Title: Methods for using elk-L to enhance neuronal
United States Patent: 6,540,992
Issued: April 1, 2003
Inventors: Lyman; Stewart (Seattle, WA); Beckmann; M.
Patricia (Poulsbo, WA); Baum; Peter R. (Seattle, WA); Carpenter; Melissa K.
Assignee: Genentech, Inc. (South San Francisco, CA)
Appl. No.: 039642
Filed: March 16, 1998
Elk ligand (Elk-L) polypeptides as well as DNA sequences, vectors and
transformed host cells useful in providing elk-L polypeptides are used in
methods for enhancing the survival or inhibiting the death of neurons,
particularly hippocampal neurons. The elk-L polypeptides bind to a cell
surface receptor that is a member of the tyrosine kinase receptor family.
DETAILED DESCRIPTION OF THE INVENTION
A cDNA encoding a novel protein ligand that binds to the rat cell surface
protein known as elk has been isolated in accordance with the present
invention. Also provided are expression vectors comprising the elk ligand
(elk-L) cDNA and methods for producing recombinant elk-L polypeptides by
cultivating host cells containing the expression vectors under conditions
appropriate for expression of elk-L, and recovering the expressed elk-L.
Purified elk-L protein is also encompassed by the present invention,
including soluble forms of the protein comprising the extracellular
The present invention also provides elk-L or antigenic fragments thereof
that can act as immunogens to generate antibodies specific to the elk-L
immunogens. Monoclonal antibodies specific for elk-L or antigenic
fragments thereof thus can be prepared.
The novel cytokine disclosed herein is a ligand for elk, a rat cell
surface receptor that is a member of the tyrosine kinase receptor family.
Binding of elk-L to elk on the cell surface is believed to initiate a
biological signal mediated by elk. One use of the elk ligand of the
present invention is as a research tool for studying the nature of this
biological signal and the role that elk-L, in conjunction with elk, may
play in growth or differentiation of cells bearing the elk receptor.
Expression of elk mRNA has been detected in the brain and testis of rats (Lhotok
et al., supra), and the possibility that elk is capable of oncogenic
activation has been suggested (Letwin et al., supra). The elk-L
polypeptides of the present invention also may be employed in in vitro
assays for detection of elk or elk-L or the interactions thereof. The
human elk-L disclosed herein also finds use in identifying the putative
human homolog of rat elk.
The elk-L protein exhibits neuroprotective and neurotrophic properties, as
described in example 10. In one embodiment of the invention, elk-L
inhibits neuronal death caused at least in part by the mechanism known as
excitotoxicity. The use of elk-L in treating neurodegenerative diseases or
injury to neurons is described in more detail below.
To identify cells suitable for use as nucleic acid sources in the cloning
attempt, over 30 different types of murine and human cells were screened
for the ability to bind elk (in the form of a fusion protein comprising
rat elk and an antibody Fc polypeptide). As described in example 2, none
of the cell types exhibited detectable elk binding. Since placental tissue
is rich in growth and differentiation factors, a human placental cDNA
expression library was screened with rat elk/Fc in an attempt to isolate
an elk-L clone. Although it was not known whether or not placenta
expressed an elk-L, and the ability of rat elk to bind to human elk-L also
was unknown, human elk-L cDNA was successfully isolated as described in
example 3. The DNA sequence and encoded amino acid sequence of the coding
region of a human elk-L cDNA clone are set forth in SEQ ID NO:1 and SEQ ID
Human elk-L cDNA comprising the coding region was isolated from the
positive clone and inserted into the Sma I site (in the multiple cloning
site region of cloning vector pBLUESCRIPT.RTM. SK(-), available from
Stratagene Cloning Systems, La Jolla, Calif. The resulting recombinant
vector, designated tele 7 in pBLUESCRIPT.RTM. SK(-), in E. coli DH5.alpha.
cells, was deposited with the American Type Culture Collection on Oct. 9,
1992, and assigned accession no. ATCC 69085. The deposit was made under
the terms of the Budapest Treaty.
Comparison of both the nucleotide and encoded amino acid sequences of the
human elk-L cDNA clone with the Genbank and Swissport databases showed
that the sequence of the elk ligand was unique. One amino acid sequence
was identified in this search that did share limited sequence identity
with the elk ligand. That sequence was for the B61 protein, which has
previously been identified as the product of a novel immediate-early
response gene induced by TNF in human umbilical vein endothelial cells (Holzman
et al., Mol. Cell. Biol. 10:5830, 1990). All four of the cysteine residues
in the extracellular domain of the two proteins are conserved, and the
overall amino acid identity between human elk-L and B61 is 33%. In
contrast to the elk ligand, the B61 protein has been reported to be
secreted, but terminates with a hydrophobic tail and has been suggested to
be associated with the membrane through a glycosylphosphatidyl inositol
linkage (Holzman et al., supra). The function of the B61 protein is
The term "elk-L" as used herein refers to a genus of polypeptides which
are capable of binding elk. Human elk-L is within the scope of the present
invention, as are elk-L proteins derived from other mammalian species
including but not limited to murine, rat, bovine, porcine, or various
primate cells. As used herein, the term "elk-L" includes membrane-bound
proteins (comprising a cytoplasmic domain, a transmembrane region, and an
extracellular domain) as well as truncated proteins that retain the
elk-binding property. Such truncated proteins include, for example,
soluble elk-L comprising only the extracellular (receptor binding) domain.
The human elk-L cDNA may be radiolabeled and used as a probe to isolate
other mammalian elk-L cDNAs by cross-species hybridization. For example, a
cDNA library prepared from placental tissue of other mammalian species may
be screened with radiolabeled human elk-L cDNA to isolate a positive
clone. Alternatively, mRNAs isolated from various cell lines can be
screened by Northern hybridization to determine a suitable source of
mammalian elk-L mRNA for use in cloning an elk-L gene.
Although an elk/Fc fusion protein was employed in the screening procedure
described in Example 3 below, elk can be used to screen clones and
candidate cell lines for expression of elk-L proteins. The elk/Fc fusion
protein, however, offers the advantage of being easily purified. In
addition, disulfide bonds form between the Fc regions of two separate
fusion protein chains, creating dimers. The dimeric elk/Fc receptor was
chosen for the potential advantage of higher affinity binding of the elk
ligand, in view of the possibility that the ligand being sought would be
Other antibody Fc regions may be substituted for the human IgG1 Fc region
described in Example 1. Other suitable Fc regions are those that can bind
with high affinity to protein A or protein G, and include the Fc region of
murine IgG1 or fragments of the human IgG1 Fc region, e.g., fragments
comprising at least the hinge region so that interchain disulfide bonds
One embodiment of the present invention provides soluble elk-L
polypeptides. Soluble elk-L polypeptides comprise all or part of the
extracellular domain of a native elk-L but lack the transmembrane region
that would cause retention of the polypeptide on a cell membrane. Soluble
elk-L polypeptides advantageously comprise the native (or a heterologous)
signal peptide when initially synthesized to promote secretion, but the
signal peptide is cleaved upon secretion of elk-L from the cell. The
soluble elk-L polypeptides that may be employed retain the ability to bind
the elk receptor. Soluble elk-L may also include part of the transmembrane
region or part of the cytoplasmic domain or other sequences, provided that
the soluble elk-L protein is capable of being secreted.
Soluble elk-L may be identified (and distinguished from its non-soluble
membrane-bound counterparts) by separating intact cells which express the
desired protein from the culture medium, e.g., by centrifugation, and
assaying the medium (supernatant) for the presence of the desired protein.
The presence of elk-L in the medium indicates that the protein was
secreted from the cells and thus is a soluble form of the desired protein.
Soluble elk-L may be a naturally-occurring form of this protein.
The use of soluble forms of elk-L is advantageous for certain
applications. Purification of the proteins from recombinant host cells is
facilitated, since the soluble proteins are secreted from the cells.
Further, soluble proteins are generally more suitable for intravenous
Examples of soluble elk-L polypeptides include those comprising the entire
extracellular domain of a native elk-L protein. One such soluble elk-L
protein comprises amino acids 1 (Ala) through 213 (Lys) of SEQ ID NO:2.
When initially expressed within a host cell, the soluble protein may
additionally comprise one of the heterologous signal peptides described
below that is functional within the host cells employed. Alternatively,
the protein may comprise the native signal peptide, such that the elk-L
comprises amino acids -24 (Met) through 213 (Lys) of SEQ ID NO:2. In one
embodiment of the invention, soluble elk-L is initially expressed as a
fusion protein comprising (from N- to C-terminus) the yeast a factor
signal peptide, the FLAG.RTM. peptide (SEQ ID NO:3) described below and in
U.S. Pat. No. 5,011,912, and soluble elk-L comprising amino acids 1-213 of
SEQ ID NO:2. This recombinant fusion protein is expressed in and secreted
from yeast cells. The FLAG.RTM. peptide (SEQ ID NO:3) facilitates
purification of the protein, and subsequently may be cleaved from the
soluble elk-L using bovine mucosal enterokinase. DNA sequences encoding
soluble elk-L proteins are encompassed by the present invention.
Truncated elk-L, including soluble polypeptides, may be prepared by any of
a number of conventional techniques. A desired DNA sequence may be
chemically synthesized using known techniques. DNA fragments also may be
produced by restriction endonuclease digestion of a full length cloned DNA
sequence, and isolated by electrophoresis on agarose gels. Linkers
containing restriction endonuclease cleavage site(s) may be employed to
insert the desired DNA fragment into an expression vector, or the fragment
may be digested at cleavage sites naturally present therein. The well
known polymerase chain reaction procedure also may be employed to isolate
a DNA sequence encoding a desired protein fragment. As a further
alternative, known mutagenesis techniques may be employed to insert a stop
codon at a desired point, e.g., immediately downstream of the codon for
the last amino acid of the extracellular domain.
In another approach, enzymatic treatment (e.g., using Bal 31 exonuclease)
may be employed to delete terminal nucleotides from a DNA fragment to
obtain a fragment having a particular desired terminus. Among the
commercially available linkers are those that can be ligated to the blunt
ends produced by Bal 31 digestion, and which contain restriction
endonuclease cleavage site(s). Alternatively, oligonucleotides that
reconstruct the N- or C-terminus of a DNA fragment to a desired point may
be synthesized. The oligonucleotide may contain a restriction endonuclease
cleavage site upstream of the desired coding sequence and position an
initiation codon (ATG) at the N-terminus of the coding sequence.
The present invention provides purified elk-L polypeptides, both
recombinant and non-recombinant. Variants and derivatives of native elk-L
proteins that retain the desired biological activity (e.g., the ability to
bind elk) are also within the scope of the present invention. elk-L
variants may be obtained by mutations of nucleotide sequences coding for
native elk-L polypeptides. An elk-L variant, as referred to herein, is a
polypeptide substantially homologous to a native elk-L, but which has an
amino acid sequence different from that of native elk-L (human, murine or
other mammalian species) because of one or more deletions, insertions or
The variant amino acid sequence preferably is at least 80% identical to a
native elk-L amino acid sequence, most preferably at least 90% identical.
The percent identity may be determined, for example, by comparing sequence
information using the GAP computer program, version 6.0 described by
Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the
University of Wisconsin Genetics Computer Group (UWGCG). The GAP program
utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol.
48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482,
1981). The preferred default parameters for the GAP program include: (1) a
unary comparison matrix (containing a value of 1 for identities and 0 for
non-identities) for nucleotides, and the weighted comparison matrix of
Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by
Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,
National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty
of 3.0 for each gap and an additional 0.10 penalty for each symbol in each
gap; and (3) no penalty for end gaps.
Alterations of the native amino acid sequence may be accomplished by any
of a number of known techniques. Mutations can be introduced at particular
loci by synthesizing oligonucleotides containing a mutant sequence,
flanked by restriction sites enabling ligation to fragments of the native
sequence. Following ligation, the resulting reconstructed sequence encodes
an analog having the desired amino acid insertion, substitution, or
Alternatively, oligonucleotide-directed site-specific mutagenesis
procedures can be employed to provide an altered gene having particular
codons altered according to the substitution, deletion, or insertion
required. Exemplary methods of making the alterations set forth above are
disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73,
1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic
Engineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc.
Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methods in Enzymol.
154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are
incorporated by reference herein.
Variants may comprise conservatively substituted sequences, meaning that a
given amino acid residue is replaced by a residue having similar
physiochemical characteristics. Examples of conservative substitutions
include substitution of one aliphatic residue for another, such as Ile,
Val, Leu, or Ala for one another, or substitutions of one polar residue
for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn.
Other such conservative substitutions, for example, substitutions of
entire regions having similar hydrophobicity characteristics, are well
elk-L also may be modified to create elk-L derivatives by forming covalent
or aggregative conjugates with other chemical moieties, such as glycosyl
groups, lipids, phosphate, acetyl groups and the like. Covalent
derivatives of elk-L may be prepared by linking the chemical moieties to
functional groups on elk-L amino acid side chains or at the N-terminus or
C-terminus of a elk-L polypeptide or the extracellular domain thereof.
Other derivatives of elk-L within the scope of this invention include
covalent or aggregative conjugates of elk-L or its fragments with other
proteins or polypeptides, such as by synthesis in recombinant culture as
N-terminal or C-terminal fusions. For example, the conjugate may comprise
a signal or leader polypeptide sequence (e.g. the .alpha.-factor leader of
Saccharomyces) at the N-terminus of a elk-L polypeptide. The signal or
leader peptide co-translationally or post-translationally directs transfer
of the conjugate from its side of synthesis to a site inside or outside of
the cell membrane or cell wall.
elk-L polypeptide fusions can comprise peptides added to facilitate
purification and identification of elk-L. Such peptides include, for
example, poly-His or the antigenic identification peptides described in
U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1998.
One such peptide is the FLAG.RTM. peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
(DYKDDDDK) (SEQ ID NO:3), which is highly antigenic and provides an
epitope reversibly bound by a specific monoclonal antibody enabling rapid
assay and facile purification of expressed recombinant protein. This
sequence is also specifically cleaved by bovine mucosal enterokinase at
the residue immediately following the Asp-Lys pairing. Fusion proteins
capped with this peptide may also be resistant to intracellular
degradation in E. coli. A murine hybridoma designated 4E 11 produces a
monoclonal antibody that binds the peptide DYKDDDDK (SEQ ID NO:3) in the
presence of certain divalent metal cations (as described in U.S. Pat. No.
5,011,912, hereby incorporated by reference) and has been deposited with
the American Type Culture Collection under accession no. HB 9259.
The present invention further includes elk-L polypeptides with or without
associated native-pattern glycosylation. elk-L expressed in yeast or
mammalian expression systems (e.g., COS-7 cells) may be similar to or
significantly different from a native elk-L polypeptide in molecular
weight and glycosylation pattern, depending upon the choice of expression
system. Expression of elk-L polypeptides in bacterial expression systems,
such as E. coli, provides non-glycosylated molecules.
DNA constructs that encode various additions or substitutions of amino
acid residues or sequences, or deletions of terminal or internal residues
or sequences not needed for biological activity or binding can be
prepared. For example, N-glycosylation sites in the elk-L extracellular
domain can be modified to preclude glycosylation, allowing expression of a
more homogeneous, reduced carbohydrate analog in mammalian and yeast
expression systems. N-glycosylation sites in eukaryotic polypeptides are
characterized by an amino acid triplet Asn-X-Y, wherein X is any amino
acid except Pro and Y is Ser or Thr. The human elk-L protein comprises one
such triplet, at amino acids 115-117 of SEQ ID NO:2. Appropriate
modifications to the nucleotide sequence encoding this triplet will result
in substitutions, additions or deletions that prevent attachment of
carbohydrate residues at the Asn side chain. Alteration of a single
nucleotide, chosen so that Asn is replaced by a different amino acid, for
example, is sufficient to inactivate an N-glycosylation site. Known
procedures for inactivating N-glycosylation sites in proteins include
those described in U.S. Pat. No. 5,071,972 and EP 276,846, hereby
incorporated by reference.
In another example, sequences encoding Cys residues that are not essential
for biological activity can be altered to cause the Cys residues to be
deleted or replaced with other amino acids, preventing formation of
incorrect intramolecular disulfide bridges upon renaturation. Other
variants are prepared by modification of adjacent dibasic amino acid
residues to enhance expression in yeast systems in which KEX2 protease
activity is present. EP 212,914 discloses the use of site-specific
mutagenesis to inactivate KEX2 protease processing sites in a protein.
KEX2 protease processing sites are inactivated by deleting, adding or
substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to
eliminate the occurrence of these adjacent basic residues. Lys-Lys
pairings are considerably less susceptible to KEX2 cleavage, and
conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and
preferred approach to inactivating KEX2 sites. Human elk-L contains three
KEX2 protease processing sites at amino acids 242-243, 243-244, and
246-247 of SEQ ID NO:2.
Naturally occurring elk-L variants are also encompassed by the present
invention. Examples of such variants are proteins that result from
alternative mRNA splicing events or from proteolytic cleavage of the elk-L
protein, wherein the elk-binding property is retained. Alternative
splicing of mRNA may yield a truncated but biologically active elk-L
protein, such as a naturally occurring soluble form of the protein, for
example. Variations attributable to proteolysis include, for example,
differences in the N- or C-termini upon expression in different types of
host cells, due to proteolytic removal of one or more terminal amino acids
from the elk-L protein (generally from 1-5 terminal amino acids).
Nucleic acid sequences within the scope of the present invention include
isolated DNA and RNA sequences that hybridize to the native elk-L
nucleotide sequences disclosed herein under conditions of moderate or
severe stringency, and which encode biologically active elk-L. Moderate
stringency hybridization conditions refer to conditions described in, for
example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed.
Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989).
Conditions of moderate stringency, as defined by Sambrook et al., include
use of a prewashing solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH
8.0) and hybridization conditions of about 55oC., 5.times.SSC,
overnight. Conditions of severe stringency include higher temperatures of
hybridization and washing. The skilled artisan will recognize that the
temperature and wash solution salt concentration may be adjusted as
necessary according to factors such as the length of the probe.
Due to the known degeneracy of the genetic code wherein more than one
codon can encode the same amino acid, a DNA sequence may vary from that
presented in SEQ ID NO:1, and still encode an elk-L protein having the
amino acid sequence of SEQ ID NO:1. Such variant DNA sequences may result
from silent mutations (e.g., occurring during PCR amplification), and may
be the product of deliberate mutagenesis of a native sequence.
The present invention thus provides isolated DNA sequences encoding
biologically active elk-L, selected from: (a) DNA derived from the coding
region of a native mammalian elk-L gene (e.g., cDNA comprising the
nucleotide sequence presented in SEQ ID NO:1); (b) DNA capable of
hybridization to a DNA of (a) under moderately stringent conditions and
which encodes biologically active elk-L; and (c) DNA which is degenerate
as a result of the genetic code to a DNA defined in (a) or (b) and which
encodes biologically active elk-L. The elk-L proteins encoded by such DNA
sequences are encompassed by the present invention.
Examples of elk-L proteins encoded by DNA that varies from the native DNA
sequence of SEQ ID NO:1, wherein the variant DNA will hybridize to the
native DNA sequence under moderately stringent conditions, include, but
are not limited to, elk-L fragments (soluble or membrane-bound) and elk-L
proteins comprising inactivated N-glycosylation site(s), inactivated KEX2
protease processing site(s), or conservative amino acid substitution(s),
as described above. Elk-L proteins encoded by DNA drived from other
mammalian species, wherein the DNA will hybridize to the human DNA of SEQ
ID NO:1, are also encompassed.
Variants possessing the requisite ability to bind elk may be identified by
any suitable assay. Biological activity of elk-L may be determined, for
example, by competition for binding to the ligand binding domain of elk
(i.e. competitive binding assays).
One type of a competitive binding assay for elk-L polypeptide uses a
radiolabeled, soluble human elk-L and intact cells expressing cell surface
elk. Instead of intact cells, one could substitute soluble elk (such as an
elk/Fc fusion protein) bound to a solid phase through a Protein A or
Protein G interaction with the Fc region of the fusion protein. Another
type of competitive binding assay utilizes radiolabeled soluble elk such
as an elk/Fc fusion protein, and intact cells expressing elk-L.
Alternatively, soluble elk-L could be bound to a solid phase.
Competitive binding assays can be performed using standard methodology.
For example, radiolabeled elk-L can be used to compete with a putative
elk-L homolog to assay for binding activity against surface-bound elk.
Qualitative results can be obtained by competitive autoradiographic plate
binding assays, or Scatchard plots may be utilized to generate
Alternatively, soluble elk can be bound to a solid phase such as a column
chromatography matrix or a similar substrate suitable for analysis for the
presence of a detectable moiety such as 125 I. Binding to a solid
phase can be accomplished, for example, by binding an elk/Fc fusion
protein to a protein A or protein G-containing matrix.
The binding characteristics of elk-L (including variants) may also be
determined using the conjugated, soluble elk (for example, 125 I-elk/Fc)
in competition assays similar to those described above. In this case,
however, intact cells expressing elk-L, or soluble elk-L bound to a solid
substrate, are used to measure the extent to which a sample containing a
putative elk variant competes for binding of a conjugated soluble elk to
The elk-L of the present invention can be used in a binding assay to
detect cells expressing elk. For example, elk-L or an extracellular domain
or a fragment thereof can be conjugated to a detectable moiety such as
1251. Radiolabeling with 125 I can be performed by any of several
standard methodologies that yield a functional 125 I-elk-L molecule
labeled to high specific activity. Alternatively, another detectable
moiety such as an enzyme that can catalyze a colorometric or fluorometric
reaction, biotin or avidin may be used. Cells to be tested for elk
expression can be contacted with labeled elk-L. After incubation, unbound
labeled elk-L is removed and binding is measured using the detectable
The elk ligand proteins disclosed herein also may be employed to measure
the biological activity of elk protein in terms of binding affinity for
elk-L. To illustrate, elk-L may be employed in a binding affinity study to
measure the biological activity of an elk protein that has been stored at
different temperatures, or produced in different cell types. The
biological activity of an elk protein thus can be ascertained before it is
used in a research study, for example.
Elk-L proteins find use as reagents that may be employed by those
conducting "quality assurance" studies, e.g., to monitor shelf life and
stability of elk protein under different conditions. Elk ligands may be
used in determining whether biological activity is retained after
modification of an elk protein (e.g., chemical modification, truncation,
mutation, etc.). The binding affinity of the modified elk protein for an
elk-L is compared to that of an unmodified elk protein to detect any
adverse impact of the modifications on biological activity of elk.
A different use of an elk ligand is as a reagent in protein purification
procedures. Elk-L or elk-L/Fc fusion proteins may be attached to a solid
support material by conventional techniques and used to purify elk by
The elk-L protein exhibits neuroprotective properties. This property has
been demonstrated in an assay in which neural death caused by treatment
with glutamate (and believed to involve the mechanism known as
excitotoxicity) was inhibited by elk-L. A trophic effect on neurons was
also demonstrated, as described in example 10.
One embodiment of the present invention is thus directed to a method of
treating disorders of neural tissue, such as injury and chronic or acute
neurologic diseases, involving contacting the injured or diseased neurons
with elk-L. Elk-L may be administered to a mammal to treat such an injury
or disease. In one embodiment of the invention, elk-L is employed in
treating an injury or disorder in which excitotoxicity plays a role, as
Elk-L exhibits a trophic effect on neurons, whether or not the neurons are
injured or afflicted with disease, and can be administered to a mammal to
exert a trophic effect on neural tissue. In a patient suffering loss of or
damage to neurons due to injury or disease, elk-L can be administered to
enhance the viability of those neurons that have survived, regardless of
the mechanism by which the loss or damage of neural tissue occurred.
Examples of conditions that may be treated with elk-L include, but are not
limited to, neuropathies such as diabetic, hereditary, and nutritional
neuropathies, neurodegenerative diseases, and other disorders
characterized by degeneration or loss of function of neurons.
Elk-L also finds use as a tissue culture reagent. An elk-L protein can be
added to neurons cultured in vitro to enhance the viability and prolong
the lifespan of the cultured neurons, thus facilitating research studies
of neural tissue.
Certain acidic or sulfur-containing amino acids have been demonstrated to
depolarize and excite neurons, such that prolonged exposure of neurons to
high concentrations of such amino acids results in neural death (Olney et
al. Expl. Brain Res. 14:61, 1971). This mechanism of neural death is known
as excitotoxicity. A number of receptors for excitatory amino acids have
been identified and characterized. In one embodiment of the invention,
elk-L is administered to a mammal afflicted with a neurodegenerative
condition characterized or mediated, at least in part, by excitotoxicity.
The major excitatory neurotransmitter in the central nervous system (CNS)
is glutamate. Responsiveness to glutamate is a normal function in the
developing and mature CNS. In addition to its normal role in excitatory
synaptic transmission and plasticity, however, glutamate can also mediate
or otherwise participate in a number of CNS dysfunctional states,
including, but not limited to, measles, Alzheimer's disease, Huntington's
Disease, Parkinsonism, stroke (ischemia), epilepsy, and AIDS-related
dementia (reviewed in Meldrum and Garthwaite, Trends Pharmacol. Sci.
11:379, 1990; Choi, J. Neurosci. 10:2493, 1990; Lipton et al., Neuron
7:111, 1991; and Andersson et al., Eur. J. Neurosci. 3:66, 1991).
The involvement of an excitotoxic component in ischemic or hypoxic brain
damage (e.g., resulting from a stroke) is well established (Choi, Neuron
1:623, 1988). Regarding AIDS related dementia, the HIV envelope
glycoprotein gp120 has been suggested to directly or indirectly enhance
neuronal sensitivity to glutamate (Lipton, Trends Neurol. Sci. 15:75,
1992). Long term rat and primate models have been developed with
excitotoxic insult that closely mimics the behavioral, neurochemical, and
pathological defects associated with Huntington's disease (Beal, Ann.
Neurol. 31:119, 1992). Deposition of amyloid plaques is a hallmark of
Alzheimer's Disease (AD). Demonstration of an enhancing effect of .beta.-amyloid
on excitotoxicity in vitro (Koh et al., Brain Res. 533:315, 1990 and
Mattson et al., J. Neurosci. 12:376, 1992) has led to the suggestion that
.beta.-amyloid potentiates slow excitotoxic neuronal death in vivo in AD
patients. The accumulation of amyloid plaques in other conditions,
including but not limited to Down's Syndrome and the aging process,
likewise suggests a role for excitotoxicity.
Regarding Parkinson's Disease, the compound MPTP
(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its metabolite MPP+
have been used to induce experimental parkinsonism. MPP+ kills
dopaminergic neurons in the substantia nigra, yielding a reasonable model
of late parkinsonism. Turski et al., (Nature 349:414, 1991) reported that
antagonists of NMDA (an excitatory amino acid receptor) could protect the
substantia nigra from MPP+ -mediated toxicity. This result has been
confirmed and extended to demonstrate selective damage mediated by MPP+,
consistent with excitotoxicity (Storey et al., J. Neurochem. 58:1975,
1992). A role for excitotoxicity in amyotrophic lateral sclerosis (ALS)
has been proposed, based on extrapolation from data demonstrating a role
for an excitatory amino acid in lathyrism (Spencer et al., Lancet 1:1066,
1986). The role of excitotoxicity in a number of neurological disorders,
both acute and chronic, is further discussed in Albin, R. and J.
Greenamyre, Neurology 42:733, 1992, and Beal, Current Opinion in Neurobiol.
Several non-exclusive models for the precise mechanism of excitotoxic
damage to neurons have been proposed, including aberrant fluxes in
intracellular Ca++ leading to death, possibly through a nitric oxide
intermediate. The production of free radicals is suggested to play a role
in DNA damage leading to death (Garthwaite, Trends Neurol. Sci. 14:60,
1991). Another proposed mechanism involves defects in energy metabolism
that cause neuronal death after excitotoxic injury (Lipton, Trends Neurol.
Sci. 15:75, 1992). It is recognized that excitotoxic neuronal damage may
occur via different mechanisms, depending on the nature of the condition
involved. For example, defective receptors for excitatory amino acids may
explain certain (e.g., inherited) disorders, whereas disorders
characterized by late onset and slow progression may be attributable to
impairment of cellular metabolism or membrane potential, wherein
excitotoxicity is the final common pathway of neuronal death (Albin and
The present invention provides pharmaceutical compositions comprising an
effective amount of a purified elk-L polypeptide and a suitable diluent,
excipient, or carrier. Such carriers will be nontoxic to patients at the
dosages and concentrations employed. Ordinarily, the preparation of such
compositions entails combining a mammalian elk-L polypeptide or derivative
thereof with buffers, antioxidants such as ascorbic acid, low molecular
weight (less than about 10 residues) peptides, proteins, amino acids,
carbohydrates including glucose, sucrose, or dextrans, chelating agents
such as EDTA, glutathione, or other stabilizers and excipients. Neutral
buffered saline is one appropriate diluent.
For therapeutic use, the compositions are administered in a manner and
dosage appropriate to the indication and the patient. As will be
understood by one skilled in the pertinent field, a therapeutically
effective dosage will vary according to such factors as the nature and
severity of the condition, the location of damaged neural tissue within
the body in the case of an injury, and the age, condition and size of the
patient. Administration may be by any suitable route, including but not
limited to continuous infusion, local infusion during surgery,
intraventricular infusion (which may involve use of an intraventricular
catheter), sustained release from implants (gels, membranes, and the
like), or injection (e.g., injection at the site of an injury or injection
into the central nervous system).
The compositions of the present invention may contain an elk-L protein in
any form described above, including variants, derivatives, and
biologically active fragments thereof. In one embodiment of the invention
the composition comprises a soluble human elk-L protein. Such protein may
comprise the extracellular domain of human elk-L fused to an Fc
polypeptide, as described above.
Elk-L derived from the same mammalian species as the patient is generally
preferred for use in pharmaceutical compositions. However, elk-L appears
to be highly conserved between species and has demonstrated cross-species
reactivity for certain mammalian species.
Oligomeric Forms of Elk-L
Elk-L polypeptides may exist as oligomers, such as dimers or trimers.
Oligomers are linked by disulfide bonds formed between cysteine residues
on different elk-L polypeptides. In one embodiment of the invention, an
elk-L dimer is created by fusing elk-L to the Fc region of an antibody
(IgG1) in a manner that does not interfere with binding of elk-L to the
elk ligand binding domain. The Fc polypeptide preferably is fused to the
C-terminus of a soluble elk-L (comprising only the extracellular domain).
Preparation of fusion proteins comprising heterologous polypeptides fused
to various portions of antibody-derived polypeptides (including the Fc
domain) has been described, e.g., by Ashkenazi et al., (PNAS USA 88:10535,
1991) and Byrn et al., (Nature 344:677, 1990), hereby incorporated by
reference. A gene fusion encoding the elk-L/Fc fusion protein is inserted
into an appropriate expression vector. The elk-L/Fc fusion proteins are
allowed to assembly much like antibody molecules, whereupon interchain
disulfide bonds from between Fc polypeptides, yielding divalent elk-L. If
fusion proteins are made with both heavy and light chains of an antibody,
it is possible to form an elk-L oligomer with as many as four elk-L
extracellular regions. Alternatively, one can link two soluble elk-L
domains with a peptide linker such as the Gly4 SerGly5 Ser (SEQ
ID NO:4) linker sequence described in U.S. Pat. No. 5,073,627.
The present invention provides oligomers of elk-L extracellular domains or
fragments thereof, linked by disulfide interactions, or expressed as
fusion polymers with or without spacer amino acid linking groups. For
example, a dimer of the elk-L extracellular domain can be linked by an IgG
Fc region linking group.
The present invention provides recombinant expression vectors for
expression of elk-L, and host cells transformed with the expression
vectors. Any suitable expression system may be employed. The vectors
include an elk-L DNA sequence operably linked to suitable transcriptional
or translational regulatory nucleotide sequences, such as those derived
from a mammalian, microbial, viral, or insect gene. Examples of regulatory
sequences include transcriptional promoters, operators, or enhancers, an
mRNA ribosomal binding site, and appropriate sequences which control
transcription and translation initiation and termination. Nucleotide
sequences are operably linked when the regulatory sequence functionally
relates to the elk-L DNA sequence. Thus, a promoter nucleotide sequence is
operably linked to a elk-L DNA sequence if the promoter nucleotide
sequence controls the transcription of the elk-L DNA sequence. The ability
to replicate in the desired host cells, usually conferred by an origin of
replication, and a selection gene by which transformants are identified,
may additionally be incorporated into the expression vector.
In addition, sequences encoding appropriate signal peptides that are not
native to the elk-L gene can be incorporated into expression vectors. For
example, a DNA sequence for a signal peptide (secretory leader) may be
fused in frame to the elk-L sequence so that the elk-L is initially
translated as a fusion protein comprising the signal peptide. A signal
peptide that is functional in the intended host cells enhances
extracellular secretion of the elk-L polypeptide. The signal peptide is
cleaved from the elk-L polypeptide upon secretion of elk-L from the cell.
Suitable host cells for expression of elk-L polypeptides include
prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and
expression vectors for use with bacterial, fungal, yeast, and mammalian
cellular hosts are described, for example, in Pouwels et al. Cloning
Vectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-free
translation systems could also be employed to produce elk-L polypeptides
using RNAs derived from DNA constructs disclosed herein.
Prokaryotes include gram negative or gram positive organisms, for example,
E. coli or Bacilli. Suitable prokaryotic host cells for transformation
include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium,
and various other species within the genera Pseudomonas, Streptomyces, and
Staphylococcus. In a prokaryotic host cell, such as E. coli, an elk-L
polypeptide may include an N-terminal methionine residue to facilitate
expression of the recombinant polypeptide in the prokaryotic host cell.
The N-terminal Met may be cleaved from the expressed recombinant elk-L
Expression vectors for use in prokaryotic host cells generally comprise
one or more phenotypic selectable marker genes. A phenotypic selectable
marker gene is, for example, a gene encoding a protein that confers
antibiotic resistance or that supplies an autotrophic requirement.
Examples of useful expression vectors for prokaryotic host cells include
those derived from commercially available plasmids such as the cloning
vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides simple means for identifying
transformed cells. An appropriate promoter and a elk-L DNA sequence are
inserted into the pBR322 vector. Other commercially available vectors
include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden)
and pGEM1 (Promeca Biotec, Madison, Wis., USA).
Promoter sequences commonly used for recombinant prokaryotic host cell
expression vectors include .beta.-lactamase (penicillinase), lactose
promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al.,
Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,
Nucl. Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p.
412, 1982). A particularly useful prokaryotic host cell expression system
employs a phage .lambda. PL promoter and a cI857ts thermolabile
repressor sequence. Plasmid vectors available from the American Type
Culture Collection which incorporate derivatives of the .lambda. PL
promoter include plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC
37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).
elk-L alternatively may be expressed in yeast host cells, preferably from
the Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, such
as Pichia or Kluyveromyces, may also be employed. Yeast vectors will often
contain an origin of replication sequence from a 2.mu. yeast plasmid, an
autonomously replicating sequence (ARS), a promoter region, sequences for
polyadenylation, sequences for transcription termination, and a selectable
marker gene. Suitable promoter sequences for yeast vectors include, among
others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman
et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess
et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem.
17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phosphoglucose isomerase, and glucolinase.
Other suitable vectors and promoters for use in yeast expression are
further described in Hitzeman, EPA-73,657. Another alternative is the
glucose-repressible ADH2 promoter described by Russell et al. (J. Biol.
Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982). Shuttle
vectors replicable in both yeast and E. coli may be constructed by
inserting DNA sequences from pBR322 for selection and replication in E.
coli (Ampr gene and origin of replication) into the above-described
The yeast .alpha.-factor leader sequence may be employed to direct
secretion of the elk-L polypeptide. The .alpha.-factor leader sequence is
often inserted between the promoter sequence and the structural gene
sequence. See, e.g., Kuran et al., Cell 30:933, 1982; Bitter et al., Proc.
Natl. Acad. Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP
324,274. Other leader sequences suitable for facilitating secretion of
recombinant polypeptides from yeast hosts are known to those of skill in
the art. A leader sequence may be modified near its 3' end to contain one
or more restriction sites. This will facilitate fusion of the leader
sequence to the structural gene.
Yeast transformation protocols are known to those of skill in the art. One
such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA
75:1929, 1978. The Hinnen et al. protocol selects for Trp+
transformants in a selective medium, wherein the selective medium consists
of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 .mu.g/ml
adenine and 20 .mu.g/ml uracil.
Yeast host cells transformed by vectors containing ADH2promoter sequence
may be grown for inducing expression in a "rich" medium. An example of a
rich medium is one consisting of 1% yeast extract, 2% peptone, and 1%
glucose supplemented with 80 .mu.g/ml adenine and 80 .mu.g/ml uracil.
Derepression of the ADH2 promoter occurs when glucose is exhausted from
Mammalian or insect host cell culture systems could also be employed to
express recombinant elk-L polypeptides. Baculovirus systems for production
of heterologous proteins in insect cells are reviewed by Luckow and
Summers, Bio/Technology 6:47 (1988). Established cell lines of mammalian
origin also may be employed. Examples of suitable mammalian host cell
lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman
et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163),
Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell
lines, and the CV-1/EBNA-1 cell line derived from the African green monkey
kidney cell line CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J.
10: 2821, 1991).
Transcriptional and translational control sequences for mammalian host
cell expression vectors may be excised from viral genomes. Commonly used
promoter sequences and enhancer sequences are derived from Polyoma virus,
Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA
sequences derived from the SV40 viral genome, for example, SV40 origin,
early and late promoter, enhancer, splice, and polyadenylation sites may
be used to provide other genetic elements for expression of a structural
gene sequence in a mammalian host cell. Viral early and late promoters are
particularly useful because both are easily obtained from a viral genome
as a fragment which may also contain a viral origin of replication (Fiers
et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also
be used, provided the approximately 250 bp sequence extending from the
Hind III site toward the Bgl I site located in the SV40 viral origin of
replication site is included.
Exemplary expression vectors for use in mammalian host cells can be
constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280,
1983). A useful system for stable high level expression of mammalian cDNAs
in C127 murine mammary epithelial cells can be constructed substantially
as described by Cosman et al. (Mol. Immunol. 23:935, 1986). A useful high
expression vector, PMLSV N1/N4, described by Cosman et al., Nature
312:768, 1984 has been deposited as ATCC 39890. Additional useful
mammalian expression vectors are described in EP-A-0367566, and in U.S.
patent application Ser. No. 07/701,415, filed May 16, 1991, incorporated
by reference herein. The vectors may be derived from retroviruses. In
place of the native signal sequence, a heterologous signal sequence may be
added, such as the signal sequence for interleukin-7 (IL-7) described in
U.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptor
described in Cosman et al., Nature 312:768 (1984); the interleukin-4
signal peptide described in EP 367,566; the type I interleukin-1 receptor
signal peptide described in U.S. Pat. No. 4,968,607; and the type II
interleukin-1 receptor signal peptide described in EP 460,846.
Elk Ligand Protein
The present invention provides substantially homogeneous elk-L protein,
which may be produced by recombinant expression systems as described above
or purified from naturally occurring cells. The elk-L is purified to
substantial homogeneity, as indicated by a single protein band upon
analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
One process for producing the elk-L protein comprises culturing a host
cell transformed with an expression vector comprising a DNA sequence that
encodes elk-L under conditions such that elk-L is expressed. The elk-L
protein is then recovered from culture medium or cell extracts, depending
upon the expression system employed. As the skilled artisan will
recognize, procedures for purifying the recombinant elk-L will vary
according to such factors as the type of host cells employed and whether
or not the elk-L is secreted into the culture medium.
For example, when expression systems that secrete the recombinant protein
are employed, the culture medium first may be concentrated using a
commercially available protein concentration filter, for example, an
Amicon or Millipore Pellicon ultrafiltration unit. Following the
concentration step, the concentrate can be applied to a purification
matrix such as a gel filtration medium. Alternatively, an anion exchange
resin can be employed, for example, a matrix or substrate having pendant
diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose,
dextran, cellulose or other types commonly employed in protein
purification. Alternatively, a cation exchange step can be employed.
Suitable cation exchangers include various insoluble matrices comprising
sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.
Finally, one or more reversed-phase high performance liquid chromatography
(RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica gel
having pendant methyl or other aliphatic groups) can be employed to
further purify elk-L. Some or all of the foregoing purification steps, in
various combinations, can be employed to provide a substantially
homogeneous recombinant protein.
It is also possible to utilize an affinity column comprising the ligand
binding domain of elk to affinity-purify expressed elk-L polypeptides.
elk-L polypeptides can be removed from an affinity column in a high salt
elution buffer and then dialyzed into a lower salt buffer for use.
Alternatively, the affinity column may comprise an antibody that binds
elk-L. Example 4 describes a procedure for employing the elk-L protein of
the present invention to generate monoclonal antibodies directed against
Recombinant protein produced in bacterial culture is usually isolated by
initial disruption of the host cells, centrifugation, extraction from cell
pellets if an insoluble polypeptide, or from the supernatant fluid if a
soluble polypeptide, followed by one or more concentration, salting-out,
ion exchange, affinity purification or size exclusion chromatography
steps. Finally, RP-HPLC can be employed for final purification steps.
Nicrobial cells can be disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of cell
Transformed yeast host cells are preferably employed to express elk-L as a
secreted polypeptide. This simplifies purification. Secreted recombinant
polypeptide from a yeast host cell fermentation can be purified by methods
analogous to those disclosed by Urdal et al. (J. Chromatog. 296:171,
1984). Urdal et al. describe two sequential, reversed-phase HPLC steps for
purification of recombinant human IL-2 on a preparative HPLC column.
Nucleic Acid Fragments
The present invention further provides fragments of the elk-L nucleotide
sequences presented herein. Such fragments desirably comprise at least
about 14 nucleotides of the sequence presented in SEQ ID NO:1. DNA and RNA
complements of said fragments are provided herein, along with both
single-stranded and double-stranded forms of the elk-L DNA.
Among the uses of such elk-L nucleic acid fragments is use as a probe.
Such probes may be employed in cross-species hybridization procedures to
isolate elk-L DNA from additional mammalian species. As one example, a
probe corresponding to the extracellular domain of elk-L may be employed.
The probes also find use in detecting the presence of elk-L nucleic acids
in in vitro assays and in such procedures as Northern and Southern blots.
Cell types expressing elk-L can be identified. Such procedures are well
known, and the skilled artisan can choose a probe of suitable length,
depending on the particular intended application.
Other useful fragments of the elk-L nucleic acids are antisense or sense
oligonucleotides comprising a single-stranded nucleic acid sequence
(either RNA or DNA) capable of binding to target elk-L mRNA (sense) or
elk-L DNA (antisense) sequences. Antisense or sense oligonucleotides,
according to the present invention, comprise a fragment of the coding
region of elk-L cDNA. Such a fragment generally comprises at least about
14 nucleotides, preferably from about 14 to about 30 nucleotides. The
ability to create an antisense or a sense oligonucleotide, based upon a
cDNA sequence for a given protein is described in, for example, Stein and
Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al., BioTechniques
Binding of antisense or sense oligonucleotides to target nucleic acid
sequences results in the formation of duplexes that block translation
(RNA) or transcription (DNA) by one of several means, including enhanced
degradation of the duplexes, premature termination of transcription or
translation, or by other means. The antisense oligonucleotides thus may be
used to block expression of elk-L proteins. Antisense or sense
oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester
backbones (or other sugar linkages, such as those described in WO91/06629)
and wherein such sugar linkages are resistant to endogenous nucleases.
Such oligonucleotides with resistant sugar linkages are stable in vivo
(i.e., capable of resisting enzymatic degradation) but retain sequence
specificity to be able to bind to target nucleotide sequences. Other
examples of sense or antisense oligonucleotides include those
oligonucleotides which are covalently linked to organic moieties, such as
those described in WO90/10448, and other moieties that increases affinity
of the oligonucleotide for a target nucleic acid sequence, such as
poly-(L-lysine). Further still, intercalating agents, such as ellipticine,
and alkylating agents or metal complexes may be attached to sense or
antisense oligonucleotides to modify binding specificities of the
antisense or sense oliginucleotide for the target nucleotide sequence.
Antisense or sense oligonucleotides may be introduced into a cell
containing the target nucleic acid sequence by any gene transfer method,
including, for example, CaPO4 -mediated DNA transfection,
electroporation, or by using gene transfer vectors such as Epstein-Barr
virus. Antisense or sense oligonucleotides are preferably introduced into
a cell containing the target nucleic acid sequence by insertion of the
antisense or sense oligonucleotide into a suitable retroviral vector, then
contacting the cell with the retrovirus vector containing the inserted
sequence, either in vivo or ex vivo. Suitable retroviral vectors include,
but are not limited to, the murine retrovirus M-MuLV, N2 (a retrovirus
derived from M-MuLV), or or the double copy vectors designated DCT5A,
DCT5B and DCT5C (see PCT application Ser. No. 90/02656).
Sense or antisense oligonucleotides also may be introduced into a cell
containing the target nucleotide sequence by formation of a conjugate with
a ligand binding molecule, as described in WO 91/04753. Suitable ligand
binding molecules include, but are not limited to, cell surface receptors,
growth factors, other cytokines, or other ligands that bind to cell
surface receptors. Preferably, conjugation of the ligand binding molecule
does not substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or block entry
of the sense or antisense oligonucleotide or its conjugated version into
Alternatively, a sense or an antisense oligonucleotide may be introduced
into a cell containing the target nucleic acid sequence by formation of an
oligonucleotide-lipid complex, as described in WO 90/10448. The sense or
antisense oligonucleotide-lipid complex is preferably dissociated within
the cell by an endogenous lipase.
Claim 1 of 7 Claims
What is claimed is:
1. A method for enhancing survival of a hippocampal neuron or inhibiting
hippocampal neuronal death from excitotoxicity, comprising contacting said
neuron with an effective amount of a substantially homogenous purified
elk-L protein, wherein said protein (1) is at least 80% identical to at
least amino acids 1 to 213 of SEQ ID NO: 2, (2) is characterized by the
N-terminal amino acid sequence Ala-Thr-Pro-Leu-Ala-Lys-Asn-Leu-Glu-Pro-Val-Ser-
(SEQ ID NO: 5), (3) is capable of binding elk, and (4) is capable of
inhibiting death of rat hippocampal neurons resulting from excitotoxicity.
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