. Five different ~ t~n~e pararneters were considered:
1) the number of amino acids s~lJdldlillg the conserved cysteines which form thecysteine (S-S) bridges of each Ig-like domain (Table 3. columns Igl, Ig2, Ig4, Ig5,
20 and Ig6);
2) the number of amino acids between the second cysteine of one Ig-like domain
and the first cysteine of the next Ig-like domain reflecting the distance between two
adjacent Ig-like domains (Table 3, columns 1-2, 2-3, 3-4, 4-5, and 5-6):
3) the number of amino acids between the last conserved cysteine of the sixth Ig-
25 like domain and the conserved tryptophan of the first FN-like repeat reflecting the
t~nce between the Ig-like domain-module and the FN-like repeat-module (Table
3, columns IgG-FNl):
4) the number of amino acids between the conserved tr,vptophan,
tyrosine/phenylalanine of each individual FN-like repeat (Table 3, columns FN1,
30 FN2, FN3, and FN4):
S) the number of amino acids between the tyrosine/phenyl~l~nine of one FN-like
repeat and the tryptophan of the next FN-like repeat reflecting the distance between
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two z~ ont FN~ ;e repeats (Table ,. colu~Tms FNl-FN'7. FN~-FN3. and FN3-
FN4).
To obtain highly stringent conditions for comparison of the Ll-like molecules, we
did not consider a few values clearly deviating from the average, most probably
S due to alternative splicing, for the calculation of the average distances and standard
deviations (neuroglian: Ig2, 2-3: Nr-CAM and ABGP: Ig6-FN1; F3: Ig4, FN3; Ng-
CAM; FN2, FN3 (Table 3, mzlrk~cl with an *)). Whereas the number of amino
acids between the S-S bridges for the first and sixth Ig-like domain (Igl; standard
deviation (SD) = 3: Ig6: SD = 4) and the number of amino acids between tne
10 conserved tryptophan, tyrosine/phenyl~l~nin~ of FN-like repeats three and four
. (FN3: SD - 4; FN4: SD = 3) are slightly variable, all other distance parameters
remain remarkablv constant for the different molecules (FNl-FN2: SD = 0; FN2
and 2-3: SD = 2 (Table 3)). Based on these criteria we calculated a similarity
index (see Material and Methods) for several Ig-like molecules in relation to the
15 average values listed in Table 3. For Ll, CHLl. Ng-CAM, Nr-Cam, ABGP, Ll.l.
TAG-l and BIG-l a similarity index of 74-95% was obtained (Table 3). For
Drosophila neuroglian a slightly lower value was determined (66%), most probablyreflecting the evolutionary ~ t~n~e between vertebrates and insects. F3 and its
species homologs are loss conserved, particularly in their Ig-like domains, but still
20 show a similarity, index of 63%. However, some conserved amino acid stretches underlying the strongly conserved colinearity (e.g.
FxVxAxNxxG(8x)S(4x)TxxAxPxxxP at the end of the first FN-like repeat or
NxxGxGPxS between the last two ~3-strands of the third FN-like repeat (not shown)
support the notion that F3 belongs to the Ll family. Interestingly, the number of
25 amino acids between adjacent domains (Ig-like domains or FN-like repeats) is even
more conserved among these molecules. indicating that the distance between the
individual domains is an important structural feature, i.e. critical for functioning of
neural recognition molecules (Table 3. columns 1-2, 2-3, 3-4, 4-5. 5-6, FNl-FN2,FN2-FN3, and FN3-FN4). Thus, this high conservation of the order (colinearity)
30 and spacing may be used to define more generally the extracellular domain of the
Ll family members. With the criteria just defined. these contain a module of six
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Ig-like domains at the N-terrninus followed by four FN-like repeats. We would
like to call this structural feature the Ll family cz~csette.
Thus, all members of the Ll family share the characteristic features of the Ll
family c~Csette and. additionally, highly conserved arnino acids. These results
suggest that these molecules derive from a common ancestral Ll-like molecule
co~ the Ll farnily c~sette, which might have spread its function potential
via gene duplication to accommodate the evolving demands for diversifying cell
interactions in more complex nervous systems. The general L1 family may thus be
subdi~rided into the "classical" Ll farnily members (Ll, CHLl, Ng-CAM, Nr-CAM.
neurofascin, neuroglian, Ll.l) which contain a variable fifth FN-like repeat, a
tr~n~membrane domain, and a highly conserved intracellular domain, and the
F3/F11 subgroup (F3/F l l/CNTNl, BIG-l/PANG, and TAG-1/Axonin- l/TAX-1),
the common feature of which is the linkage by GPI to the membrane and for which
a variable fifth FN-like repeat has so far not been identified. The extracellular
domains of both subgroups contain the Ll family r~cettP.
Other members of the Ig-sul,~.r~llily, e.g. N-CAM (Cl~nningh~m et al., 1987,
Barthels et al., 1987). MAG (Arquint et al., 1987; Lai et al., 1987; Salzor et al.,
1987), neuromusculin (Kanla et al., 1993), and rse (Mark et al., 1994) or
fibronectin (Kornblihtt et al., 1985) which contain Ig-like domains and/or FN-like
repeats, show clearly distinct distance parameters~ indicating that they are much less
related to each other and to the members of the Ll family (Table 3). Ill~ele~Lillgly.
the human leukocyte cornrnon antigen-related gene (HLAR) (Streull et al., 1988)
and the tumor ~u~l~lcssor gene product (DCC) deleted in colorectal cancer (Fearon
et al., 1990) are closely related to the Ll family according to the flict~nre
parameters (42% and 50% similarity index~ respectively). Inspection of conservedamino acids reveals that DCC is indeed related more closely to the Ll family than
to N-CAM as previously suggested by Fearon et al. (1990) and Pierceall et al.
(1994), although it seems to have lost the fifth and sixth Ig-like domain. Recent
studies show that DCC is expressed predomin~ntly in brain and that neurite
,~ 30 outgrowth of rat PC12 cells is stimulated on a substrate of DCC-transfected
fibroblasts ex~les~ing the protein on its cell surface (Pierceall et al., 1994).
CA 02218599 1997-10-16
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Although HLAR also shows a relatively high similarity index its relationship to the
Ll family is not so obvious.
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WO 961329S9 85 PCT/U~
EXAMPLE 2~
Tissue distribution of CHLl mRNA and Protein
To investig~tÇ whether CHL1 shares the predominant expression in the nervous
system with other members of the L1 family~ we analyzed the expression of CHLl
5 in various tissues at the mRNA and protein levels. In Northern blot analysis the
CHL1 riboprobe hybridized with a predominant mRNA band of approxim~tely 8 kb
(Figure 21) which is significantly larger than the size of the mRNA ~etectecl with
Ll probes (approximately 6 kb) (Tacke et al., 1987). The smaller and weaker
RNA band (Figure 21a, lane 3) is most probably due to the cross-hybridization
10 with ribosomal RNA. The 8 kb RNA was detected in cerebellum. brain minus
cerebella, and spinal cord but not in dorsal root ganglia (DRG) ~Figure 21a). Incontrast, the Ll riboprobe showed a strong signal with RNA from DRG (Figure
21a). CHLl mRNA was also detectable in nine-day-old rat cerebellum and six-
day-old rat spinal cord but not in rat PC12 cells m~int~ined v~rith and without NFG
15 or in COS-l cells (Figure 21b). In all other tissues analyzed (thymus, lung, liver,
intestin~, spleen. and kidney) no signal was detectable (Figure 21a).
To identify the CHLl protein, antibodies to a bacterially expressed fragment of the
CHLl protein were genc,ated. Excluding regions of high homology to other
known Ll family members, e.g. tr~n~memhrane s~ or intracellular regions, a
20 1.7 kD cDNA fragment representin,, part of the sixth Ig-like domain and the four
FN-like repeats (Figures 18 and l9b) was cloned into the pET t:xplession vector.Expression of the resl-ltin~ protein fragment led to a 70 kD band ~itotectecl byCoomassle blue st~inin~ after SDS-PAGE which was purified by anion exchange
chronomatography. Western blot analysis (Figure 22a) and ELISA (not shown)
25 showed that the antisera from two rabbits reacted with the CHLl protein fragment
but not with purified Ll, N-CAM, or MAG. Some reaction with bacterial proteins
copurified with the CHLl peptide or degradation products of the CHLI fragment
was observed (Figure 22a. lane 4).
To further exarnine the specificity of the antibodies and to determine whether they
30 recognize the native cell surface expressed CHLl. transiently transfected COS-l
cells were e~minPcl with the antibodies. Immunocytochemistry revealed cell
CA 02218~99 1997- lo- 16
WO 96J329Sg 86 PCr/u~.3GI;~JS434
surface c~ es~.ion of CHLI on CHL1-transfected cells, but not on cells mock-
transfected with the vector (Figure 23). These results also demonstrate that theputative signal sequence is functional and that the open reading frarne is correct.
Although the first CHLl cDNA clone was isolated from an expression library by
5 screening with immllnt~affinity purified polyclonal antibodies against brain derived
L1, reaction of a different ~ aldLion of L1 antibodies against the recombinantlyexpressed Ig-like domains of Ll with CHLl-transfected cells was not observed (not
shown). Also CHL1 antibodies directed against the extracellular part of the
molecule (Figure 19) did not react with Ll-transfected cells (not shown). It is
10 therefore likely that the Ll polyclonal antibodies used for screenin~ the e~-ession
library were reactive with the C-terminal, intracellular part of CHL1 which is most
homologous between CHLl and Ll.
The CHLl antisera were used to identify immlln~reactive proteins in several tissues
(brain, liver~ lung, kidney, and intPstine from nine-day old mice. Figure 23b).
Crude membrane fractions, soluble and insoluble in 0.5 % Triton X-100, were
analyzed by Western blotting. Polyclonal antibodies against Ll were used as a
control. The CHL1 antibodies recognized three distinct bands of 18~, 165, and 125
kD in lhe insoluble and soluble fractions of brain membranes. The 185 kD band
was only weakly detectable in the soluble fraction and the 125 kD band was less
20 prominent in the insoluble fraction (Figure 23b, lane 1 and 2), indicating that the
185 kD band is probably the membrane bound form of CHLl, whereas the 125 and
165 kD forms are probably proteolytically cleaved fr~gme~lt~ A similar pattern of
immunoreactive bands was observed after Western blot analysis of CHLl
transfected COS cells and total brain tissue (not shown). A similar pattern of
25 bands has been observed for L1 (Faissner et al., 1985: Sadoul et al.~ 1988), Ng-
CAM (Grumet et al.. 1984), and NrCAM (Kayyem et al., 1992). However, a
dibasic consensus sequence for proteolytic cleavage in tne third FN-like domain
(Ll: "SKR": Ng-CAM: "SRR": Nr-CAM: "SRR": Nr-CAM: "SRRSKR") is not
present in CHLI. Like the other members of the L1 family. CHLl was found to
30 be expressed only at later stages of development. It was not detectable in brain
before embryonic dav 15 by Western blot analysis (not shown). A 50 kD
CA 02218~99 1997-10-16
WO 96/329S9 87 PCT/US96~05434
imml~nQreactive band was ~1et~ct~cl in the detergent soluble fraction of whole liver
tissue. This band is most probably due to some cross-reactivity of the CHLI
antibodies with a CHL1-related protein. since no CHL1-specific mRNA was seen in
liver by Northern blot analysis (Figure ~2a). No CHL1 immllnoreactivity could bedetected in the other tissues that were tested (Figure 23b).
In the CNS, members of the Ll family are predomin~ntly t~leaaed by neurons.
Therefore, we were i,llcre.Led if CHLl shares this pattern of e~ saion with L1.
In situ hybridization ex~ ents were pclrc~ ed to identify the cells synth~si7ingCHL1 and Ll in the retina, optic nerve. and cerebellar cortex of young po.stnatal
mice. In the retina of 7-day-old mice, L1 (Fig. 24a) and CHLl mRNA (Fig. 24b)
. are e2~ cssed by ganglion cells. Ll transcripts were additionally detectable in
amacrine and hol;~onL~l cells located in the inner nuclear layer (Fig. 24a). CHLl
mRNA, in contrast. was only occasionally detect~ble in a few cells located at the
inner ~i.e. vitread) margin of the inter nuclear layer (Fig. 24b). Glial cells in the
optic nerve did not contain ~letect~ble levels of Ll transcripts (Fig. 24a). In
striking collhdsl, CHL1 mRNA was strongly e~lcsaed by glial cells located in
proxirnal (i.e. retina-near) regions of the optic nerve (Fig. 24b) and low levels of
CHL1 e~ aion were visible in glial cells located in more distal regions of the
nerve (Fig. 24b).
In the cerebellar cortex of two-week-old mice, Ll mRNA was detectable in stellate
and basket cells located in the molecular layer and in Golgi and granule cells
located in the intern~l gr~nnl~r layer (Fig. 24d). The same cells types were labeled
when sections were hybridized with the CHL1 ~nti~n~e cRNA probe (Fig. 24e),
with the exception that CHL1 transcripts were hardly detectable in cells located in
2~ the inter part of the molecular layer (colll~alc Fig. 24d and e). As a negative
control, sections were hybridized with the corresponding sense cRNA probes and
no labeling of cells was detectable (for a retina and optic nerve hybridized with a
" CHL1 sense cRNA probe, see Fig. 24c). In order to address whether gilal cells
express CHL1 in vitro, cultures of purified astrocytes or oligodendrocytes were
30 ~lep~ed from the forebrain of young postnatal mice or rats. The same polyclonal
CHLI antibodies which specifically detected CHLl at the cell surface of
CA 02218~99 1997-10-16
WO 961329S9 88 PCTIUS96/OS434
transfected COS-l cells were used. Astrocytes and oligodendrocytes were
identified with antibodies to GFAP or with antibodies to the Ol antigen,
l~sl,c~,lively. Astrocyte cultures contained some cells which were double-labeled by
polyclonal CHLl (Fig. 25a, d) and monoclonal GFAP (Fig. 25b, e) antibodies.
5 Analysis of oligodendrocyte cultures. however, revealed no co-localization of
CHLl and the Ol antigen, indicatin~ that mature oligodendroc~tes in vitro do notexpress detectable levels of CHL1. The combined observations indicate that CHL1
and Ll show overlapping but also distinct patterns of ~ cs~ion. Most strikingly
CHL1, but not Ll, is ~ essed by certain glial cells of the nervous system in vivo,
10 suggesting that different members of the L1 family perform different functions.
Analysis of glycosyla~ion and detection of the HNK-1 carbohydrate by the
CHLl glycoprotein
Since the observed molecular weight of CHL1 (185 kD) is considerably larger thanthe calculated molecular mass (134.9 kD), the carbohydrate contribution to the
lS molecular mass and the type of carbohydrate modification was analyzed. The
de~r~ soluble and insoluble fractions from crude brain membranes of seven-day-
old mice were subjected to enzymatic deglycosylation. After N-glycosidase F
(PNGasoF) treatment the molecular mass of all CHL1 immunoreactive proteins was
reduced (Figure 26): The 185 kD band shifted to 150 kD, the 165 kD band to 135
20 kD, and the 125 kD band to 110 kD. Tre~trnent with O-glycosidase, an enzyme
known to cleave serine/threonine linked galactosyl ~3(1-3)N-acetylgalactosaminyl~ic~-ch~rides (Glasgow et al., 1977) resulted in a slightly increased mobility: The
185 and 165 kD bands shifted to about 180 and 160 kD, respectively, whereas the
125 kD band did not shift. These observations indicate that most of the
2~ carbohydrate molecular mass is due to N-linked carbohydrates. Treatment with
both er ymes together (Figure 26) led to a larger shift than seen with tre~tn~ent
with individual enzymes from 185 to 145 kD, suggesting that not all glycosylation
sites. most probably the O-glycosylation sites were cleaved by O-glycosidase alone.
The results show that CHL1 contains approximately 30 % of its molecular mass as
30 N-glycosidally linked carbohydrates.
CA 02218~99 1997-10-16
WO 961329S9 89 PCTIUS96105434
Several neural cell adhesion molecules carry the HNK-I carbohydrate, such as L1
(Kruse et al., 198~)~ TAG-1 (Dodd et al.~ 1988)~ Nr-CA~I (Grumet et al.~ 1991),
F3 (Gennarini et al.. 1989), N-CA M (Kruse et al.~ 1984). the myelin associated
glycoprotein MAG (McGarry et al., 1983; Kruse et al., 1984), and PO (Bollenson
5 and Sçh~hner, 1987). Therefore, we analyzed whether CHLl carries the HNK-l
carbohydrate. CHLI was immtlno~le~ iL~led from detergent Iysates of whole
brain tissue from nine-day-old mice with CHL1 antibodies. As control, L1 was
similarly imm~mopleci~ Led with polyclonal antibodies from the same brain
extract. Western blot analysis with monoclonal antibody ~12 directed against the10 HNK-l carbohydrate epitope showed that both immunoprecipitates contained bands
which were recognized by the monoclonal antibody 412 at molecular masses
- expected for CHLl (Figure 27) or Ll (not shown). Since the HNK-l carbohydrate
is involved in cell-to-cell adhesion and binds to l~minin (Keilhauer et al., 1985;
Kfln~rn-ln~l et al., 1988; Hall et al., 1993, 1995), CHLl, like the other members of
15 the L1 family, may interact with l~minin via the HNK-l carbohydrate.
CONCLUSIONS
The above t:x~,,hllents has added CHLl as another member of the Ll family of
neural recognition molecules found in such diverse species as human, rat, mouse,chicken, zebrafish, and Drosophila. thus con~liluling a phylogenetically conserved
20 family of molecules all of which are e,~ essed late in development at the onset of
axogenesis by neurons and subsets of neurons. The fact that many Ll related
molecules exist points to nature's re4~ llt for structurally similar, but
functionally most likely distinct neurite outgrowth promoting molecules, and to the
evolution of the L 1 family as a group of molecules that may determine the fine-
25 tuning of axonal p~thfin~ling
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wo 96)329Sg go PCT/US96/0543
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While the invention has been described and illustrated herein by references to
various specific material, procedures and examples. it is understood that the
invention is not restricted to the particular material combinations of material. and
procedures selected for that purpose. Numerous variations of such details can be25 implied as will be appreciated by those skilled in the art.
