Note: Descriptions are shown in the official language in which they were submitted.
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CHORDIN-LIFE-2 MOLECULES AND USES THEREOF-
Field of the Invention
The present invention relates to Chordin-Like-2 (CHL2) polypeptides and
nucleic acid molecules encoding the same. The invention also relates to
selective
binding agents, vectors, host cells, and methods for producing CHL2
polypeptides. The invention further relates to pharmaceutical compositions and
methods for the diagnosis, treatment, amelioration, and/or prevention of
diseases,
disorders, and conditions associated with CHL2 polypeptides.
Background of the Invention
Technical advances in the identification, cloning, expression, and
manipulation of nucleic acid molecules and the deciphering of the human genome
have greatly accelerated the discovery of novel therapeutics. Rapid nucleic
acid
sequencing techniques can now generate sequence information at unprecedented
rates and, coupled with computational analyses, allow the assembly of
overlapping sequences into partial and entire genomes and the identification
of
polypeptide-encoding regions. A comparison of a predicted amino acid sequence
against a database compilation of known amino acid sequences allows one to
2 0 determine the extent of homology to previously identified sequences and/or
structural landmarks. The cloning and expression of a polypeptide-encoding
region of a nucleic acid molecule provides a polypeptide product for
structural
and functional analyses. The manipulation of nucleic acid molecules and
encoded
polypeptides may confer advantageous properties on a product for use as a
2 5 therapeutic.
In spite of the significant technical advances in genome research over the
past decade, the potential for the development of novel therapeutics based on
the
human genome is still largely unrealized. Many genes encoding potentially
beneficial polypeptide therapeutics or those encoding polypeptides, wluch may
3 0 act as "targets" for therapeutic molecules, have still not been
identified.
Accordingly, it is an object of the invention to identify novel polypeptides,
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and nucleic acid .molecules encoding the same, which have diagnostic or
therapeutic benefit.
CHL2 is structurally related to the bone morphogenetic protein (BMP)
inhibitor known as chordin (CHD), (Sasai et al., 1994, Cell 79:779-90), or
short
gastrulation (SOG; Francois, et al., 1994, Genes Dev. 8:2602-16). The CHL2
gene is believed to be a member of CHD/SOG family.
Bone morphogenetic protein (BMP) is a member of the transforming
growth factor-beta family, which was originally identified as a factor
promoting
bone formation from a cartridge implant (Wozney et al., 1988, Science 242:1528
34; Celeste et al., 1990, Pf~oc. Nat. Acad. Sci. USA 87:9843-47). BMP is also
known to play an essential role during the early embryogenesis of the frog,
the fly,
and in mammals. The precise concentration of active BMP seems to be important
for the specification of particular cell types (Dale et al., 1992, Development
115:573-85; Dosch et al., 1997 Development 124:2325-34). An activity gradient
of BMP2/4 is observed in, for example, Xenopus embryos in which the lowest
expression is detected at the dorsal tip and the highest expression at the
ventral tip
- establishing the dorsoventral axis determination in the embryo. In another
example, the control of BMP concentration at specific sites of tissue
development
2 0 suggests a role for BMP in organogenesis. Control of BMP expression is
achieved by either localized expression of the BMP gene products or through
the
influence of the BMP inhibitor chordin (CHD) (Sasai et al., 1994, Cell 79:779-
90)
- or short gastrulation (SOG) (Francois et al., 1994, Genes Dev. 8:2602-16).
CHD/SOG is a large secreted protein produced from the Spemann's
2 5 organizer, the master-controhhing region for the dorsoventral axis
specification at
the gastrulation stage of Xeraopus embryogenesis. CHD/SOG fiulctions as a
dorsalization factor, as does Noggin (Smith and Harland, 1992, Cell 70:829-
40),
which is also secreted from the organizer. The D~osophila SOG has a
transmembrane domain at its amino-terminus, suggesting that it may be a type
II
3 0 transmembrane protein (Francois et al., 1994, Genes Dev. 8:2602-16). It
has been
proposed that the carboxyl-terminal side (extracelluhar domain) of the
D~osophila
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SOG is cleaved off. However, Xehopus CHD (Sasai et al., 1994, Cell 79:779-90),
Zebrafish CHD (Schulte-Merker et al., 1997, NatuYe 387:862-63), and marine
CHD (Pappano et al., 1998, GefZOmics 52:236-39) do not contain the
transmembrane domain. Instead, these proteins have a signal peptide, and are
, secreted directly. The CHD/SOG polypeptide contains four repeats of the
cysteine-rich domain (CRl-4) that is also found in a variety of extracellular
matrix
proteins such as collagen and thrombospondin.
CHD/SOG is known to bind to one of the ventralizing factors, BMP4
(Piccolo et al., 1996, Cell 86:589-98). BMP4 has been shown to be essential
for
~ embryonic development of posterior-ventral mesoderm in mice (Winnier et al.,
1995, Genes Dev. 9:2105-16). The binding of CHD/SOG to BMP4 inhibits
BMP4 activity by preventing BMP4 from binding to its receptor (Piccolo et al.,
1996, Cell 86:589-98). In this respect, the functional relationship between
CHD/SOG and BMP4 resembles that between OPG and OPGL, although
CHD/SOG is not structurally related to the BMP receptors. The binding affnuty
of CHD/SOG to BMP4 is specific and tight (Kd = 3 x 10-1° M (Piccolo et
al.,
1996, Cell 86:589-98), and seems to require proteolysis in order to effectuate
the
release of bound BMP4. This proteolysis is aclueved by a specific
metalloprotease - Tolloid (TLD) or BMP1 - that cleaves CHD/SOG to liberate
2 0 either, or both, the first (CRl) and last (CR4) CR motifs (Piccolo et al.,
1997, Cell
91: 407-16). Whether or not CHD/SOG has other functions or an independent
function through its own receptor remains to be determined.
One of the most important roles of CHD/SOG is to establish a BMP4
morphogen gradient (Jones and Smith, 1998, Dev. Biol. 194:12-17). BMP4 itself
2 5 only migrates a short distance and seems to act essentially on the cell
autonomously (Jones et al., 1996, Curr. Biol. 6:1468-75). In contrast, the
BMP4
inhibitors Noggin and CHD/SOG appear to exert a long-range effect, thereby
forming an activity gradient of BMP4.
BMPs also play important roles outside of early embryogenesis, for
3 0 example in the organogenesis of lung, gut, kidney, skin, heart and teeth,
as well as
in the later stages of embryogenesis (Hogan, 1996, Genes Dev. 10:1580-94).
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Some BMPs are expressed in a very localized fashion while others are expressed
widely in a tissue. The importance of the localized action of BMP for
organogenesis has been supported by transgenic mouse experiments using
constructs by which BMP concentration is artificially elevated throughout the
target tissue. In the case of lung, BMP4 is expressed in the distal tips of
epithelium in the developing lung, and when overexpressed with the surfactant
protein C promoter, the development of a small lung in which the structural
organization (i.e., branching) has been severely disrupted is observed
(Bellusci et
al., 1996, Development 122:1693-702). Since the putative BMP-activity gradient
could also be disrupted by the transgene expression, BMPs expressed widely in
the tissue could also play a role in the determination of the structural
organization
of a tissue.
Noggin is another BMP2/4 inhibitor secreted from Spemann's organizer
(Zimmerman et al., 1996, Cell 86:599-606). The biological role of Noggin and
its
mode of action are similar to CHD/SOG in Xezzopus. Although the most notable
function of Noggin is, like CHD/SOG, dorsahization, Noggin null-mutant mice
have shown a bone phenotype (hyperplasia of chondrocytes) instead of an early
embryonic phenotype (McMahon et al., 1998, Genes Dev. 12:1438-52; Brunet et
al., 1998, Sciezzce 280:1455-57). This suggests that CHL2 or even CHD might
2 o have a non-dispensable function in the later stage of embryogenesis.
Summary of the Invention
The present invention relates to novel CHL2 nucleic acid molecules and
encoded polypeptides.
2 5 The invention provides for an isolated nucleic acid molecule comprising a
nucleotide sequence selected from the group consisting of
(a) the nucleotide sequence as set forth in either SEQ ID NO: 1 or
SEQ ID NO: 4;
(b) the nucleotide sequence of the DNA insert either ATCC Deposit
3 0 Nos. PTA-1479 or PTA-1480;
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(c) a nucleotide sequence encoding the polypeptide as set forth in
either SEQ 1D NO: 2 or SEQ ID NO: 5;
(d) a nucleotide sequence which hybridizes under moderately or
highly stringent conditions to the complement of any of (a) - (c); and
(e) a nucleotide sequence complementary to any of (a) - (c).
The invention also provides for an isolated nucleic acid molecule
comprising a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence encoding a polypeptide which is at least
about 70 percent identical to the polypeptide as set forth in either SEQ ID
NO: 2
or SEQ ID NO: 5, wherein the encoded polypeptide has an activity of the
polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;
(b) a nucleotide sequence encoding am allelic variant or splice variant
of the nucleotide sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO:
4,
the nucleotide sequence of the DNA insert in either ATCC Deposit Nos. PTA-
1479 or PTA-1480, or (a);
(c) a region of the nucleotide sequence of any of SEQ ID NO: 1 or
SEQ ID NO: 4, the DNA insert in either ATCC Deposit Nos. PTA-1479 or PTA-
1480, (a), or (b) encoding a polypeptide fragment of at least about 25 amino
acid
2 0 residues, wherein the polypeptide fragment has an activity of the
polypeptide~ set
forth in either SEQ ID NO: 2~or SEQ ID NO: 5, or is antigenic;
(d) a region of the nucleotide sequence of any of SEQ ID NO: 1 or
SEQ ID NO: 4, the DNA insert in either ATCC Deposit Nos. PTA-1479 or PTA-
1480, or any of (a) - (c) comprising a fragment of at least about 16
nucleotides;
2 5 (e) a nucleotide sequence which hybridizes under moderately or
highly stringent conditions to the complement of any of (a) - (d); and
(f) a nucleotide sequence complementary to any of (a) - (d).
The invention further provides for an isolated nucleic acid molecule
3 0 comprising a nucleotide sequence selected from the group consisting of:
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(a) a nucleotide sequence encoding a polypeptide as set forth in either
SEQ ID NO: 2 or SEQ ID NO: 5 with at least one conservative amino acid
substitution, wherein the encoded polypeptide has an activity of the
polypeptide
set forth in either SEQ ID NO: 2 or SEQ ID NO: 5;
(b) a nucleotide sequence encoding a polypeptide as set forth in either
SEQ ID NO: 2 or SEQ 1D NO: 5 with at least one amino acid insertion, wherein
the encoded polypeptide has an activity of the polypeptide set forth in either
SEQ
ID NO: 2 or SEQ ID NO: 5;
(c) a nucleotide sequence encoding a polypeptide as set forth in either
Z 0 SEQ ID NO: 2 or SEQ ID NO: 5 with at least one amino acid deletion,
wherein
the encoded polypeptide has an activity of the polypeptide set forth in either
SEQ
ID NO: 2 or SEQ ID NO: 5;
(d) a nucleotide sequence encoding a polypeptide as set forth in either
SEQ ID NO: 2 or SEQ ID NO: S which has a C- and/or N- terminal truncation,
l5 wherein the encoded polypeptide has an activity of the polypeptide set
forth in
either SEQ ID NO: 2 or SEQ ID NO: 5;
(e) a nucleotide sequence encoding a polypeptide as set forth in either
SEQ ID NO: 2 or SEQ ID NO: 5 with at least one modification selected from the
group consisting of amino acid substitutions, amino acid insertions, amino
acid
2 0 deletions, C-terminal truncation, and N-terminal truncation, wherein the
encoded
polypeptide has an activity of the polypeptide set forth in either SEQ D7 NO:
2 or
SEQ ID NO: 5;
(f) a nucleotide sequence of any of (a) - (e) comprising a fragment of
at least about 16 nucleotides;
2 5 (g) a nucleotide sequence which hybridizes under moderately or
highly stringent conditions to the complement of any of (a) - (f); and
(h) a nucleotide sequence complementary to any of (a) - (e).
The present invention provides for an isolated polypeptide comprising an
3 0 amino acid sequence selected from the group consisting of
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(a) the amino acid sequence as set forth in either SEQ ID NO: 2 or
SEQ ID NO: S; and
(b) the amino acid sequence encoded by the DNA insert in either
ATCC Deposit Nos. PTA-1479 or PTA-1480.
The invention also provides for an isolated polypeptide comprising the
amino acid sequence selected from the group consisting of:
(a) the amino acid sequence as set forth in either SEQ ID NO: 3 or
SEQ ID NO: 6, optionally further comprising an amino-terminal methionine;
(b) an amino acid sequence for an ortholog of any of SEQ ID NO: 2 or
SEQ ID NO: 5;
(c) an amino acid sequence which is at least about 70 percent identical
to the amino acid sequence of any of SEQ ID NO: 2 or SEQ ID NO: 5, wherein
the polypeptide has an activity of the polypeptide set forth in either SEQ ID
NO:
2 or SEQ ID NO: 5;
(d) a fragment of the amino acid sequence set forth in either SEQ ID
NO: 2 or SEQ ID NO: 5 comprising at least about 25 amino acid residues,
wherein the fragment has an activity of the polypeptide set forth in either
SEQ ID
NO: 2 or SEQ ID NO: 5, or is antigenic; and
2 0 (e) an amino acid sequence for an allelic variant or splice variant of
the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5,
the amino acid sequence encoded by the DNA insert in either ATCC Deposit Nos.
PTA-1479 or PTA-1480, or any of (a) - (c).
2 5 The invention further provides for an isolated polypeptide comprising the
amino acid sequence selected from the group consisting of:
(a) the amino acid sequence as set forth in either SEQ ID NO: 2 or
SEQ ID NO: 5 with at least one conservative amino acid substitution, wherein
the
polypeptide has an activity of the polypeptide set forth in either SEQ ID NO:.
2 or
3 o SEQ ID NO: 5;
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(b) the amino acid sequence as set forth in either SEQ ID NO: 2 or
SEQ ID NO: 5 with at least one amino acid insertion, wherein the polypeptide
has
an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ TD NO:
5;
(c) the amino acid sequence as set forth in either SEQ ID NO: 2 or
SEQ 117 NO: 5 with at Ieast one amino acid deletion, wherein the polypeptide
has
an activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ JD NO:
5;
(d) the amino acid sequence as set forth in either SEQ ID NO: 2 or
SEQ ID NO: 5 which has a C- and/or N-terminal truncation, wherein the
polypeptide has an activity of the polypeptide set forth in either SEQ ID NO:
2 or
l o SEQ ID NO: 5; and
(e) the amino acid sequence as set forth in either SEQ ID NO: 2 or
SEQ ID NO: 5 with at least one modification selected from the group consisting
of amino acid substitutions, amino acid insertions, amino acid deletions, C-
terminal truncation, and N-terminal truncation, wherein the polypeptide has an
activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.
The invention still further provides for an isolated polypeptide comprising
the amino acid sequence as set forth in SEQ ID NO: 5 with at least one
conservative amino acid substitution selected from the group consisting of:
2 0 leucine or methionine at position 2; methionine at position 5; lysine at
position 6;
alanine at position 7; isoleucine at position 8; phenylalanine at position 14;
leucine at position 15; threonine at position 23; leucine at position 25;
valine at
position 27; glutamic acid at position 30; tyrosine at position 32; methionine
at
position 34; glutamine at position 36; lysine at position 39; alanine at
position 41;
2 5 threonine at position 45; valine at position 55; valine at position 59;
asparagine at
position 60; proline at position 66; asparagine at position 68; serine or
threonine
at position 72; valine at position 74; arginine at position 75; arginine at
position
94; asparagine at position 99; serine at position 100; lysine at position 105;
valine
at position 106; tyrosine at position 113; serine at position 116; serine at
position
3 0 118; arginine at position 120; leucine at position 123; alanine at
position 125;
alanine at position 129; alanine at position 139; threonine at position 142;
serine
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at position 144; asparagine at position 147; valine at position 148; serine at
position 149; alanine at position 159; alanine at position 160; alanine at
position
161; valine at position 164; valine at position 166; valine at position 173;
arginine
at position 175; aspartic acid at position 177; alanine at position 190;
. phenylalanine at position 191; arginine at position 192; leucine at position
194;
asparagine at position 196; leucine at position 205; alanine at position 210;
alanine at position 212; serine at position 213; alanine at position 216;
serine at
position 217; alanine at position 218; isoleucine at position 219; alanine at
position 222; leucine at position 225; phenylalanine at position 226; leucine
at
position 230; glutamine or arginine at position 233; glutamine at position
241;
leucine at position 242; isoleucine at position 244; glutamine or asparagine
at
position 245; glutamine at position 249; leucine or valine at position 251;
alanine
at position 256; asparagine at position 257; serine at position 259; alanine
at
position 260; glutamine at position 261; phenylalanine at position 265; valine
at
position 268; leucine at position 269; leucine at position 272; valine at
position
275; valine at position 278; glutamic acid at position 284; glutamic acid at
position 288; alanine or isoleucine at position 292; serine at position 300;
isoleucine at position 306; valine at position 313; serine at position 314;
leucine at
position 319; glutamine at position 323; threonine at position 324; alanine at
2 0 position 326; alanine at position 327; serine at position 329; serine at
position
331; leucine at position 334; asparagine at position 337; valine at position
339;
leucine at position 340; serine at position 342; phenylalanine at position
344;
glutamic acid at position 349; isoleucine or valine at position 354;
methionine at
position 356; valine at position 366; methionine or valine at position 368;
2 5 isoleucine at position 371; leucine at position 375; leucine at position
376;
glutamine at position 377; phenylalanine at position 381; asparagine at
position
383; isoleucine at position 384; leucine at position 393; arginine at position
395;
valine at position 398; alanine or valine at position 399; tyrosine at
position 403;
asparagine at position 406; isoleucine at position 409; alanine or valine at
position
3 0 415; isoleucine at position 417; and leucine at position 421; wherein the
polypeptide has an activity of the polypeptide as set forth in SEQ ID NO: 5.
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Also provided are fusion polypeptides comprising CHL2 amino acid
sequences.
The present invention also provides for an expression vector comprising
the isolated nucleic acid molecules as set forth herein, recombinant host
cells
comprising the recombinant nucleic acid molecules as set forth herein, and a
method of producing a CHL2 polypeptide comprising culturing the host cells and
optionally isolating the polypeptide so produced.
A transgenic non-human animal comprising a nucleic acid molecule
encoding a CHL2 polypeptide is also encompassed by the invention. The CHL2
nucleic acid molecules are introduced into the animal in a manner that allows
expression and increased levels of a CHL2 polypeptide, which may include
increased circulating levels. Alternatively, the CHLZ nucleic acid molecules
are
introduced into the animal in a manner that prevents expression of endogenous
CHL2 polypeptide (i.e., generates a transgenic animal possessing a CHL2
polypeptide gene knockout). The transgenic non-human animal is preferably a
mammal, and more preferably a rodent, such as a rat or a mouse.
Also provided are derivatives of the CHL2 polypeptides of the present
invention.
2 o Additionally provided are selective binding agents such as antibodies and
peptides capable of specifically binding the CHL2 polypeptides of the
invention.
Such antibodies and peptides may be agonistic or antagonistic.
Pharmaceutical compositions comprising the nucleotides, polypeptides, or
selective binding agents of the invention and one or more pharmaceutically
2 5 acceptable formulation agents are also encompassed by the invention. The
pharmaceutical compositions are used to provide therapeutically effective
amounts of the nucleotides or polypeptides of the present invention. The
invention is also directed to methods of using the polypeptides, nucleic acid
molecules, and selective binding agents.
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The CHL2 polypeptides and nucleic acid molecules of the present
invention may be used to treat, prevent, ameliorate, and/or detect diseases
and
disorders, including those recited herein.
The present invention also provides a method of assaying test molecules to
identify a test molecule that binds to a CHL2 polypeptide. The method
comprises
contacting a CHL2 polypeptide with a test molecule to determine the extent of
binding of the test molecule to the polypeptide. The method further comprises
determining whether such test molecules are agonists or antagoiusts of a CHL2
polypeptide. The present invention further provides a method of testing the
impact of molecules on the expression of CHL2, polypeptide or on the activity
of
CHL2 polypeptide.
Methods of regulating expression and modulating (i.e., increasing or
decreasing) levels of a CHL2 polypeptide are also encompassed by the
invention.
One method comprises administering to an anmal a nucleic acid molecule
encoding a CHL2 polypeptide. In another method, a nucleic acid molecule
comprising elements that regulate or modulate the expression of a CHL2
polypeptide may be administered. Examples of these methods include gene
therapy, cell therapy, and anti-sense therapy as further described herein.
In another aspect of the present invention, the CHL2 polypeptides may be
2 o used for identifying receptors thereof ("CHL2 polypeptide receptors").
Various
forms of "expression cloning" have been extensively used to clone receptors
for
protein ligands. See, e.g., Simonsen and Lodish, 1994, T~e~ds PhaYmacol. Sci.
15:437-41 and Tartaglia et al., 1995, Cell X3:1263-71. The isolation of a CHL2
polypeptide receptor is useful for identifying or developing novel agonists
and
2 5 antagonists of the CHL2 polypeptide signaling pathway. Such agonists and
antagonists include soluble CHL2 polypeptide receptors, anti-CHL2 polypeptide
receptor-selective binding agents (such as antibodies and derivatives
thereof),
small molecules, and antisense oligonucleotides, any of which can be used for
treating one or more disease or disorder, including those disclosed herein.
Brief Description of the Figures
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Figures lA-1C illustrate the nucleotide sequence of the marine CHL2 gene (SEQ
ID NO: 1) and the deduced amino acid sequence of marine CHL2 polypeptide
(SEQ ID NO: 2). The predicted signal sequence is indicated (underline ;
Figure 2 illustrates an amino acid sequence alignment of marine CHL2
polypeptide (mouse CHL2; SEQ m NO: 2) and marine chordin (Af069501; SEQ
ID NO: 7);
Figures 3A-3C illustrate the nucleotide sequence of the human CHL2 gene (SEQ
TD NO: 4) and the deduced amino acid sequence of human CHL2 polypeptide
(SEQ ID NO: 5). The predicted signal sequence is indicated (underline);
Figure 4 illustrates an amino acid sequence alignment of human chordin (huCHD;
SEQ ID NO: ~), human CHL1 polypeptide (huCHL; SEQ ID NO: 9), and human
CHL2 polypeptide (huCHL2; SEQ ID NO: 5);
Figure 5 illustrates a schematic representation of marine chordin (Chordin),
CHL1
polypeptide, and CHL2 polypeptide. Pro-collagen repeats (CR; homologous CR
domains are indicated by gray boxes), signal peptides (SP), putative
BMP1/Tolloid cleavage sites (~), and sites of amino acid sequence variation in
CHL1 (dE and d5) are indicated;
Figure 6 illustrates the expression of marine CHL2 mRNA as detected by in situ
hybridization in E17.5 mouse hip joint (panels A and B) and costal-chondral
2 5 articulation (panel C; showing signal in articular chondrocytes on both
sides of the
articulation);
Figure 7 illustrates the expression of marine CHL2 mRNA as detected by iyz
situ
hybridization in normal adult vertebral articulation (panels A, B, and C;
showing
3 0 signal in articular chondrocytes at the surface of the articular cartilage
on both
sides of the zygapophyseal or facet joint between the processes of adjacent
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vertebrae) and the fibrocartilage of the anulus fbrosus of a vertebral disc
(panel
D);
Figure 8 illustrates the expression of marine CHL2 mRNA as detected by iya
situ
hybridization in E18.5 mouse sternum and placenta and normal adult mouse
uterus, colon, and small intestine;
Figure 9 illustrates the secondary axis-forming activity of marine CHL2
polypeptide;
Figure 10 illustrates the effect of CHL2 polypeptide on the BMP4-dependent
generation of CD34+/CD31+ erythro-myeloid progenitor cells (R7) and CD34-
/CD31+ cells (R3);
Z 5 Figure 11 illustrates the effect of CHL2 polypeptide on the BMP2-dependent
induction of alkaline phosphatase in C2C12 myoblastic cells;
Figure 12 illustrates the results of Western blot analysis using mCHL2-FLAG
proteins. In panel A, mCHL2-FLAG was mixed with BMPS or activin A, treated
2 0 with an anti-mCHL2 antiserum, and precipitated with protein A agarose
beads. In
panel B, mCHL2-FLAG was mixed with BMP2, BMP4, BMPS, BMP6, GDFS
(BMP 14), or activin A, treated with an anti-mCHL2 antiserum, and precipitated
with protein A agarose beads.
2 5 Detailed Description of the Invention
The section headings used herein are for organizational purposes only and
are not to be construed as limiting the subject matter described. All
references
cited in this application are expressly incorporated by reference herein.
3 0 Definitions
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The terms "CHL2 gene" or "CHL2 nucleic acid molecule" or "CHL2
polynucleotide" refer to a nucleic acid molecule comprising or consisting of a
nucleotide sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO: 4, a
nucleotide sequence encoding the polypeptide as set forth in either SEQ ID NO:
2
or SEQ ID NO: 5, a nucleotide sequence of the DNA insert in either ATCC
Deposit Nos. PTA-1479 or PTA-1480, and nucleic acid molecules as defined
herein.
The term "CHL2 polypeptide allelic variant" refers to one of several
possible naturally occurring alternate forms of a gene occupying a.given locus
on
a chromosome of an organism or a population of organisms.
The term "CHL2 polypeptide splice variant" refers to a nucleic acid
molecule, usually RNA, which is generated by alternative processing of intron
sequences in an RNA transcript of CHL2 polypeptide amino acid sequence as set
forth in either SEQ ID NO: 2 or SEQ ID NO: 5.
The term "isolated nucleic acid molecule" refers to a nucleic acid
molecule of the invention that (1) has been separated from at least about 50
percent of proteins, lipids, carbohydrates, or other materials with which it
is
naturally found when total nucleic acid is isolated from the source cells, (2)
is not
linked to all or a portion of a polynucleotide to which the "isolated nucleic
acid
2 0 molecule" is linlced in nature, (3) is operably linked to a polynucleotide
which it is
not linked to in nature, or (4) does not occur in nature as part of a larger
polynucleotide sequence. Preferably, the isolated nucleic acid molecule of the
present invention is substantially free from any other contaminating nucleic
acid
molecules) or other contaminants that are found in its natural enviromnent
that
2 5 would interfere with its use in polypeptide production or its therapeutic,
diagnostic, prophylactic or research use.
The term "nucleic acid sequence" or "nucleic acid molecule" refers to a
DNA or RNA sequence. The term encompasses molecules formed from any of
the known base analogs of DNA and RNA such as, but not limited to 4-
3 0 acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinyl-cytosine,
pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-
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bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy-
methylaminomethyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1-
methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-
dimethyl-guanine, 2-rnethyladenine, 2-methylguanine, 3-methylcytosine, 5-
methylcytosine, N6-methyladenine, 7-methylguanine, 5-
methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D-
mannosylqueosine, 5' -methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-
methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-
5-
oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-
methyl-
2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic
acid
methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine,
and
2,6-diaminopurine.
The term "vector" is used to refer to any molecule (e.g., nucleic acid,
plasmid, or virus) used to transfer coding information to a host cell.
The term "expression vector" refers to a vector that is suitable for
transformation of a host cell and contains nucleic acid sequences that direct
and/or
control the expression of inserted heterologous nucleic acid sequences.
Expression includes, but is not limited to, processes such as transcription,
translation, and RNA splicing, if introns are present.
2 o The teen "operably linked" is used herein to refer to an arrangement of
flanking sequences wherein the flanking sequences so described are configured
or
assembled so as to perform their usual function. Thus, a flanking sequence
operably linked to a coding sequence may be capable of effecting the
replication,
transcription and/or translation of the coding sequence. For example, a coding
2 5 sequence is operably linked to a promoter when the promoter is capable of
directing transcription of that coding sequence. A flanking sequence need not
be
contiguous with the coding sequence, so long as it functions correctly. Thus,
for
example, intervening untranslated yet transcribed sequences can be present
between a promoter sequence and the coding sequence and the promoter sequence
3 0 can still be considered "operably linked" to the coding sequence.
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The term "host cell" is used to refer to a cell which has been transformed,
or is capable of being transformed with a nucleic acid sequence and then of
expressing a selected gene of interest. The term includes the progeny of the
parent cell, whether or not the progeny is identical in morphology or in
genetic
make-up to the original parent, so long as the selected gene is present.
The teen "CHL2 polypeptide" refers to a polypeptide comprising the
amino acid sequence of any of SEQ ID NO: 2 or SEQ ID NO: 5 and related
polypeptides. Related polypeptides include CHL2 polypeptide fragments, CHL2
polypeptide orthologs, CHL2 polypeptide variants, and CHL2 polypeptide
1 o derivatives, which possess at Ieast one activity of the polypeptide as set
forth in
either SEQ ID NO: 2 or SEQ ID NO: 5. CHL2 polypeptides may be mature
polypeptides, as defined herein, and may or may not have an amino-tenninal
methionine residue, depending on the method by which they are prepared.
The term "CHL2 polypeptide fragment" refers to a polypeptide that
comprises a truncation at the amino-terminus (with or without a leader
sequence)
and/or a truncation at the carboxyl-terminus of the polypeptide as set forth
in
either SEQ ID NO: 2 or SEQ ff~ NO: 5. The term "CHL2 polypeptide fragment"
also refers to amino-terminal and/or carboxyl-terminal truncations of CHL2
polypeptide orthologs, CHL2 polypeptide derivatives, or CHL2 polypeptide ,
2 0 variants, or to amino-terminal and/or carboxyl-terminal truncations of the
polypeptides encoded by CHL2 polypeptide allelic variants or CHL2 polypeptide
splice variants. CHLZ polypeptide fragments may result from alternative RNA
splicing or from in vivo protease activity. Membrane-bound forms of a CHL2
polypeptide are also contemplated by the present invention. In preferred
2 5 embodiments, truncations and/or deletions comprise about 10 amino acids,
or
about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or
about
100 amino acids, or more than about 100 amino acids. The polypeptide fragments
so produced will comprise about 25 contiguous amino acids, or about 50 amino
acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino
3 0 acids, or about 200 amino acids, or more than about 200 amino acids. Such
CHL2
polypeptide fragments may optionally comprise an amino-terminal methionine
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residue. It will be appreciated that such fragments can be used, for example,
to
generate antibodies to CHL2 polypeptides.
The term "CHL2 polypeptide ortholog" refers to a polypeptide from
another species that corresponds to CHL2 polypeptide amino acid sequence as
set
forth in either SEQ ID NO: 2 or SEQ ID NO: 5. For example, mouse and human
CHL2 polypeptides are considered orthologs of each other.
The term "CHLZ polypeptide variants" refers to CHL2 polypeptides
comprising amino acid sequences having one or more amino acid sequence
substitutions, deletions (such as internal deletions and/or CHL2 polypeptide
1 o fragments), and/or additions (such as internal additions and/or CHL2
fusion
polypeptides) as compared to the CHL2 polypeptide amino acid sequence set
forth in either SEQ ID NO: 2 or SEQ ID NO: 5 (with or without a leader
sequence). Variants may be naturally occurring (e.g., CHL2 polypeptide allelic
variants, CHL2 polypeptide orthologs, and CHLZ polypeptide splice variants) or
artificially constructed. Such CHL2 polypeptide variants rnay be prepared from
the corresponding nucleic acid molecules having a DNA sequence that varies
accordingly from the DNA sequence as set forth in either SEQ ID NO: 1 or SEQ
ID NO: 4. In preferred embodiments, the variants have from 1 to 3, or from 1
to
5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from
1 to
2 0 50, or from 1 to 75, or from 1 to 100, or more than 100 amino acid
substitutions,
insertions, additions and/or deletions, wherein the substitutions may be
conservative, or non-conservative, or any combination thereof.
The term "CHL2 polypeptide derivatives" refers to the polypeptide as set
forth in either SEQ ID NO: 2 or SEQ ID NO: 5, CHL2 polypeptide fragments,
2'5 CHL2 polypeptide orthologs, or CHL2 polypeptide variants, as defined
herein,
that have been chemically modified. The term "CHL2 polypeptide derivatives"
also refers to the polypeptides encoded by CHL2 polypeptide allelic variants
or
CHL2 polypeptide splice variants, as defined herein, that have been chemically
modified.
3 o The term "mature CHL2 polypeptide" refers to a CHL2 polypeptide
lacking a leader sequence. A mature CHL2 polypeptide may also include other
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modifications such as proteolytic processing of the amino-terminus (with or
without a leader sequence) and/or the carboxyl-terminus, cleavage of a smaller
polypeptide from a larger precursor, N-linked and/or O-linked glycosylation,
and
the like. Exemplary mature CHL2 polypeptides are depicted by the amino acid
sequences of SEQ ID NO: 3 and SEQ ID NO: 6.
The term "CHL2 fusion polypeptide" refers to a fusion of one or more
amino acids (such as a heterologous protein or peptide) at the amino- or
carboxyl-
terminus of the polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO:
5,
CHL2 polypeptide fragments, CHL2 polypeptide orthologs, CHL2 polypeptide
1 o variants, or CHL2 derivatives, as deEned herein. The term "CHL2 fusion
polypeptide" also refers to a fusion of one or more amino acids at the amino-
or
carboxyl-terminus of the polypeptide encoded by CHL2 polypeptide allelic
variants or CHL2 polypeptide splice variants, as defined herein.
The term "biologically active CHL2 polypeptides" refers to CHLZ
polypeptides having at least one activity characteristic of the polypeptide
comprising the amino acid sequence of any of SEQ ID NO: 2 or SEQ ID NO: 5.
In addition, a CHL2 polypeptide may be active as an immmlogen; that is, the
CHL2 polypeptide contains at least one epitope to which antibodies may be
raised.
2 0 The term "isolated polypeptide" refers to a polypeptide of the present
invention that (1) has been separated from at least about 50 percent of
polynucleotides, lipids, carbohydrates, or other materials with which it is
naturally
found when isolated from the source cell, (2) is not linked (by covalent or
noncovalent interaction) to all or a portion of a polypeptide to which the
"isolated
2 5 polypeptide" is linked in nature, (3) is operably linked (by covalent or
noncovalent interaction) to a polypeptide with which it is not linked in
nature, or
(4) does not occur in nature. Preferably, the isolated polypeptide is
substantially
free from any other contaminating polypeptides or other contaminants that are
found in its natural environment that would interfere with its therapeutic,
3 0 diagnostic, prophylactic or research use.
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The term "identity," as known in the art, refers to a relationship between
the sequences of two or more polypeptide molecules or two or more nucleic acid
molecules, as determined by comparing the sequences. In the art, "identity"
also
means the degree of sequence relatedness between nucleic acid molecules or
polypeptides, as the case may be, as determined by the match between strings
of
two or more nucleotide or two or more amino acid sequences. "Identity"
measures the percent of identical matches between the smaller of two or more
sequences with gap aligmnents (if any) addressed by a particular mathematical
model or computer program (i.e., "algoritlnns").
The term "similarity" is a related concept, but in contrast to "identity,"
"similarity" refers to a measure of relatedness which includes both identical
matches and conservative substitution matches. If two polypeptide sequences
have, for example, 10/20 identical amino acids, and the remainder are all non-
conservative substitutions, then the percent identity and similarity would
both be
50%. If in the same example, there are five more positions where there are
conservative substitutions, then the percent identity remains 50%, but the
percent
similarity would be 75% (15/20). Therefore, in cases where there are
conservative substitutions, the percent similarity between two polypeptides
will be
higher than the percent identity between those two polypeptides.
2 0 The term "naturally occurring" or "native" when used in comlection with
biological materials such as nucleic acid molecules, polypeptides, host cells,
and
the like, refers to materials which are found in nature and are not
manipulated by
man. Similarly, "non-naturally occurring" or "non-native" as used herein
refers to
a material that is not found in nature or that has been structurally modified
or
2 5 synthesized by man.
The teens "effective amount" and "therapeutically effective amount" each
refer to the amount of a CHL2 polypeptide or CHL2 nucleic acid molecule used
to
support an observable level of one or more biological activities of the CHL2
polypeptides as set forth herein.
3 o The term "pharmaceutically acceptable carrier" or "physiologically
acceptable carrier" as used herein refers to one or more formulation materials
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suitable for accomplishing or enhancing the delivery of the CHL2 polypeptide,
CHL2 nucleic acid molecule, or CHL2 selective binding agent as a
pharmaceutical composition.
The term "antigen" refers to a molecule or a portion of a molecule capable
of being bound by a selective binding agent, such as an antibody, and
additionally
capable of being used in an animal to produce antibodies capable of binding to
an
epitope of that antigen. An antigen may have one or more epitopes.
The term "selective binding agent" refers to a molecule or molecules
having specificity for a CHL2 polypeptide. As used herein, the terms,
"specific"
and "specificity" refer to the ability of the selective binding agents to bind
to
human CHL2 polypeptides and not to bind to human non-CHL2 polypeptides. It
will be appreciated, however, that the selective binding agents may also bind
orthologs of the polypeptide as set forth in either SEQ 117 NO: 2 or SEQ ID
NO:
5, that is, interspecies versions thereof, such as mouse and rat CHL2
polypeptides.
The term "transduction" is used to refer to the transfer of genes from one
bacterium to another, usually by a phage. "Transduction" also refers to the
acquisition and transfer of eukaryotic cellular sequences by retroviruses.
The term "transfection" is used to refer to the uptake of foreign or
exogenous DNA by a cell, and a cell has been "transfected" when the exogenous
2 o DNA has been introduced inside the cell membrane. A number of transfection
techniques are well known in the art and are disclosed herein. See, e.g.,
Graham
et al., 1973, Virology 52:456; Sambrook et al., Molecular Cloning, A
Labo~ato~y
Manual (Cold Spring Harbor Laboratories, 1989); Davis et al., Basic Methods
iya
MoleculaY Biology (Elsevier, 1986); and Chu et al., 1981, Gehe 13:197. Such
2 5 techniques can be used to introduce one or more exogenous DNA moieties
into
suitable host cells.
The term "transformation" as used herein refers to a change in a cell's
genetic characteristics, and a cell has been transformed when it has been
modified
to contain a new DNA. Fox example, a cell is transformed where it is
genetically
3 0 modified from its native state. Following transfection or transduction,
the
transforming DNA may recombine with that of the cell by physically integrating
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into a chromosome of the cell, may be maintained transiently as an episomal
element without being replicated, or may replicate independently as a plasmid.
A
cell is considered to have been stably transformed when the DNA is replicated
with the division of the cell.
Relatedness of Nucleic Acid Molecules and/or Polypeptides
It is understood that related nucleic acid molecules include allelic or splice
variants of the nucleic acid molecule of any of SEQ ID NO: 1 or SEQ ID NO: 4,
and include sequences which are complementary to any of the above nucleotide
l0 sequences. Related nucleic acid molecules also include a nucleotide
sequence
encoding a polypeptide comprising or consisting essentially of a substitution,
modification, addition and/or deletion of one or more amino acid residues
compared to the polypeptide in either SEQ ID NO: 2 or SEQ ID NO: 5. Such
related CHL2 polypeptides may comprise, for example, an addition and/or a
deletion of one or more N-linked or O-linked glycosylation sites or an
addition
and/or a deletion of one or more cysteine residues.
Related nucleic acid molecules also include fragments of CHL2 nucleic
acid molecules which encode a polypeptide of at least about 25 contiguous
amino
acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino
2 0 acids, or about 150 amino acids, or about 200 amino acids, or more than
200
amino acid residues of the CHL2 polypeptide of any of SEQ ID NO: 2 or SEQ ID
NO: 5.
In addition, related CHL2 .nucleic acid molecules also include those
molecules which comprise nucleotide sequences which hybridize under
2 5 moderately or highly stringent conditions as defined herein with the fully
complementary sequence of the CHL2 nucleic acid molecule of any of SEQ ID
NO: 1 or SEQ ID NO: 4, or of a molecule encoding a polypeptide, which
polypeptide comprises the amino acid sequence as shown in either SEQ ID NO: 2
or SEQ ID NO: 5, or of a nucleic acid fragment as defined herein, or of a
nucleic
3 0 acid fragment encoding a polypeptide as defined herein. Hybridization
probes
may be prepared using the CHL2 sequences provided herein to screen cDNA,
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genomic or synthetic DNA libraries for related sequences. Regions of the DNA
and/or amino acid sequence of CHL2 polypeptide that exhibit significant
identity
to known sequences are readily determined using sequence alignment algorithms
as described herein and those regions may be used to design probes for
screening.
The term "highly stringent conditions" refers to those conditions that are
designed to permit hybridization of DNA strands whose sequences are highly
complementary, and to exclude hybridization of significantly mismatched DNAs.
Hybridization stringency is principally determined by temperature, ionic
strength,
and the concentration of denaturing agents such as fonnamide. Examples of
"highly stt-ingent conditions" for hybridization and washing are 0.015 M
sodium
CHL2oride, 0.0015 M sodium citrate at 65-68°C or 0.015 M sodium
CHL2oride,
0.0015 M sodium citrate, and 50% formamide at 42°C. See Sambrook,
Fritsch &
Maniatis, Moleculaf° Clonif2g: A Laboratory Manual (2nd ed., Cold
Spring Harbor
Laboratory, 1989); Anderson et al., Nucleic Acid Hybridisation: A Practical
Appy~oach Ch. 4 (IRL Press Limited).
More stringent conditions (such as higher temperature, lower ionic
strength, higher fonnamide, or other denaturing agent) may also be used -
however, the rate of hybridization will be affected. Other agents may be
included
in the hybridization and washing buffers for the purpose of reducing non-
specific
2 0 and/or background hybridization. Examples are 0.1 % bovine serum albumin,
0.1 % polyvinyl-pyrrolidone, 0.1 % sodium pyrophosphate, 0.1 % sodium
dodecylsulfate, NaDodS04, (SDS), ficoll, Denhardt's solution, sonicated salmon
sperm DNA (or another non-complementary DNA), and dextran sulfate, although
other suitable agents can also be used. The concentration and types of these
2 5 additives can be changed without substantially affecting the stringency of
the
hybridization conditions. Hybridization experiments are usually carried out at
pH
6.8-7.4; however, at typical ionic strength conditions, the rate of
hybridization is
nearly independent of pH. See Anderson et al., Nucleic Acid Hyby~idisatiosa: A
P~°actical AppYOach Ch. 4 (IRL Press Limited).
3 0 Factors affecting the stability of DNA duplex include base composition,
length, and degree of base pair mismatch. Hybridization conditions can be
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adjusted by one skilled in the art in order to accommodate these variables and
allow DNAs of different sequence relatedness to form hybrids. The melting
temperature of a perfectly matched DNA duplex can be estimated by the
following equation:
Tn,(°C) = 81.5 + 16.6(log[Na+]) + 0.41(%G+C) - 600/N -
0.72(%formamide)
where N is the length of the duplex formed, [Na+] is the molar concentration
of
the sodium ion in the hybridization or washing solution, %G+C is the
percentage
of (guanine+cytosine) bases in the hybrid. For imperfectly matched hybrids,
the
melting temperature is reduced by approximately 1°C for each 1%
mismatch.
1 o The term "moderately stringent conditions" refers to conditions under
which a DNA duplex with a greater degree of base pair mismatching than could
occur under "highly stringent conditions" is able to form. Examples of typical
"moderately stringent conditions" are 0.015 M sodium CHL2oride, 0.0015 M
sodium citrate at 50-65°C or 0.015 M sodium CHL2oride, 0.0015 M sodium
citrate, and 20% formamide at 37-50°C. By way of example, "moderately
stringent conditions" of 50°C in 0.015 M sodium ion will allow about a
21
mismatch.
It will be appreciated by those skilled in the art that there is no absolute
distinction between "highly stringent conditions" and "moderately stringent
2 0 conditions." For example, at 0.015 M sodium ion (no formamide), the
melting
temperature of perfectly matched long DNA is about 71°C. With a wash at
65°C
(at the same ionic strength), this would allow for approximately a 6%
mismatch.
To capture more distantly related sequences, one skilled in the art can simply
lower the temperature or raise the ionic strength.
2 5 A good estimate of the melting temperature in 1M NaCl* for
oligonucleotide probes up to about 20nt is given by:
Tm = 2°C per A-T base pair + 4°C per G-C base pair
*The sodium ion concentration in 6X salt sodium citrate (SSC) is 1M. See Suggs
et al., Developmental Biology Usirag Purified Gehes 683 (Brown and Fox, eds.,
3 0 1981).
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High stringency washing conditions for oligonucleotides are usually at a
temperature of 0-5°C below the Tm of the oligonucleotide in 6X SSC,
0.1% SDS.
In another embodiment, related nucleic acid molecules comprise or consist
of a nucleotide sequence that is at least about 70 percent identical to the
nucleotide sequence as shown in either SEQ ID NO: 1 or SEQ ID NO: 4, or
comprise or consist essentially of a nucleotide sequence encoding a
polypeptide
that is at least about 70 percent identical to the polypeptide as set forth in
either
SEQ ID NO: 2 or SEQ ID NO: 5. In preferred embodiments, the nucleotide
sequences are about 75 percent, or about 80 percent, or about 85 percent, or
about
l0 90 percent, or about 95, 96, 97, 98, or 99 percent identical to the
nucleotide
sequence as shown in either SEQ ID NO: 1 or SEQ ID NO: 4, or the nucleotide
sequences encode a polypeptide that is about 75 percent, or about 80 percent,
or
about 85 percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percent
identical to the polypeptide sequence as set forth in either SEQ ID NO: 2 or
SEQ
ID NO: 5. Related nucleic acid molecules encode polypeptides possessing at
least
one activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ m NO:
5.
Differences in the nucleic acid sequence may result in conservative and/or
non-conservative modifications of the amino acid sequence relative to the
amino
acid sequence of any of SEQ ID NO: 2 or SEQ ID NO: 5.
2 0 Conservative modifications to the amino acid sequence of any of SEQ ID
NO: 2 or SEQ ID NO: 5 (and the corresponding modifications to the encoding
nucleotides) will produce a polypeptide having functional and chemical
characteristics similar to those of CHL2 polypeptides. In contrast,
substantial
modifications in the functional and/or chemical characteristics of CHL2,
2 5 polypeptides may be accomplished by selecting substitutions in the amino
acid
sequence of any of SEQ ID NO: 2 or SEQ ID NO: 5 that differ significantly in
their effect on maintaining (a) the structure of the molecular backbone in the
area
of the substitution, for example, as a sheet or helical conformation, (b) the
charge
or hydrophobicity of the molecule at the target site, or (c) the bulle of the
side
3 0 chain.
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For example, a "conservative amino acid substitution" may involve a
substitution of a native amino acid residue with a nonnative residue such that
there is little or no effect on the polarity or charge of the amino acid
residue at
that position. Furthermore, any native residue in the polypeptide may also be
substituted with alanine, as has been previously described for "alanine
scanning
mutagenesis."
Conservative amino acid substitutions also encompass non-naturally
occurring amino acid residues that are typically incorporated by chemical
peptide
synthesis rather than by synthesis in biological systems. These include
peptidomimetics, and other reversed or inverted forms of amino acid moieties.
Naturally occurring residues may be divided into classes based on
conunon side chain properties:
1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
2) neutral hydrophilic: Cys, Ser, Thr;
3) acidic: Asp, Glu;
4) basic: Asn, Gln, His, Lys, Arg;
5) residues that influence chain orientation: Gly, Pro; and
6) aromatic: Trp, Tyr, Phe.
For example, non-conservative substitutions may involve the exchange of
2 0 a member of one of these classes for a member from another class. Such
substituted residues may be introduced into regions of the human CHL2
polypeptide that are homologous with non-human CHL2 polypeptides, or into the
non-homologous regions of the molecule.
In making such changes, the hydropathic index of amino acids may be
2 5 considered. Each amino acid has been assigned a hydropathic index on the
basis
of its hydrophobicity and charge characteristics. The hydropathic indices are:
isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);
cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-
1.6);
3 0 histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5);
asparagine (-
3.5); lysine (-3.9); and arginine (-4.5).
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The importance of the hydropathic amino acid index in conferring
interactive biological function on a protein is generally understood in the
art (Kyte
et al., 1982, J. Mol. Biol. 157:105-31). It is known that certain amino acids
may
be substituted for other amino acids having a similar hydropathic index or
score
and still retain a similar biological activity. In making changes based upon
the
hydropathic index, the substitution of amino acids whose hydropathic indices
are
within ~2 is preferred, those which are within ~1 are particularly preferred,
and
those within X0.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can
be made effectively on the basis of hydrophilicity, particularly where the
biologically functionally equivalent protein or peptide thereby created is
intended
for use in irmnunological embodiments, as in the present case. The greatest
local
average hydrophilicity of a protein, as governed by the hydrophilicity of its
adjacent amino acids, correlates with its immunogenicity and antigenicity,
i.e.,
with a biological property of the protein.
The following hydroplulicity values have been assigned to these amino
acid residues: arginine (+3.0); lysine (+3.0); aspaxtate (+3.0 ~ 1); glutamate
(+3.0
~ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);
threonine (-
0.4); proline (-0.5 ~ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine
2 0 (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); and tryptophan (-3.4). In malting changes based upon
similar hydrophilicity values, the substitution of amino acids whose
hydrophilicity
values are within +2 is preferred, those which are within ~1 are particularly
preferred, and those within +0.5 are even more particularly preferred. One may
2 5 also identify epitopes from primary amino acid sequences on the basis of
hydrophilicity. These regions are also referred to as "epitopic core regions."
Desired amino acid substitutions (whether conservative or non-
conservative) can be determined by those skilled in the art at the time such
substitutions are desired. For example, amino acid substitutions can be used
to
3 0 identify important residues of the CHL2 polypeptide, or to increase or
decrease
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the affinity of the CHL2 polypeptides described herein. Exemplary amino acid
substitutions are set forth in Table I.
Table I
Amino Acid Substitutions
Original ResiduesExemplary SubstitutionsPreferred Substitutions
Ala Val, Leu, Ile Val
Arg Lys, Gln, Asn Lys
Asn Gln Gln
Asp Glu Glu
Cys Ser, Ala Ser
Gln Asn Asn
Glu Asp Asp
Gly Pro, Ala Ala
His Asn, Gln, Lys, Arg Arg
Ile Leu, Val, Met, Ala, Leu
Phe, Norleucine
Leu Norleucine, Ile, Ile
Val, Met, Ala, Phe
Lys Arg, 1,4 Diamino-butyricArg
Acid, Ghl, Asn
Met Leu, Phe, Ile Leu
Phe Leu, Val, Ile, Ala, Leu
Tyr
Pro Ala Gly
Ser Thr, Ala, Cys Thr
Thr Ser Ser
Trp Tyr, Phe Tyr
Tyr Trp, Phe, Thr, Ser Phe
~
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Val ~ Ile, Met, Leu, Phe, ~ Leu
Ala, Norleucine
.- A skilled artisan will be able to determine suitable variants of the
polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5 using well-
known techniques. For identifying suitable areas of the molecule that may be
changed without destroying biological activity, one skilled in the art may
target
areas not believed to be important for activity. For example, when similar
polypeptides with similar activities from the same species or from other
species
are known, one skilled in the art may compare the amino acid sequence of a
CHL2 polypeptide to such similar polypeptides. With such a comparison, one can
identify residues and portions of the molecules that are conserved among
similar
polypeptides. It will be appreciated that changes in areas of the CHL2
molecule
that are not conserved relative to such similar polypeptides would be less
likely to
adversely affect the biological activity and/or structure of a CHL2
polypeptide.
One slcilled in the art would also know that, even in relatively conserved
regions,
one may substitute chemically similar amino acids for the naturally occurring
residues while retaining activity (conservative amino acid residue
substitutions).
Therefore, even areas that may be important for biological activity or for
structure
may be subject to conservative amino acid substitutions without destroying the
biological activity or without adversely affecting the polypeptide structure.
2 0 Additionally, one skilled in the art can review structure-function studies
identifying residues in similar polypeptides that are important for activity
or
structure. In view of such a comparison, one can predict the importance of
amino
acid residues in a CHL2 polypeptide that correspond to amino acid residues
that
are important for activity or structure in similar polypeptides. One skilled
in the
2 5 art may opt for chemically similar amino acid substitutions for such
predicted
important amino acid residues of CHL2 polypeptides.
One skilled in the art can also analyze the three-dimensional structure and
amino acid sequence in relation to that structure in similar polypeptides. In
view
of such information, one skilled in the art may predict the alignment of amino
acid
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residues of CHL2 polypeptide with respect to its three dimensional structure.
One
skilled in the art may choose not to make radical changes to amino acid
residues
predicted to b'e on the surface of the protein, since such residues may be
involved
in important interactions with other molecules. Moreover, one skilled in the
art
may generate test variants containing a single amino acid substitution at each
amino acid residue. The variants could be screened using activity assays known
to those with skill in the art. Such variants could be used to gather
information
about suitable variants. For example, if one discovered that a change to a
particular amino acid residue resulted in destroyed, undesirably reduced, or
unsuitable activity, variants with such a change would be avoided. In other
words, based on information gathered from such routine experiments, one
skilled
in the art can readily determine the amino acids where further substitutions
should
be avoided either alone or in combination with other mutations.
A number of scientific publications have been devoted to the prediction of
secondary structure. See Moult, 1996, Curs. OpifZ. Biotechfaol. 7:422-27; Chou
et
al., 1974, Biochemists~y 13:222-45; Chou et al., 1974, Biochemistry 113:211-
22;
Chou et al., 1978, Adv. Ehzymol. Relat. A~°eas Mol. Biol. 47:45-48;
Chou et al.,
1978, Anh. Rev. Biochem. 47:251-276; and Chou et al., 1979, Biophys. J. 26:367-
84. Moreover, computer programs are currently available to assist with
predicting
2 o secondary structure. One method of predicting secondary structure is based
upon
homology modeling. For example, two polypeptides or proteins which have a
sequence identity of greater than 30%, or similarity greater than 40%, often
have
similar structural topologies. The recent growth of the protein structural
database
(PDB) has provided enhanced predictability of secondary structure, including
the
2 5 potential number of folds within the structure of a polypeptide or
protein. See
Holm et al., 1999, Nucleic Acids Res. 27:244-47. It has been suggested that
there
are a limited number of folds in a given polypeptide or protein and that once
a
critical number of structures have been resolved, structural prediction will
become
dramatically more accurate (Brenner et al., 1997, Curr. Opin. StYUCt. Biol.
7:369
3 0 76).
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Additional methods of predicting secondary structure include "threading"
(Jones, 1997, Cur. Opih. St~uct. Biol. 7:377-87; Sippl et al., 1996,
Stj°uctu~e
4:15-19), "profile analysis" (Bowie et al., 1991, Science, 253:164-70;
Gribskov et
al., 1990, Methods Erzzymol. 183:146-59; Gribskov et al., 1987, Py°oc.
Nat. Acad.
Sci. ZJ.S.A. 84:4355-58), and "evolutionary linkage" (See Holin et al., supra,
and
Brenner et al., supra).
Preferred CHL2 polypeptide variants include glycosylation variants
wherein the number andlor type of glycosylation sites have been altered
compared
to the amino acid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.
In
one embodiment, CHL2 polypeptide variants comprise a greater or a lesser
number of N-linked glycosylation sites than the amino acid sequence set forth
in
either SEQ ID NO: 2 or SEQ ID NO: 5. An N-linked glycosylation site is
characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid
residue designated as X may be any amino acid residue except proline. The
substitution of amino acid residues to create this sequence provides a
potential
new site for the addition of an N-linked carbohydrate chain. Alternatively,
substitutions that eliminate this sequence will remove an existing N-linked
carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate
chains wherein one or more N-linked glycosylation sites (typically those that
are
2 0 naturally occurring) are eliminated and one or more new N-linked sites are
created. Additional preferred CHL2 variants include cysteine variants, wherein
one or more cysteine residues are deleted~or substituted with another amino
acid
(e.g., serine) as compared to the amino acid sequence set forth in either SEQ
m
NO: 2 or SEQ ID NO: 5. Cysteine variants are useful when CHL2 polypeptides
2 5 must be refolded into a biologically active conformation such as after the
isolation
of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine
residues than the native protein, and typically have an even number to
minimize
interactions resulting from unpaired cysteines.
In other embodiments, related nucleic acid molecules comprise or consist
3 0 of a nucleotide sequence encoding a polypeptide as set forth in either SEQ
ID
NO: 2 or SEQ m NO: 5 with at least one amino acid insertion and wherein the
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polypeptide has an activity of the polypeptide set forth in either SEQ ID NO:
2 or
SEQ ID NO: 5, or a nucleotide sequence encoding a polypeptide as set forth in
either SEQ ID NO: 2 or SEQ ID NO: 5 with at least one amino acid deletion and
wherein the polypeptide has an activity of the polypeptide set forth in either
SEQ
ID NO: 2 or SEQ ID NO: 5. Related nucleic acid molecules also comprise or
consist of a nucleotide sequence encoding a polypeptide as set forth in either
SEQ
ID NO: 2 or SEQ ID NO: 5 wherein the polypeptide has a carboxyl- and/or
amino-terminal truncation and further wherein the polypeptide has an activity
of
the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5. Related
nucleic acid molecules also comprise or consist of a nucleotide sequence
encoding a polypeptide as set forth in either SEQ ID NO: 2 or SEQ ID NO: 5
with
at least one modif canon selected from the group consisting of amino acid
substitutions, amino acid insertions, amino acid deletions, carboxyl-terminal
truncations, and amino-terminal truncations and wherein the polypeptide has an
activity of the polypeptide set forth in either SEQ ID NO: 2 or SEQ ID NO: 5.
Tn addition, the polypeptide comprising the amino acid sequence of any of
SEQ ID NO: 2 or SEQ ID NO: S, or other CHL2 polypeptide, may be fused to a
homologous polypeptide to form a homodimer or to a heterologous polypeptide to
form a heterodimer. Heterologous peptides and polypeptides include, but are
not
2 0 limited to: an epitope to allow for the detection and/or isolation of a
CHL2 fusion
polypeptide; a transmembrane receptor protein or a portion thereof, such as an
extracellular domain or a transmembrane and intracellular domain; a ligand or
a
portion thereof which binds to a transmembrane receptor protein; an enzyme or
portion thereof which is catalytically active; a polypeptide or peptide which
2 5 promotes oligomerization, such as a leucine zipper domain; a polypeptide
or
peptide which increases stability, such as an immunoglobulin constant region;
and
a polypeptide which has a therapeutic activity different from the polypeptide
comprising the amino acid sequence as set forth in either SEQ ID NO: 2 or SEQ
ID NO: 5, or other CHL2 polypeptide.
3 0 Fusions can be made either at the amino-terminus or at the carboxyl-
terminus of the polypeptide comprising the amino acid sequence set forth in
either
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SEQ ID NO: 2 or SEQ ID NO: 5, or other CHL2 polypeptide. Fusions may be
direct with no linker or adapter molecule or may be through a linker or
adapter
molecule. A linker or adapter molecule may be one or more amino acid residues,
typically from about 20 to about 50 amino acid residues. A linlcer or adapter
molecule may also be designed with a cleavage site for a DNA restriction
endonuclease or for a protease to allow for the separation of the fused
moieties. It
will be appreciated that once constructed, the fusion polypeptides can be
derivatized according to the methods described herein.
In a further embodiment of the invention, the polypeptide comprising the
amino acid sequence of any of SEQ ID NO: 2 OR SEQ ID NO: 5, or other CHL2
polypeptide, is fused to one or more domains of an Fc region of human IgG.
Antibodies comprise two functionally independent parts, a variable domain
known as "Fab," that binds an antigen, and a constant domain known as "Fc,"
that
is involved in effector functions such as complement activation and attack by
phagocytic cells. An Fc has a long serum half life, whereas an Fab is short-
lived.
Capon et al., 19$9, Natuf a 337:525-31. When constructed together with a
therapeutic protein, an Fc domain can provide longer half life or incorporate
such
functions as Fc receptor binding, protein A binding, complement fixation, and
perhaps even placental transfer. Id. Table II summarizes the use of certain Fc
2 0 fusions known in the art.
Table II
Fc Fusion with Therapeutic Proteins
Form of Fc Fusion artnerThera eutic im licationsReference
IgG1 N-terminus Hodgkin's disease; U.S. Patent
of No.
CD30-L anaplastic lymphoma;5,480,981
T-
cell leukemia
Murine Fcy2aIL-10 anti-inflammatory; Zheng et al.,
199
trans lant re'ectionImmuyaol.
154:55S
IgGl TNF receptor septic shock Fisher et
al., 1996
Engl. J. Med.
334
1702; Van
Zee et
1996, J. Inamuhol.
156:2221-30
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IgG, IgA, TNF receptor inflammation, U.S. Patent
IgM, No.
or IgE autoimmune disorders5,808,029
(excluding
the
first domain
IgG1 CD4 receptor AIDS Capon et al.,
198
Nature 337:
525-:
IgGI, N-terminus anti-cancer, antiviralHarvill et
al., 199
I G3 of IL-2 Immunotech.
1:95
IgG1 C-terminus osteoarthritis; WO 97/23614
of
OPG bone densit
IgGl N-terminus anti-obesity PCTlCTS 97/2318:
of
1e tin December 1
l, 19f
Human Ig CTLA-4 ~ autoimmune disordersLinsley, 1991,
Cyl ~ J. .
Med., 174:561-69
In one example, a human IgG hinge, CH2, and CH3 region may be fused
at either the amino-terminus or carboxyl-terminus of the CHL2 polypeptides
using
methods known to the skilled artisan. In another example, a human IgG hinge,
CH2, and CH3 region may be fused at either the amino-terminus or carboxyl-
terminus of a CHL2 polypeptide fragment (e.g., the predicted extracellular
portion
of CHL2 polypeptide).
The resulting CHL2 fusion polypeptide may be purified by use of a
Protein A affinity column. Peptides and proteins fused to an Fc region have
been
l0 found to exhibit a substantially greater half life in vivo than the unfused
counterpart. Also, a fusion to an Fc region allows for
dimerization/multimerization of the fusion polypeptide. The Fc region may be a
naturally occurring Fc region, or may be altered to improve certain qualities,
such
as therapeutic qualities, circulation time, or reduced aggregation.
Identity and similarity of related nucleic acid molecules and polypeptides
are readily calculated by known methods. Such methods include, but are not
limited to those described in Computational Molecular Biology (A.M. Lesk, ed.,
Oxford University Press 1988); Biocomputing: Informatics and Genome Projects
(D.W. Smith, ed., Academic Press 1993); Computer Analysis of Sequence Data
2 0 (Part 1, A.M. Griffin and H.G. Griffin, eds., Humana Press 1994); G. von
Heinle,
Sequence Analysis in Molecular Biology (Academic Press 1987); Sequence
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Analysis Primer (M. Gribskov and J. Devereux, eds., M. Stockton Press 1991);
and Carillo et al., 1988, SIAMJ. Applied Math., 48:1073.
Preferred methods to determine identity and/or similarity are designed to
give the largest match between the sequences tested. Methods to determine
identity and similarity are described in publicly available computer programs.
Preferred computer program methods to determine identity and similarity
between
two sequences include, but are not limited to, the GCG program package,
including GAP (Devereux et al., 1984, Nucleic Acids Res. 12:387; Genetics
Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN,
1 o and FASTA (Altschul et al., 1990, J. Mol. Biol. 215:403-10). The BLASTX
program is publicly available from the National Center for Biotechnology
Information (NCBI) and other sources (Altschul et al., BLAST Manual (NCB
NLM NIH, Bethesda, MD); Altschul et al., 1990, sups°a). The well-
known Smith
Waterman algorithm may also be used to determine identity.
Certain alignment schemes for aligning two amino acid sequences may
result in the matching of only a short region of the two sequences, and this
small
aligned region may have very high sequence identity even though there is no
significant relationship between the two full-length sequences. Accordingly,
in a
preferred embodiment, the selected alignment method (GAP program) will result
2 0 in an alignment that spans at least 50 contiguous amino acids of the
claimed
polypeptide.
For example, using the computer algorithm GAP (Genetics Computer
Group, University of Wisconsin, Madison, WI), two polypeptides for which the
percent sequence identity is to be determined are aligned for optimal matching
of
2 5 their respective amino acids (the "matched span," as determined by the
algorithm). A gap opening penalty (which is calculated as 3X the average
diagonal; the "average diagonal" is the average of the diagonal of the
comparison
matrix being used; the "diagonal" is the score or number assigned to each
perfect
amino acid match by the particular comparison matrix) and a gap extension
3 0 penalty (which is usually O.1X the gap opening penalty), as well as a
comparison
matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the
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algorithm. A standard comparison matrix is also used by the algorithm (see
Dayhoff et al., 5 Atlas of Protein Sequence afZd St~uctuYe (Supp. 3
1978)(PAM250 comparison matrix); Henikoff et al., 1992, Proc. Natl. Acad. Sci
USA 89:10915-19 (BLOSLTM 62 comparison matrix)).
Preferred parameters for polypeptide sequence comparison include the
following:
Algorithm: Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-53;
Comparison matrix: BLOSUM 62 (Henikoff et al., supra);
l0 Gap Penalty: 12
Gap Length Penalty: 4
Threshold.of Similarity: 0
The GAP program is useful with the above parameters. The aforementioned
parameters are the default parameters for polypeptide comparisons (along with
no
penalty for end gaps) using the GAP algorithm.
Preferred parameters for nucleic acid molecule sequence comparison
include the following:
2 0 Algoritlnn: Needleman and Wunsch, supra;
Comparison matrix: matches = +10, mismatch = 0
Gap Penalty: 50
Gap Length Penalty: 3
2 5 The GAP program is also useful with the above parameters. The
aforementioned
parameters are the default parameters for nucleic acid molecule comparisons.
Other exemplary algorithms, gap opening penalties, gap extension
penalties, comparison matrices, and thresholds of similarity may be used,
including those set forth in the Program Manual, Wisconsin Package, Version 9,
3 o September, 1997. The particular choices to be made will be apparent to
those of
skill in the art and will depend on the specific comparison to be made, such
as
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DNA-to-DNA, protein-to-protein, protein-to-DNA; and additionally, whether the
comparison is between given pairs of sequences (in which case GAP or BestFit
are generally preferred) or between one sequence and a large database of
sequences (in which case FASTA or BLASTA are preferred).
Nucleic Acid Molecules
The nucleic acid molecules encoding a polypeptide comprising the amino
acid sequence of a CHL2 polypeptide can readily be obtained in a variety of
ways
including, without limitation, chemical synthesis, cDNA or genomic library
screening, expression library screening, and/or PCR amplification of cDNA.
Recombinant DNA methods used herein are generally those set forth in
Sambrook et al., MoleculaY Cloyaihg: A Laborato~ Manual (Cold Spring Harbor
Laboratory Press, 1989) and/or Current Protocols in Molecular Biology (Ausubel
et al., eds., Green Publishers Inc. and Wiley and Sons 1994). The invention
provides for nucleic acid molecules as described herein and methods for
obtaining
such molecules.
Where a gene encoding the amino acid sequence of a CHL2 polypeptide
has been identified from one species, all or a portion of that gene may be
used as a
probe to identify orthologs or related genes from the same species. The probes
or
2 0 primers may be used to screen cDNA libraries from various tissue sources
believed to express the CHL2 polypeptide. In addition, part or all of a
nucleic
acid molecule having the sequence as set forth in either SEQ ID NO: 1 or SEQ
ID
NO: 4 may be used to screen a genomic library to identify and isolate a gene
encoding the amino acid sequence of a CHL2 polypeptide. Typically, conditions
2 5 of moderate or high stringency will be employed for screening to minimize
the
number of false positives obtained from the screening.
Nucleic acid molecules encoding the amino acid sequence of CHL2
polypeptides may also be identified by expression cloning which employs the
detection of positive clones based upon a property of the expressed protein.
3 0 Typically, nucleic acid libraries are screened by the binding an antibody
or other
binding partner (e.g., receptor or ligand) to cloned proteins that are
expressed and
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displayed on a host cell surface. The antibody or binding partner is modified
with
a a detectable label to identify those cells expressing the desired clone.
Recombinant expression techniques conducted in accordance with the
descriptions set forth below may be followed to produce these polynucleotides
and to express the encoded polypeptides. For example, by inserting a nucleic
acid
sequence that encodes the amino acid sequence of a CHL2 polypeptide into an
appropriate vector, one skilled in the art can readily produce large
quantities of the
desired nucleotide sequence. The sequences can then be used to generate
detection probes or amplification primers. Alternatively, a polynucleotide
encoding the amino acid sequence of a CHL2 polypeptide can be inserted into an
expression vector. By introducing the expression vector into an appropriate
host,
the encoded CHL2 polypeptide may be produced in large amounts.
Another method for obtaining a suitable nucleic acid sequence is the
polymerase chain reaction (PCR). In this method, cDNA is prepared from
poly(A)+RNA or total RNA using the enzyme reverse transcriptase. Two
primers, typically complementary to two separate regions of cDNA encoding the
amino acid sequence of a CHL2 polypeptide, are then added to the cDNA along
with a polymerase such as Taq polymerase, and the polymerase amplifies the
cDNA region between the two primers.
2 0 Another means of preparing a nucleic acid molecule encoding the amino
acid sequence of a CHL2 polypeptide is chemical synthesis using methods well
known to the skilled artisan such as those described by Engels et al., 1989,
Ahgew. Ch.em. Ihtl. Ed. 28:716-34. These methods include, ihte~ alia, the
phosphotriester, phosphoramidite, and H-phosphonate methods for nucleic acid
2 5 synthesis. A preferred method for such chemical synthesis is polymer-
supported
synthesis using standard phosphoramidite chemistry. Typically, the DNA
encoding the amino acid sequence of a CHL2 polypeptide will be several hundred
nucleotides in length. Nucleic acids larger than about 100 nucleotides can be
synthesized as several fragments using these methods. The fragments can then
be
3 0 ligated together to form the full-length nucleotide sequence of a CHL2
gene.
Usually, the DNA fragment encoding the amino-terminus of the polypeptide will
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have an ATG, which encodes a methionine residue. This methionine may or may
not be present on the mature form of the CHL2 polypeptide, depending on
whether the polypeptide produced in the host cell is designed to be secreted
from
that cell. Other methods known to the skilled artisan may be used as well.
In certain embodiments, nucleic acid variants contain codons which have
been altered for optimal expression of a CHL2 polypeptide in a given host
cell.
Particular codon alterations will depend upon the CHL2 polypeptide and host
cell
selected for expression. Such "codon optimization" can be carried out by a
variety of methods, for example, by selecting codons which are preferred for
use
in highly expressed genes in a given host cell. Computer algorithms which
incorporate codon frequency tables such as "Eco high.Cod" for codon preference
of highly expressed bacterial genes may be used and are provided by the
University of Wisconsin Package Version 9.0 (Genetics Computer Group,
Madison, WI). Other useful codon frequency tables include
"Celegans high.cod," "Celegans low.cod," "Drosophila high.cod,"
"Human high.cod," "Maize high.cod," and "Yeast high.cod."
In some cases, it may be desirable to prepare nucleic acid molecules
encoding CHL2 polypeptide variants. Nucleic acid molecules encoding variants
may be produced using site directed mutagenesis, PCR amplification, or other
2 0 appropriate methods, where the primers) have the desired point mutations
(see
Sambrook et al., supra, and Ausubel et al., supra, for descriptions of
mutagenesis
techniques). Chemical synthesis using methods described by Engels et al.,
supra,
may also be used to prepare such variants. Other methods known to the skilled
artisan may be used as well.
Vectors and Host Cells
A nucleic acid molecule encoding the amino acid sequence of a CHL2
polypeptide is inserted into an appropriate expression vector using standard
ligation techniques. The vector is typically selected to be functional in the
particular host cell employed (i.e., the vector is compatible with the host
cell
machinery such that amplification of the gene and/or expression of the gene
can
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occur). A nucleic acid molecule encoding the amino acid sequence of a CHL2
polypeptide may be amplified/expressed in prokaryotic, yeast, insect
(baculovirus
systems) and/or eukaryotic host cells. Selection of the host cell will depend
in
part on whether a CHL2 polypeptide is to be post-translationally modified
(e.g.,
glycosylated and/or phosphorylated). If so, yeast, insect, or mammalian host
cells
are preferable. For a review of expression vectors, see Meth. Ehz., vol. 185
(D.V.
Goeddel, ed., Academic Press 1990).
Typically, expression vectors used in any of the host cells will contain
sequences for plasmid maintenance and for cloning and expression of exogenous
nucleotide sequences. Such sequences, collectively referred to as "flanking
sequences" in certain embodiments will typically include one or more of the
following nucleotide sequences: a promoter, one or more enhancer sequences, an
origin of replication, a transcriptional termination sequence, a complete
intron
sequence containing a donor and acceptor splice site, a sequence encoding a
leader sequence for polypeptide secretion, a ribosome binding site, a
polyadenylation sequence, a polylinker region for inserting the nucleic acid
encoding the polypeptide to be expressed, and a selectable marker element.
Each
of these sequences is discussed below.
Optionally, the vector may contain a "tag"-encoding sequence, i.e., an
2 0 oligonucleotide molecule located at the 5' or 3' end of the CHL2
polypeptide
coding sequence; the oligonucleotide sequence encodes polyHis (such as
hexaHis), or another "tag" such as FLAG, HA (hemaglutinin influenza virus), or
rrayc for which commercially available antibodies exist. This tag is typically
fused
to the polypeptide upon expression of the polypeptide, and can serve as a
means
2 5 for affinity purification of the CHL2 polypeptide from the host cell.
Affinity
purification can be accomplished, for example, by column chromatography using
antibodies against the tag as an affinity matrix. Optionally, the tag can
subsequently be removed from the purified CHL2 polypeptide by various means
such as using certain peptidases for cleavage.
30 Flanking sequences may be homologous (i.e., from the same species
and/or strain as the host cell), heterologous (i.e., from a species other than
the host
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cell species or strain), hybrid (i.e., a combination of flanking sequences
from
more than one source), or synthetic, or the flanking sequences may be native
sequences which normally function to regulate CHL2 polypeptide expression. As
such, the source of a flanking sequence may be any prokaryotic or eukaryotic
organism, any vertebrate or invertebrate organism, or any plant, provided that
the
flanking sequence is functional in, and can be activated by, the host cell
machinery.
Flanking sequences useful in the vectors of this invention may be obtained
by any of several methods well known in the art. Typically, flanking sequences
useful herein - other than the CHL2 gene flanking sequences - will have been
previously identified by mapping and/or by restriction endonuclease digestion
and
can thus be isolated from the proper tissue source using the appropriate
restriction
endonucleases. In some cases, the full nucleotide sequence of a flanking
sequence may be known. Here, the flanking sequence may be synthesized using
the methods described herein for nucleic acid synthesis or cloning.
Where all or only a portion of the flanking sequence is known, it may be
obtained using PCR and/or by screening a genomic library with a suitable
oligonucleotide and/or flanking sequence fragment from the same or another
species. Where the flanking sequence is not knovnnz, a fragment of DNA
2 0 containing a flanking sequence may be isolated from a larger piece of DNA
that
may contain, for example, a coding sequence or even another gene or genes.
Isolation may be accomplished by restriction endonuclease digestion to produce
the proper DNA fragment followed by isolation using agarose gel purification,
Qiagen'~ column chromatography (Chatsworth, CA), or other methods known to
2 5 the skilled artisan. The selection of suitable enzymes to accomplish this
purpose
will be readily apparent to one of ordinary skill in the art.
An origin of replication is typically a part of those prokaryotic expression
vectors purchased commercially, and the origin aids in the amplification of
the
vector in a host cell. Amplification of the vector to a certain copy number
can, in
3 0 some cases, be important for the optimal expression of a CHL2 polypeptide.
If
the vector of choice does not contain an origin of replication site, one may
be
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chemically synthesized based on a known sequence, and ligated into the vector.
For example, the origin of replication from the plasmid pBR322 (New England
Biolabs, Beverly, MA) is suitable for most gram-negative bacteria and various
origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV), or
papillomaviruses such as HPV or BPV) are useful for closing vectors in
mammalian cells. Generally, the origin of replication component is not needed
for mammalian expression vectors (for example, the SV40 origin is often used
only because it contains the early promoter).
A transcription termination sequence is typically located 3' of the end of a
polypeptide coding region and serves to terminate transcription. Usually, a
transcription termination sequence in prokaryotic cells is a G-C rich fragment
followed by a poly-T sequence. While the sequence is easily cloned from a
library or even purchased commercially as part of a vector, it can also be
readily
synthesized using methods for nucleic acid synthesis such as those described
herein.
A selectable marker gene element encodes a protein necessary for the
survival and growth of a host cell grown in a selective culture medium.
Typical
selection marker genes encode proteins that (a) confer resistance to
antibiotics or
other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic
host cells;
2 0 (b) complement auxotrophic deficiencies of the cell; or (c) supply
critical
nutrients not available from complex media. Preferred selectable markers are
the
kanamycin resistance gene, the ampicillin resistance gene, and the
tetracycline
resistance gene. A neomycin resistance gene may also be used for selection in
prokaryotic and eukaryotic host cells.
2 5 Other selection genes may be used to amplify the gene that will be
expressed. Amplification is the process wherein genes that are in greater
demand
for the production of a protein critical for growth are reiterated in tandem
within
the chromosomes of successive generations of recombinant cells. Examples of
suitable selectable markers for mammalian cells include dihydrofolate
reductase
3 0 (DHFR) and thymidine kinase. The mammalian cell transformants are placed
under selection pressure wherein only the transformants are uniquely adapted
to
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survive by virtue of the selection gene present in the vector. Selection
pressure is
imposed by culturing the transformed cells under conditions in which the
concentration of selection agent in the medium is successively changed,
thereby
leading to the amplification of both the selection gene and the DNA that
encodes a
CHL2 polypeptide. As a result, increased quantities of CHL2 polypeptide are
synthesized from the amplified DNA.
A ribosome binding site is usually necessary for translation initiation of
inRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a
I~ozak sequence (eukaryotes). The element is typically located 3' to the
promoter
1 o and 5' to the coding sequence of a CHL2 polypeptide to be expressed. The
Shine-
Dalgarno sequence is varied but is typically a polypurine (i.e., having a high
A-G
content). Many Shine-Dalgarno sequences have been identified, each of which
can be readily synthesized using methods set forth herein and used in a
prokaryotic vector.
A leader, or signal, sequence may be used to direct a CHL2 polypeptide
out of the host cell. Typically, a nucleotide sequence encoding the signal
sequence is positioned in the coding region of a CHL2 nucleic acid molecule,
or
directly at the 5' end of a CHL2 polypeptide coding region. Many signal
sequences have been identified, and any of those that are functional in the
selected
2 0 host cell may be used in conjunction with a CHL2 nucleic acid molecule.
Therefore, a signal sequence may be homologous (naturally occurring) or
heterologous to the CHL2 nucleic acid molecule. Additionally, a signal
sequence
may be chemically synthesized using methods described herein. In most cases,
the secretion of a CHL2 polypeptide from the host cell via the presence of a
signal
2 5 peptide will result in the removal of the signal peptide from the secreted
CHL2
polypeptide. The signal sequence may be a component of the vector, or it may
be
a part of a CHL2 nucleic acid molecule that is inserted into the vector.
Included within the scope of this invention is the use of either a nucleotide
sequence encoding a native CHL2 polypeptide signal sequence joined to a CHL2
3 0 polypeptide coding region or a nucleotide sequence encoding a heterologous
signal sequence joined to a CHL2 polypeptide coding region. The heterologous
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signal sequence selected should be one that is recognized and processed, i.e.,
cleaved by a signal peptidase, by the host cell. For prokaryotic host cells
that do
not recognize and process the native CHL2 polypeptide signal sequence, the
signal sequence is substituted by a prokaryotic signal sequence selected, for
example, from the group of the alkaline phosphatase, penicillinase, or heat-
stable
enterotoxin II leaders. For yeast secretion, the native CHL2 polypeptide
signal
sequence may be substituted by the yeast invertase, alpha factor, or acid
phosphatase leaders. In mammalian cell expression the native signal sequence
is
satisfactory, although other mammalian signal sequences may be suitable.
1 o In some cases, such as where glycosylation is desired in a eukaryotic host
cell expression system, one may manipulate the various presequences to improve
glycosylation or yield. For example, one may alter the peptidase cleavage site
of
a particular signal peptide, or add pro-sequences, which also may affect
glycosylation. The final protein product may have, in the -1 position
(relative to
the first amino acid of the mature protein) one or more additional amino acids
incident to expression, which may not have been totally removed. For example,
the final protein product may have one or two amino acid residues found in the
peptidase cleavage site, attached to the amino-terminus. Alternatively, use of
some enzyne cleavage sites may result in a slightly truncated form of the
desired
2 o CHL2 polypeptide, if the enzyme cuts at such area within the mature
polypeptide.
In many cases, transcription of a nucleic acid molecule is increased by the
presence of one or more introns in the vector; this is particularly true where
a
polypeptide is produced.in eukaryotic host cells, especially mammalian host
cells.
The introns used may be naturally occurring within the CHL2 gene especially
2 5 where the gene used is a full-length genomic sequence or a fragment
thereof.
Where the intron is not naturally occurring witlun the gene (as for most
cDNAs),
the intron may be obtained from another source. The position of the intron
with
respect to flanking sequences and the CHL2 gene is generally important, as the
intron must be transcribed to be effective. Thus, when a CHL2 cDNA molecule is
3 0 being transcribed, the preferred position for the intron is 3' to the
transcription
start site and 5' to the poly-A transcription termination sequence.
Preferably, the
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intron or introns will be located on one side or the other (i.e., 5' or 3') of
the
cDNA such that it does not interrupt the coding sequence. Any intron from any
source, including viral, prokaryotic and eukaryotic (plant or animal)
organisms,
may be used to practice tlus invention, provided that it is compatible with
the host
cell into which it is inserted. Also included herein are synthetic introns.
Optionally, more than one intron may be used in the vector.
The expression and cloning vectors of the present invention will typically
contain a promoter that is recognized by the host organism and operably linked
to
the molecule encoding the CHL2 polypeptide. Promoters are untranscribed
1 o sequences located upstream (i.e., 5') to the start codon of a structural
gene
(generally within about 100 to 1000 bp) that control the transcription of the
structural gene. Promoters are conventionally grouped into one of two classes:
inducible promoters and constitutive promoters. Inducible promoters initiate
increased levels of transcription from DNA under their control in response to
some change in culture conditions, such as the presence or absence of a
nutrient or
a change in temperature. Constitutive promoters, on the other hand, initiate
continual gene product production; that is, there is little or no control over
gene
expression. A laxge number of promoters, recognized by a variety of potential
host cells, are well known. A suitable promoter is operably linked to the DNA
2 0 encoding CHL2 polypeptide by removing the promoter from the source DNA by
restriction enzyme digestion and inserting the desired promoter sequence into
the
vector. The native CHL2 promoter sequence may be used to direct amplification
and/or expression of a CHL2 nucleic acid molecule. A heterologous promoter is
preferred, however, if it permits greater transcription and higher yields of
the
2 5 expressed protein as compared to the native promoter, and if it is
compatible with
the host cell system that has been selected for use.
Promoters suitable for use with prokaryotic hosts include the beta-
lactamase and lactose promoter systems; alkaline phosphatase; a tryptophan
(trp)
promoter system; and hybrid promoters such as the tac promoter. Other known
3 0 bacterial promoters are also suitable. Their sequences have been
published,
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thereby enabling one skilled in the art to ligate them to the desired DNA
sequence,
using linkers or adapters as needed to supply any useful restriction sites.
Suitable promoters for use with yeast hosts are also well known in the art.
Yeast enhancers are advantageously used with yeast promoters. Suitable
promoters for use with mammalian host cells are well known and include, but
are
not limited to, those obtained from the genomes of viruses such as polyoma
virus,
fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus,
avian
sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most
preferably Simian Virus 40 (SV40). Other suitable mammalian promoters include
heterologous mammalian promoters, for example, heat-shock promoters and the
actin promoter.
Additional promoters which may be of interest in controlling CHL2 gene
expression include, but are not limited to: the SV40 early promoter region
(Bernoist and Chambon, 1981, Nature 290:304-10); the CMV promoter; the
promoter contained in the 3' long terminal repeat of Rous sarcoma virus
(Yamamoto, et al., 1980, Cell 22:787-97); the herpes thymidine kinase promoter
(Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1444-45); the
regulatory
sequences of the metallothionine gene (Brinster et al., 1982, Nature 296:39-
42);
prokaryotic expression vectors such as the beta-lactamase promoter (Villa-
2 o Kamaroff et al., 1978, Pr~oc. Natl. Acad. Sci. U.S.A., 75:3727-31); or the
tac
promoter (DeBoer et al., 1983, P~oc. Natl. Acad. Sci. U.S.A., 80:21-25). Also
of
interest are the following animal transcriptional control regions, which
exhibit
tissue specificity and have been utilized in transgenic animals: the elastase
I gene
control region which is active in pancreatic acinar cells (Swift et al., 1984,
Cell
2 5 38:639-46; Ornitz et al., 1986, Cold .Spying Ha~bo~ Symp. Quaht. Biol.
50:399-
409 (1986); MacDonald, 1987, Hepatology 7:425-515); the insulin gene control
region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-
22); the iinmunoglobulin gene control region which is active in lymphoid cells
(Grosschedl et al., 1984, Cell 38:647-58; Adames et al., 1985, Nature 318:533-
38;
3 o Alexander et al., 1987, Mol. Cell. Biol., 7:1436-44); the mouse mammary
tumor
virus control region which is active in testicular, breast, lymphoid and mast
cells
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(Leder et al., 1986, Cell 45:485-95); the albumin gene control region which is
active in liver (Pinkert et.al., 1987, Geyaes and Devel. 1:268-76); the alpha-
feto-
protein gene control region which is active in liver (Krumlauf et al., 1985,
Mol.
Cell. Biol., 5:1639-48; Hammer et al., 1987, Science 235:53-58); the alpha 1-
antitrypsin gene control region which is active in the liver (Kelsey et al.,
1987,
Genes afZd Devel. 1:161-71); the beta-globin gene control region which is
active in
myeloid cells (Mogram et al., 1985, Nature 315:338-40; Kollias et al., 1986,
Cell
46:89-94); the myelin basic protein gene control region which is active in
oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-12);
the
1 o myosin light chain-2 gene control region which is active in skeletal
muscle (Sani,
1985, Nature 314:283-86); and the gonadotropic releasing hormone gene control
region which is active in the hypothalamus (Mason et al., 1986, Science
234:1372-
78).
An enhancer sequence may be inserted into the vector to increase the
transcription of a DNA encoding a CHL2 polypeptide of the present invention by
higher eulcaryotes. Enhancers are cis-acting elements of DNA, usually about 10-
300 by in length, that act on the promoter to increase transcription.
Enhancers are
relatively orientation and position independent. They have been found 5' and
3'
to the transcription unit. Several enhancer sequences available from mammalian
2 0 genes are knomn (e.g., globin, elastase, albumin, alpha-feto-protein and
insulin).
Typically, however, an enhancer from a virus will be used. The SV40 enhancer,
the cytomegalovirus early promoter enhancer, the polyoma enhancer, and
adenovirus enhancers are exemplary enhancing elements for the activation of
eulcaryotic promoters. While an enhancer may be spliced into the vector at a
2 5 position 5' or 3' to a CHL2 nucleic acid molecule, it is typically located
at a site
5' from the promoter.
Expression vectors of the invention may be constructed from a starting
vector such as a commercially available vector. Such vectors may or may not
contain all of the desired flanking sequences. Where one or more of the
flanl~ing
3 0 sequences described herein are not already present in the vector, they may
be
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individually obtained and ligated into the vector. Methods used for obtaining
each of the flanking sequences are well known to one skilled in the art.
Preferred vectors for practicing this invention are those which are
compatible with bacterial, insect, and mammalian host cells. Such vectors
include, ihteY alia, pCRII, pCR3, and pcDNA3.l (W vitrogen, San Diego, CA),
pBSII (Stratagene, La Jolla, CA), pETlS (Novagen, Madison, WI), pGEX
(Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA),
pETL (BlueBacII, Invitrogen), pDSR-alpha (PCT Pub. No. WO 90/14363) and
pFastBacDual (Gibco-BRL, Grand Island, NY).
Additional suitable vectors include, but are not limited to, cosmids,
plasmids, or modified viruses, but it will be appreciated that the vector
system
must be compatible with the selected host cell. Such vectors include, but axe
not
limited to plasmids such as Bluescript~ plasmid derivatives (a high copy
number
ColEl-based phagemid, Stratagene Cloning Systems, La Jolla CA), PCR cloning
plasmids designed for cloning Taq-amplified PCR products (e.g., TOPOTM TA
Cloning~ I~it, PCR2.1~ plasmid derivatives, Invitrogen, Carlsbad, CA), and
mammalian, yeast or virus vectors such as a baculovirus expression system
(pBacPAK plasmid derivatives, Clontech, Palo Alto, CA).
After the vector has been constructed and a nucleic acid molecule
2 0 encoding a CHL2 polypeptide has been inserted into the proper site of the
vector,
the completed vector may be inserted into a suitable host cell for
amplification
and/or polypeptide expression. The transformation of an expression vector for
a
CHL2 polypeptide into a selected host cell may be accomplished by well known
methods including methods such as transfection, infection, calcium CHL2oride,
2 5 electroporation, microinj ection, lipofection, DEAE-dextran method, or
other
known techniques. The method selected will in part be a function of the type
of
host cell to be used. These methods and other suitable methods are well known
to
the skilled artisan, and are set forth, for example, in Sambrook et al.,
supra.
Host cells may be prokaryotic host cells (such as E. coli) or eukaryotic
3 0 host cells (such as a yeast, insect, or vertebrate cell). The host cell,
when cultured
under appropriate conditions, synthesizes a CHL2 polypeptide which can
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subsequently be collected from the culture medium (if the host cell secretes
it into
the medium) or directly from the host cell producing it (if it is not
secreted). The
selection of an appropriate host cell will depend upon various factors, such
as
desired expression levels, polypeptide modifications that are desirable or
necessary for activity (such as glycosylation or phosphorylation) and ease of
folding into a biologically active molecule.
A number of suitable host cells are known in the art and many are
available from the American Type Culture Collection (ATCC), Manassas, VA.
Examples include, but are not limited to, mammalian cells, such as Chinese
l0 hamster ovary cells (CHO), CHO DHFR(-) cells (Urlaub et al., 1980, P~oc.
Natl.
Acad. Sci. U.SA. 97:4216-20), human embryonic kidney (HEK) 293 or 293T
cells, or 3T3 cells. The selection of suitable mammalian host cells and
methods
for transformation, culture, amplification, screening, product production, and
purification are known in the art. Other suitable mammalian cell lines, are
the
monkey COS-l and COS-7 cell lines, and the CV-1 cell line. Further exemplary
mammalian host cells include primate cell lines and rodent cell lines,
including
transformed cell lines. Normal diploid cells, cell strains derived from in
vitro
culture of primary tissue, as well as primary explants, are also suitable.
Candidate
cells may be genotypically def cient in the selection gene, or may contain a
2 0 dominantly acting selection gene. Other suitable marmnalian cell lines
include
but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929
cells,
3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell
lines. Each of these cell lines is known by and available to those skilled in
the art
of protein expression.
2 5 Similarly useful as host cells suitable for the present invention are
bacterial cells. For example, the various strains of E. coli (e.g., HB101,
DHSa.,
DH10, and MC1061) are well-known as host cells in the field of biotechnology.
Various strains of B. subtilis, Pseudomonas spp., other Bacillus spp.,
Streptomyces spp., and the like may also be employed in this method.
3 0 Many strains of yeast cells known to those spilled in the art are also
available as host cells for the expression of the polypeptides of the present
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invention. Preferred yeast cells include, for example, Saccha~omyces ce~ivisae
and Pichia pasto~is.
Additionally, where desired, insect cell systems may be utilized in the
methods of the present invention. Such systems are described, for example, in
Kitts et al., 1993, Biotechhiques, 14:810-17; Lucklow, 1993, Cu~~. Opih.
BioteclZnol. 4:564-72; and Lucklow et al., 1993, J. hi~ol., 67:4566-79.
Preferred
insect cells are Sf 9 and Hi5 (Invitrogen).
One may also use transgenic animals to express glycosylated CHL2
polypeptides. For example, one may use a transgenic milk-producing animal (a
cow or goat, for example) and obtain the present glycosylated polypeptide in
the
animal milk. One may also use plants to produce CHL2 polypeptides, however,
in general, the glycosylation occurring in plants is different from that
produced in
mammalian cells, and may result in a glycosylated product which is not
suitable
for human therapeutic use.
Polypeptide Production
Host cells comprising a CHL2 polypeptide expression vector may be
cultured using standard media well known to the skilled artisan. The media
will
usually contain all nutrients necessary for the growth and survival of the
cells.
2 0 Suitable media for culturing E. coli cells include, for example, Luria
Broth (LB)
and/or Terrific Broth (TB). Suitable media for culturing eukaryotic cells
include
Roswell Park Memorial Institute medium 1640 (RPMI 1640), Minimal Essential
Medium (MEM) and/or Dulbecco's Modified Eagle Medium (DMEM), all of
which may be supplemented with serum and/or growth factors as necessary for
2 5 the particular cell line being cultured. A suitable medium for insect
cultures is
Grace's medium supplemented with yeastolate, lactalbumin hydrolysate, and/or
fetal calf serum as necessary.
Typically, an antibiotic or other compound useful for selective growth of
transfected or transformed cells is added as a supplement to the media. The
3 0 compound to be used will be dictated by the selectable marker element
present on
the plasmid with which the host cell was transformed. For example, where the
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selectable marker element is kanamycin resistance, the compound added to the
culture medium will be kanamycin. Other compounds for selective growth
include ampicillin, tetracycline, and neomycin.
The amount of a CHL2 polypeptide produced by a host cell can be
evaluated using standard methods known in the art. Such methods include,
without limitation, Western blot analysis, SDS-polyacrylamide gel
electrophoresis, non-denaturing gel electrophoresis, High Performance Liquid
Chromatography (HPLC) separation, immunoprecipitation, and/or activity assays
such as DNA binding gel shift assays.
l0 If a CHL2 polypeptide has been designed to be secreted from the host
cells, the majority of polypeptide may be found in the cell culture medium. If
however, the CHL2 polypeptide is not secreted from the host cells, it will be
present in the cytoplasm and/or the nucleus (for eukaryotic host cells) or in
the
cytosol (for gram-negative bacteria host cells).
For a CHL2 polypeptide situated in the host cell cytoplasm and/or nucleus
(for eukaryotic host cells) or in the cytosol (for bacterial host cells), the
intracellular material (including inclusion bodies for gram-negative bacteria)
can
be extracted from the host cell using any standard technique known to the
skilled
artisan. For example, the host cells can be lysed to release the contents of
the
2 0 periplasm/cytoplasm by French press, homogenization, and/or sonication
followed by centrifugation.
If a CHL2 polypeptide has formed inclusion bodies in the cytosol, the
inclusion bodies can often bind to the firmer and/or outer cellular membranes
and
thus will be found primarily in the pellet material after centrifugation. The
pellet
2 5 material can then be treated at pH extremes or with a chaotropic agent
such as a
detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the
presence of a reducing agent such as dithiothreitol at alkaline pH or tris
carboxyethyl phosphine at acid pH to release, break apart, and solubilize the
inclusion bodies. The solubilized CHL2 polypeptide can then be analyzed using
3 0 gel electrophoresis, immunoprecipitation, or the like. If it is desired to
isolate the
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CHL2 polypeptide, isolation may be accomplished using standard methods such
as those described herein and in Marston et al., 1990, Meth. Enz., 1 X2:264-
75.
In some cases, a CHL2 polypeptide may not be biologically active upon
isolation. Various methods for "refolding" or converting the polypeptide to
its
tertiary structure and generating disulfide linkages can be used to restore
biological activity. Such methods include exposing the solubilized polypeptide
to
a pH usually above 7 and in the presence of a particular concentration of a
chaotrope. The selection of chaotrope is very similar to the choices used for
inclusion body solubilization, but usually the chaotrope is used at a lower
l0 concentration and is not necessarily the same as chaotropes used for the
solubilization. In most cases the refolding/oxidation solution will also
contain a
reducing agent or the reducing agent plus its oxidized form in a specific
ratio to
generate a particular redox potential allowing for disulfide shuffling to
occur in
the formation of the protein's cysteine bridges. Some of the commonly used
redox couples include cysteine/cystamine, glutathione (GSH)/dithiobis GSH,
cupric CHL2oride, dithiothreitol(DTT)/dithiane DTT, and 2-2
mercaptoethanol(bME)/dithio-b(ME). In many instances, a cosolvent may be
used or may be needed to increase the efficiency of the refolding, and the
more
common reagents used for this purpose include glycerol, polyethylene glycol of
2 0 various molecular weights, arginine and the like.
If inclusion bodies are not formed to a significant degree upon expression
of a CHL2 polypeptide, then the polypeptide will be found primarily in the
supernatant after centrifugation of the cell homogenate. The polypeptide may
be
further isolated from the supernatant using methods such as those described
2 5 herein.
The purification of a CHL2 polypeptide from solution can be
accomplished using a variety of techniques. If the polypeptide has been
synthesized such that it contains a tag such as Hexahistidine (CHL2
polypeptide/hexaHis) or other small peptide such as FLAG (Eastman Kodak Co.,
3 0 New Haven, CT) or myc (Invitrogen, Carlsbad, CA) at either its carboxyl-
or
amino-terminus, it may be purified in a one-step process by passing the
solution
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through an affinity column where the column matrix has a high affinity for the
tag.
For example, polyhistidine binds with great affinity and specificity to
nickel. Thus, an affinity column of nickel (such as the Qiagen~ nickel
columns)
can be used for purification of CHL2 polypeptide/polyHis. See, e.g., Cu~reht
Protocols ih Molecular Biology ~ 10.11.8 (Ausubel et al., eds., Green
Publishers
Inc. and Wiley and Sons 1993).
Additionally, CHL2 polypeptides may be purified through the use of a
monoclonal antibody that is capable of specifically recognizing and binding to
a
CHL2 polypeptide.
Other suitable procedures for purification include, without limitation,
affinity chromatography, immunoaffinity chromatography, ion exchange
chromatography, molecular sieve chromatography, HPLC, electrophoresis
(including native gel electrophoresis) followed by gel elution, and
preparative
isoelectric focusing ("Isoprime" machine/teclniique, Hoefer Scientific, San
Francisco, CA). In some cases, two or more purification techniques may be
combined to achieve increased purity.
CHL2 polypeptides may also be prepared by chemical synthesis methods
(such as solid phase peptide synthesis) using techniques known in the art such
as
2 0 those set forth by Merrifield et al., 1963, J. Am. Chem. Soc. 85:2149;
Houghten et
al., 1985, P~oc Natl Acad. ,Sci. USA 82:5132; and Stewart and Young, Solid
Phase Peptide .Synthesis (Pierce Chemical Co. 1984). Such polypeptides may be
synthesized with or without a methionine on the amino-terminus. Chemically
synthesized CHL2 polypeptides may be oxidized using methods set forth in these
2 5 references to form disulfide bridges. Chemically synthesized CHL2
polypeptides
are expected to have comparable biological activity to the corresponding CHL2
polypeptides produced recombinantly or purified from natural sources, and thus
may be used interchangeably with a recombinant or natural CHL2 polypeptide.
Another means of obtaining CHL2 polypeptide is via purification from
3 0 biological samples such as source tissues and/or fluids in which the CHL2
polypeptide is naturally found. Such purification can be conducted using
methods
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for protein purification as described herein. The presence of the CHL2
polypeptide during purification may be monitored, for example, using an
antibody
prepared against recombinantly produced CHL2 polypeptide or peptide fragments
thereof.
A number of additional methods for producing nucleic acids and
polypeptides are known in the art, and the methods can be used to produce
polypeptides having specificity for CHL2 polypeptide. See, e.g., Roberts et
al.,
1997, P~~oc. Natl. Acad. Sci. U.S.A. 94:12297-303, which describes the
production
of fusion proteins between an mRNA and its encoded peptide. See also, Roberts,
1999, Cur. Opih. Che~z. Biol. 3:268-73. Additionally, U.S. Patent No.
5,824,469
describes methods for obtaining oligonucleotides capable of carrying out a
specific biological function. The procedure involves generating a
heterogeneous
pool of oligonucleotides, each having a 5' randomized sequence, a central
preselected sequence, and a 3' randomized sequence. The resulting
heterogeneous pool is introduced into a population of cells that do not
exhibit the
desired biological function. Subpopulations of the cells are then screened for
those that exhibit a predetermined biological function. From that
subpopulation,
oligonucleotides capable of carrying out the desired biological function are
isolated.
U.S. Patent Nos. 5,763,192; 5,814,476; 5,723,323; and 5,817,483 describe
processes for producing peptides or polypeptides. This is done by producing
stochastic genes or fragments thereof, and then introducing these genes into
host
cells which produce one or more proteins encoded by the stochastic genes. The
host cells are then screened to identify those clones producing peptides or
2 5 polypeptides having the desired activity.
Another method for producing peptides or polypeptides is described in
PCT/LTS98/20094 (W099/15650) filed by Athersys, Inc. Known as "Random
Activation of Gene Expression for Gene Discovery" (RAGE-GD), the process
involves the activation of endogenous gene expression or over-expression of a
3 0 gene by ih situ recombination methods. For example, expression of an
endogenous gene is activated or increased by integrating a regulatory sequence
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into the target cell which is capable of activating expression of the gene by
non
homologous or illegitimate recombination. The target DNA is first subjected to
radiation, and a genetic promoter inserted. The promoter eventually locates a
break at the front of a gene, initiating transcription of the gene. This
results in
expression of the desired peptide or polypeptide.
It will be appreciated that these methods can also be used to create
comprehensive CHL2, polypeptide expression libraries, which can subsequently
be used for high throughput phenotypic screening in a variety of assays, such
as
biochemical assays, cellular assays, and whole organism assays (e.g., plant,
1. 0 mouse, etc.).
Synthesis
It will be appreciated by those skilled in the art that the nucleic acid and
polypeptide molecules described herein may be produced by recombinant and
other means.
Selective Binding Agents
The term "selective binding agent" refers to a molecule that has specificity
for one or more CHL2 polypeptides. Suitable selective binding agents include,
2 0 but are not limited to, antibodies and derivatives thereof, polypeptides,
and small
molecules. Suitable selective binding agents may be prepared using methods
known in the art. An exemplary CHL2 polypeptide selective binding agent of the
present invention is capable of binding a certain portion of the CHL2
polypeptide
thereby inhibiting the binding of the polypeptide to a CHL2 polypeptide
receptor.
2 5 Selective binding agents such as antibodies and antibody fragments that
bind CHLZ polypeptides are within the scope of the present invention. The
antibodies may be polyclonal including monospecific polyclonal; monoclonal
(MAbs); recombinant; chimeric; humanized, such as CDR-grafted; human; single
chain; and/or bispecific; as well as fragments; variants; or derivatives
thereof.
3 o Antibody fragments include those portions of the antibody that bind to an
epitope
on the CHL2 polypeptide. Examples of such fragments include Fab and F(ab')
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fragments generated by enzymatic cleavage of fiill-length antibodies. Other
binding fragments include those generated by recombinant DNA techniques, such
as the expression of recombinant plasmids containing nucleic acid sequences
encoding antibody variable regions.
Polyclonal antibodies directed toward a CHL2 polypeptide generally are
produced in animals (e.g., rabbits or mice) by means of multiple subcutaneous
or
intraperitoneal injections of CHL2 polypeptide and an adjuvant. It may be
useful
to conjugate a CHL2 polypeptide to a carrier protein that is immunogenic in
the
species to be immunized, such as keyhole Iimpet hemocyanin, serum, albumin,
bovine thyroglobulin, or soybean trypsin inhibitor. Also, aggregating agents
such
as alum are used to enhance the immune response. After immunization, the
animals are bled and the serum is assayed for anti-CHL2 antibody titer.
Monoclonal antibodies directed toward CHL2 polypeptides are produced
using any method that provides for the production of antibody molecules by
continuous cell lines in culture. Examples of suitable methods for preparing
monoclonal antibodies include the hybridoma methods of Kohler et al., 1975,
Nature 256:495-97 and the human B-cell hybridoma method (Kozbor, 1984, J.
Imfnuaol. 133:3001; Brodeur et al., Monoclonal Antibody P~oductioh Techniques
and Applications S1-63 (Marcel Dekker, Inc., 1987). Also provided by the
2 o invention are hybridoma cell lines that produce monoclonal antibodies
reactive
with CHL2 polypeptides.
Monoclonal antibodies of the invention may be modified for use as
therapeutics. One embodiment is a "chimeric" antibody in which a portion of
the
heavy (H) and/or light (L) chain is identical with or homologous to a
2 5 corresponding sequence in antibodies derived from a particular species or
belonging to a particular antibody class or subclass, while the remainder of
the
chains) is/are identical with or homologous to a corresponding sequence in
antibodies derived from another species or belonging to another antibody class
or
subclass. Also included are fragments of such antibodies, so long as they
exhibit
3 o the desired biological activity. See U.S. Patent No. 4,816,567; Morrison
et al.,
1985, Proc. Natl. Acad. Sci. 81:6851-SS.
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In another embodiment, a monoclonal antibody of the invention is a
"humasuzed" antibody. Methods for humanizing non-human antibodies are well
k~zown in the art. See U.S. Patent Nos. 5,585,089 and 5,693,762. Generally, a
humanized antibody has one or more amino acid residues introduced into it from
a source that is non-human. Humaiuzation can be performed, for example, using
methods described in the art (Jones et al., 1986, Nature 321:522-25; Riechmann
et al., 1998, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-36),
by substituting at least a portion of a rodent complementarity-determining
region
(CDR) for the corresponding regions of a human antibody.
Also encompassed by the invention are human antibodies that bind CHL2
polypeptides. Using transgenic animals (e.g., mice) that are capable of
producing
a repertoire of human antibodies in the absence of endogenous immunoglobulin
production such antibodies are produced by immunization with a CHL2
polypeptide antigen (i.e., having at least 6 contiguous amino acids),
optionally
conjugated to a carrier. See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad.
Sci.
90:2551-55; Jakobovits et al., 1993, Nature 362:255-58; Bruggermann et al.,
1993, Year iya Imfnuho. 7:33. In one method, such transgenic animals are
produced by incapacitating the endogenous loci encoding the heavy and light
immunoglobulin chains therein, and inserting loci encoding human heavy and
2 0 light chain proteins into the genome thereof. Partially modified animals,
that is
those having less than the full complement of modifications, are then cross-
bred
to obtain an animal having all of the desired immune system modifications.
When administered an immunogen, these transgenic animals produce antibodies
with huma~i (rather than, e.g., marine) amino acid sequences, including
variable
2 5 regions which are immunospecific for these antigens. See PCT App. Nos.
PCT/LJS96/05928 and PCT/LTS93/06926. Additional methods are described in
U.S. Patent No. 5,545,807, PCT App. Nos. PCT/LTS91/245 and
PCT/GB89/01207, and in European Patent Nos. 546073B1 and 546073A1.
Human antibodies can also be produced by the expression of recombinant DNA in
3 0 host cells or by expression in hybridoma cells as described herein.
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In an alternative embodiment, human antibodies can also be produced
from phage-display libraries (Hoogenboom et al., 1991, J. Mol. Biol. 227:381;
Marks et al., 1991, J. Mol. Biol. 222:581). These processes mimic immune
selection through the display of antibody repertoires on the surface of
filamentous
bacteriophage, and subsequent selection of phage by their binding to an
antigen of
choice. One such technique is described in PCT App. No. PCT/US98/17364,
which describes the isolation of high affinity and functional agonistic
antibodies
for MPL- and msk- receptors using such an approach.
Chimeric, CDR grafted, and humanized antibodies are typically produced
by recombinant methods. Nucleic acids encoding the antibodies are introduced
into host cells and expressed using materials and procedures described herein.
In
a preferred embodiment, the antibodies are produced in mammalian host cells,
such as CHO cells. Monoclonal (e.g., human) antibodies may be produced by the
expression of recombinant DNA in host cells or by expression in hybridoma
cells
as described herein.
The anti-CHL2 antibodies of the invention may be employed in any
known assay method, such as competitive binding assays, direct and indirect
sandwich assays, and immunoprecipitation assays (Sola, Monoclonal Antibodies:
A Manual of Techniques 147-158 (CRC Press, Inc., 1987)) for the detection and
2 0 quantitation of CHL2 polypeptides. The antibodies will bind CHL2
polypeptides
with an affinity that is appropriate for the assay method being employed.
For diagnostic applications, in certain embodiments, anti-CHL2 antibodies
may be labeled with a detectable moiety. The detectable moiety can be any one
that is capable of producing, either directly or indirectly, a detectable
signal. For
2 5 example, the detectable moiety may be a radioisotope, such as 3H, 14C,
32P, 3sS,
lash 99TC' 111In, or ~7Ga; a fluorescent or chemiluminescent compound, such as
fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as
alkaline
phosphatase, (3-galactosidase, or horseradish peroxidase (Bayer, et al., 1990,
Meth. Enz. 184:138-63).
3 0 Competitive binding assays rely on the ability of a labeled standard
(e.g., a
CHL2 polypeptide, or an immunologically reactive portion thereof) to compete
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with the test sample analyte (an CHL2 polypeptide) for binding with a limited
amount of anti-CHL2 antibody. The amount of a CHL2 polypeptide in the test
sample is inversely proportional to the amount of standard that becomes bound
to
the antibodies. To facilitate determining the amount of standard that becomes
bond, the antibodies typically are insolubilized before or after the
competition,
so that the standard and analyte that are bound to the antibodies may
conveuently
be separated from the standard and analyte which remain unbound.
Sandwich assays typically involve the use of two antibodies, each capable
of binding to a different immunogenic portion, or epitope, of the protein to
be
detected and/or quantitated. In a sandwich assay, the test sample analyte is
typically bound by a first antibody which is immobilized on a solid support,
and
thereafter a second antibody binds to the analyte, thus forming an insoluble
three-
part complex. See, e.g., U.S. Patent No. 4,376,110. The second antibody may
itself be labeled with a detectable moiety (direct sandwich assays) or may be
measured using an anti-immunoglobulin antibody that is labeled with a
detectable
moiety (indirect sandwich assays). For example, one type of sandwich assay is
an
enzyme-linked immunosorbent assay (ELISA), in which case the detectable
moiety is an enzyme.
The selective binding agents, including anti-CHL2 antibodies, are also
2 0 useful for ih vivo imaging. An antibody labeled with a detectable moiety
may be
administered to an animal, preferably into the bloodstream, and the presence
and
location of the labeled antibody in the host assayed. The antibody may be
labeled
with any moiety that is detectable in an animal, whether by nuclear magnetic
resonance, radiology, or other detection means known in the art.
2 5 Selective binding agents of the invention, including antibodies, may be
used as therapeutics. These therapeutic agents are generally agonists or
antagonists, in that they either enhance or reduce, respectively, at least one
of the
biological activities of a CHL2 polypeptide. In one embodiment, antagonist
antibodies of the invention are antibodies or binding fragments thereof which
are
3 0 capable of specifically binding to a CHL2 polypeptide and which are
capable of
inhibiting or eliminating the functional activity of a CHL2 polypeptide isa
vivo or
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ih vitro. In preferred embodiments, the selective binding agent, e.g., an
antagonist
antibody, will inhibit the functional activity of a CHL2 polypeptide by at
least
about 50%, and preferably by at least about 80%. In another embodiment, the
selective binding agent may be an anti-CHL2 polypeptide antibody that is
capable
of interacting with a CHL2 polypeptide binding partner (a ligand or receptor)
thereby inhibiting or eliminating CHL2 polypeptide activity i~ vitro or ih
vivo.
Selective binding agents, including agonist and antagonist anti-CHL2
polypeptide
antibodies, are identified by screening assays that are well known in the art.
The invention also relates to a kit comprising CHL2 selective binding
agents (such as antibodies) and other reagents useful for detecting CHL2
polypeptide levels in biological samples. Such reagents may include a
detectable
label, blocking serum, positive and negative control samples, and detection
reagents.
Microarrays
It will be appreciated that DNA microarray technology can be utilized in
accordance with the present invention. DNA microarrays are miniature, high-
density arrays of nucleic acids positioned on a solid support, such as glass.
Each
cell or element within the array contains numerous copies of a single nucleic
acid
2 o species that acts as a target for hybridization with a complementary
nucleic acid
sequence (e.g., mRNA). In expression profiling using DNA microarray
technology, mRNA is first extracted from a cell or tissue sample and then
converted enzymatically to fluorescently labeled cDNA. This material is
hybridized to the microarray and unbomzd cDNA is removed by washing. The
2 5 expression of discrete genes represented on the array is then visualized
by
quantitating the amount of labeled cDNA that is specifically bound to each
target
nucleic acid molecule. In this way, the expression of thousands of genes can
be
quantitated in a high throughput, parallel manner from a single sample of
biological material.
3 0 This high throughput expression profiling has a broad range of
applications with respect to the CHL2 molecules of the invention, including,
but
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not limited to: the identification and validation of CHL2 disease-related
genes as
targets for therapeutics; molecular toxicology of related CHL2 molecules and
inhibitors thereof; stratification of populations and generation of surrogate
markers for clinical trials; and enhancing related CHL2 polypeptide small
molecule drug discovery by aiding in the identification of selective compounds
in
high throughput screens.
Chemical Derivatives
Chemically modified derivatives of CHL2 polypeptides may be prepared
by one skilled in the art, given the disclosures described herein. CHL2
polypeptide derivatives are modified in a mamler that is different - either in
the
type or location of the molecules naturally attached to the polypeptide.
Derivatives may include molecules formed by the deletion of one or more
naturally-attached chemical groups. The polypeptide comprising the amino acid
sequence of any of SEQ ID NO: 2 or SEQ ID NO: 5, or other CHL2 polypeptide,
may be modified by the covalent attachment of one or more polymers. For
example, the polymer selected is typically water-soluble so that the protein
to
which it is attached does not precipitate in an aqueous environment, such as a
physiological environment. Included within the scope of suitable polymers is a
2 0 mixture of polymers. Preferably, for therapeutic use of the end-product
preparation, the polymer will be pharmaceutically acceptable.
The polymers each may be of any molecular weight and may be branched
or unbranched. The polymers each typically have an average molecular weight of
between about 2 kDa to about 100 kDa (the term "about" indicating that in
2 5 preparations of a water-soluble polymer, some molecules will weigh more,
some
less, than the stated molecular weight). The average molecular weight of each
polymer is preferably between about 5 kDa and about 50 kDa, more preferably
between about 12 kDa and about 40 kDa and most preferably between about 20
kDa and about 35 kDa.
3 0 Suitable water-soluble polymers or mixtures thereof include, but are not
limited to, N-linked or O-linked carbohydrates, sugars, phosphates,
polyethylene
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glycol (PEG) (including the forms of PEG that have been used to derivatize
proteins, including mono-(Cl-Clo), alkoxy-, or aryloxy-polyethylene glycol),
monomethoxy-polyethylene glycol, dextran (such as low molecular weight
dextran of, for example, about 6 kD), cellulose, or other carbohydrate based
polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol
homopolymers, polypropylene oxide/ethylene oxide co-polymers,
polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol. Also
encompassed by the present invention are bifunctional crosslinking molecules
which may be used to prepare covalently attached CHL2 polypeptide multimers.
In general, chemical derivatization may be performed under any suitable
condition used to react a protein with an activated polymer molecule. Methods
for preparing chemical derivatives of polypeptides will generally comprise the
steps of (a) reacting the polypeptide with the activated polymer molecule
(such as
a reactive ester or aldehyde derivative of the polymer molecule) under
conditions
whereby the polypeptide comprising the amino acid sequence of any of SEQ ID
NO: 2 or SEQ ID NO: 5, or other CHL2 polypeptide, becomes attached to one or
more polymer molecules, and (b) obtaining the reaction products. The optimal
reaction conditions will be determined based on known parameters and the
desired result. For example, the larger the ratio of polymer molecules to
protein,
2 0 the greater the percentage of attached polymer molecule. In one
embodiment, the
CHL2 polypeptide derivative may have a single polymer molecule moiety at the
amino-terminus. See, e.g., U.S. Patent No. 5,234,784.
The pegylation of a polypeptide may be specifically carried out using any
of the pegylation reactions known in the art. Such reactions are described,
for
2 5 example, in the following references: Francis et al., 1992, Focus on
Growth
Factors 3:4-10; European Patent Nos. 0154316 and 0401384; and U.S. Patent No.
4,179,337. For example, pegylation may be carried out via an acylation
reaction
or an alkylation reaction with a reactive polyethylene glycol molecule (or an
analogous reactive water-soluble polymer) as described herein. For the
acylation
3 0 reactions, a selected polymer should have a single reactive ester group.
For
reductive alkylation, a selected polymer should have a single reactive
aldehyde
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group. A reactive aldehyde is, for example, polyethylene glycol
propionaldehyde,
which is water stable, or mono C1-Clo alkoxy or aryloxy derivatives thereof
(see
U.S. Patent No. 5,252,714).
In another embodiment, CHL2 polypeptides may be chemically coupled to
biotin. The biotin/CHL2 polypeptide molecules are then allowed to bind to
avidin, resulting in tetravalent avidin/biotin/CHL2 polypeptide molecules.
CHL2
polypeptides may also be covalently coupled to dinitrophenol (DNP) or
trinitrophenol (TNP) and the resulting conjugates precipitated with anti-DNP
or
anti-TNP-IgM to form decasneric conjugates with a valency of 10.
Generally, conditions that may be alleviated or modulated by the
administration of the present CHL2 polypeptide derivatives include those
described herein for CHL2 polypeptides. However, the CHL2 polypeptide
derivatives disclosed herein may have additional activities, enhanced or
reduced
biological activity, or other characteristics, such as increased or decreased
half
life, as compared to the non-derivatized molecules.
Geneticall~~ineered Non-Human Animals
Additionally included within the scope of the present invention are non
human animals such as mice, rats, or other rodents; rabbits, goats, sheep, or
other
2 0 farm animals, in which the genes encoding native CHL2 polypeptide have
been
disrupted (i.e., "knocked out") such that the level of expression of CHL2
polypeptide is significantly decreased or completely abolished. Such animals
may be prepared using techniques and methods such as those described in U.S.
Patent No. 5,557,032.
2 5 The present invention further includes non-human animals such as mice,
rats, or other rodents; rabbits, goats, sheep, or other farm animals, in which
either
the native form of a CHL2 gene for that animal or a heterologous CHL2 gene is
over-expressed by the animal, thereby creating a "transgenic" animal. Such
transgenic animals may be prepared using well known methods such as those
3 o described in U.S. Patent No 5,489,743 and PCT Pub. No. WO 94/28122.
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The present invention further includes non-human animals in which the
promoter for one or more of the CHL2 polypeptides of the present invention is
either activated or inactivated (e.g., by using homologous recombination
methods)
to alter the level of expression of one or more of the native CHL2
polypeptides.
These non-human animals may be used for drug candidate screening. In
such screening, the impact of a drug candidate on the asumal may be measured.
For example, drug candidates may decrease or increase the expression of the
CHL2 gene. In certain embodiments, the amount of CHL2 polypeptide that is
produced may be measured after the exposure of the animal to the drug
candidate.
l0 Additionally, in certain embodiments, one may detect the actual impact of
the
drug candidate on the animal. For example, over-expression of a particular
gene
may result in, or be associated with, a disease or pathological condition. In
such
cases, one may test a drug candidate's ability to decrease expression of the
gene
or its ability to prevent or inhibit a pathological condition. In other
examples, the
production of a particular metabolic product such as a fragment of a
polypeptide,
may result in, or be associated with, a disease or pathological condition. In
such
cases, one may test a drug candidate's ability to decrease the production of
such a
metabolic product or its ability to prevent or inhibit a pathological
condition.
2 0 Assayin~ for Other Modulators of CHL2 Polypeptide Activity
In some situations, it may be desirable to identify molecules that are
modulators, i.e., agonists or antagonists, of the activity of CHL2
polypeptide.
Natural or synthetic molecules that modulate CHL2 polypeptide may be
identified
using one or more screening assays, such as those described herein. Such
2 5 molecules may be administered either in an ex vivo manner or in an i~ vivo
manner by injection, or by oral delivery, implantation device, or the like.
"Test molecule" refers to a molecule that is raider evaluation for the ability
to modulate (i.e., increase or decrease) the activity of a CHL2 polypeptide.
Most
commonly, a test molecule will interact directly with a CHL2 polypeptide.
3 0 However, it is also contemplated that a test molecule may also modulate
CHL2
polypeptide activity indirectly, such as by affecting CHL2 gene expression, or
by
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binding to a CHL2 polypeptide binding partner (e.g., receptor or ligand). In
one
embodiment, a test molecule will bind to a CHL2 polypeptide with an affinity
constant of at least about 10-~ M, preferably about 10-8 M, more preferably
about
10-~ M, and even more preferably about 10-1° M.
Methods for identifying compounds that interact with CHL2 polypeptides
are encompassed by the present invention. In certain embodiments, a CHL2
polypeptide is incubated with a test molecule under conditions that permit the
interaction of the test molecule with a CHL2 polypeptide, and the extent of
the
interaction is measured. The test molecule can be screened in a substantially
purified form or in a crude mixture.
In certain embodiments, a CHL2 polypeptide agonist or antagonist may be
a protein, peptide, carbohydrate, lipid, or small molecular weight molecule
that
interacts with CHL2 polypeptide to regulate its activity. Molecules which
regulate CHL2 polypeptide expression include nucleic acids which are
complementary to nucleic acids encoding a CHL2 polypeptide, or are
complementary to nucleic acids sequences which direct or control the
expression
of CHL2 polypeptide, and which act as anti-sense regulators of expression.
Once a test molecule has been identified as interacting with a CHL2
polypeptide, the molecule may be further evaluated for its ability to increase
or
2 0 decrease CHL2 polypeptide activity. The measurement of the interaction of
a test
molecule with CHL2 polypeptide may be carried out in several formats,
including
cell-based binding assays, membrane binding assays, solution-phase assays, and
immunoassays. In general, a test molecule is incubated with a CHL2 polypeptide
for a specified period of time, and CHL2 polypeptide activity is determined by
2 5 one or more assays for measuring biological activity.
The interaction of test molecules with CHL2 polypeptides may also be
assayed directly using polyclonal or monoclonal antibodies in an immunoassay.
Alternatively, modified forms of CHL2 polypeptides containing epitope tags as
described herein may be used in solution and immunoassays.
3 0 In the event that CHL2 polypeptides display biological activity through an
interaction with a binding partner (e.g., a receptor or a ligand), a variety
of in vitf°o
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assays may be used to measure the binding of a CHL2 polypeptide to the
corresponding binding partner (such as a selective binding agent, receptor, or
ligand). These assays may be used to screen test molecules for their ability
to
increase or decrease the rate and/or the extent of binding of a CHL2
polypeptide
to its binding partner. In one assay, a CHL2 polypeptide is immobilized in the
wells of a microtiter plate. Radiolabeled CHL2 polypeptide binding partner
(for
example, iodinated CHLZ polypeptide binding partner) and a test molecule can
then be added either one at a time (in either order) or simultaneously to the
wells.
After incubation, the wells can be washed and counted fox radioactivity, using
a
scintillation counter, to determine the extent to which the binding partner
bound to
the CHL2 polypeptide. Typically, a molecule will be tested over a range of
concentrations, and a series of control wells lacking one or more elements of
the
test assays can be used for accuracy in the evaluation of the results. An
alternative to this method involves reversing the "positions" of the proteins,
i.e.,
immobilizing CHL2 polypeptide binding partner to the microtiter plate wells,
incubating with the test molecule and radiolabeled CHL2 polypeptide, and
determining the extent of CHL2 polypeptide binding. See, e.g., CuYr~ent
Protocols
iu Molecular Biology, chap. 18 (Ausubel et al., eds., Green Publishers Inc.
and
Wiley and Sons 1995).
2 o As an alternative to radiolabeling, a CHL2 polypeptide or its binding
partner may be conjugated to biotin, and the presence of biotinylated protein
can
then be detected using streptavidin linked to an enzyme, such as horse radish
peroxidase (HRP) or alkaline phosphatase (AP), ~ which can be detected
colorometrically, or by fluorescent tagging of streptavidin. An antibody
directed
2 5 to a CHL2 polypeptide or to a CHL2 polypeptide binding partner, and which
is
conjugated to biotin, may also be used for purposes of detection following
incubation of the complex with enzyme-linked streptavidin linked to AP or HRP.
A CHL2 polypeptide or a CHL2 polypeptide binding partner can also be
immobilized by attachment to agarose beads, acrylic beads, or other types of
such
3 0 inert solid phase substrates. The substrate-protein complex can be placed
in a
solution containing the complementary protein and the test compound. After
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incubation, the beads can be precipitated by centrifugation, and the amount of
binding between a CHL2 polypeptide and its binding partner can be assessed
using the methods described herein. Alternatively, the substrate-protein
complex
can be immobilized in a column with the test molecule and complementary
protein passing through the column. The formation of a complex between a
CHL2 polypeptide and its binding partner can then be assessed using any of the
techniques described herein (e.g., radiolabelling or antibody binding).
Another in vitro assay that is useful for identifying a test molecule which
increases or decreases the formation of a complex between a CHL2 polypeptide
binding protein and a CHL2 polypeptide binding partner is a surface plasmon
resonance detector system such as the BIAcore assay system (Pharmacia,
Piscataway, NJ). The BIAcore system is utilized as specified by the
manufacturer. This assay essentially involves the covalent binding of either
CHL2 polypeptide or a CHL2 polypeptide binding partner to a dextran-coated
sensor chip that is located in a detector. The test compound and the other
complementary protein can then be injected, either simultaneously or
sequentially,
into the chamber containing the sensor chip. The amount of complementary
protein that binds can be assessed based on the change in molecular mass that
is
physically associated with the dextran-coated side of the sensor chip, with
the
2 0 change in molecular mass being measured by the detector system.
In some cases, it may be desirable to evaluate two or more test compounds
together for their ability to increase or decrease the formation of a complex
between a CHL2 polypeptide and a CHL2 polypeptide binding partner. In these
cases, the assays set forth herein can be readily modified by adding such
2 5 additional test compounds) either simultaneously with, or subsequent to,
the first
test compound. The remainder of the steps in the assay are as set forth
herein.
In vitro assays such as those described herein may be used advantageously
to screen large numbers of compounds for an effect on the formation of a
complex
between a CHL2 polypeptide and CHL2 polypeptide binding partner. The assays
3 0 may be automated to screen compounds generated in phage display, synthetic
peptide, and chemical synthesis libraries.
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Compounds which increase or decrease the formation of a complex
between a CHL2 polypeptide and a CHL2 polypeptide binding partner may also
be screened in cell culture using cells and cell lines expressing either CHL2
polypeptide or CHL2 polypeptide binding partner. Cells and cell lines may be
obtained from any mammal, but preferably will be from human or other primate,
canine, or rodent sources. The binding of a CHL2 polypeptide to cells
expressing
CHL2 polypeptide binding partner at the surface is evaluated in the presence
or
absence of test molecules, and the extent of binding may be determined by, for
example, flow cytometry using a biotinylated antibody to a CHL2 polypeptide
l0 binding partner. Cell culture assays can be used advantageously to further
evaluate compounds that score positive in protein binding assays described
herein.
Cell cultures can also be used to screen the impact of a drug candidate.
For example, drug candidates may decrease or increase the expression of the
CHL2 gene. In certain embodiments, the amount of CHL2 polypeptide or a
CHL2 polypeptide fragment that is produced may be measured after exposure of
the cell culture to the drug candidate. W certain embodiments, one may detect
the
actual impact of the drug candidate on the cell culture. For example, the over-
expression of a particular gene may have a particular impact on the cell
culture.
2 0 In such cases, one may test a drug candidate's ability to increase or
decrease the
expression of the gene or its ability to prevent or inhibit a particular
impact on the
cell culture. In other examples, the production of a particular metabolic
product
such as a fragment of a polypeptide, may result in, or be associated with, a
disease
or pathological condition. In such cases, one may test a drug candidate's
ability to
2 5 decrease the production of such a metabolic product in a cell culture.
Internalizing Proteins
The tat protein sequence (from HIV) can be used to internalize proteins
into a cell. See, e.g., Falwell et al., 1994, Proc. Natl. Acad. Sci. U.S.A.
91:664-68.
3 0 For example, an 11 amino acid sequence (Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID
NO: 10) of the HIV tat protein (termed the "protein transduction domain," or
TAT
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PDT) has been described as mediating delivery across the cytoplasmic membrane
and the nuclear membrane of a cell. See Schwarze et al., 1999, Scieyace
285:1569-
72; and Nagahaxa et al., 1998, Nat. Med. 4:1449-S2. In these procedures, FITC-
constructs (FITC-labeled G-G-G-G-Y-G-R-K-K-R-R-Q-R-R-R; SEQ 1D NO: 11),
which penetrate tissues following intraperitoneal administration, are
prepared, and
the binding of such constructs to cells is detected by fluorescence-activated
cell
sorting (FACS) analysis. Cells treated with a tat-~3-gal fusion protein will
demonstrate (3-gal activity. Following inj ection, expression of such a
construct
can be detected in a number of tissues, including liver, kidney, lung, heas-t,
and
I O brain tissue. It is believed that such constructs undergo some degree of
unfolding
in order to enter the cell, and as such, may require a refolding following
entry into
the cell.
It will thus be appreciated that the tat protein sequence may be used to
internalize a desired polypeptide into a cell. For example, using the tat
protein
sequence, a CHL2 antagonist (such as an anti-CHL2 selective binding agent,
small molecule, soluble receptor, or antisense oligonucleotide) can be
administered intracellularly to inhibit the activity of a CHL2 molecule. As
used
herein, the term "CHL2 molecule" refers to both CHL2 nucleic acid molecules
and CHL2 polypeptides as defined herein. Where desired, the CHL2 protein
itself
2 o may also be internally administered to a cell using these procedures. See
also,
Straus, 1999, Science 285:1466-67.
Cell Source Identification Using CHL2 Poly,~eptide
In accordance with certain embodiments of the invention, it may be useful
2 5 to be able to determine the source of a certain cell type associated with
a CHL2
polypeptide. For example, it may be useful to determine the origin of a
disease or
pathological condition as an aid in selecting an appropriate therapy. In
certain
embodiments, nucleic acids encoding a CHL2 polypeptide can be used as a probe
to identify cells described herein by screening the nucleic acids of the cells
with
3 0 such a probe. In other embodiments, one may use anti-CHL2 polypeptide
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antibodies to test for the presence of CHL2 polypeptide in cells, and thus,
determine if such cells are of the types described herein.
CHL2 PolXpeptide Compositions and Administration
Therapeutic compositions are within the scope of the present invention.
Such CHL2 polypeptide pharmaceutical compositions may comprise a
therapeutically effective amount of a CHL2 polypeptide or a CHL2 nucleic acid
molecule in admixture with a pharmaceutically or physiologically acceptable
formulation agent selected for suitability with the mode of administration.
l0 Pharmaceutical compositions may comprise a therapeutically effective amount
of
one or more CHL2 polypeptide selective binding agents in admixture with a
pharmaceutically or physiologically acceptable formulation agent selected for
suitability with the mode of administration.
Acceptable formulation materials preferably are nontoxic to recipients at
the dosages and concentrations employed.
The pharmaceutical composition may contain formulation materials for
modifying, maintaining, or preserving, for example, the pH, osmolarity,
viscosity,
clarity, color, isotonicity, odor, sterility, stability, rate of dissolution
or release,
adsorption, or penetration of the composition. Suitable formulation materials
2 0 include, but are not limited to, amino acids (such as glycine, glutamine,
asparagine, arginine, or lysine), antimicrobials, antioxidants (such as
ascorbic
acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate,
bicarbonate, Tris-HCI, citrates, phosphates, or other organic acids), bulking
agents
(such as mannitol or glycine), chelating agents (such as ethylenediamine
2 5 tetraacetic acid (EDTA)), complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin),
fillers, monosaccharides, disaccharides, and other carbohydrates (such as
glucose,
mannose, or dextrins), proteins (such as serum albumin, gelatin, or
immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents,
3 0 hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight
polypeptides, salt-forming counterions (such as sodium), preservatives (such
as
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benzalkonium CHL2oride, benzoic acid, salicylic acid, thimerosal, phenethyl
alcohol, methylparaben, propylparaben, CHL2orhexidine, sorbic acid, or
hydrogen peroxide), solvents (such as glycerin, propylene glycol, or
polyethylene
glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents,
surfactants or wetting agents (such as pluronics; PEG; sorbitan esters;
polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine;
lecithin; cholesterol or tyloxapal), stability enhancing agents (such as
sucrose or
sorbitol), tonicity enhancing agents (such as alkali metal halides -
preferably
sodium or potassium CHL2oride - or mannitol sorbitol), delivery vehicles,
diluents, excipients and/or pharmaceutical adjuvants. See Remington's
PIZarmaceutical Sciences (18th Ed., A.R. Gennaro, ed., Mack Publislung
Company 1990.
The optimal pharmaceutical composition will be determined by a skilled
artisan depending upon, for example, the intended route of achninistration,
delivery format, and desired dosage. See, e.g., Remington's Pharmaceutical
Sciences, supra. Such compositions may influence the physical state,
stability,
rate of in vivo release, and rate of in vivo clearance of the CHL2 molecule.
The primary vehicle or carrier in a pharmaceutical composition may be
either aqueous or non-aqueous in nature. For example, a suitable vehicle or
2 o carrier for injection may be water, physiological saline solution, or
artificial
cerebrospinal fluid, possibly supplemented with other materials common in
compositions for parenteral administration. Neutral buffered saline or saline
mixed with serum albumin are further exemplary vehicles. Other exemplary
pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or
acetate
2 5 buffer of about pH 4.0-5.5, which may further include sorbitol or a
suitable
substitute. In one embodiment of the present invention, CHL2 polypeptide
compositions may be prepared for storage by mixing the selected composition
having the desired degree of purity with optional formulation agents
(Ren2ington's
Pharmaceutical Sciences, supra) in the form of a lyophilized calve or an
aqueous
3 0 solution. Further, the CHL2 polypeptide product may be formulated as a
lyophilizate using appropriate excipients such as sucrose.
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The CHL2 polypeptide pharmaceutical compositions can be selected for
parenteral delivery. Alternatively, the compositions may be selected for
inhalation or for delivery through the digestive tract, such as orally. The
preparation of such pharmaceutically acceptable compositions is within the
skill
of the art.
The formulation components are present in concentrations that are
acceptable to the site of administration. For example, buffers are used to
maintain
the composition at physiological pH or at a slightly lower pH, typically
within a
pH range of from about 5 to about 8.
When parenteral administration is contemplated, the therapeutic
compositions for use in this invention may be in the form of a pyrogen-free,
parenterally acceptable, aqueous solution comprising the desired CHL2 molecule
in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for
parenteral injection is sterile distilled water in which a CHL2 molecule is
formulated as a sterile, isotonic solution, properly preserved. Yet another
preparation can involve the formulation of the desired molecule with an agent,
such as injectable microspheres, bio-erodible particles, polymeric compounds
(such as polylactic acid or polyglycolic acid), beads, or liposomes, that
provides
for the controlled or sustained release of the product which may then be
delivered
2 0 via a depot injection. Hyaluronic acid may also be used, and this may have
the
effect of promoting sustained duration in the circulation. Other suitable
means for
the introduction of the desired molecule include implantable drug delivery
devices.
In one embodiment, a pharmaceutical composition may be formulated for
2 5 inhalation. For example, CHL2 polypeptide may be formulated as a dry
powder
for inhalation. CHL2 polypeptide or nucleic acid molecule inhalation solutions
may also be formulated with a propellant for aerosol delivery. In yet another
embodiment, solutions may be nebulized. Pulmonary administration is further
described in PCT Pub. No. WO 94/20069, which describes the pulmonary
3 0 delivery of chemically modified proteins.
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It is also contemplated that certain formulations may be administered
orally. In one embodiment of the present invention, CHL2 polypeptides that are
administered in this fashion can be formulated with or without those carriers
customarily used in the compounding of solid dosage forms such as tablets and
capsules. For example, a capsule may be designed to release the active portion
of
the formulation at the point in the gastrointestinal tract when
bioavailability is
maximized and pre-systemic degradation is miumized. Additional agents can be
included to facilitate absorption of the CHLZ polypeptide. Diluents,
flavorings,
low melting point waxes, vegetable oils, lubricants, suspending agents, tablet
1 o disintegrating agents, and binders may also be employed.
Another pharmaceutical composition may involve an effective quantity of
CHL2 polypeptides in a mixture with non-toxic excipients that are suitable for
the
manufacture of tablets. By dissolving the tablets in sterile water, or another
appropriate vehicle, solutions can be prepared in unit-dose form. Suitable
excipients include, but are not limited to, inert diluents, such as calcium
carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or
binding agents, such as starch, gelatin, or acacia; or lubricating agents such
as
magnesium stearate, stearic acid, or talc.
Additional CHL2 polypeptide pharmaceutical compositions will be
2 o evident to those skilled in the art, including formulations involving CHL2
polypeptides in sustained- or controlled-delivery formulations. Techniques for
formulating a variety of other sustained- or controlled-delivery means, such
as
liposome carriers, bio-erodible microparticles or porous beads and depot
injections, are also known to those skilled in the art. See, e.g.,
PCT/US93/00829,
2 5 which describes the controlled release of porous polymeric microparticles
for the
delivery of pharmaceutical compositions.
Additional examples of sustained-release preparations include
semipermeable polymer matrices in the form of shaped articles, e.g. films, or
microcapsules. Sustained release matrices may include polyesters, hydrogels,
3 0 polylactides (IJ.S. Patent No. 3,773,919 and European Patent No. 058481),
copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,
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1983, Biopolymers 22:547-56), poly(2-hydroxyethyl-methacrylate) (Larger et
al.,
1981, J. Biomed. Mater. Res. 15:167-277 and Larger, 1982, Chena. Tech. 12:98-
105), ethylene vinyl acetate (Larger et al., supra) or poly-D(-)-3-
hydroxybutyric
acid (European Patent No. 133988). Sustained-release compositions may also
include liposomes, which can be prepared by any of several methods known in
the
art. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. LISA 82:3688-92;
and
European Patent Nos. 036676, 088046, and 143949.
The CHL2 pharmaceutical composition to be used for in vivo
administration typically must be sterile. This may be accomplished by
filtration
1 o through sterile filtration membranes. Where the composition is
lyophilized,
sterilization using this method may be conducted either prior to, or
following,
lyophilization and reconstitution. The composition for parenteral
administration
may be stored in lyophilized form or in a solution. In addition, parenteral
compositions generally are placed into a container having a sterile access
port, for
example, an intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
Once the pharmaceutical composition has been formulated, it may be
stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as
a
dehydrated or lyophilized powder. Such formulations may be stoxed either in a
2 0 ready-to-use form or in a form (e.g., lyophilized) requiring
reconstitution prior to
administration.
In a specific embodiment, the present invention is directed to kits for
producing a single-dose administration unit. The kits may each contain both a
first container having a dried protein and a second container having an
aqueous
2 5 formulation. Also included within the scope of this invention are kits
containing
single and mufti-chambered pre-filled syringes (e.g., liquid syringes and
lyosyringes).
The effective amount of a CHL2 pharmaceutical composition to be
employed therapeutically will depend, for example, upon the therapeutic
context
3 0 and objectives. One skilled in the art will appreciate that the
appropriate dosage
levels for treatment will thus vary depending, in part, upon the molecule
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delivered, the indication for which the CHL2 molecule is being used, the route
of
administration, and the size (body weight, body surface, or organ size) and
condition (the age and general health) of the patient. Accordingly, the
clinician
may titer the dosage and modify the route of administration to obtain the
optimal
therapeutic effect. A typical dosage may range from about 0.1 ~,g/kg to up to
about 100 mg/kg or more, depending on the factors mentioned above. In other
embodiments, the dosage may range from 0.1 ~,g/kg up to about 100 mg/kg; or 1
~g/kg up to about 100 mg/kg; or 5 gg/kg up to about 100 mg/kg.
The frequency of dosing will depend upon the pharmacokinetic parameters
of the CHL2 molecule in the formulation being used. Typically, a clinician
will
administer the composition until a dosage is reached that achieves the desired
effect. The composition may therefore be administered as a single dose, as two
or
more doses (which may or may not contain the same amount of the desired
molecule) over time, or as a continuous infusion via an implantation device or
catheter. Further refinement of the appropriate dosage is routinely made by
those
of ordinary skill in the art and is within the ambit of tasks routinely
performed by
them. Appropriate dosages may be ascertained through use of appropriate dose-
response data.
The route of administration of the pharmaceutical composition is in accord
with known methods, e.g., orally; through injection by intravenous,
intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular,
intramuscular, intraocular, intraarterial, intraportal, or intralesional
routes; by
sustained release systems; or by implantation devices. Where desired, the
compositions may be administered by bolus injection or continuously by
infusion,
2 5 or by implantation device.
Alternatively or additionally, the composition may be administered locally
via implantation of a membrane, sponge, or other appropriate material onto
which
the desired molecule has been absorbed or encapsulated. Where an implantation
device is used, the device may be implanted into any suitable tissue or organ,
and
3 o delivery of the desired molecule may be via diffusion, timed-release
bolus, or
continuous administration.
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In some cases, it may be desirable to use CHL2 polypeptide
pharmaceutical compositions in an ex vivo manner. In such instances, cells,
tissues, or organs that have been removed from the patient are exposed to CHL2
polypeptide pharmaceutical compositions after which the cells, tissues, or
organs
are subsequently implanted back into the patient.
In other cases, a CHL2 polypeptide can be delivered by implanting certain
cells that have been genetically engineered, using methods such as those
described herein, to express and secrete the CHL2 polypeptide. Such cells may
be animal or human cells, and may be autologous, heterologous, or xenogeneic.
Optionally, the cells may be immortalized. In order to decrease the chance of
an
immunological response, the cells may be encapsulated to avoid infiltration of
surrounding tissues. The encapsulation materials are typically biocompatible,
semi-permeable polymeric enclosures or membranes that allow the release of the
protein products) but prevent the destruction of the cells by the patient's
immune
system or by other detrimental factors from the surrounding tissues.
As discussed herein, it may be desirable to treat isolated cell populations
(such as stem cells, lymphocytes, red blood cells, chondrocytes, neurons, and
the
like) with one or more CHL2 polypeptides. This can be accomplished by
exposing the isolated cells to the polypeptide directly, where it is in a form
that is
2 0 permeable to the cell membrane.
Additional embodiments of the present invention relate to cells and
methods (e.g., homologous recombination and/or other recombinant production
methods) for both the in vitro production of therapeutic polypeptides and for
the
production and delivery of therapeutic polypeptides by gene therapy or cell
2 5 therapy. Homologous and other recombination methods may be used to modify
a
cell that contains a normally transcriptionally-silent CHL2 gene, or an under-
expressed gene, and thereby produce a cell which expresses therapeutically
efficacious amounts of CHL2 polypeptides. .
Homologous recombination is a technique originally developed for
3 0 targeting genes to induce or correct mutations in transcriptionally active
genes.
Kucherlapati, 1989, Py~og. iya Nucl. Acid Res. & Mol. Biol. 36:301. The basic
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technique was developed as a method for introducing specific mutations into
specific regions of the mammalian genome (Thomas et al., 1986, Cell 44:419-28;
Thomas and Capecchi, 1987, Cell S1:S03-12; Doetschman et al., 1988, P~oc.
Natl.
Acad. Sci. U.S.A. 8S:8S83-87) or to correct specific mutations within
defective
genes (Doetschman et al., 1987, Nature 330:576-78). Exemplary homologous
recombination techniques are described in U.S. Patent No. 5,272,071; European
Patent Nos. 9193051 and SOSS00; PCT/US90/07642, and PCT Pub No. WO
91/099SS).
Through homologous recombination, the DNA sequence to be inserted
into the genome can be directed to a specific region of the gene of interest
by
attaching it to targeting DNA. The targeting DNA is a nucleotide sequence that
is
complementary (homologous) to a region of the genomic DNA. Small pieces of
targeting DNA that are complementary to a specific region of the genome are
put
in contact with the parental strand during the DNA replication process. It is
a
general property of DNA that has been inserted into a cell to hybridize, and
therefore, recombine with other pieces of endogenous DNA through shared
homologous regions. If this complementary strand is attached to an
oligonucleotide that contains a mutation or a different sequence ox an
additional
nucleotide, it too is incorporated into the newly synthesized strand as a
result of
2 0 the recombination. As a result of the proofreading function, it is
possible for the
new sequence of DNA to serve as the template. Thus, the transferred DNA is
incorporated into the genome.
Attached to these pieces of targeting DNA are regions of DNA that may
interact with or control the expression of a CHL2 polypeptide, e.g., flanking
2 5 sequences. For example, a promoter/enhancer element, a suppressor, or an
exogenous transcription modulatory element is inserted in the genome of the
intended host cell in proximity and orientation sufficient to influence the
transcription of DNA encoding the desired CHL2 polypeptide. The control
element controls a portion of the DNA present in the host cell genome. Thus,
the
3 0 expression of the desired CHL2 polypeptide may be achieved not by
transfection
of DNA that encodes the CHL2 gene itself, but rather by the use of targeting
DNA
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(containing regions of homology with the endogenous gene of interest) coupled
with DNA regulatory segments that provide the endogenous gene sequence with
recognizable signals for transcription of a CHL2 gene.
In an exemplary method, the expression of a desired targeted gene in a cell
(i. e., a desired endogenous cellular gene) is altered via homologous
recombination
into the cellular genome at a preselected site, by the introduction of DNA
which
includes at least a regulatory sequence, an exon, and a splice donor site.
These
components are introduced into the chromosomal (genomic) DNA in such a
manner that this, in effect, results in the production of a new transcription
unit (in
1 o which the regulatory sequence, the exon, and the splice donor site present
in the
DNA construct are operatively linked to the endogenous gene). As a result of
the
introduction of these components into the chromosomal DNA, the expression of
the desired endogenous gene is altered.
Altered gene expression, as described herein, encompasses activating (or
causing to be expressed) a gene which is normally silent (unexpressed) in the
cell
as obtained, as well as increasing the expression of a gene which is not
expressed
at physiologically significant levels in the cell as obtained. The embodiments
further encompass changing the pattern of regulation or induction such that it
is
different from the pattern of regulation or induction that occurs in the cell
as
2 0 obtained, and reducing (including eliminating) the expression of a gene
which is
expressed in the cell as obtained.
One method by which homologous recombination can be used to increase,
or cause, CHL2 polypeptide production from a cell's endogenous CHL2 gene
involves first using homologous recombination to place a recombination
sequence
2 5 from a site-specific recombination system (e.g., Cre/loxP, FLP/FRT)
(Sauer,
1994, Curf°. Opih. Biotechhol., 5:521-27; Sauer, 1993, Metlz.ods
Enzymol.,
225:890-900) upstream of (i.e., 5' to) the cell's endogenous genomic CHL2
polypeptide coding region. A plasmid containing a recombination site
homologous to the site that was placed just upstream of the genomic CHL2
3 0 polypeptide coding region is introduced into the modified cell line along
with the
appropriate recombinase enzyme. This recombinase causes the plasmid to
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integrate, via the plasmid's recombination site, into the recombination site
located
just upstream of the genomic CHL2 polypeptide coding region in the cell line
(Baubonis and Sauer, 1993, Nucleic Acids Res. 21:2025-29; O'Gorman et al.,
1991, Science 251:1351-55). Any flanking sequences known to increase
transcription (e.g., enhancer/promoter, intron, translational enhancer), if
properly
positioned in this plasmid, would integrate in such a manner as to create a
new or
modified transcriptional unit resulting in de novo or increased CHL2
polypeptide
production from the cell's endogenous CHL2 gene.
A fwther method to use the cell line in which the site specific
recombination sequence had been placed just upstream of the cell's endogenous
genomic CHL2 polypeptide coding region is to use homologous recombination to
introduce a second recombination site elsewhere in the cell line's genome. The
appropriate recombinase enzyme is then introduced into the two-recombination-
site cell line, causing a recombination event (deletion, inversion, and
translocation) (Sauer, 1994, CuYr. Opih. Biotechhol., 5:521-27; Sauer, 1993,
Methods Ehzymol., 225:890-900) that would create a new or modified
transcriptional unit resulting in de yZOVO or increased CHL2 polypeptide
production from the cell's endogenous CHL2 gene.
An additional approach for increasing, or causing, the expression of CHL2
2 o polypeptide from a cell's endogenous CHL2 gene involves increasing, or
causing,
the expression of a gene or genes (e.g., transcription factors) and/or
decreasing the
expression of a gene or genes (e.g., transcriptional repressors) in a manner
which
results in de novo or increased CHL2 polypeptide production from the cell's
endogenous CHL2 gene. This method includes the introduction of a non-naturally
2 5 occurring polypeptide (e.g., a polypeptide comprising a site specific DNA
binding
domain fused to a transcriptional factor domain) into the cell such that de
hovo or
increased CHL2 polypeptide production from the cell's endogenous CHL2 gene
results.
The present invention further relates to DNA constructs useful in the
3 0 method of altering expression of a target gene. In certain embodiments,
the
exemplary DNA constructs comprise: (a) one or more targeting sequences, (b) a
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regulatory sequence, (c) an axon, and (d) an unpaired splice-donor site. The
targeting sequence in the DNA construct directs the integration of elements
(a) -
(d) into a target gene in a cell such that the elements (b) - (d) are
operatively
linked to sequences of the endogenous target gene. Tiz another embodiment, the
DNA constructs comprise: (a) one or more targeting sequences, (b) a regulatory
sequence, (c) an axon, (d) a splice-donor site, (e) an intron, and (f) a
splice-
acceptor site, wherein the targeting sequence directs the integration of
elements
(a) - (f) such that the elements of (b) - (f) are operatively linked to the
endogenous
gene. The targeting sequence is homologous to the preselected site in the
cellular
Z 0 chromosomal DNA with which homologous recombination is to occur. In the
construct, the axon is generally 3' of the regulatory sequence and the splice-
donor
site is 3' of the axon.
If the sequence of a particular gene is known, such as the nucleic acid
sequence of CHL2 polypeptide presented herein, a piece of DNA that is
complementary to a selected region of the gene can be synthesized or otherwise
obtained, such as by appropriate restriction of the native DNA at specific
recognition sites bounding the region of interest. This piece serves as a
targeting
sequence upon insertion into the cell and will hybridize to its homologous
region
within the genome. If this hybridization occurs during DNA replication, this
piece
2 0 of DNA, and any additional sequence attached thereto, will act as an
Okazaki
fragment and will be incorporated into the newly synthesized daughter strand
of
DNA. The present invention, therefore, includes nucleotides encoding a CHL2
polypeptide, which nucleotides may be used as targeting sequences.
CHL2 polypeptide cell therapy, e.g., the implantation of cells producing
2 5 CHL2 polypeptides, is also contemplated. This embodiment involves
implanting
cells capable of synthesizing and secreting a biologically active form of CHLZ
polypeptide. Such CHL2 polypeptide-producing cells can be cells that are
natural
producers of CHL2 polypeptides or may be recombinant cells whose ability to
produce CHL2 polypeptides has been augmented by transformation with a gene
3 0 encoding the desired CHL2 polypeptide or with a gene augmenting the
expression
of CHL2 polypeptide. Such a modification may be accomplished by means of a
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vector suitable for delivering the gene as well as promoting its expression
and
secretion. In order to minimize a potential immunological reaction in patients
being administered a CHL2 polypeptide, as may occur with the administration of
a polypeptide of a foreign species, it is preferred that the natural cells
producing
CHL2 polypeptide be of human origin and produce human CHL2 polypeptide.
Likewise, it is preferred that the recombinant cells producing CHL2
polypeptide
be transformed with an expression vector containing a gene encoding a human
CHL2 polypeptide.
Implanted cells may be encapsulated to avoid the infiltration of
1 o surrounding tissue. Human or non-human animal cells may be implanted in
patients in biocompatible, semipermeable polymeric enclosures or membranes
that allow the release of CHL2 polypeptide, but that prevent the destruction
of the
cells by the patient's immune system or by other detrimental factors from the
surrounding tissue. Alternatively, the patient's own cells, transformed to
produce
CHL2 polypeptides ex vivo, may be implanted directly into the patient without
such encapsulation.
Techniques for the encapsulation of living cells are known in the art, and
the preparation of the encapsulated cells and their implantation in patients
may be
routinely accomplished. For example, Baetge et al. (PCT Pub. No. WO 95/05452
2 0 and PCT/IJS94/09299) describe membrane capsules containing genetically
engineered cells for the effective delivery of biologically active molecules.
The
capsules are biocompatible and are easily retrievable. The capsules
encapsulate
cells transfected with recombinant DNA molecules comprising DNA sequences
coding for biologically active molecules operatively linked to promoters that
are
not subject to down-regulation in vivo upon implantation into a mammalian
host.
The devices provide for the delivery of the molecules from living cells to
specific
sites within a recipient. In addition, see U.S. Patent Nos. 4,892,538;
5,011,472;
and 5,106,627. A system for encapsulating living cells is described in PCT
Pub.
No. WO 91/10425 (Aebischer et al.). See also, PCT Pub. No. WO 91/10470
3 0 (Aebischer et al.); Winn et al., 1991, Expe~. NeuYOI. 113:322-29;
Aebischer et al.,
1991, Expe~. Neu~ol. 111:269-75; and Tresco et al., 1992, ASAIO 38:17-23.
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Ifz vivo and iyz vitro gene therapy delivery of CHL2 polypeptides is also
envisioned. One example of a gene therapy technique is to use the CHL2 gene
(either genomic DNA, cDNA, and/or synthetic DNA) encoding a CHL2
polypeptide which may be operably linked to a constitutive or inducible
promoter
to form a "gene therapy DNA construct." The promoter may be homologous or
heterologous to the endogenous CHL2 gene, provided that it is active in the
cell or
tissue type into which the construct will be inserted. Other components of the
gene therapy DNA construct may optionally include DNA molecules designed for
site-specific integration (e.g., endogenous sequences useful for homologous
1 o recombination), tissue-specific promoters, enhancers or silencers, DNA
molecules
capable of providing a selective advantage over the parent cell, DNA molecules
useful as labels to identify transformed cells, negative selection systems,
cell
specific binding agents (as, for example, for cell targeting), cell-specific
internalization factors, transcription factors enhancing expression from a
vector,
and factors enabling vector production.
A gene therapy DNA construct can then be introduced into cells (either ex
vivo or ifa vivo) using viral or non-viral vectors. One means for introducing
the
gene therapy DNA construct is by means of viral vectors as described herein.
Certain vectors, such as retroviral vectors, will deliver the DNA construct to
the
2 0 chromosomal DNA of the cells, and the gene can integrate into the
chromosomal
DNA. Other vectors will function as episomes, and the gene therapy DNA
construct will remain in the cytoplasm.
In yet other embodiments, regulatory elements can be included for the
controlled expression of the CHL2 gene in the target cell. Such elements are
2 5 turned on in response to an appropriate effector. In this way, a
therapeutic
polypeptide can be expressed when desired. One conventional control means
involves the use of small molecule dimerizers or rapalogs to dimerize chimeric
proteins which contain a small molecule-binding domain and a domain capable of
initiating a biological process, such as a DNA-binding protein or
transcriptional
3 0 activation protein (see PCT Pub. Nos. WO 96/41865, WO 97/31898, and WO
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97/31899). The dimerization of the proteins can be used to initiate
transcription
of the transgene.
An alternative regulation technology uses a method of storing proteins
expressed from the gene of interest inside the cell as an aggregate or
cluster. The
gene of interest is expressed as a fusion protein that includes a conditional
aggregation domain that results in the retention of the aggregated protein in
the
endoplasmic reticulum. The stored proteins are stable and inactive inside the
cell.
The proteins can be released, however, by administering a drug (e.g., small
molecule ligand) that removes the conditional aggregation domain and thereby
l o specifically breaks apart the aggregates or clusters so that the proteins
may be
secreted from the cell. See Aridor et al., 2000, Scieyace 287:816-17 and
Rivera et
al., 2000, Science 287:826-30. '
Other suitable control means or gene switches include, but are not limited
to, the systems described herein. Mifepristone (RU486) is used as a
progesterone
l5 antagonist. The binding of a modified progesterone receptor ligand-binding
domain to the progesterone antagonist activates transcription by forming a
dimer
of two transcription factors that then pass into the nucleus to bind DNA. The
ligand-binding domain is modified to eliminate the ability of the receptor to
bind
to the natural ligand. The modified steroid hormone receptor system is further
z 0 described in U.S. Patent No. 5,364,791 and PCT Pub. Nos. WO 96/40911 and
WO 97/10337.
Yet another control system uses ecdysone (a fruit fly steroid hormone)
which binds to and activates an ecdysone receptor (cytoplasmic receptor). The
receptor then translocates to the nucleus to bind a specific DNA response
element
2 5 (promoter from ecdysone-responsive gene). The ecdysone receptor includes a
transactivation domain, DNA-binding domain, and ligand-binding domain to
initiate transcription. The ecdysone system is further described in U.S.
Patent No.
5,514,578 and PCT Pub. Nos. WO 97/38117, WO 96/37609, and WO 93/03162.
Another control means uses a positive tetracycline-controllable
3 o transactivator. This system involves a mutated tet repressor protein DNA-
binding
domain (mutated tet R-4 amino acid changes which resulted in a reverse
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tetracycline-regulated transactivator protein, i.e., it binds to a tet
operator in the
presence of tetracycline) linked to a polypeptide which activates
transcription.
Such systems are described in U.S. Patent Nos. 5,464,758, 5,650,298, and
5,654,168.
Additional expression control systems and nucleic acid constructs are
described in U.S. Patent Nos. 5,741,679 and 5,834,186, to Innovir Laboratories
Inc.
Ih vivo gene therapy may be accomplished by introducing the gene
encoding CHL2 polypeptide into cells via local injection of a CHL2 nucleic
acid
molecule or by other appropriate viral or non-viral delivery vectors. Hefti
1994,
Neurobiology 25:1418-35. For example, a nucleic acid molecule encoding a
CHL2 polypeptide may be contained in an adeno-associated virus (AAV) vector
for delivery to the targeted cells (see, e.g., Johnson, PCT Pub. No. WO
95134670;
PCT App. No. PCT/US95/07178). The recombinant AAV genome typically
contains AAV inverted terminal repeats flanking a DNA sequence encoding a
CHL2 polypeptide operably linked to functional promoter and polyadenylation
sequences.
Alternative suitable viral vectors include, but are not limited to,
retrovirus,
adenovirus, herpes simplex virus, lentivirus, hepatitis virus, parvovirus,
2 0 papovavirus, poxvirus, alphavirus, coronavirus, rhabdovirus,
paramyxovirus, and
papilloma virus vectors. U.S. Patent No. 5,672,344 describes an ifa vivo viral-
mediated gene transfer system involving a recombinant neurotrophic HSV-1
vector. U.S. Patent No. 5,399,346 provides examples of a process for providing
a
patient with a therapeutic protein by the delivery of human cells which have
been
2 5 treated ih vitro to insert a DNA segment encoding a therapeutic protein.
Additional methods and materials for the practice of gene therapy techniques
are
described in U.S. Patent Nos. 5,631,236 (involving adenoviral vectors),
5,672,510
(involving retroviral vectors), 5,635,399 (involving retroviral vectors
expressing
cytokines).
3 0 Nonviral delivery methods include, but are not limited to, liposome-
mediated transfer, naked DNA delivery (direct injection), receptor-mediated
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transfer (ligand-DNA complex), electroporation, calcium phosphate
precipitation,
and microparticle bombardment (e.g., gene gun). Gene therapy materials and
methods may also include inducible promoters, tissue-specific enhancer-
promoters, DNA sequences designed for site-specific integration, DNA sequences
capable of providing a selective advantage over the parent cell, labels to
identify
transformed cells, negative selection systems and expression control systems
(safety measures), cell-specific binding agents (for cell targeting), cell-
specific
internalization factors, and transcription factors to enhance expression by a
vector
as well as methods of vector manufacture. Such additional methods and
materials
1 o for the practice of gene therapy techniques are described in U.S. Patent
Nos.
4,970,154 (involving electroporation techniques), 5,679,559 (describing a
lipoprotein-containing system for gene delivery), 5,676,954 (involving
liposome
carriers), 5,593,875 (describing methods for calcium phosphate transfection),
and
4,945,050 (describing a process wherein biologically active particles are
propelled
at cells at a speed whereby the particles penetrate the surface of the cells
and
become incorporated into the interior of the cells), aald PCT Pub. No. WO
96/40958 (involving nuclear ligands).
It is also contemplated that CHL2 gene therapy or cell therapy can further
include the delivery of one or more additional polypeptide(s) in the same or a
2 0 different cell(s). Such cells may be separately introduced into the
patient, or the
cells may be contained in a single implantable device, such as the
encapsulating
membrane described above, or the cells may be separately modified by means of
viral vectors.
A means to increase endogenous CHL2 polypeptide expression in a cell
2 5 via gene therapy is to insert one or more enhancer elements into the CHL2
polypeptide promoter, where the enhancer elements can serve to increase
transcriptional activity of the CHL2 gene. The enhancer elements used will be
selected based on the tissue in which one desires to activate the gene -
enhancer
elements known to confer promoter activation in that tissue will be selected.
For
3 0 example, if a gene encoding a CHL2 polypeptide is to be "turned on" in T-
cells,
the lck promoter enhancer element may be used. Here, the functional portion of
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the transcriptional element to be added may be inserted into a fragment of DNA
containing the CHL2 polypeptide promoter (and optionally, inserted into a
vector
and/or 5' and/or 3' flanking sequences) using standard cloning techniques.
This
construct, known as a "homologous recombination construct," can then be
introduced into the desired cells either ex vivo or i~ vivo.
Gene therapy also can be used to decrease CHL2 .polypeptide expression
by modifying the nucleotide sequence of the endogenous promoter. Such
modification is typically accomplished via homologous recombination methods.
For example, a DNA molecule containing all or a portion of the promoter of the
CHL2 gene selected for inactivation can be engineered to remove and/or replace
pieces of the promoter that regulate transcription. For example, the TATA box
and/or the binding site of a transcriptional activator of the promoter may be
deleted using standard molecular biology techniques; such deletion can inlubit
promoter activity thereby repressing the transcription of the corresponding
CHL2
gene. The deletion of the TATA box or the transcription activator binding site
in
the promoter may be accomplished by generating a DNA construct comprising all
or the relevant portion of the CHL2 polypeptide promoter (from the same or a
related species as the CHL2 gene to be regulated) in which one or more of the
TATA box and/or transcriptionah activator binding site nucleotides are mutated
2 0 via substitution, deletion and/or insertion of one or more nucleotides. As
a result,
the TATA box and/or activator binding site has decreased activity or is
rendered
completely inactive. This construct, which also will typically contain at
least
about 500 bases of DNA that correspond to the native (endogenous) 5' and 3'
DNA sequences adjacent to the promoter segment that has been modified, may be
2 5 introduced into the appropriate cells (either ex vivo or in vivo) either
directly or
via a viral vector as described herein. Typically, the integration of the
construct
into the genomic DNA of the cells will be via homologous recombination, where
the 5' and 3' DNA sequences in the promoter construct can serve to help
integrate
the modified promoter region via hybridization to the endogenous chromosomal
3 o DNA.
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Therapeutic Uses
CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists
thereof can be used to treat, diagnose, ameliorate, or prevent a number of
diseases,
disorders, or conditions, including those recited herein.
CHL2 polypeptide agonists and antagonists include those molecules which
regulate CHL2 polypeptide activity and either increase or decrease at least
one
activity of the mature form of the CHL2 polypeptide. Agonists or antagonists
may be co-factors, such as a protein, peptide, carbohydrate, lipid, or small
molecular weight molecule, which interact with CHL2 polypeptide and thereby
l0 regulate its activity. Potential polypeptide agonists or antagousts include
antibodies that react with either soluble or membrane-bound forms of CHL2
polypeptides that comprise part or all of the extracellular domains of the
said
proteins. Molecules that regulate CHL2 polypeptide expression typically
include
nucleic acids encoding CHL2 polypeptide that can act as anti-sense regulators
of
expression.
One of the major roles of the BMP-family of gene products, specifically
BMP2 and BMP4, is the regulation of bone-mass in the adult. Since BMP1 has
been shown to cleave a~zd inactivate CHD, and has been isolated with BMP2 and
BMP3 from bone (Wozney et al., 1988, Science 242:1528-34; Celeste et al.,
1990,
2 0 Proc. Nat. Acad. Sci. USA 87: 9843-47), CHL2 polypeptides may play a key
regulatory role in osteogenesis. Accordingly, CHL2 nucleic acid molecules,
polypeptides, and agonists and antagonists thereof (including, but not limited
to,
anti-CHL2 selective binding agents) may be useful in diagnosing or treating
diseases and conditions affecting bone density. Examples of such diseases and
2 5 conditions include, but are not limited to, osteopetrosis and
osteoporosis. Other
diseases and conditions affecting bone density are encompassed within the
scope
of this invention.
The direct delivery of BMP4 or other BMP-family members to the
regenerating bone through the blood stream appears to be a straightforward
3 o therapeutic concept for treatment of osteopetrosis. However, it may be
difficult to
accomplish since BMP4 is known to travel only a short distance in vivo (Jones
et
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al., 1996, Curs. Biol. 6:1468-75). As is the case with CHD during
embryogenesis,
the formation of a complex between BMP and CHLII polypeptide may result in
the further migration of BMP and formation of a BMP concentration gradient
(Jones and Smith, 1998, Dev. Biol. 194:12-17).
Based on the tight spatial regulation of CHL2 gene expression at the
surface of the articular cartilage - where the first sign of cartilage damage
is
detected during the pathogenesis of osteoarthritis - changes of expression
levels
of CHL2 polypeptide rnay play a role in the pathogenesis of osteoarthritis or
rheumatoid arthritis. Accordingly, CHL2 nucleic acid molecules, polypeptides,
and agonists and antagonists thereof may be useful in preventing cartilage
fibrosis
during the early phase of osteoarthritis, regenerating the once-disrupted
superficial
zone in the later phases of osteoarthritis or rheumatoid arthritis, or
generating a
proper superficial zone (i.e. surface) in transplanted cartilages.
BMP polypeptides have been shown to function in organ formation during
late embryogenesis. It has been shown that organ formation in embryonic
kidney,
Lung, and gut are affected by BMP4 expression (Hogan, 1996, Genes Dev.
10:1580-94). A combination of BMP4 and CHL2 polypeptides may be useful for
controlling the proliferation and differentiation of progenitor cells, thus
permitting
the regulation of tissue regeneration or wound healing i~ vivo. Accordingly,
2 0 CHL2 nucleic acid molecules, polypeptides, and agonists and antagonists
thereof
may be useful in promoting tissue regeneration or wound healing.
The CHL2 nucleic acid molecules, polypeptides, agonists and antagonists
thereof may also be used in hematopoietic stem cell-genesis and expansion. It
may be possible to use BMP/CHL2 polypeptide complexes to regulate primitive
2 5 hematopoietic stem cells and thereby control adult-marrow repopulating
stem
cells. Alternatively, it may be possible to control stem cell genesis from a
mesodermal stem cell. Thus, the CHL2 polypeptides and nucleic acids of the
present invention, along with BMP, may be useful for ex vivo expansion of
hematopoietic stern cells and gene therapy performed through suchcells.
3 0 BMP4 is an essential factor for generating hematopoietic progenitor cells
from the mouse ES cells. However, the effective concentration of BMP4 falls
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into a narrow range (0.5 ng/ml to 5 ng/ml), which is consistent with the idea
that
the difference in the active concentration of BMP correlates with the
difference in
the resulting cell-type from the totipotent epiblast. A system for the
reproducible
ih vitro generation of hematopoietic stem cells from ES cells has not yet been
disclosed. However, it may be achieved by precise control of the concentration
of
BMP4. The CHL2 nucleic acid molecules and polypeptides of the present
invention may be useful in optimizing the culturing conditions for the in
vitro
generation of hematopoietic stem cells from ES cells.
Primitive hematopoietic stem cells have been recently defined in the
mouse yolk sac (Yoder et al., 1997, Proc. Natl. Acad. Sci. USA 94:6776-80).
This
subclass of hematopoietic stem cells does not exhibit a marrow-repopulating
activity in adults. However, when exposed to a newborn liver environment, the
primitive stem cells are converted to long-term marrow-repopulating stem cells
(i.e., definitive stem cells). This can also be accomplished by culturing the
primitive stem cells on certain stroma cell lines. The long-term survival of
the
definitive stem cells in culture and the long-term maintenance of the
primitive
stem cells that are spontaneously differentiated into definitive stem cells
have not
yet been established. Interactions between BMP and CHL2 polypeptide might
function in the regulation of definitive stem cells. The recombinant CHL2
2 o polypeptides and CHL2 antibodies of the present invention may be useful
tools
for ih vitro long-term maintenance of hematopoietic stem cells and ih vitro
generation of definitive stem cells from primitive stem cells. Alternatively,
BMP4 or putative, novel CHL2-interacting molecules may be useful for
controlling these processes.
2 5 In addition, primitive hematopoietic stem cells have yet to be fully
characterized. While primitive stem cells may be of a lymphocytic cell type,
such
cells may also be mesodermal precursors that are able to generate
hematopoietic
cell types as well as other mesodermal progeny. In support of this idea, adult
bone marrow has recently been shown to contain endothelial progenitor cells,
3 0 cells that regenerate liver (Petersen et al., 1999, Science 284:1168-70),
and a
common stem cell that has a capability of deriving endothelial cells, muscle
cells
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and hematopoietic cells in vivo (Ferrari et al., 1998, Science 279:1528-30).
Furthermore, the osteoblast cell lineage, which consists of the bone marrow
stroma, is known to be derived from a mesenchymal stem cell that is present in
bone marrow. The possibility that a common mesodermal stem cell is responsible
for the generation of both stroma and hematopoietic cells has also been
previously
speculated.
Since CHL2 polypeptide expression has been detected in skeletal muscle,
CHL2 polypeptides may also play a role in the development and function of
skeletal muscle. Accordingly, CHL2 nucleic acid molecules, polypeptides, and
agonists and antagonists thereof may be useful in diagnosing or treating
diseases
and conditions affecting skeletal muscle. Examples of such diseases and
conditions include, but are not limited to, cachexia and muscular dystrophy.
Other diseases and conditions associated with skeletal muscle development and
function are encompassed within the scope of this invention.
Since CHL2 polypeptide expression has been detected in the heart, CHL2
polypeptides may play a role in the development and function of the heart.
Accordingly, CHL2 nucleic acid molecules, polypeptides, and agonists and
antagonists thereof may also be useful in diagnosing or treating diseases and
conditions affecting the heart. Examples of such diseases and conditions
include,
2 o but are not limited to, arhythmias, angina, hypertension, myocardial
infarction,
and congestive heart failure. Other diseases and conditions associated with
the
heart are encompassed within the scope of this invention.
Since CHL2 polypeptide expression has been detected in the stomach,
CHL2 polypeptides may play a role in the development and function of the
2 5 stomach. Accordingly, CHL2 nucleic acid molecules, polypeptides, and
agonists
and antagonists thereof may also be useful in diagnosing or treating diseases
and
conditions involving the stomach. Examples of such diseases and conditions
include, but are not limited to, stomach cancer and stomach ulcer. Other
diseases
and conditions associated with the stomach are encompassed within the scope of
3 0 this invention.
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Since CHL2 polypeptide expression has been detected in the liver, CHL2
polypeptides may play a role in the development and function of the liver.
Accordingly, CHLZ nucleic acid molecules, polypeptides, and agonists and
antagonists thereof may also be useful in diagnosing or treating diseases and
conditions involving the liver. Examples of such diseases and conditions
include,
but are not limited to, hepatitis and hepatoma. Other diseases and conditions
associated with the liver are encompassed within the scope of this invention.
Agonists or antagonists of CHL2 polypeptide function may be used
(simultaneously or sequentially) in combination with one or more cytokines,
growth factors, antibiotics, anti-inflammatories, and/or chemotherapeutic
agents
as is appropriate for the condition being treated.
Other diseases caused by or mediated by undesirable levels of CHL2
polypeptides are encompassed within the scope of the invention. Undesirable
levels include excessive levels of CHL2 polypeptides and sub-normal levels of
CHL2 polypeptides.
Uses of CHL2 Nucleic Acids and Polypeptides
Nucleic acid molecules of the invention (including those that do not
themselves encode biologically active polypeptides) may be used to map the
2 0 locations of the CHL2 gene and related genes on chromosomes. Mapping may
be
done by techniques known in the art, such as PCR amplification and ih situ
hybridization.
CHL2 nucleic acid molecules (including those that do not themselves
encode biologically active polypeptides), may be useful as hybridization
probes in
2 5 diagnostic assays to test, either qualitatively or quantitatively, for the
presence of
a CHL2 nucleic acid molecule in mammalian tissue or bodily fluid samples.
Other methods may also be employed where it is desirable to inhibit the
activity of one or more CHL2 polypeptides. Such inhibition may be effected by
nucleic acid molecules that are complementary to and hybridize to expression
3 o control sequences (triple helix formation) or to CHL2 mRNA. For example,
antisense DNA or RNA molecules, which have a sequence that is complementary
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to at least a portion of a CHL2 gene can be introduced into the cell. Anti-
sense
probes may be designed by available techniques using the sequence of the CHL2
gene disclosed herein. Typically, each such antisense molecule will be
complementary to the start site (5' end) of each selected CHL2 gene. When the
antisense molecule then hybridizes to the corresponding CHL2 mRNA, translation
of this mRNA is prevented or reduced. Anti-sense inhibitors provide
information
relating to the decrease or absence of a CHL2 polypeptide in a cell or
organism.
Alternatively, gene therapy may be employed to create a dominant
negative inhibitor of one or more CHL2 polypeptides. In this situation, the
DNA
l0 encoding a mutant polypeptide of each selected CHL2 polypeptide can be
prepared and introduced into the cells of a patient using either viral or non-
viral
methods as described herein. Each such mutant is typically designed to compete
with endogenous polypeptide in its biological role.
In addition, a CHL2 polypeptide, whether biologically active or not, may
be used as an immunogen, that is, the polypeptide contains at least one
epitope to
which antibodies may be raised. Selective binding agents that bind to a CHL2
polypeptide (as described herein) may be used for ih vivo and in vitro
diagnostic
purposes, including, but not limited to, use in labeled form to detect the
presence
of CHL2 polypeptide in a body fluid or cell sample. The antibodies may also be
2 0 used to prevent, treat, or diagnose a number of diseases and disorders,
including
those recited herein. The antibodies may bind to a CHL2 polypeptide so as to
diminish or block at least one activity characteristic of a CHL2 polypeptide,
or
may bind to a polypeptide to increase at least one activity characteristic of
a
CHL2 polypeptide (including by increasing the pharmacokinetics of the CHL2
2 5 polypeptide).
The CHL2 polypeptides of the present invention can be used to clone
CHL2 polypeptide receptors, using an expression cloning strategy. Radiolabeled
(izsIodine) CHL2 polypeptide or affinity/activity-tagged CHL2 polypeptide
(such
as an Fc fusion or an alkaline phosphatase fusion) can be used in binding
assays
3 0 to identify a cell type or cell line or tissue that expresses CHL2
polypeptide
receptors. RNA isolated from such cells or tissues can be converted to cDNA,
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cloned into a mammalian expression vector, and transfected into mammalian
cells
(such as COS or 293 cells) to create an expression library. A radiolabeled or
tagged CHL2 polypeptide can then be used as an affinity ligand to identify and
isolate from this library the subset of cells that express the CHL2
polypeptide
receptors on their surface. DNA can then be isolated from these cells and
transfected into mammalian cells to create a secondary expression library in
which the fraction of cells expressing CHL2 polypeptide receptors is many-fold
higher than in the original library. This enrichment process can be repeated
iteratively until a single recombinant clone containing a CHL2 polypeptide
receptor is isolated. Isolation of the CHL2 polypeptide receptors is useful
for
identifying or developing novel agonists and antagonists of the CHL2
polypeptide
signaling pathway. Such agonists and antagonists include soluble CHL2
polypeptide receptors, anti-CHL2 polypeptide receptor antibodies, small
molecules, or antisense oligonucleotides, and they may be used for treating,
preventing, or diagnosing one or more of the diseases or disorders described
herein.
The marine and human CHL2 nucleic acids of the present invention are
also useful tools for isolating the corresponding chromosomal CHL2 polypeptide
genes. For example, mouse chromosomal DNA containing CHL2 sequences can
2 0 be used to construct knockout mice, thereby permitting an examination of
the in
vivo role for CHL2 polypeptide. The human CHL2 genomic DNA can be used to
identify heritable tissue-degenerating diseases.
Deposits of cDNA encoding marine and human CHL2 polypeptide,
subcloned into pSPORTI (Gibco BRL), having Accession Nos. PTA-1479 and
2 5 PTA-1480, were made with the American Type Culture Collection, 10801
University Boulevard, Manassas, VA 20110-2209 on March 14, 2000.
The following examples are intended for illustration purposes only, and
should not be construed as limiting the scope of the invention in any way.
3 0 . Example 1: Cloning of the Marine CHL2 Polypeptide Gene
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Generally, materials and methods as described in Sambrook et al. supra
were used to clone and analyze the gene encoding marine CHL2 polypeptide.
Two marine placenta cDNA libraries were prepared in order to isolate
sequences encoding marine CHL2 polypeptide. Total RNA was extracted from
mouse placenta and poly-A+ RNA selected using standard extraction and
isolation
techniques. Random-primed cDNA was then synthesized from poly-A+ RNA
using the Superscript Plasmid System for cDNA Synthesis (Gibco-BRL,
Rockville, MD). The resulting cDNA was digested with Not I and fractionated on
a 0.8% agarose gel. Electrophoresed cDNA of 300-1000 by was isolated and
l0 ligated into the signal trap vector kFGF7 and cDNA of greater than 1.5 kb
was
isolated and ligated into pSPORT 1. Ligation reactions were introduced into E.
coli using standard transformation techniques, transformants selected on
ampicillin-containing media, and the transformants collected to generate the
two
cDNA libraries.
Clones containing signal peptide sequences were enriched from the IcFGF-
based library using kFGF signal trapping technology (U.S. Patent App. No.
09/026,959). Plasmid DNA from this cDNA library was prepared from 10 pools
of 100,000 colonies each using standard techniques. This DNA was introduced
into NIH 3T3 cells by calcium phosphate transfection and transfected cells
were
2 0 then grown for 14 days in selective media supplemented with 0.5% fetal
bovine
serum. Only transformants containing plasmids with signal peptide sequences
generated colonies. These colonies were harvested by trypsinization and total
RNA from the colonies was isolated using TRIzon reagent (Gibco-BRL)
according to the manufacturer's recommended protocol. Poly-A+ RNA was
2 5 isolated from the total RNA using an mRNA Purification Kit (Amersham
Pharmacia Biotech, Piscataway, NJ).
To generate a cDNA library enriched for molecules containing signal
peptide sequences, first strand cDNA was initially prepared from the poly-A+
RNA isolated above using the SuperScriptTM preamplification system (Gibco-
3 0 BRL). The first strand cDNA reaction was performed by combining 1 ~,g of
poly-
A+ RNA and 2 pmole of the primer 1605-21 (5'-A-A-T-C-C-G-A-T-G-C-C-C-A-
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C-G-T-T-G-C-A-G-T-A-3'; SEQ ID NO: 12) in a total volume of 15 ~,L and
heating the template-primer mixture at 70°C for 10 minutes. The
template-primer
mixture was transferred to 50°C, and a reaction premixture consisting
of 2.5 ~,L of
lOX buffer, 2.5 ~L of 25 mM MgCl2, 1.3 ~L 10 mM dNTPs, and 2.5 ~,L 0.1 M
dithiothreitol was added. Reverse transcriptase (250 U) was then added and the
reaction incubated at 50°C for 1 hour. The first strand cDNA reaction
was
stopped by heating at 70°C for 15 minutes, and the RNA digested by
treatment
with 2 U of RNAse H for 20 minutes at 37°C.
Following first strand cDNA synthesis, double strand cDNA was
generated by PCR amplification in a reaction containing 2 ~L of first strand
cDNA, the primers 1239-08 (5'-A-A-A-A-T-C-T-T-A-G-A-C-C-G-A-C-G-A-C-
T-G-T-G-T-T-T-3'; SEQ ID NO: 13) and 1605-22 (5'-C-G-T-A-A-A-A-G-A-T-
C-C-T-G-C-G-C-T-A-G-A-T-G-C-G-3'; SEQ ID NO: 14) at a final concentration
of 0.5 ~,M each, 200 ~,M of dNTPs, and 2.5 U of Pfu polymerase (PE Biosystems,
Norwalk, CT), in a volume of 100 ~,L. The PCR reaction was perfonned at
95°C
for 1 minute for one cycle; 95°C for 30 seconds, 66°C for 45
seconds, and 72°C
for 2 minutes for 30 cycles; and 72°C for 10 minutes for one cycle.
Amplification
products were digested with Not I and Sal I and ligated into kFGF7. The
ligation
reaction was introduced into E. coli using standard transformation techniques
to
2 0 generate a signal enriched cDNA library. Plasmid DNA was prepared from 400
selected clones and analyzed by sequencing.
The sequence of one clone (designated ymkz5-00011-c10) was found to
share significant homology with the chordin precursor from Xeszopus (GenBank
accession no. Q91713). The identified clone was found to contain an insert of
418
2 5 by encoding the N-terminal 115 amino acids of murine CHL2. A full-length
cDNA clone was isolated from the cDNA library cloned into pSPORT 1 using the
ymkz5-00011-c10 clone as a probe.
To screen the pSPORT 1 library, 1 x 10~ clones were plated on 150 mm
plates at approximately 5 x 104 clones per plate and the clones were then
lifted
3 0 from the plates on nitrocellulose filters. Filters were prehybridized in
ExpressHyb
hybridization solution (Clontech) for 30 minutes at 68°C and then
hybridized
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overnight at 68°C in the same hybridization solution containing the 32P-
dCTP-
labeled probe. Hybridized filters were washed twice in 2X SSC and 0.1% SDS
for 10 minutes at room temperature and twice in 0.1X SSC and 0.1% SDS for 30
minutes at 65°C. Following washing, filters were subjected to
autoradiography
overnight at -80°C in the presence of intensifying screens. Positive
clones were
identified and re-screened. Two positive clones were identified following a
secondary screen and DNA fiom these two clones was isolated and sequenced.
One clone (designated pSPORTmCHL2) contained an insert of approximately 1.8
lcb, which encoded the complete marine CHL2 polypeptide.
Sequence analysis of the full-length cDNA for marine CHL2 polypeptide
indicated that the gene comprises a 1278 by open reading frame encoding a
protein of 426 amino acids. Figures lA-1C illustrate the nucleotide sequence
of
the marine CHL2 gene (SEQ ID NO: 1) and the deduced amino acid sequence of
marine CHL2 polypeptide (SEQ ID NO: 2). In Figure 1A, the signal peptide
sequence, as predicted by the von Heijne algorithm, is underlined.
Computer analysis of the marine CHL2 amino acid sequence indicated
that this polypeptide, like CHL (CHL1) polypeptide and CHLdS polypeptide (co-
pending arid co-owned U.S. Patent App. No. 09/724,915), possesses three pro-
collagen repeats (CR motifs) - in contrast to the four repeats observed in CHD
2 0 (Figure 5). The amino acid sequence of CHL2 was found to share 24.4%
identity
(or 28% identity by a GAP search) with marine chordin. Figure 2 illustrates
the
amino acid sequence aligmnent of marine CHL2 polypeptide (mouse CHL2; SEQ
ID NO: 2) and marine chordin (Afl)69501; SEQ ID NO: 7). The amino acid
sequence of marine CHL2 polypeptide was also found to share 50% identity with
2 5 CHL1 and structural similarity with BMP ' and SOG, specifically in the CR
domains where the similarity is high. A BLAST search of the Celera human
genome database indicated that the CHL2 gene shared the greatest homology with
the CHL1 gene. These results suggest that the CHL2 gene is a novel member of
the CHD/SOG gene family.
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Example 2: Cloning of the Human CHL2 Polxpeptide Gene
Generally, materials and methods as described in Sambrook et al. supYa
were used to clone and analyze the gene encoding human CHL2 polypeptide.
A human placenta cDNA library was prepared in order to isolate
sequences encoding human CHL2 polypeptide. Total RNA was extracted from
human placenta and poly-A+ RNA selected using standard extraction and
isolation techniques. Oligo d(T)-primed cDNA was then synthesized from poly
A+ RNA using the Superscript Plasmid System for cDNA Synthesis (Gibco
BRL). The resulting cDNA was digested with Not I and fractionated on a 0.8%
agarose gel. Electrophoresed cDNA of greater than 1.5 kb was isolated and
ligated into pSPORT 1. Ligation reactions were introduced into E. coli using
standard transformation techniques, transformants selected on ampicillin-
containing media, and the transformants collected to generate the human
placenta
cDNA library.
A 32P-dCTP-labeled murine CHL2 cDNA fragment was used to screen the
human placenta cDNA library. Plasmid DNA was isolated from 12 pools of
100,000 clones each, 1 wg of plasmid DNA from each pool was digested with Not
I and Sal I, electrophoresed on a 0.8% agarose gel, and then transferred to a
nitrocellulose filter. Filters were prehybridized in ExpressHyb hybridization
2 0 solution (Clontech) for 30 minutes at 60°C and then hybridized
overnight at 60°C
in the same solution containing the murine CHL2 cDNA probe. Hybridized filters
were washed twice in 2X SSC and 0.1% SDS for 10 minutes at room temperature
and twice in O.SX SSC and 0.1% SDS for 30 minutes at 60°C. Following
washing, filters were subj ected to autoradiography overnight at -80°C
in the
2 5 presence of intensifying screens. Positive cDNA fragments were identified
in two
of the twelve plasmid pools.
An individual clone containing sequences encoding human CHL2
polypeptide was isolated from the two plasmid pools identified above by
plating 3
x 105 clones from each pool on 150 mm plates at approximately 5 x 104 clones
per
3 0 plate. The clones were then lifted from the plates on nitrocellulose
filters and the
filters analyzed as described above. Two positive clones were identified, and
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each was subjected to sequence analysis. One clone (designated pSPORThCHL2)
contained an insert of approximately 1.5 kb, which encoded the complete human
CHL2 polypeptide.
Sequence analysis of the full-length cDNA for human CHL2 polypeptide
indicated that the gene comprises a 1287 by open reading frame encoding a
protein of 429 amino acids. Figures 3A-3C illustrate the nucleotide sequence
of
the human CHL2 gene (SEQ ID NO: 4) and the deduced amino acid sequence of
human CHL2 polypeptide (SEQ ID NO: 5). The amino acid sequence of CHL2
was found to share 26.7% identity with human chordin. Figure 4 illustrates the
amino acid sequence alignment of human CHL2 polypeptide (human CHL2; SEQ
ID NO: 5) and human chordin (Af076612; SEQ ID NO: 8).
Example 3: CHL2 mRNA Expression
Multiple human or marine tissue Northern blots (Clontech) were probed
using a 32P-dCTP-labeled human or marine CHL2 cDNA fragment, respectively.
Northern blots were prehybridized in ExpressHyb hybridization solution
(Clontech) for 30 minutes at 68°C and then were hybridized in the same
solution
with the addition of labeled probe overnight at 68°C. Following
hybridization, the
filters were washed twice in 2X SSC and 0.1% SDS for 10 minutes at room
temperature and twice in O.1X SSC and 0.1% SDS for 30 minutes at 68°C.
Following washing, the blots were subjected to autoradiography for 72 hours at
-
80°C in the presence of intensifying screens.
Northern blot analysis of human tissue blots revealed predominant CHL2
transcripts of approximately 2 kb in the prostate, testis, uterus (very
abundant
2 5 expression), colon, small intestine, heart, skeletal muscle, and stomach.
Wealc
expression was detected in the trachea, placenta, and bone marrow, and vary
weak
expression was detected in the liver. Northern blot analysis of marine tissue
blots
revealed predominant CHL2 transcripts of approximately 1.8 kb in liver and
kidney. Very weak expression was detected in the heart. Weak expression of an
3 0 approximately 2 kb transcript was detected in the testis and skeletal
muscle.
Expression of CHL2 in marine stomach tissue was not analyzed. Very weak
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expression was detected in mouse embryonic tissue from day 7, 11, 15, and 17
embryos.
The Northern bot analysis strengthens the relationship between CHD/SOG
and CHL2 polypeptide. CHD/SOG is expressed at a relatively high level in E7
embryos and at decreased levels in E11, E15, and E17 embryos (Pappano et al.,
1998, Genofnics 52:236-39). CHL2 mRNA was similarly detected in E7, E11,
E15, and E17 embryos, although at lower levels than are CHD/SOG transcripts.
CHD/SOG is expressed in spleen, liver, and kidney (Pappano et al., 1998). CHL2
mRNA was similarly detected in liver and kidney. The similarity in expression
pattern, coupled with the similarity in structure, ' suggest that CHL2 and
CHD/SOG may have similar biological activity, but that these proteins may
function at different developmental stages.
The expression of CHL2 mRNA was localized by in situ hybridization. In
situ hybridization to embryonic and adult mouse tissue sections was performed
as
described (Wilcox, 1993, J. Histochenz. Cytochena. 41:1725-33). A marine CHL2
probe was prepared by first removing the 1.2-kb Nco I - Sal I fragment from
pSPORTmCHL2 to generate pSPmCHL2COOH, which was then linearized with
Eco RI. In addition, the Hind III fragment was removed to obtain
2 o pSPmCHL2NH2. Antisense-RNAs were synthesized with SP6 RNA polymerase.
The RNAs synthesized from these plasmids contained non-overlapping
sequences.
In situ hybridization analysis demonstrated that CHL2 mRNA expression
began in mice at about embryonic day 12.5 in the sternum and persisted in a
2 5 restricted area of adult articular cartilage. CHL2 mRNA expression was not
detected in the sections examined before E10.5. At E13.5, strong CHL2 mRNA
expression was detected over the chondrocytes in the limb bones (Figures 6 and
7)
and sternum, as well as in the perichondrial mesenchymal cells adjacent to the
developing joints. Strong but highly restricted CHL2 mRNA expression was alos
3 0 detected in the superficial zone chondrocytes within the developing
articular
cartilage of vertebra and the epiphyses of the long bones (Figures 6 - 8). By
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E18.5, and through to adulthood, the skeletal expression of CHL2 mRNA was
restricted to a single layer of superficial zone chondrocytes in articular
cartilage.
The epiphyseal growth plate did not show CHL2 mRNA expression at any point
in mouse development.
Among non-cartilagenous tissues, significant CHL2 mRNA expression
were observed in ovary, oviduct, and uterus of female mice, and in testis,
epididymus and possibly other accessory glands (e.g., seminal vesicle,
coagulating gland, and prostate) of male mice. The strong signal observed in
uterine wall was somewhat analogous to the CHL1 mRNA expression detected in
1 o uterus. A weak expression of CHL2 mRNA was also detected on the colon
surface. However, the location of CHL2 expression in the colon clearly
differed
from that of CHL1, wherein CHL2 mRNA was detected in the
fibroblast/connective tissue cells dividing the submucosa and muscularis
(Nalcayama et al., 2001). In contrast with the CHL1 gene, CHL2 mRNA
expression was not detected in stomach or small intestine. Thus, among soft
tissues, the CHL2 gene was expressed specifically in the reproductive organs.
Example 4: Chromosomal Mapping of the Marine CHL2 Polypeptide Gene
Fluorescence i~c situ hybridization (FISH) analysis was used to determine
2 o the chromosomal localization of the marine CHL2 gene (Shi et al., 1997,
Ge~comics 45:42-47). A FISH probe was prepared from a BAC clone (F1067)
isolated from the Mouse ES-129/SvJ II BAC chromosome DNA library (Genome
Systems) by PCR using standard techniques and primers corresponding to the 5'
untranslated region of the marine CHL2 gene (5'-T-C-C-T-C-T-C-A-T-C-C-T-C-
2 5 A-C-C-T-T-A-G-3'; SEQ ID NO: 15 and 5'-G-G-A-G-A-A-A-G-T-G-A-G-A-T-
A-A-G-G-A-C-A-C-3'; SEQ ID NO: 16). The marine CHL2 gene was localized
to chromosome 7 using the chromosome 7 centromere specific P1 clone as a co-
hybridization probe (Shi et al., 1997). A total of 80 metaphase cells were
analyzed, and 10 of the 77 that exhibited specific labeling were used for co-
3 0 hybridization experiments. As a result, the mCHL2 gene was located at a
position
66% of the distance from the heterochromatic-euchromatic boundary to the
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telomere of chromosome 7, which corresponds to band 7E2-E3. Thus, the CHL2
gene differs from the CHL1 gene in that the CHL2 gene is autosomal.
Example 5: Production of CHL2 Polypeptides
A. Expression of CHL2 Polypeptides in Bacteria
PCR is used to amplify template DNA sequences encoding a CHL2
polypeptide using primers corresponding to the 5' and 3' ends of the sequence.
The amplified DNA products may be modified to contain restriction enzyme sites
to allow for insertion into expression vectors. PCR products are gel purified
and
l0 inserted into expression vectors using standard recombinant DNA
methodology.
An exemplary vector, such as pAMG21 (ATCC no. 98113) containing the lux
promoter and a gene encoding kanamycin resistance is digested with Bam HI and
Nde I for directional cloning of inserted DNA. The ligated mixture is
transformed
into an E. coli host strain by electroporation and transformants are selected
for
kanamycin resistance. Plasmid DNA from selected colonies is isolated and
subjected to DNA sequencing to confirm the.presence of the insert.
Transformed host cells are incubated in 2xYT medium containing 30
~,g/mL kanamycin at 30°C prior to induction. Gene expression is induced
by the
addition of N-(3-oxohexanoyl)-dl-homoserine lactone to a final concentration
of
2 0 30 ng/mL followed by incubation at either 30°C or 37°C for
six hours. The
expression of CHL2 polypeptide is evaluated by centrifugation of the culture,
resuspension and lysis of the bacterial pellets, and analysis of host cell
proteins by
SDS-polyacrylamide gel electrophoresis.
Inclusion bodies containing CHL2 polypeptide are purified as follows.
2 5 Bacterial cells are pelleted by centrifugation and resuspended in water.
The cell
suspension is lysed by sonication and pelleted by centrifugation at 195,000 xg
for
5 to 10 minutes. The supernatant is discarded, and the pellet is washed and
transferred to a homogenizer. The pellet is homogenized in 5 mL of a Percoll
solution (75% liquid Percoll and 0.15 M NaCI) until uniformly suspended and
3 0 then diluted and centrifuged at 21,600 xg for 30 minutes. Gradient
fractions
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containing the inclusion bodies are recovered and pooled. The isolated
inclusion
bodies are analyzed by SDS-PAGE.
A single band on an SDS polyacrylamide gel corresponding to E. coli
produced CHL2 polypeptide is excised from the gel, and the N-terminal amino
acid sequence is determined essentially as described by Matsudaira et al.,
1987, J.
Biol. Chem. 262:10-35.
B. Construction of CHL2 Polypeptide Mammalian Expression Vectors
Marine CHL2 was transiently expressed using the pSRamCHL2 vector,
l0 which was prepared as follows. The open reading frame for CHL2 polypeptide
was first arriplified by PCR using standard techniques and the primers 2360-40
(5'-G-C-T-A-T-C-T-A-G-A-G-C-C-A-C-C-A-T-G-G-T-T-C-C-C-G-G-G-G-T-G
A-G-G-A-T-C-A-T-C-3'; SEQ ID NO: 17) and 2360-41 (5'-G-C-T-A-G-T-C-G
A-C-C-T-A-T-A-A-T-G-T-C-T-T-G-G-T-C-A-C-T-T-T-G-T-C-T-G-3'; SEQ ID
NO: 18). The amplification product was digested with Xba I and Sal I and then
inserted into an SRcc-based expression plasmid (Takebe et al., 1988, Mol.
Cell.
Biol. 8:466-72) to yield pSRamCHL2.
A FLAG-tagged marine CHL2 polypeptide expression construct was
prepared as follows. A full-length marine CHL2 DNA fragment, in which the
2 0 stop codon was replaced by a Sal I site, was obtained by PCR using the
fixll-length
marine cDNA clone as a template and the primers 5'-G-C-T-A-G-C-G-G-C-C-G-
C-G-C-C-A-C-C-A-T-G-G-T-T-C-C-C-G-G-G-G-T-G-A-G-G-A-T-C-A-T-C-3'
(SEQ ID NO: 19) and 5'-G-C-T-A-G-T-C-G-A-C-T-A-A-T-G-T-C-T-T-G-G-T-
C-A-C-T-T-T-G-T-C-T-G-G-G-C-3' (SEQ ID NO: 20). The amplified PCR
2 5 product was digested with Not I and Sal I and then inserted into the pFLAG-
CMV-Sa expression vector (Sigma) with the FLAG-sequence attached in-frame
with the CHL2 sequence at its carboxyl-terminus. The resulting mCHL2-FLAG
expression plasmid was designated as pFLAGmCHL2.
The expression of the mCHL2-FLAG polypeptide was detected by direct
3 0 western blot analysis using 10 ~g/ml of the anti-FLAG mouse monoclonal
antibody, M2 (Sigma), according to the manufacturer's recommendations, with
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the exception that 5% (w/v) nonfat dry milk (Trader Joe's, Thousand Oaks CA)
was used in place of 3% (w/v) bovine serum albumin. Rabbit polyclonal
antibodies for murine CHL2 were raised using a synthetic peptide based on the
carboxyl-terminal sequence: C-P-E-D-E-A-E-D-D-H-S-E-V-I-S-T-R (SEQ ID
NO: 21), as described in Harlow and Lane, Using Antibodies: A Laboratory
Manual (Cold Spring Harbor Laboratory Press, 1988). The resulting anti-sera
were either used directly or subj ected to affinity purification with the
corresponding peptides.
To generate clones capable of stably expressing mCHL2-FLAG, 293 cells
were transfected with linearized pFLAGmCHL2, stable transfectants were
selected, and the expression level of the corresponding FLAG-tagged proteins
in
both the serum-free conditioned media and the cell lysates was compared by
western blot analysis (Nalcayama et al., 2001, Dev. Biol. in press).
Conditioned
media was concentrated before western blotting and the proteins were then
visualized. Transient transfection-based expression was carried out using 293T
cells, and ten-fold concentrated conditioned media was analyzed as described
above.
For mid-scale preparation, stable clones were cultured in CL 1000 flasles
(Integra Biosciences, Ijamsville, MD) in 293 SFM II serum-free media and
2 0 conditioned media was collected every 2-3 days. An expression level of
approximately 3-4 ~.g/ml was obtained. The FLAG-tagged proteins were purified
from 500 ml of the collected supernatant by a single-step of affinity
chromatography using an anti-FLAG M2 affinty gel (Sigma) under high-salt
conditions (Piccolo et al., 1997, Cell 91:407-16). The protein concentration
of
2 5 each preparation was determined by western blot analysis with M2 and
comparison with a FLAG-bacterial alkaline phosphatase standard (Sigma).
Example 6: Production of Anti-CHL2 Polypeptide Antibodies
Antibodies to CHL2 polypeptides may be obtained by immunization with
3 0 purified protein or with CHL2 peptides produced by biological or chemical
synthesis. Suitable procedures for generating antibodies include those
described
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in Hudson and Bay, Practical Immunology (2nd ed., Blackwell Scientific
Publications).
In one procedure for the production of antibodies, aumals (typically mice
or rabbits) are injected with a CHL2 antigen (such as a CHL2 polypeptide), and
those with sufficient serum titer levels as determined by ELISA are selected
for
hybridoma production. Spleens of immunized animals are collected and prepared
as single cell suspensions from which splenocytes are recovered. The
splenocytes
are fused to mouse myeloma cells (such as Sp2/0-Agl4 cells), are first
incubated
in DMEM with 200 U/mL penicillin, 200 ~g/mL streptomycin sulfate, and 4 mM
l0 glutamine, and are then incubated in HAT selection medium (hypoxanthine,
aminopterin, and thymidine). After selection, the tissue culture supernatants
are
taken from each fusion well and tested for anti-CHL2 antibody production by
ELISA.
Alternative procedures for obtaining anti-CHL2 antibodies may also be
employed, such as the immunization of transgenic mice harboring human Ig loci
for production of human antibodies, and the screening of synthetic antibody
libraries, such as those generated by mutagenesis of an antibody variable
domain.
Example 7: Biological Activity of Murine CHL2 in Xehopus emb os
2 o Chordin is known to dorsalize the gastrulating Xehopus embryo by
inhibiting the activity of BMP4. The effects of CHL2 on Xehopus embryo
development were examined as follows. The Eco RI - Not I fragment of
pSPORTmCHL2 was first cloned into pCS2+ (Rupp et al., 1994, Genes Dev.
8:1311-23), linearized with Not I, and capped mRNAs were synthesized with SP6
2 5 polymerase and quantified (Nishinakamura et al., 1999, Dev. Biol. 216:481-
90).
The pSPORTmCHL2 plasmid was also linearized directly with Not I and
transcribed with T7 polymerase. Both constructs induced secondary axis
formation. As a negative control, elongation factor 1 (EF1) RNA was
synthesized.
3 0 Each RNA sample was injected into two ventral blastomeres of a four-cell
stage Xenopus embryo. Following injection, the embryos were cultured in 10%
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Steinberg's solution for 48 hours and were then scored for ectopic axis
(Nishinakamura et al., 1999; Figure 9). When blastomeres were injected with 1
pg of marine CHL2 RNA, the axis duplication rates ranged from 77 to 87%,
whereas the rate for uninfected control embryos and EF1 RNA-injected embryos
was 0%. As a positive control, experiments were also performed using marine
CHL1 RNA. In these experiments, an axis duplication rate of 83% was obtained
when 10-30pg of CHLl RNA was used. Thus, marine CHL2 RNA was active in
antagonizing the endogenous ventralizing factor (presumably, BMP4).
Example 8: Marine CHL2 Polypeptide Inhibition of BMP4 Action
The formation of chordin-CHL1 complexes is known to inhibit BMP4
function. The similar axis duplication activity observed for CHL2 polypeptide
(Example 7) suggested that CHL2 polypeptide also inhibited BMP4 action
directly. CD34+/CD3lh' lymphohematopoietic progenitor cells,
CD34+/CD311°
erythro-myeloid progenitor cells, and CD45+ myelomonocytic cells have been
shown to' be dependent on the presence of 0.15 to 2 ng/ml of BMP4 during the
differentiation of mouse embryonic stem (ES) cells (Nakayama et al., 2000,
Blood
95:2275:83).
The ability of CHL2 polypeptide to iWibit BMP4 action directly was
2 0 examined as follows. Rosa26 ES cells were transferred to a fibronectin-
coated
plate, adjusted for two days in serum-free medium, and then subjected to
differentiation in serum-free methylcellulose medium in the presence of 0.9
ng/ml
BMP4 (Nalcayama et al., 2000). A BMP4 concentration of 0.9 ng/ml resulted in a
near maximal level. Embryoid bodies (EBs) were collected on day 7 and
2 5 dissociated with collagenase. The cells were stained with monoclonal
antibodies
for hematopoietic progenitor cell markers, such as CD31 and CD34, and the
stained samples were analyzed on a FACScan (Becton Dickinson, San Jose, CA).
When affinity purified mCHL2-FLAG was added over 100 ng/ml, the
CD34-/CD31+ and CD34+/CD31+ cell populations were reduced to background
3 0 levels (i.e., levels achieved without BMP4; see Figure 10). These results
suggest
that the FLAG-tagged mCHL2 protein was able to inhibit the action of BMP4 ifa
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vitt~o.
Example 9: Marine CHL2 Polypeptide Inhibition of BMP2 and BMP4 Action
Both BMP2 and BMP4 are known to induce alkaline phospatase expression
in C2C12 myoblastic cells. CHL2 polypeptide inhibition of BMP2 and BMP4
action was demonstrated in a C2C12 alkaline phospatase assay.
The promyoblast cell line C2C12 (ATCC accession no. CRL-1772) was
maintained in DMEM containing 10% fetal bovine serum (FBS) and antibiotics at
37°C in a humidified atmosphere of 5% COa. Because receptor sensitivity
may
l0 decrease in cells that have been highly passaged, cells were discarded
after being
passaged between 10 and 15 times. C2C12 cells were plated in 96-well
microtiter
plates in 100 ~,1 of DMEM containing 2% calf serum and antibiotics at a
density
of 3 x 104 cells/well. To avoid excessive drying, the peripheral wells of the
microtiter plates were filled with 200 w1 of DMEM alone. Cells were then
incubated overnight at 37°C in a humidified atmosphere of 5% C02.
Following plating, C2C12 cells were exposed to serial dilutions (1.47,
2.93, 5.9, 11.7, 23.4, 46.9, 93.8, 187.5, 375, 750, and 1500 ng/ml) of marine
CHL2-FLAG in the presence of either 909 ng/ml (35 nM) BMP2 or 566 ng/ml (22
nM) BMP4. Cells were then incubated for three days at 37°C in a
humidified
2 0 atmosphere of 5% CO~. Following incubation, the media was removed, the
cells
were rinsed with 0.1 M Tris, pH 7.4, and 150 ~,1 of glycine buffer (0.1 M
glycine,
1 mM MgCl2, pH 10.5) contaiiung 0.1% IGEPAL CA-630 (Sigma), was added to
the wells. The cells were then frozen at -80°C and thawed, 50 ~,l of
cell
supernatant was removed for use in Bradford protein assays, and 100 ~,1 of p-
2 5 Nitrophenyl phosphate, disodium (Sigma; diluted to 4 mg/ml in glycine
buffer)
was added to the remaining cell supenlatant. This mixture was incubated at
37°C
for 30 minutes and 50 ~,1 of O.SN NaOH was added to stop the reaction. The
plates were then read at 405-410 nm. CHL2-FLAG was found to inhibit both
BMP2 and BMP4 action in a dose dependent manner (Figure 11).
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Example 10: Direct Interaction of CHL2 PolXpeptide and BMPs
The direct interaction of marine CHL1 polypeptide with human BMP4,
BMPS, BMP6, and TGF(32 has been previously demonstrated (Nakayama et al.,
2001). Similar immunoprecipitation experiments were performed using marine
CHL2 polypeptide, with the exception that mCHL2-FLAG protein was used at
600 ng/ml. The mCHL2-FLAG protein was found to co-immiuloprecipitate with
BMP2, BMP4, BMPS, BMP6, GDFS (BMP14), or activin A (Figure 12).
However, a high concentration of BMPS was required to show detectable levels
of
interaction with mCHL2-FLAG. CHL2 might have weaker affinities with BMPS
than with other BMPs. Similar to mCHLl, activin A - another TGF(3 superfamily
member - showed no sign of interaction with CHL2 under either set of
conditions.
However, in contrast to marine CHL1, TGF[3 interaction was never observed.
BMP2 and BMP4 form a separate subfamily from BMPS, BMP6, and BMP7
(Celeste et al., 1990, P~oc. Natl. Acad. Sci. U.S.A. 87:9843-47). Thus, both
CHLl
and CHL2 polypeptides may be pan-BMP binding proteins.
Example 11: Expression of CHL2 Polypeptide in Transgenic Mice
To assess the biological activity of CHL2 polypeptide, a construct
encoding a CHL2 polypeptide/Fc fusion protein under the control of a liver
2 0 specific ApoE promoter is prepared. The delivery of this construct is
expected to
cause pathological changes that are informative as to the function of CHL2
polypeptide. Similarly, a construct containing the full-length CHL2
polypeptide
under the control of the beta actin promoter is prepared. The delivery of this
construct is expected to result in ubiquitous expression.
2 5 To generate these constructs, PCR is used to amplify template DNA
sequences encoding a CHL2 polypeptide using primers that correspond to the 5'
and 3' ends of the desired sequence and which incorporate restriction enzyme
sites to permit insertion of the amplified product into an expression vector.
Following amplification, PCR products are gel purified, digested with the
3 0 appropriate restriction enzymes, and ligated into an expression vector
using
standard recombinant DNA techniques. For example, amplified CHL2
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polypeptide sequences can be cloned into an expression vector under the
control
of the human (3-actin promoter as described by Graham et al., 1997, Nature
Gefaetics, 17:272-74 and Ray et al., 1991, Gehes Dev. 5:2265-73.
Following ligation, reaction mixtures are used to transform an E. coli host
strain by electroporation and transformants are selected for drug resistance.
Plasmid DNA from selected colonies is isolated and subjected to DNA
sequencing to confirm the presence of an appropriate insert and absence of
mutation. The CHL2 polypeptide expression vector is purified through two
rounds of CsCI density gradient centrifugation, cleaved with a suitable
restriction
enzyme, and the linearized fragment containing the CHL2 polypeptide transgene
is purified by gel electrophoresis. The purified fragment is resuspended in 5
mM
Tris, pH 7.4, and 0.2 mM EDTA at a concentration of 2 mg/mL.
Single-cell embryos from BDF1 x BDF1 bred mice are injected as
described (PCT Pub. No. WO 97/23614). Embryos are cultured overnight in a
COz incubator and 15-20 two-cell embryos are transferred to the oviducts of a
pseudopregnant CD1 female mice. Offspring obtained from the implantation of
microinjected embryos are screened by PCR amplification of the integrated
transgene in genomic DNA samples as follows. Ear pieces are digested in 20 mL
ear buffer (20 mM Tris, pH 8.0, 10 mM EDTA, 0.5% SDS, and 500 mg/mL
2 0 proteinase K) at 55°C overnight. The sample is then diluted with
200 mL of TE,
and 2 mL of the ear sample is used in a PCR reaction using appropriate
primers.
At 8 weeks of age, transgenic founder animals and control animals are
sacrificed for necropsy and pathological analysis. Portions of spleen are
removed
and total cellular RNA isolated from the spleens using the Total RNA
Extraction
2 5 Kit (Qiagen) and transgene expression determined by RT-PCR. RNA recovered
from spleens is converted to cDNA using the SuperScriptTM Preamplification
System (Gibco-BRL) as follows. A suitable primer, located in the expression
vector sequence and 3' to the CHL2 polypeptide transgene, is used to prime
cDNA synthesis from the transgene. transcripts. Ten mg of total spleen RNA
from
3 0 transgenic founders and controls is incubated with 1 mM of primer for 10
minutes
at 70°C and placed on ice. The reaction is then supplemented with 10 mM
Tris-
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HCI, pH 8.3, 50 mM ICI, 2.5 mM MgCl2, 10 mM of each dNTP, 0.1 mM DTT,
and 200 U of Superscript II reverse transcriptase. Following incubation for 50
minutes at 42°C, the reaction is stopped by heating for 15 minutes at
72°C and
digested with 2U of RNase H for 20 minutes at 37°C. Samples are then
amplified
by PCR using primers specific for CHL2 polypeptide.
Example 12: Biological Activity of CHL2 Polypeptide in Trans~enic Mice
Prior to euthanasia, transgenic animals are weighed, anesthetized by
isofluorane and blood drawn by cardiac puncture. The samples are subjected to
hematology and serum chemistry analysis. Radiography is performed after
terminal exsanguination. Upon gross dissection, major visceral organs are
subject
to weight analysis.
Following gross dissection, tissues (i.e., liver, spleen, pancreas, stomach,
the entire gastrointestinal tract, kidney, reproductive organs, skin and
mammary
glands, bone, brain, heart, lung, thymus, trachea, esophagus, thyroid,
adrenals,
urinary bladder, lymph nodes and skeletal muscle) are removed and fixed in 10%
buffered Zn-Formalin for histological examination. After fixation, the tissues
are
processed into paraffin blocks, and 3 mm sections are obtained. All sections
are
stained with hematoxylin and exosin, and are then subjected to histological
2 0 analysis.
The spleen, lymph node, and Peyer's patches of both the transgenic and
the control mice are subjected to immunohistology analysis with B cell and T
cell
specific antibodies as follows. The formalin fixed paraffin embedded sections
are
deparaffinized and hydrated in deionized water. The sections are quenched with
3% hydrogen peroxide, blocked with Protein Block (Lipshaw, Pittsburgh, PA),
and incubated in rat monoclonal anti-mouse B220 and CD3 (Harlan, Indianapolis,
Il~. Antibody binding is detected by biotinylated rabbit anti-rat
immunoglobulins
and peroxidase conjugated streptavidin (BioGenex, San Ramon, CA) with DAB
as a chromagen (BioTek, Santa Barbara, CA). Sections are counterstained with
3 0 hematoxylin.
CA 02401175 2002-08-22
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- 109 -
After necropsy, MLN and sections of spleen and thymus from transgenic
animals and control littermates are removed. Single cell suspensions are
prepared
by gently grinding the tissues with the flat end of a syringe against the
bottom of a
100 mm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). Cells are
washed twice, comited, and approximately 1 x 106 cells from each tissue are
then
incubated for 10 minutes with 0.5 ~,g CD16/32(FcyIII/II) Fc block in a 20 ~L
voltune. Samples axe then stained for 30 minutes at 2-8°C in a 100 ~L
volume of
PBS (lacking Ca~ and Mg+), 0.1% bovine serum albumin, and 0.01% sodium
azide with 0.5 ~,g antibody of FITC or PE-conjugated monoclonal antibodies
against CD90.2 (Thy-1.2), CD45R (B220), CDllb(Mac-1), Gr-1, CD4, or CD8
(PharMingen, San Diego, CA). Following antibody binding, the cells are washed
and then analyzed by flow cytometry on a FACScan (Becton Dickinson).
While the present invention has been described in terms of the preferred
embodiments, it is understood that variations and modifications will occur to
those skilled in the art. Therefore, it is intended that the appended claims
cover
all such equivalent variations that come within the scope of the invention as
claimed.
CA 02401175 2002-08-22
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SEQUENCE LISTING
<110> Zhang, Ke
Cam, Linh
Nakayama, Naoki
<120> Chordin-Like-2 Molecules and Uses Thereof
<130> 01-005
<140> 60/186,462
<141> 2000-03-02
<160> 21
<170> PatentIn Ver. 2.0
<210> 1
<211> 1839
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (471)..(1748)
<220>
<221> sig-peptide
<222> (471) . . (530)
<400> 1
cctgccgagg cgtgcacagc ggCagCgCtC aaCCtCCCCC gcgccgccac cgagggtctt 60
gtCgCCCCa.C CgCgCCCCag aCCCgCgCCg gaCCCCgCgC CgCCgCgCCg ccgccagcca 120
gCgCCdCagg gaCaCtgCaC CCCggtgaCC gcaccccgca gatcccggtt ctctagctag 180
CaCCttCtCC CtCtCtgCCa tagCCttttt CttCatttCC CCaaCtaatt tCtCtCtCtC 240
tCtCtCtCt C tCt CtCtCtC tCtClCtCaC tCtCtCtCtC ttCtCCtCgt CCCCCCCCCa 3OO
CCgtCCtCtC atCCtCaCCt tagaCC'tCtC CtgtCCttgg CtCCtCttCa tCtttgCttt 360
tCCgaCtCCt CaagCagCgg tCCtaCttgg tCCtCtgagg aCttaCttgt gtCCttatCt 420
cactttctcc cggctcatcc cggggttgtc tgaccttggg acaaggaagg atg gtt 476
Met Val
1
ccc ggg gtg agg atc atc ccc tct ttg ctg gga ctc gtg atg ttc tgg 524
Pro Gly Val Arg Ile Ile Pro Ser Leu Leu Gly Leu Val Met Phe Trp
10 15
CtC CCg ttg gaC tcg caa gca cta tcc cgc tcg ggc aaa gtc tgc ctt 572
Leu Pro Leu Asp Ser Gln Ala Leu Ser Arg Ser Gly Lys Val Cys Leu
20 25 30
ttc ggt gaa aag ata tat acc ccc ggc cag agc tgg Cac CCC tac ttg 620
Phe Gly Glu Lys Ile Tyr Thr Pro Gly Gln Ser Trp His Pro Tyr Leu
35 40 45 50
1
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gaa cca caa ggc acg ata tac tgc gtg cgc tgt acc tgc tct gag aat 668
Glu Pro Gln Gly Thr Ile Tyr Cys Val Arg Cys Thr Cys Ser Glu Asn
55 60 65
gga cat gtg aat tgt taC CgC CtC CgC tgC CCa CCC Ctt CdC tgC tCa 716
Gly His Val Asn Cys Tyr Arg Leu Arg Cys Pro Pro Leu His Cys Ser
70 75 80
cag cct gtg atg gag cca cag caa tgc tgt ccc agg tgt gtg gat cct 764
Gln Pro Val Met Glu Pro Gln Gln Cys Cys Pro Arg Cys Val Asp Pro
85 90 95
cat gtc CCC tCt ggc ctc cga gtt CCC Cta aag tCC tgc cag Ctc aat 812
His Val Pro Ser Gly Leu Arg Val Pro Leu Lys Ser Cys Gln Leu Asn
100 105 110
gag acc aca tac caa cat gga gag atc ttc agt gcc cag gag ctg ttc 860
Glu Thr Thr Tyr Gln His Gly Glu Ile Phe Ser Ala Gln Glu Leu Phe
115 120 125 130
cct gcc cgc ctg tcc aac cag tgt gtc ctg tgt agc tgt att gaa ggc 908
Pro Ala Arg Leu Ser Asn Gln Cys Val Leu Cys Ser Cys Ile Glu Gly
135 140 145
cac act tac tgt ggt ctc atg acc tgt cct gaa ccc agc tgc ccc acc 956
His Thr Tyr Cys Gly Leu Met Thr Cys Pro Glu Pro Ser Cys Pro Thr
150 155 160
aca ctc cct ctg cct gat tcc tgc tgt cag acc tgc aaa gac agg aca 1004
Thr Leu Pro Leu Pro Asp Ser Cys Cys Gln Thr Cys Lys Asp Arg Thr
165 170 175
act gag agt tcc aca gaa gaa aac ttg aca cag ctg cag cat gga gag 1052
Thr Glu Ser Ser Thr Glu Glu Asn Leu Thr Gln Leu Gln His Gly Glu
180 185 190
aga cat tcc cag gat cca tgc tcg gag agg aga ggc ccc.agc acg cca 1100
Arg His Ser Gln Asp Pro Cys Ser Glu Arg Arg Gly Pro Ser Thr Pro
195 200 205 210
gCC CCC aCC agC Ctc agc tCC CCt Ctg ggc ttC atC CCt CgC CaC ttC 1148
Ala Pro Thr Ser Leu Ser Ser Pro Leu Gly Phe Ile Pro Arg His Phe
215 220 225
cag tca gta gga atg ggc agc aca acc atc aag att atc ttg aag gag 1196
Gln Ser Val Gly Met Gly Ser Thr Thr Ile Lys Ile Ile Leu Lys Glu
230 235 240
aaa cat aaa aaa get tgc aca cac aat ggg aag aca tac tcc cat ggg 1244
Lys His Lys Lys Ala Cys Thr His Asn Gly Lys Thr Tyr Ser His Gly
245 250 255
gag gtg tgg cac ccc act gtg ctc tcc ttt ggc CCC atg ccc tgc atc 1292
Glu Val Trp His Pro Thr Val Leu Ser Phe Gly Pro Met Pro Cys Ile
260 265 270
ctg tgc aca tgt att gat ggc tac cag gac tgc cac cgt gtg acc tgc 1340
Leu Cys Thr Cys Ile Asp Gly Tyr Gln Asp Cys His Arg Val Thr Cys
275 280 285 290
ccc acc caa tat ccc tgc agt caa ccc aag aaa gtg get ggg aag tgc 1388
2
CA 02401175 2002-08-22
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Pro Thr Gln Tyr Pro Cys Ser Gln Pro Lys Lys Val Ala Gly Lys Cys
295 300 305
tgc aag atc tgc cca gag gac gag gcg gaa gat gac cac agt gag gtc 1436
Cys Lys Ile Cys Pro Glu Asp Glu Ala Glu Asp Asp His Ser Glu Val
310 315 320
att tcc acc cgg tgt ccc aag gta cca ggc cag ttc cag gtg tac acg 1484
Ile Ser Thr Arg Cys Pro Lys Val Pro Gly Gln Phe Gln Val Tyr Thr
325 330 335
ttg gca tct cca agc cca gac agc cta cac cgc ttt gtc ctg gag cat 1532
Leu Ala Ser Pro Ser Pro Asp Ser Leu His Arg Phe Val Leu Glu His
340 345 350
gaa gcc tct gac cag gta gag atg tac att tgg aag ctg gtg aaa gga 1580
Glu Ala Ser Asp Gln Val Glu Met Tyr Ile Trp Lys Leu Val Lys Gly
355 360 365 370
atc tac cac ttg gtt cag atc aag aga gtc agg aag caa gat ttc cag 1628
Ile Tyr His Leu Val Gln Ile Lys Arg Val Arg Lys Gln Asp Phe Gln
375 380 385
aaa gag get cag aac ttc cgg ctg ctc acc ggc acc cat gaa ggt tac 1676
Lys Glu Ala Gln Asn Phe Arg Leu Leu Thr Gly Thr His Glu Gly Tyr
390 395 400
tgg acc gtc ttc cta gcc cag act cca gag ctg aaa gtt aca gcc agc 1724
Trp Thr Val Phe Leu Ala Gln Thr Pro Glu Leu Lys Val Thr Ala Ser
405 410 415
cca gac aaa gtg acc aag aca tta tagcaaggac ctaaagagtt gcagatacga 1778
Pro Asp Lys Val Thr Lys Thr Leu
420 425
gttttattgg ttttgttatt atatattaat aaagaagtcg cattaccctc tCCCCCCCaC 1838
t 1839
<210> 2
<211> 426
<212> PRT
<213> Mus musculus
<400> 2
Met Val Pro Gly Val Arg Ile Ile Pro Ser Leu Leu Gly Leu Val Met
1 5 10 15
Phe Trp Leu Pro Leu Asp Ser Gln Ala Leu Ser Arg Ser Gly Lys Val
20 25 30
Cys Leu Phe Gly Glu Lys Ile Tyr Thr Pro Gly Gln Ser Trp His Pro
35 40 45
Tyr Leu Glu Pro Gln Gly Thr Ile Tyr Cys Val Arg Cys Thr Cys Ser
50 55 60
Glu Asn Gly His Val Asn Cys Tyr Arg Leu Arg Cys Pro Pro Leu His
65 70 75 80
3
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Cys Ser Gln Pro Val Met Glu Pro Gln Gln Cys Cys Pro Arg Cys Val
85 90 95
Asp Pro His Val Pro Ser Gly Leu Arg Val Pro Leu Lys Ser Cys Gln
100 105 110
Leu Asn Glu Thr Thr Tyr Gln His Gly Glu I1e Phe Ser Ala Gln Glu
115 120 125
Leu Phe Pro Ala Arg Leu Ser Asn Gln Cys Val Leu Cys Ser Cys Ile
130 135 140
Glu Gly His Thr Tyr Cys Gly Leu Met Thr Cys Pro Glu Pro Ser Cys
145 150 155 160
Pro Thr Thr Leu Pro Leu Pro Asp Ser Cys Cys Gln Thr Cys Lys Asp
165 170 175
Arg Thr Thr Glu Ser Ser Thr Glu Glu Asn Leu Thr Gln Leu Gln His
180 185 190
Gly Glu Arg His Ser Gln Asp Pro Cys Ser Glu Arg Arg Gly Pro Ser
195 200 205
Thr Pro Ala Pro Thr Ser Leu Ser Ser Pro Leu Gly Phe Ile Pro Arg
210 215 220
His Phe Gln Ser Val Gly Met Gly Ser Thr Thr Ile Lys Ile Ile Leu
225 230 235 240
Lys Glu Lys His Lys Lys Ala Cys Thr His Asn Gly Lys Thr Tyr Ser
245 250 255
His Gly Glu Val Trp His Pro Thr Val Leu Ser Phe Gly Pro Met Pro
260 265 270
Cys Ile Leu Cys Thr Cys Ile Asp Gly Tyr Gln Asp Cys His Arg Val
275 280 285
Thr Cys Pro Thr Gln Tyr Pro Cys Ser Gln Pro Lys Lys Val Ala Gly
290 295 300
Lys Cys Cys Lys Ile Cys Pro Glu Asp Glu Ala Glu Asp Asp His Ser
305 310 315 320
Glu Val Ile Ser Thr Arg Cys Pro Lys Val Pro Gly Gln Phe Gln Val
325 330 335
Tyr Thr Leu Ala Ser Pro Ser Pro Asp Ser Leu His Arg Phe Val Leu
340 345 350
Glu His Glu Ala Ser Asp Gln Val Glu Met Tyr Ile Trp Lys Leu Val
355 360 365
Lys Gly Ile Tyr His Leu Val Gln Ile Lys Arg Val Arg Lys Gln Asp
370 375 380
Phe Gln Lys Glu Ala Gln Asn Phe Arg Leu Leu Thr Gly Thr His Glu
385 390 395 400
Gly Tyr Trp Thr Val Phe Leu Ala Gln Thr Pro Glu Leu Lys Val Thr
4
CA 02401175 2002-08-22
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Ala Ser Pro Asp Lys Val Thr Lys Thr Leu
420 425
<210> 3
<211> 405
<212> PRT
<213> Mus musculus
<400> 3
Asp Ser Gln Ala Leu Ser Arg Ser Gly Lys Val Cys Leu Phe Gly Glu
1 5 10 15
Lys Ile Tyr Thr Pro Gly Gln Ser Trp His Pro Tyr Leu Glu Pro Gln
20 25 30
Gly Thr Ile Tyr Cys Val Arg Cys Thr Cys Ser Glu Asn Gly His Val
35 40 45
Asn Cys Tyr Arg Leu Arg Cys Pro Pro Leu His Cys Ser Gln Pro Val
50 55 60
Met Glu Pro Gln Gln Cys Cys Pro Arg Cys Val Asp Pro His Val Pro
65 70 75 80
Ser Gly Leu Arg Val Pro Leu Lys Ser Cys Gln Leu Asn Glu Thr Thr
85 90 95
Tyr Gln His Gly Glu Ile Phe Ser Ala Gln Glu Leu Phe Pro Ala Arg
100 105 110
Leu Ser Asn Gln Cys Val Leu Cys Ser Cys Ile Glu Gly His Thr Tyr
115 l20 125
Cys Gly Leu Met Thr Cys Pro Glu Pro Ser Cys Pro Thr Thr Leu Pro
130 135 140
Leu Pro Asp Ser Cys Cys Gln Thr Cys Lys Asp Arg Thr Thr Glu Ser
145 150 155 160
Ser Thr Glu Glu Asn Leu Thr Gln Leu Gln His Gly Glu Arg His Ser
165 170 175
Gln Asp Pro Cys Ser Glu Arg Arg Gly Pro Ser Thr Pro Ala Pro Thr
180 185 190
Ser Leu Ser Ser Pro Leu Gly Phe Ile Pro Arg His Phe Gln Ser Val
195 200 205
Gly Met Gly Ser Thr Thr Ile Lys Ile Ile Leu Lys Glu Lys His Lys
210 215 220
Lys Ala Cys Thr His Asn Gly Lys Thr Tyr Ser His Gly Glu Val Trp
225 230 235 240
His Pro Thr Val Leu Ser Phe Gly Pro Met Pro Cys Ile Leu Cys Thr
245 250 255
Cys Ile Asp Gly Tyr Gln Asp Cys His Arg Val Thr Cys Pro Thr Gln
CA 02401175 2002-08-22
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Tyr Pro Cys Ser Gln Pro Lys Lys Val Ala Gly Lys Cys Cys Lys Ile
275 280 285
Cys Pro Glu Asp G1u Ala Glu Asp Asp His Ser Glu Val Ile Ser Thr
290 295 300
Arg Cys Pro Lys Val Pro Gly Gln Phe Gln Val Tyr Thr Leu Ala Ser
305 310 315 320
Pro Ser Pro Asp Ser Leu His Arg Phe Val Leu Glu His Glu Ala Ser
325 330 335
Asp Gln Val G1u Met Tyr Ile Trp Lys Leu Val Lys Gly Ile Tyr His
340 345 350
Leu Val Gln Ile Lys Arg Val Arg Lys Gln Asp Phe Gln Lys Glu Ala
355 360 365
Gln Asn Phe Arg Leu Leu Thr Gly Thr His Glu Gly Tyr Trp Thr Val
370 375 380
Phe Leu Ala Gln Thr Pro Glu Leu Lys Val Thr Ala Ser Pro Asp Lys
385 390 395 400
Val Thr Lys Thr Leu
405
<210> 4
<211> 1570
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (184)..(1470)
<220>
<221> sig~eptide
<222> (184)..(243)
<400> 4
agaCCtCCCt tCCtgCCCtC CtttCCtgCC CICCgCtgCt tCCtggCCCt tCtCCgaCCC 60
cgctctagca gcagacctcc tggggtctgt gggttgatct gtggcccctg tgcctccgtg 120
tCCttttCgt CtCCCttCCt ~CCCgaC'tCCg CtCCCggaCC agcggcctga ccctggggaa 180
agg atg gtt ccc gag gtg agg gtc ctc tcc tcc ttg ctg gga ctc gcg 228
Met Val Pro Glu Val Arg Val Leu Ser Ser Leu Leu Gly Leu Ala
1 5 10 15
Ctg Ctc tgg ttC CCC Ctg gac tcc cac get cga gcc cgc CCa gac atg 276
Leu Leu Trp Phe Pro Leu Asp Ser His Ala Arg Ala Arg Pro Asp Met
20 25 30
ttc tgc ctt ttc cat ggg aag aga tac tcc ccc ggc gag agc tgg cac 324
Phe Cys Leu Phe His Gly Lys Arg Tyr Ser Pro Gly Glu Ser Trp His
35 40 45
6
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ccc tac ttg gag cca caa ggc ctg atg tac tgc ctg cgc tgt acc tgc 372
Pro Tyr Leu Glu Pro Gln Gly Leu Met Tyr Cys Leu Arg Cys Thr Cys
50 55 60
tca gag ggc gcc cat gtg agt tgt tac cgc ctc cac tgt ccg cct gtc 420
Ser Glu Gly Ala His Val Ser Cys Tyr Arg Leu His Cys Pro Pro Val
65 70 75
CdC tgC CCC Cag CCt gtg acg gag cca cag Caa tgc tgt ccc aag tgt 468
His Cys Pro Gln Pro Val Thr Glu Pro Gln Gln Cys Cys Pro Lys Cys
80 85 90 95
gtg gaa cct cac act CCC tct gga ctc cgg gcc cca cca aag tCC tgc 516
Val Glu Pro His Thr Pro Ser Gly Leu Arg Ala Pro Pro Lys Ser Cys
100 105 110
cag cac aac ggg acc atg tac caa cac gga gag atc ttc agt gcc cat 564
Gln His Asn Gly Thr Met Tyr Gln His Gly Glu Ile Phe Ser Ala His
115 120 125
gag Ctg ttC CCC tCC CgC Ctg ccc aac cag tgt gtc ctc tgc agc tgc 612
Glu Leu Phe Pro Ser Arg Leu Pro Asn Gln Cys Val Leu Cys Ser Cys
130 135 140
aca gag ggc cag atc tac tgc ggc ctc aca acc tgc CCC gaa cca ggc 660
Thr Glu Gly Gln Ile Tyr Cys Gly Leu Thr Thr Cys Pro Glu Pro Gly
145 150 155
tgC CCa gca CCC CtC CCg Ctg CCa gaC tCC tgC tgc caa gcc tgc aaa 708
Cys Pro Ala Pro Leu Pro Leu Pro Asp Ser Cys Cys Gln Ala Cys Lys
160 165 170 175
gat gag gca agt gag caa tcg gat gaa gag gac agt gtg cag tcg ctc 756
Asp Glu Ala Ser Glu Gln Ser Asp Glu Glu Asp Ser Val Gln Ser Leu
180 185 190
cat ggg gtg aga cat cct cag gat cca tgt tcc agt gat get ggg aga 804
His Gly Val Arg His Pro Gln Asp Pro Cys Ser Ser Asp Ala Gly Arg
195 200 205
aag aga ggC CCg ggC aCC CCa gCC CCC aCt ggc ctc agc gcc CCt ctg 852
Lys Arg Gly Pro Gly Thr Pro Ala Pro Thr Gly Leu Ser Ala Pro Leu
210 215 220
agc ttc atc cct CgC CaC ttc aga ccc aag gga gca ggc agc aca act 900
Ser Phe Ile Pro Arg His Phe Arg Pro Lys Gly Ala Gly Ser Thr Thr
225 230 235
gtc aag atc gtc ctg aag gag aaa cat aag aaa gcc tgt gtg cat ggc 948
Val Lys Ile Val Leu Lys Glu Lys His Lys Lys Ala Cys Val His Gly
240 245 250 255
ggg aag acg tac tcc cac ggg gag gtg tgg Cac ccg gcc ttc cgt gcc 996
Gly Lys Thr Tyr Ser His Gly Glu Val Trp His Pro Ala Phe Arg Ala
260 265 270
ttc ggc ccc ttg ccc tgc atc cta tgc acc tgt gag gat ggc cgc cag 1044
Phe Gly Pro Leu Pro Cys Ile Leu Cys Thr Cys Glu Asp Gly Arg Gln
275 280 285
7
CA 02401175 2002-08-22
gaWO 01/64885 cgt gtg acc tgt CCC acc gag tac ccc tgc cgt cac PCs /USO x/06891
Asp Cys Gln Arg Val Thr Cys Pro Thr Glu Tyr Pro Cys Arg His Pro
290 295 300
gag aaa gtg get ggg aag tgc tgc aag att tgc cca gag gac aaa gca 1140
Glu Lys Val Ala Gly Lys Cys Cys Lys Ile Cys Pro Glu Asp Lys Ala
305 310 315
gac cct ggc cac agt gag atc agt tct acc agg tgt ccc aag gca ccg 1188
Asp Pro Gly His Ser Glu Ile Ser Ser Thr Arg Cys Pro Lys Ala Pro
320 325 330 335
ggc cgg gtc ctc gtc cac aca tcg gta tcc cca agc cca gac aac ctg 1236
Gly Arg Val Leu Val His Thr Ser Val Ser Pro Ser Pro Asp Asn Leu
340 345 350
cgt cgc ttt gcc ctg gaa cac gag gcc tcg gac ttg gtg gag atc tac 1284
Arg Arg Phe Ala Leu Glu His Glu Ala Ser Asp Leu Val Glu Ile Tyr
355 360 365
ctc tgg aag ctg gtg aaa gga atc ttc cac ttg act cag atc aag aaa 1332
Leu Trp Lys Leu Val Lys Gly Ile Phe His Leu Thr Gln Ile Lys Lys
370 375 380
gtc agg aag caa gac ttc cag aaa gag gca cag cac ttc cga ctg ctc 1380
Val Arg Lys Gln Asp Phe Gln Lys Glu Ala Gln His Phe Arg Leu Leu
385 390 395
get ggC CCC CdC gaa ggt cac tgg aac gtc ttc cta gcc cag acc ctg 1428
Ala Gly Pro His Glu Gly His Trp Asn Val Phe Leu Ala Gln Thr Leu
400 405 410 415
gag ctg aag gtc acg gcc agt cca gac aaa gtg acc aag aca 1470
Glu Leu Lys Val Thr Ala Ser Pro Asp Lys Val Thr Lys Thr
420 425
taacaaagac ctaacagttg cagatatgag ctgtataatt gttgttatta tatattaata 1530
aataagaagt tgcattaccc tcaaaaaaaa aaaaaaaaaa 1570
<210> 5
<211> 429
<212> PRT
<213> Homo Sapiens
<400> 5
Met Val Pro Glu Val Arg Val Leu Ser Ser Leu Leu Gly Leu Ala Leu
1 5 10 15
Leu Trp Phe Pro Leu Asp Ser His Ala Arg Ala Arg Pro Asp Met Phe
20 25 30
Cys Leu Phe His Gly Lys Arg Tyr Ser Pro Gly Glu Ser Trp His Pro
35 40 45
Tyr Leu Glu Pro Gln Gly Leu Met Tyr Cys Leu Arg Cys Thr Cys Ser
50 55 60
Glu Gly Ala His Val Ser Cys Tyr Arg Leu His Cys Pro Pro Val His
65 70 75 80
8
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Cys Pro Gln Pro Val Thr Glu Pro Gln Gln Cys Cys Pro Lys Cys Val
85 90 95
Glu Pro His Thr Pro Ser Gly Leu Arg Ala Pro Pro Lys Ser Cys Gln
100 105 110
His Asn Gly Thr Met Tyr Gln His Gly Glu Ile Phe Ser Ala His Glu
115 120 125
Leu Phe Pro Ser Arg Leu Pro Asn Gln Cys Val Leu Cys Ser Cys Thr
130 135 140
Glu Gly Gln Ile Tyr Cys Gly Leu Thr Thr Cys Pro Glu Pro Gly Cys
145 150 155 160
Pro Ala Pro Leu Pro Leu Pro Asp Ser Cys Cys Gln Ala Cys Lys Asp
165 170 175
Glu Ala Ser Glu Gln Ser Asp Glu Glu Asp Ser Val Gln Ser Leu His
180 185 190
Gly Val Arg His Pro Gln Asp Pro Cys Ser Ser Asp Ala Gly Arg Lys
195 200 205
Arg Gly Pro Gly Thr Pro Ala Pro Thr Gly Leu Ser Ala Pro Leu Ser
210 215 220
Phe Ile Pro Arg His Phe Arg Pro Lys Gly Ala Gly Ser Thr Thr Val
225 230 235 240
Lys Ile Val Leu Lys Glu Lys His Lys Lys Ala Cys Val His Gly Gly
245 250 255
Lys Thr Tyr Ser His Gly Glu Val Trp His Pro Ala Phe Arg Ala Phe
260 265 270
Gly Pro Leu Pro Cys Ile Leu Cys Thr Cys Glu Asp Gly Arg Gln Asp
275 280 ' 285
Cys Gln Arg Val Thr Cys Pro Thr Glu Tyr Pro Cys Arg His Pro Glu
290 295 300
Lys Val Ala Gly Lys Cys Cys Lys Ile Cys Pro Glu Asp Lys Ala Asp
305 310 315 320
Pro Gly His Ser Glu Ile Ser Ser Thr Arg Cys Pro Lys Ala Pro Gly
325 330 335
Arg Val Leu Val His Thr Ser Val Ser Pro Ser Pro Asp Asn Leu Arg
340 345 350
Arg Phe Ala Leu Glu His Glu Ala Ser Asp Leu Val Glu Ile Tyr Leu
355 360 365
Trp Lys Leu Val Lys Gly Ile Phe His Leu Thr Gln Ile Lys Lys Val
370 375 380
Arg Lys Gln Asp Phe Gln Lys Glu Ala Gln His Phe Arg Leu Leu Ala
385 390 . 395 400
9
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Gly Pro His Glu Gly His Trp Asn Val Phe Leu Ala Ulri TYir Z;eu "Glu
405 410 415
Leu Lys Val Thr Ala Ser Pro Asp Lys Val Thr Lys Thr
420 425
<210> 6
<211> 408
<212> PRT
<213> Homo Sapiens
<400> 6
Asp Ser His Ala Arg Ala Arg Pro Asp Met Phe Cys Leu Phe His Gly
Z 5 10 15
Lys Arg Tyr Ser Pro Gly Glu Ser Trp His Pro Tyr Leu Glu Pro Gln
20 25 30
Gly Leu Met Tyr Cys Leu Arg Cys Thr Cys 5er Glu Gly Ala His Val
35 40 45
Ser Cys Tyr Arg Leu His Cys Pro Pro Val His Cys Pro Gln Pro Val
50 55 60
Thr Glu Pro Gln Gln Cys Cys Pro Lys Cys Val Glu Pro His Thr Pro
65 70 75 80
Ser Gly Leu Arg Ala Pro Pro Lys Ser Cys Gln His Asn Gly Thr Met
85 90 95
Tyr Gln His Gly Glu Ile Phe Ser Ala His Glu Leu Phe Pro Ser Arg
100 105 110
Leu Pro Asn Gln Cys Val Leu Cys Ser Cys Thr Glu Gly Gln Ile Tyr
115 120 125
Cys Gly Leu Thr Thr Cys Pro Glu Pro Gly Cys Pro Ala Pro Leu Pro
130 135 140
Leu Pro Asp Ser Cys Cys Gln Ala Cys Lys Asp Glu Ala Ser Glu Gln
145 150 155 160
Ser Asp Glu Glu Asp Ser Val Gln Ser Leu His Gly Val Arg His Pro
165 170 175
Gln Asp Pro Cys Ser Ser Asp Ala Gly Arg Lys Arg Gly Pro Gly Thr
180 185 190
Pro Ala Pro Thr Gly Leu Ser Ala Pro Leu Ser Phe Ile Pro Arg His
195 200 205
Phe Arg Pro Lys Gly Ala Gly Ser Thr Thr Val Lys Ile Val Leu Lys
210 215 220
Glu Lys His Lys Lys Ala Cys Val His Gly Gly Lys Thr Tyr Ser His
225 230 235 ~ 240
Gly Glu Val Trp His Pro Ala Phe Arg Ala Phe Gly Pro Leu Pro Cys
245 250 255
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Ile Leu Cys Thr Cys Glu Asp Gly Arg Gln Asp Cys ~ln'~~~A'rg~ ~3a1~"'Thr
260 265 270
Cys Pro Thr Glu Tyr Pro Cys Arg His Pro Glu Lys Val Ala Gly Lys
275 280 285
Cys Cys Lys Ile Cys Pro Glu Asp Lys Ala Asp Pro Gly His Ser Glu
290 295 300
Ile Ser Ser Thr Arg Cys Pro Lys Ala Pro Gly Arg Val Leu Val His
305 310 315 320
Thr Ser Val Ser Pro Ser Pro Asp Asn Leu Arg Arg Phe Ala Leu Glu
325 330 335
His Glu Ala Ser Asp Leu Val Glu Ile Tyr Leu Trp Lys Leu Val Lys
340 345 350
Gly Ile Phe His Leu Thr Gln Ile Lys Lys Val Arg Lys Gln Asp Phe
355 360 365
Gln Lys Glu Ala Gln His Phe Arg Leu Leu Ala Gly Pro His Glu Gly
370 375 380
His Trp Asn Val Phe Leu Ala Gln Thr Leu Glu Leu Lys Val Thr Ala
385 390 395 400
Ser Pro Asp Lys Val Thr Lys Thr
405
<210> 7
<2l1> 283
<212> PRT
<213> Mus musculus
<400> 7 '
Asn Gly Glu Ala Ala Thr Ser Pro Met Leu Pro Ala Gly Pro Gly Pro
1 5 10 15
Glu Ala Pro Val Pro Ala Lys His Gly Ser Pro Gly Arg Pro Arg Asp
20 25 30
Pro Asn Thr Cys Phe Phe Glu Gly Gln Gln Arg Pro His Gly Ala Arg
35 40 45
Trp Ala Pro Asn Tyr Asp Pro Leu Cys Ser Leu Cys Ile Cys Gln Arg
50 55 60
Arg Thr Val Ile Cys Asp Pro Vah Val Cys Pro Pro Pro Ser Cys Pro
65 70 75 80
His Pro Val Gln Ala Leu Asp Gln Cys Cys Pro Val Cys Pro Glu Lys
85 90 95
Gln Arg Ser Arg Asp Leu Pro Ser Leu Pro Asn Leu Glu Pro Gly Glu
100 105 110
Gly Cys Tyr Phe Asp Gly Asp Arg Ser Trp Arg Ala Ala Gly Thr Arg
115 120 125
11
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Trp His Pro Val Val Pro Pro Phe Gly Leu Ile Lys ~ys-~A~a V'a~l""Cys
130 135 140
Thr Cys Lys Gly Ala Thr Gly Glu Val His Cys Glu Lys Val Gln Cys
145 150 155 160
Pro Arg Leu Ala Cys Ala Gln Pro Val Arg Ala Asn Pro Thr Asp Cys
165 170 175
Cys Lys Gln Cys Pro Val Gly Ser Gly Thr Asn Ala Lys Leu Gly Asp
180 185 190
Pro Met Gln Ala Asp Gly Pro Arg Gly Cys Arg Phe Ala Gly Gln Trp
195 200 205
Phe Pro Glu Asn Gln Ser Trp His Pro Ser Val Pro Pro Phe Gly Glu
210 215 220
Met Ser Cys Ile Thr Cys Arg Cys Gly Ala GIy VaI Pro His Cys Glu
225 230 235 240
Arg Asp Asp Cys Ser Pro Pro Leu Ser Cys Gly Sex Gly Lys Glu Ser
245 250 255
Arg Cys Cys Ser His Cys Thr Ala Gln Arg Ser Ser Glu Thr Arg Thr
260 265 270
Leu Pro Glu Leu Glu Lys Glu Ala Glu His Ser
275 280
<210> 8
<211> 955
<212> PRT
<213> Homo sapiens
<400> 8
Met Pro Ser Leu Pro Ala Pro Pro Ala Pro Leu Leu Leu Leu Gly Leu
1 5 10 15
Leu Leu Leu Gly Ser Arg Pro Ala Arg Gly Ala Gly Pro Glu Pro Pro
20 25 30
Val Leu Pro Ile Arg Ser Glu Lys Glu Pro Leu Pro Val Arg Gly Ala
35 40 45
Ala Gly Cys Thr Phe Gly Gly Lys Val Tyr Ala Leu Asp Glu Thr Trp
50 55 60
His Pro Asp Leu Gly Glu Pro Phe Gly Val Met Arg Cys Val Leu Cys
65 70 75 80
Ala Cys Glu Ala Pro Gln Trp Gly Arg Arg Thr Arg Gly Pro Gly Arg
85 90 95
Val Ser Cys Lys Asn Ile Lys Pro Glu Cys Pro Thr Pro Ala Cys Gly
100 105 110
Gln Pro Arg Gln Leu Pro Gly His Cys Cys Gln Thr Cys Pro Gln Glu
115 120 125
12
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Arg Ser Ser Ser Glu Arg Gln Pro Ser Gly Leu Ser I~he"'Glu'' T~"'Pro'
130 135 ~ 140
Arg Asp Pro Glu His Arg Ser Tyr Ser Asp Arg Gly Glu Pro Gly Ala
I45 150 I55 160
Glu Glu Arg Ala Arg Gly Asp Gly His Thr Asp Phe Val Ala Leu Leu
165 170 175
Thr Gly Pro Arg Ser Gln Ala Val Ala Arg Ala Arg Val Ser Leu Leu
I80 185 190
Arg Ser Ser Leu Arg Phe Ser Ile Ser Tyr Arg Arg Leu Asp Arg Pro
195 200 205
Thr Arg Ile Arg Phe Ser Asp Ser Asn Gly Ser Val Leu Phe Glu His
210 215 220
Pro Ala Ala Pro Thr Gln Asp Gly Leu Val Cys Gly Val Trp Arg Ala
225 230 235 240
Val Pro Arg Leu Ser Leu Arg Leu Leu Arg Ala Glu Gln Leu His Val
245 250 255
Ala Leu Val Thr Leu Thr His Pro Ser Gly Glu Val Trp Gly Pro Leu
260 265 270
Ile Arg His Arg Ala Leu Ala Ala Glu Thr Phe Ser Ala Ile Leu Thr
275 280 285
Leu Glu Gly Pro Pro Gln Gln Gly Val Gly Gly Tle Thr Leu Leu Thr
290 295 300
Leu Ser Asp Thr Glu Asp Ser Leu His Phe Leu Leu Leu Phe Arg Gly
305 310 315 320
Leu Leu G1u Pro Arg Ser Gly Gly Leu Thr Gln Val Pro Leu Arg Leu
325 330 335
Gln Ile Leu His Gln Gly Gln Le.u Leu Arg Glu Leu Gln Ala Asn Val
340 345 350
Ser Ala Gln Glu Pro Gly Phe Ala Glu Val Leu Pro Asn Leu Thr Val
355 360 365
Gln Glu Met Asp Trp Leu Val Leu Gly Glu Leu Gln Met Ala Leu Glu
370 375 380
Trp Ala Gly Arg Pro Gly Leu Arg Ile Ser Gly His Ile Ala Ala Arg
385 390 395 400
Lys Ser Cys Asp Val Leu Gln Ser Val Leu Cys Gly Ala Asp Ala Leu
405 410 415
Ile Pro Val Gln Thr Gly Ala Ala Gly Ser Ala Ser Leu Thr Leu Leu
420 425 430
Gly Asn Gly Ser Leu Ile Tyr Gln Val Gln Val Val Gly Thr Ser Ser
435 440 445
Glu Val Val Ala Met Thr Leu Glu Thr Lys Pro Gln Arg Arg Asp Gln
13
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450 455 460
Arg Thr Val Leu Cys His Met Ala Gly Leu Gln Pro Gly Gly His Thr
465 470 475 480
Ala Val Gly Ile Cys Pro Gly Leu Gly Ala Arg Gly Ala His Met Leu
485 490 495
Leu Gln Asn Glu Leu Phe Leu Asn Val Gly Thr Lys Asp Phe Pro Asp
500 505 510
Gly Glu Leu Arg Gly His Val Ala Ala Leu Pro Tyr Cys Gly His Ser
515 520 525
Ala Arg His Asp Thr Leu Pro Val Pro Leu Ala Gly Ala Leu Val Leu
530 535 540
Pro Pro Val Lys Ser Gln Ala Ala Gly His Ala Trp Leu Ser Leu Asp
545 550 555 560
Thr His Cys His Leu His Tyr Glu Val Leu Leu Ala Gly Leu Gly Gly
565 570 575
Ser Glu Gln Gly Thr Val Thr Ala His Leu Leu Gly Pro Pro Gly Thr
580 585 590
Pro Gly Pro Arg Arg Leu Leu Lys Gly Phe Tyr Gly Ser Glu Ala Gln
595 600 605
Gly Val Val Lys Asp Leu Glu Pro Glu Leu Leu Arg His Leu Ala Lys
610 615 620
Gly Met Ala Ser Leu Leu Ile Thr Thr Lys Gly Ser Pro Arg Gly Glu
625 630 635 640
Leu Arg Gly Gln Val His Ile Ala Asn Gln Cys Glu Val Gly Gly Leu
645 650 655
Arg Leu Glu Ala Ala Gly Ala Glu Gly Val Arg Ala Leu Gly Ala Pro
660 665 670
Asp Thr Ala Ser Ala Ala Pro Pro Val Val Pro Gly Leu Pro Ala Leu
675 680 685
Ala Pro Ala Lys Pro Gly Gly Pro Gly Arg Pro Arg Asp Pro Asn Thr
690 695 700
Cys Phe Phe Glu Gly Gln Gln Arg Pro His Gly Ala Arg Trp Ala Pro
705 710 715 720
Asn Tyr Asp Pro Leu Cys Ser Leu Cys Thr Cys Gln Arg Arg Thr Val
725 730 735
Ile Cys Asp Pro Val Val Cys Pro Pro Pro Ser Cys Pro His Pro Val
740 745 750
Gln Ala Pro Asp Gln Cys Cys Pro Val Cys Pro Glu Lys Gln Asp Val
755 760 765
Arg Asp Leu Pro Gly Leu Pro Arg Ser Arg Asp Pro Gly Glu Gly Cys
770 775 780
14
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Tyr Phe Asp Gly Asp Arg Ser Trp Arg Ala Ala Gly Thr Arg Trp His
785 790 795 800
Pro Val Val Pro Pro Phe Gly Leu Ile Lys Cys Ala Val Cys Thr Cys
80S 810 815
Lys Gly Gly Thr GIy Glu Val His Cys Glu Lys VaI Gln Cys Pro Arg
820 825 830
Leu Ala Cys Ala Gln Pro Val Arg Val Asn Pro Thr Asp Cys Cys Lys
835 840 845
Gln Cys Pro Val Gly Ser Gly Ala His Pro Gln Leu Gly Asp Pro Met
850 855 860
Gln Ala Asp Gly Pro Arg Gly Cys Arg Phe Ala Gly Gln Trp Phe Pro
865 870 875 880
Glu Ser Gln Ser Trp His Pro Ser Val Pro Pro Phe Gly Glu Met Ser
885 890 895
Cys Ile Thr Cys Arg Cys Gly Ala Gly Val Pro His Cys Glu Arg Asp
900 905 910
Asp Cys Ser Leu Pro Leu Ser Cys Gly Ser Gly Lys Glu Ser Arg Cys
915 920 925
Cys Ser Arg Cys Thr Ala His Arg Arg Pro Ala Pro Glu Thr Arg Thr
930 935 940
Asp Pro Glu Leu Glu Lys Glu Ala Glu Gly Ser
945 950 955
<210> 9
<211> 452
<212> PRT
<213> Homo sapiens
<400> 9
Met Gly Gly Met Lys Tyr Ile Phe Ser Leu Leu Phe Phe Leu Leu Leu
1 5 10 15
Glu Gly Gly Lys Thr Glu Gln Val Lys His Ser Glu Thr Tyr Cys Met
20 25 30
Phe Gln Asp Lys Lys Tyr Arg Val Gly Glu Arg Trp His Pro Tyr Leu
35 40 45
Glu Pro Tyr Gly Leu Val Tyr Cys Val Asn Cys Ile Cys Ser Glu Asn
50 55 60
Gly Asn Val Leu Cys Ser Arg Val Arg Cys Pro Asn Val His Cys Leu
65 70 75 80
Ser Pro Val His Ile Pro His Leu Cys Cys Pro Arg Cys Pro Glu Asp
85 90 95
Ser Leu Pro Pro Val Asn Asn Lys Val Thr Ser Lys Ser Cys Glu Tyr
100 105 110
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Asn Gly Thr Thr Tyr Gln His Gly Glu Leu Phe Val Ala Glu Gly Leu
115 120 l25
Phe Gln Asn Arg Gln Pro Asn Gln Cys Thr Gln Cys Ser Cys Ser Glu
I30 235 140
Gly Asn Val Tyr Cys Gly Leu Lys Thr Cys Pro Lys Leu Thr Cys Ala
145 150 155 160
Phe Pro Val Ser Val Pro Asp Ser Cys Cys Arg Val Cys Arg Gly Asp
165 170 175
Gly Glu Leu Ser Trp Glu His Ser Asp Gly Asp Ile Phe Arg Gln Pro
180 185 190
Ala Asn Arg Glu Ala Arg His Ser Tyr His Arg Ser His Tyr Asp Pro
195 200 205
Pro Pro Ser Arg Gln Ala Gly Gly Leu Ser Arg Phe Pro Gly Ala Arg
210 215 220
Ser His Arg Gly Ala Leu Met Asp Ser Gln Gln Ala Ser Gly Thr Ile
225 230 235 240
Val Gln Ile Val Ile Asn Asn Lys His Lys His Gly Gln Val Cys Val
245 250 255
Ser Asn Gly Lys Thr Tyr Ser His Gly Glu Ser Trp His Pro Asn Leu
260 265 270
Arg Ala Phe Gly Ile Val Glu Cys Val Leu Cys Thr Cys Asn Val Thr
275 280 285
Lys Gln Glu Cys Lys Lys Ile His Cys Pro Asn Arg Tyr Pro Cys Lys
290 295 300
Tyr Pro Gln Lys Ile Asp Gly Lys Cys Cys Lys Val Cys Pro Gly Lys
305 310 315 320
Lys Ala Lys Glu Glu Leu Pro Gly Gln Ser Phe Asp Asn Lys Gly Tyr
325 330 ' 335
Phe Cys Gly Glu Glu Thr Met Pro Val Tyr Glu Ser Val Phe Met Glu
340 345 350
Asp Gly Glu Thr Thr Arg Lys Ile Ala Leu Glu Thr Glu Arg Pro Pro
355 360 365
Gln Val Glu Val His Val Trp Thr Ile Arg Lys Gly Ile Leu Gln His
370 375 380
Phe His Ile Glu Lys Ile Ser Lys Arg Met Phe Glu Glu Leu Pro His
385 390 395 400
Phe Lys Leu Val Thr Arg Thr Thr Leu Ser Gln Trp Lys Ile Phe Thr
405 410 415
Glu Gly Glu Ala Gln Ile Ser Gln Met Cys Ser Ser Arg Val Cys Arg
420 425 430
16
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Thr Glu Leu Glu Hsp Leu val Lys val Leu wyr Leu Ulu Hrg Ser Ulu
435 440 445
Lys Gly His Cys
450
<210> 10
<211> 11
<212> PRT
<213> Human immunodeficiency virus type Z
<400> 10
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10
<210> 11
<211> 15
<2l2> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: internalizing
domain derived from HIV tat protein
<400> 11
Gly Gly Gly Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10 15
<210> 12
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide; primer 1605-21
<400> 12
aatccgatgc ccacgttgca gta 23
<210> 13
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide; PCR primer 1239-08
<400> 13
aaaatcttag accgacgact gtgttt 26
<2l0> 14
<211> 25
<212> DNA
<213> Artificial Sequence
17
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WO 01/64885 PCT/USO1/06891
<220>
<223> Description of Artificial Sequence:
oligonucleotide; PCR primer
<400> 14
cgtaaaagat cctgcgctag atgcg 25
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide; PCR primer
<400> 15
tcctctcatc ctcaccttag 20
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide; PCR primer
<400> 16
ggagaaagtg agataaggac ac 22
<210> 17
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide; PCR primer 2360-40
<400> 17
gctatctaga gccaccatgg ttcccggggt gaggatcatc 40
<210> 18
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide; PCR primer 2360-41
<400> 18
gctagtcgac ctataatgtc ttggtcactt tgtctg 36
18
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WO 01/64885 PCT/USO1/06891
<210> 19
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide; PCR primer
<400> l9
gctagcggcc gcgccaccat ggttcccggg gtgaggatca tc 42
<210> 20
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
oligonucleotide; PCR primer
<400> 20
gctagtcgac taatgtcttg gtcactttgt ctgggc 36
<210> 21
<211> 17
<212> PRT
<213> Mus musculus
<400> 21
Cys Pro Glu Asp Glu Ala Glu Asp Asp His Ser Glu Val I1e Ser Thr
Z 5 10 15
Arg
19
