Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL CHIMPANZEE ERYTHROPOIETIN ICHEP0) POLYPEPTIDES AND NUCLEIC ACIDS
ENCODING THE
SAME
FIELD OF THE INVENTION
The present invention relates generally to the identification and isolation of
novel chimpanzee erythropoietin
polypeptides, nucleic acid molecules encoding those polypeptides and to the
recombinant production of those polypeptides.
BACKGROUND OF THE INVENTION
Erythropoiesis, the production of red blood cells, occurs continuously
throughout the human life span to offset
cell destruction. Erythropoiesis is a very precisely controlled physiological
mechanism enabling sufficient numbers of red
blood cells to be available in the blood for proper tissue oxygenation, but
not so many that the cells would impede
circulation. The formation of red blood cells occurs in the bone marrow and is
under control of the hormone, erythropoietin.
Erythropoietin, an acidic glycoprotein is approximately 34,000 dalton
molecular weight, may occur in three forms:
alpha, beta and asialo. The alpha and beta forms different slightly in
carbohydrate components have the same potency,
biological activity and molecular weight. The asialo form is an alpha or beta
form with the terminal carbohydrate (sialic
acid) removed. Erythropoietin is present in a very low concentrations in
plasma when the body is in a healthy state wherein
tissues receive sufficient oxygenation from the existing number of
erythrocytes. Tbis normal low concentration is enough
to stimulate replacement of red blood cells which are lost normally through
aging.
The amount of erythropoietin in the circulation is increased under conditions
of hypoxia when oxygen transport
2 0 by blood cells in the circulation is reduced. Hypoxia may be caused by
loss of large amounts of blood through hemorrhage,
destruction of red blood cells by over-exposure to radiation, reduction in
oxygen intake due to high altitudes or prolonged
unconsciousness, or various forms of anemia. In response to tissues undergoing
hypoxic stress, erythropoietin will increase
red blood cell production by stimulating the conversion of primitive precursor
cells in the bone marrow into proerythroblasts
which subsequently mature, synthesize hemoglobin and are released into the
circulation as red blood cells. When the
2 5 number of red blood cells in circulation is greater than needed for normal
tissue oxygen requirements, erythropoietin in
circulation is decreased.
Because erythropoietin is essential in the process of red blood cell
formation, the hormone has potential useful
application in both the diagnosis and treatment of blood disorders
characterized by low or defective red blood cell
production. See, generally, Pennathur-Das, et al., Blood 63(5):1168-71 (1984)
and Haddy, Am. Jour. Ped. Hematol. Oncol..
3 0 4:191-196 (1982) relating to erythropoietin in possible therapies for
sickle cell disease, and Eschbach et al., J. Clin. Invest.
7412):434-441 (1984), describing a therapeutic regimen for uremic sheep based
on in viva response to erythropoietin-rich
plasma infusions and proposing a dosage of 10 U EOPIkg per day for 15-40 days
as corrective of anemia of the type
associated with chronic renal failure. See also, Krane, Henry Ford Hosp. Med.
J., 31(31:177-181 119831.
We describe herein the identification and characterization of a novel
erythropoietin polypeptide derived from the
3 5 chimpanzee, designated herein as ~ CHEPO .
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SUMMARY OF THE INDENTION
A cDNA clone has been identified that has homology to nucleic acid encoding
human erythropoietin that encodes
a novel chimpanzee erythropoietin polypeptide, designated in the present
application as "CHEPO".
In one embodiment, the invention provides an isolated nucleic acid molecule
comprising a nucleotide sequence
that encodes a CHEPO polypeptide.
In one aspect, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about 80%
nucleic acid sequence identity, alternatively at least about 81 % nucleic acid
sequence identity, alternatively at least about
82% nucleic acid sequence identity, alternatively at least about 83% nucleic
acid sequence identity, alternatively at least
about 84°'o nucleic acid sequence identity, alternatively at least
about 85% nucleic acid sequence identity, alternatively
at least about 86% nucleic acid sequence identity, alternatively at least
about 87% nucleic acid sequence identity,
alternatively at least about 88% nucleic acid sequence identity, alternatively
at least about 89% nucleic acid sequence
identity, alternatively at least about 909'o nucleic acid sequence identity,
alternatively at least about 91 % nucleic acid
sequence identity, alternatively at least about 92% nucleic acid sequence
identity, alternatively at least about 93% nucleic
acid sequence identity, alternatively at least about 94% nucleic acid sequence
identity, alternatively at least about 959'°
nucleic acid sequence identity, alternatively at least about 96% nucleic acid
sequence identity, alternatively at least about
979'o nucleic acid sequence identity, alternatively at least about 98% nucleic
acid sequence identity and alternatively at
least about 99~ nucleic acid sequence identity to (a) a DNA molecule encoding
a CHEPO polypeptide having the sequence
of amino acid residues from about 1 or about 28 to about 193, inclusive, of
Figure 3 (SEO ID NOS:2 and 5), or (bl the
complement of the DNA molecule of (a).
2 0 In another aspect, the isolated nucleic acid molecule comprises (a) a
nucleotide sequence encoding a CHEPO
polypeptide having the sequence of amino acid residues from about 1 or about
28 to about 193, inclusive, of Figure 3 (SEO
ID NOS:2 and 51, or (b) the complement of the nucleotide sequence of (a1.
In other aspects, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about 80%
nucleic acid sequence identity, alternatively at least about 81 % nucleic acid
sequence identity, alternatively at least about
2 5 829'o nucleic acid sequence identity, alternatively at least about 839'o
nucleic acid sequence identity, alternatively at least
about 84% nucleic acid sequence identity, alternatively at least about
859'° nucleic acid sequence identity, alternatively
at least about 869'o nucleic acid sequence identity, alternatively at least
about 87% nucleic acid sequence identity,
alternatively at least about 889'° nucleic acid sequence identity,
alternatively at least about 89% nucleic acid sequence
identity, alternatively at least about 90% nucleic acid sequence identity,
alternatively at least about 91 % nucleic acid
3 0 sequence identity, alternatively at least about 92% nucleic acid sequence
identity, alternatively at least about 93% nucleic
acid sequence identity, alternatively at least about 94% nucleic acid sequence
identity, alternatively at least about 959'0
nucleic acid sequence identity, alternatively at least about 96% nucleic acid
sequence identity, alternatively at least about
97% nucleic acid sequence identity, alternatively at least about 989'o nucleic
acid sequence identity and alternatively at
least about 99% nucleic acid sequence identity to (a) a DNA molecule having
the sequence of nucleotides from about 1 or
3 5 about 82 to about 579, inclusive, of Figure 2 (SEO ID N0:31, or (b) the
complement of the DNA molecule of (a).
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In another aspect, the isolated nucleic acid molecule comprises (a) the
nucleotide sequence of from about 1 or
about 82 to about 579, inclusive, of Figure 2 (SEO ID N0:3), or (b) the
complement of the nucleotide sequence of (a).
In another aspect, the invention concerns an isolated nucleic acid molecule
which encodes an active CHEPO
polypeptide as defined below comprising a nucleotide sequence that hybridizes
to the complement of a nucleic acid
sequence that encodes amino acids 1 or about 28 to about 193, inclusive, of
Figure 3 (SEO ID NOS:2 and 51. Preferably,
hybridization occurs under stringent hybridization and wash conditions.
In yet another aspect, the invention concerns an isolated nucleic acid
molecule which encodes an active CHEPO
polypeptide as defined below comprising a nucleotide sequence that hybridizes
to the complement of the nucleic acid
sequence between about nucleotides 1 or about 82 and about 579, inclusive, of
Figure 2 (SEO ID N0:31. Preferably,
hybridization occurs under stringent hybridization and wash conditions.
In a further aspect, the invention concerns an isolated nucleic acid molecule
which is produced by hybridizing a
test DNA molecule under stringent conditions with (a) a DNA molecule encoding
a CHEPO polypeptide having the sequence
of amino acid residues from about 1 or about 28 to about 193, inclusive, of
Figure 3 (SEO ID NOS:2 and 5), or (b) the
complement of the DNA molecule of (a), and, if the test DNA molecule has at
least about an 809'o nucleic acid sequence
identity, alternatively at least about 81 % nucleic acid sequence identity,
alternatively at least about 82% nucleic acid
sequence identity, alternatively at least about 83% nucleic acid sequence
identity, alternatively at least about 84% nucleic
acid sequence identity, alternatively at least about 85% nucleic acid sequence
identity, alternatively at least about 86~
nucleic acid sequence identity, alternatively at least about 87% nucleic acid
sequence identity, alternatively at least about
889~o nucleic acid sequence identity, alternatively at least about 89% nucleic
acid sequence identity, alternatively at least
2 0 about 909'o nucleic acid sequence identity, alternatively at least about
91 % nucleic acid sequence identity, alternatively
at least about 92% nucleic acid sequence identity, alternatively at least
about 93% nucleic acid sequence identity,
alternatively at least about 94% nucleic acid sequence identity, alternatively
at least about 95% nucleic acid sequence
identity, alternatively at least about 96% nucleic acid sequence identity,
alternatively at least about 97% nucleic acid
sequence identity, alternatively at least about 989'o nucleic acid sequence
identity and alternatively at least about 999'0
nucleic acid sequence identity to la) or (b), and isolating the test DNA
molecule.
In another aspect, the invention concerns an isolated nucleic acid molecule
comprising (a) a nucleotide sequence
encoding a polypeptide scoring at least about 809'o positives, alternatively
at least about 81 % positives, alternatively at
least about 829'° positives, alternatively at least about 839'o
positives, alternatively at least about 84% positives,
alternatively at least about 85% positives, alternatively at least about 86%
positives, alternatively at least about 87°~
3 0 positives, alternatively at least about 88% positives, alternatively at
least about 89% positives, alternatively at least about
90% positives, alternatively at least about 91 % positives, alternatively at
least about 92% positives, alternatively at least
about 93% positives, alternatively at least about 94% positives, alternatively
at least about 95% positives, alternatively
at least about 96% positives, alternatively at least about 979'°
positives, alternatively at least about 98% positives and
alternatively at least about 99% positives when compared with the amino acid
sequence of residues about 1 or about 28
3 5 to 193, inclusive, of Figure 3 (SEO ID NOS:2 and 5), or (b) the complement
of the nucleotide sequence of (al.
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In a specific aspect, the invention provides an isolated nucleic acid molecule
comprising DNA encoding a CHEPO
polypeptide without the N-terminal signal sequence andlor the initiating
methionine, or is complementary to such encoding
nucleic acid molecule. The signal peptide has been tentatively identified as
extending from about amino acid position 1
to about amino acid position 27 in the sequence of Figure 3 (SEO ID NOS:2 and
51. It is noted, however, that the C-terminal
boundary of the signal peptide may vary, but most likely by no more than about
5 amino acids on either side of the signal
peptide C-terminal boundary as initially identified herein, wherein the C-
terminal boundary of the signal peptide may be
identified pursuant to criteria routinely employed in the art for identifying
that type of amino acid sequence element (e.g.,
Nielsen et al., Prot~En4. 10:1-6 (1997) and von Heinje et al., Nucl. Acids.
Res.14:4683-4690 (1986)1. Moreover, it is also
recognized that, in some cases, cleavage of a signal sequence from a secreted
polypeptide is not entirely uniform, resulting
in more than one secreted species. These polypeptides, and the polynucleotides
encoding them, are contemplated by the
present invention. As such, for purposes of the present application, the
signal peptide of the CHEPO polypeptide shown
in Figure 3 (SEO ID NOS:2 and 5) extends from amino acids 1 to X of Figure 3
(SEO ID NOS:2 and 51, wherein X is any
amino acid from 23 to 32 of Figure 3 (SED ID NOS:2 and 5). Therefore, mature
forms of the CHEPO polypeptide which
are encompassed by the present invention include those comprising amino acids
X to 193 of Figure 3 (SEO ID NOS:2 and
5), wherein X is any amino acid from 23 to 32 of Figure 3 (SEO ID NOS:2 and 51
and variants thereof as described below.
Isolated nucleic acid molecules encoding these polypeptides are also
contemplated.
Another embodiment is directed to fragments of a CHEPO polypeptide coding
sequence that may find use as, for
example, hybridization probes or for encoding fragments of a CHEPO polypeptide
that may optionally encode a polypeptide
comprising a binding site for an anti-CHEPO antibody. Such nucleic acid
fragments are usually at least about 20
2 0 nucleotides in length, alternatively at least about 30 nucleotides in
length, alternatively at least about 40 nucleotides in
length, alternatively at least about 50 nucleotides in length, alternatively
at least about 60 nucleotides in length,
alternatively at least about 70 nucleotides in length, alternatively at least
about 80 nucleotides in length, alternatively at
least about 90 nucleotides in length, alternatively at least about 100
nucleotides in length, alternatively at least about 110
nucleotides in length, alternatively at least about 120 nucleotides in length,
alternatively at least about 130 nucleotides
2 5 in length, alternatively at least about 140 nucleotides in length,
alternatively at least about 150 nucleotides in length,
alternatively at least about 160 nucleotides in length, alternatively at least
about 170 nucleotides in length, alternatively
at least about 180 nucleotides in length, alternatively at least about 190
nucleotides in length, alternatively at least about
200 nucleotides in length, alternatively at least about 250 nucleotides in
length, alternatively at least about 300
nucleotides in length, alternatively at least about 350 nucleotides in length,
alternatively at least about 400 nucleotides
3 0 in length, alternatively at least about 450 nucleotides in length,
alternatively at least about 500 nucleotides in length,
alternatively at least about 600 nucleotides in length, alternatively at least
about 700 nucleotides in length, alternatively
at least about 800 nucleotides in length, alternatively at least about 900
nucleotides in length and alternatively at least
about 1000 nucleotides in length, wherein in this context the term about means
the referenced nucleotide sequence length
plus or minus 10% of that referenced length. In a preferred embodiment, the
nucleotide sequence fragment is derived from
35 any coding region of the nucleotide sequence shown in Figure 1 (SEO ID
N0:1). It is noted that novel fragments of a
CHEPO polypeptide-encoding nucleotide sequence may be determined in a routine
manner by aligning the CHEPO
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polypeptide-encoding nucleotide sequence with other known nucleotide sequences
using any of a number of well known
sequence alignment programs and determining which CHEPO polypeptide-encoding
nucleotide sequence fragments) are
novel. All of such CHEPO polypeptide-encoding nucleotide sequences are
contemplated herein and can be determined
without undue experimentation. Also contemplated are the CHEPO polypeptide
fragments encoded by these nucleotide
molecule fragments, preferably those CHEPO polypeptide fragments that comprise
a binding site for an anti-CHEPO
antibody.
In another embodiment, the invention provides a vector comprising a nucleotide
sequence encoding CHEPO or its
variants. The vector may comprise any of the isolated nucleic acid molecules
hereinabove identified.
A host cell comprising such a vector is also provided. By way of example, the
host cells may be CHO cells, E.
coli, or yeast. A process for producing CHEPO polypeptides is further provided
and comprises culturing host cells under
conditions suitable for expression of CHEPO and recovering CHEPO from the cell
culture.
In another embodiment, the invention provides isolated CHEPO polypeptide
encoded by any of the isolated nucleic
acid sequences hereinabove identified.
In a specific aspect, the invention provides isolated native sequence CHEPO
polypeptide, which in certain
embodiments, includes an amino acid sequence comprising residues from about 1
or about 28 to about 193 of Figure 3
(SED ID NOS:2 and 5).
In another aspect, the invention concerns an isolated CHEPO polypeptide,
comprising an amino acid sequence
having at least about 80% amino acid sequence identity, alternatively at least
about 81 % amino acid sequence identity,
alternatively at least about 82% amino acid sequence identity, alternatively
at least about 83% amino acid sequence
2 0 identity, alternatively at least about 84% amino acid sequence identity,
alternatively at least about 85% amino acid
sequence identity, alternatively at least about 86% amino acid sequence
identity, alternatively at least about 87% amino
acid sequence identity, alternatively at least about 88% amino acid sequence
identity, alternatively at least about 89%
amino acid sequence identity, alternatively at least about 90% amino acid
sequence identity, alternatively at least about
91 % amino acid sequence identity, alternatively at least about 92% amino acid
sequence identity, alternatively at least
about 93% amino acid sequence identity, alternatively at least about 94% amino
acid sequence identity, alternatively at
least about 95% amino acid sequence identity, alternatively at least about 96%
amino acid sequence identity, alternatively
at least about 97% amino acid sequence identity, alternatively at least about
98% amino acid sequence identity and
alternatively at least about 99% amino acid sequence identity to the sequence
of amino acid residues from about 1 or about
28 to about 193, inclusive, of Figure 3 (SEO ID NOS:2 and 5).
3 0 In a further aspect, the invention concerns an isolated CHEPO polypeptide
comprising an amino acid sequence
scoring at least about 80% positives, alternatively at least about 81 %
positives, alternatively at least about 82% positives,
alternatively at least about 83% positives, alternatively at least about 84%
positives, alternatively at least about 859'0
positives, alternatively at least about 869'o positives, alternatively at
least about 87% positives, alternatively at least about
88°io positives, alternatively at least about 89% positives,
alternatively at least about 90% positives, alternatively at least
3 5 about 919'o positives, alternatively at least about 92°Yo
positives, alternatively at least about 93% positives, alternatively
at least about 94% positives, alternatively at least about 95% positives,
alternatively at least about 96% positives,
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alternatively at least about 979'° positives, alternatively at least
about 98% positives and alternatively at least about 99%
positives when compared with the amino acid sequence of residues from about 1
or about 28 to about 193, inclusive, of
Figure 3 (SEO ID NOS:2 and 51.
In a specific aspect, the invention provides an isolated CHEPO polypeptide
without the N-terminal signal sequence
andlor the initiating methionine and is encoded by a nucleotide sequence that
encodes such an amino acid sequence as
hereinbefore described. Processes for producing the same are also herein
described, wherein those processes comprise
culturing a host cell comprising a vector which comprises the appropriate
encoding nucleic acid molecule under conditions
suitable for expression of the CHEPO polypeptide and recovering the CHEPO
polypeptide from the cell culture.
In yet another aspect, the invention concerns an isolated CHEPO polypeptide,
comprising the sequence of amino
acid residues from about 1 or about 28 to about 193, inclusive, of Figure 3
ISEO ID NOS:2 and 51, or a fragment thereof
which is biologically active or sufficient to provide a binding site for an
anti-CHEPO antibody, wherein the identification
of CHEPO polypeptide fragments that possess biological activity or provide a
binding site for an anti-CHEPO antibody may
be accomplished in a routine manner using techniques which are well known in
the art. Preferably, the CHEPO fragment
retains a qualitative biological activity of a native CHEPO polypeptide.
In a still further aspect, the invention provides a polypeptide produced by
(i) hybridizing a test DNA molecule
under stringent conditions with (a) a DNA molecule encoding a CHEPO
polypeptide having the sequence of amino acid
residues from about 1 or about 28 to about 193, inclusive, of Figure 3 (SEO ID
NOS:2 and 5), or (b) the complement of the
DNA molecule of (al, and if the test DNA molecule has at least about an 80%
nucleic acid sequence identity, alternatively
at least about 81 % nucleic acid sequence identity, alternatively at least
about 82% nucleic acid sequence identity,
2 0 alternatively at least about 83% nucleic acid sequence identity,
alternatively at least about 84% nucleic acid sequence
identity, alternatively at least about 859'° nucleic acid sequence
identity, alternatively at least about 86% nucleic acid
sequence identity, alternatively at least about 879'° nucleic acid
sequence identity, alternatively at least about 88°Y° nucleic
acid sequence identity, alternatively at least about 899'° nucleic acid
sequence identity, alternatively at least about 90%
nucleic acid sequence identity, alternatively at least about 91 % nucleic acid
sequence identity, alternatively at least about
2 5 92% nucleic acid sequence identity, alternatively at least about 93%
nucleic acid sequence identity, alternatively at least
about 94% nucleic acid sequence identity, alternatively at least about 95%
nucleic acid sequence identity, alternatively
at least about 96% nucleic acid sequence identity, alternatively at least
about 97% nucleic acid sequence identity,
alternatively at least about 98% nucleic acid sequence identity and
alternatively at least about 99% nucleic acid sequence
identity to (a) or (bl, (ii) culturing a host cell comprising the test DNA
molecule under conditions suitable for expression of
3 0 the polypeptide, and (iii) recovering the polypeptide from the cell
culture.
In another embodiment, the invention provides chimeric molecules comprising a
CHEPO polypeptide fused to a
heterologous polypeptide or amino acid sequence, wherein the CHEPO polypeptide
may comprise any CHEPO polypeptide,
variant or fragment thereof as hereinbefore described. An example of such a
chimeric molecule comprises a CHEPO
polypeptide fused to an epitope tag sequence or a Fc region of an
immunoglobulin.
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In another embodiment, the invention provides an antibody as defined below
which specifically binds to a CHEPO
polypeptide as hereinbefore described. Optionally, the antibody is a
monoclonal antibody, an antibody fragment or a single
chain antibody.
In yet another embodiment, the invention concerns agonists and antagonists of
a native CHEPO polypeptide as
defined below. In a particular embodiment, the agonist or antagonist is an
anti-CHEPO antibody or a small molecule.
In a further embodiment, the invention concerns a method of identifying
agonists or antagonists to a CHEPO
polypeptide which comprise contacting the CHEPO polypeptide with a candidate
molecule and monitoring a biological
activity mediated by said CHEPO polypeptide. Preferably, the CHEPO polypeptide
is a native CHEPO polypeptide.
In a still further embodiment, the invention concerns a composition of matter
comprising a CHEPO polypeptide,
or an agonist or antagonist of a CHEPO polypeptide as herein described, or an
anti-CHEPO antibody, in combination with
a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.
Another embodiment of the present invention is directed to the use of a CHEPO
polypeptide, or an agonist or
antagonist thereof as herein described, or an anti-CHEPO antibody, for the
preparation of a medicament useful in the
treatment of a condition which is responsive to the CHEPO polypeptide, an
agonist or antagonist thereof or an anti-CHEPO
antibody.
Yet another embodiment of the present invention is directed to CHEPO
polypeptides having altered glycosylation
patterns in one or more regions of the polypeptide as compared to the native
sequence CHEPO polypeptide, preferably in
the region surrounding andlor including amino acid position 84 in the CHEPO
amino acids sequence shown in Figure 3 (SEO
ID NOS:2 and 51. In various embodiments, CHEPO variant polypeptides are
prepared using well known techniques so as
2 0 to create an N- or 0-linked glycosylation site at or near amino acid
position 84 in the CHEPO polypeptide sequence. For
example, CHEPO polypeptides contemplated by the present invention include
those where la) amino acids 81-84 of the
CHEPO amino acid sequence shown in Figure 3 (SEO ID NOS:2 and 5) (i.e., Met-
Glu-Ual-Arg; SEO ID N0:6) are replaced by
the amino acid sequence Asn-X-Ser-X (SEO ID N0:7) or Asn-X-Thr-X ISEO ID
N0:81, where X is any amino acid except for
Pro; (b) amino acids 82-85 of the CHEPO amino acid sequence shown in Figure 3
(SEO ID NOS:2 and 5) (i.e., Glu-Val-Arg-
Gln; SEO ID N0:9) are replaced by the amino acid sequence Asn-X-Ser-X (SEO ID
N0:7) or AsmX-Thr-X (SEO ID N0:8),
where X is any amino acid except for Pro; (c) amino acids 83-86 of the CHEPO
amino acid sequence shown in Figure 3 (SEO
ID NOS:2 and 5) (i.e., Ual-Arg-Gln-Gln; SEO ID N0:101 are replaced by the
amino acid sequence Asn-X-Ser-X (SEO ID N0:7)
or Asn-X-Thr-X ISEO ID N0:81, where X is any amino acid except for Pro; or (d)
amino acids 84-87 of the CHEPO amino
acid sequence shown in Figure 3 (SEO ID NOS:2 and 5) (i.e., Arg-Gln-Gln-Ala;
SEO ID N0:11 ) are replaced by the amino acid
3 0 sequence Asn-X-Ser-X (SEO ID N0:7) or Asn-X-Thr-X (SEO ID N0:8), where X
is any amino acid except for Pro, thereby
creating an N-glycosylation site at those positions. Nucleic acids encoding
these variant polypeptides are also
contemplated herein as are vectors and host cells comprising those nucleic
acids.
BRIEF DESCRIPTION OF THE DRAWINGS
3 5 Figures 1 A-C show a nucleotide sequence (SEO ID N0:1 ) of an isolated
genomic DNA molecule containing a
nucleotide sequence (nucleotides 134-146, 667-812, 1071-1157, 1760-1939 and
2074-2226, exclusive of others)
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encoding native sequence CHEPO. Also presented in the genomic sequence are the
locations of the start codon, exons and
introns as well as the amino acid sequence (SEO ID N0:2) encoded by the coding
sequence of SEO ID N0:1.
Figure 2 shows the cDNA sequence of the CHEPO molecule (SEtl ID N0:3) and the
amino acid sequence encoded
thereby (SEQ ID N0:21.
Figure 3 shows a comparison of the human erythropoietin amino acid sequence
(human) (SEQ ID N0:4) and that
of the chimp erythropoietin (chepo) described herein, wherein the amino acid
designated X at amino acid position 142 of
the CHEPO sequence is either glutamine (SEl1 ID N0:2) or lysine ISEO ID N0:5).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
The terms "CHEPO polypeptide", "CHEPO protein" and "CHEPO" when used herein
encompass native sequence
CHEPO and CHEPO polypeptide variants (which are further defined hereinl. The
CHEPO polypeptide may be isolated from
a variety of sources, such as from human tissue types or from another source,
or prepared by recombinant and/or synthetic
methods.
A "native sequence CHEPO" comprises a polypeptide having the same amino acid
sequence as a CHEPO derived
from nature. Such native sequence CHEPO can be isolated from nature or can be
produced by recombinant andlor synthetic
means. The term "native sequence CHEPO" specifically encompasses naturally-
occurring truncated or secreted forms (eg.,
an extracellular domain sequenceh naturally-occurring variant forms (e.g.,
alternatively spliced forms) and naturally-
occurring allelic variants of the CHEPO. In one embodiment of the invention,
the native sequence CHEPO is a mature or
2 0 full-length native sequence CHEPO comprising amino acids 1 to 193 of
Figure 3 (SEQ ID NOS:2 and 51. Also, while the
CHEPO polypeptides disclosed in Figure 3 (SEO ID NOS:2 and 5) is shown to
begin with the methionine residue designated
herein as amino acid position 1, it is conceivable and possible that another
methionine residue located either upstream or
downstream from amino acid position 1 in Figure 3 (SEQ ID NOS:2 and 5) may be
employed as the starting amino acid
residue for the CHEPO polypeptide.
"CHEPO variant polypeptide" means an active CHEPO polypeptide as defined below
having at least about 80%
amino acid sequence identity with the amino acid sequence of (a) residues 1 or
about 28 to 193 of the CHEPO polypeptide
shown in Figure 3 (SEQ ID NOS:2 and 51, (b) X to 193 of the CHEPO polypeptide
shown in Figure 3 (SEO ID NOS:2 and 5),
wherein X is any amino acid residue from 23 to 32 of Figure 3 (SEO ID NOS:2
and 51 or (c) another specifically derived
fragment of the amino acid sequence shown in Figure 3 (SEO ID NOS:2 and 51.
Such CHEPO variant polypeptides include,
3 0 for instance, CHEPO polypeptides wherein one or more amino acid residues
are added, or deleted, at the N- andlor C-
terminus, as well as within one or more internal domains, of the sequence of
Figure 3 (SEO ID NOS:2 and 51. Ordinarily,
a CHEPO variant polypeptide will have at least about 809'o amino acid sequence
identity, alternatively at least about 81
amino acid sequence identity, alternatively at least about 82~o amino acid
sequence identity, alternatively at least about
83% amino acid sequence identity, alternatively at least about 84% amino acid
sequence identity, alternatively at least
3 5 about 859'o amino acid sequence identity, alternatively at least about 86%
amino acid sequence identity, alternatively at
least about 879'o amino acid sequence identity, alternatively at least about
88% amino acid sequence identity, alternatively
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at least about 899'° amino acid sequence identity, alternatively at
least about 90% amino acid sequence identity,
alternatively at least about 91 ~° amino acid sequence identity,
alternatively at least about 92% amino acid sequence
identity, alternatively at least about 93% amino acid sequence identity,
alternatively at least about 949'° amino acid
sequence identity, alternatively at least about 95% amino acid sequence
identity, alternatively at least about 96% amino
acid sequence identity, alternatively at least about 97% amino acid sequence
identity, alternatively at least about 989'°
amino acid sequence identity and alternatively at least about 99% amino acid
sequence identity with (a) residues 1 or about
28 to 193 of the CHEPO polypeptide shown in Figure 3 (SEO ID NOS:2 and 51, (b)
X to 193 of the CHEPO polypeptide
shown in Figure 3 (SED ID NOS:2 and 51, wherein X is any amino acid residue
from 23 to 32 of Figure 3 (SEO ID NOS:2
and 5) or (c) another specifically derived fragment of the amino acid sequence
shown in Figure 3 (SEO ID NOS:2 and 5).
CHEPO variant polypeptides do not encompass the native CHEPO polypeptide
sequence. Ordinarily, CHEPO variant
polypeptides are at least about 10 amino acids in length, alternatively at
least about 20 amino acids in length, alternatively
at least about 30 amino acids in length, alternatively at least about 40 amino
acids in length, alternatively at least about
50 amino acids in length, alternatively at least about 60 amino acids in
length, alternatively at least about 70 amino acids
in length, alternatively at least about 80 amino acids in length,
alternatively at least about 90 amino acids in length,
alternatively at least about 100 amino acids in length, alternatively at least
about 150 amino acids in length, alternatively
at least about 200 amino acids in length, alternatively at least about 300
amino acids in length, or more.
"Percent (%) amino acid sequence identity" with respect to the CHEPO
polypeptide sequences identified herein
is defined as the percentage of amino acid residues in a candidate sequence
that are identical with the amino acid residues
in a CHEPO sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent
2 0 sequence identity, and not considering any conservative substitutions as
part of the sequence identity. Alignment for
purposes of determining percent amino acid sequence identity can be achieved
in various ways that are within the skill in
the art, for instance, using publicly available computer software such as
BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign
(DNASTAR) software. Those skilled in the art can determine appropriate
parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the full-length of the
sequences being compared. For purposes
2 5 herein, however, % amino acid sequence identity values are obtained as
described below by using the sequence comparison
computer program ALIGN-2, wherein the complete source code for the ALIGN-2
program is provided in Table 1 below. The
ALIGN-2 sequence comparison computer program was authored by Genentech, Inc.
and the source code shown in Table
1 has been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered
under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is
publicly available through Genentech, Inc.,
3 0 South San Francisco, California or may be compiled from the source code
provided in Table 1. The ALIGN-2 program should
be compiled for use on a UNIX operating system, preferably digital UNIX U4.OD.
All sequence comparison parameters are
set by the ALIGN-2 program and do not vary.
For purposes herein, the % amino acid sequence identity of a given amino acid
sequence A to, with, or against
a given amino acid sequence B (which can alternatively be phrased as a given
amino acid sequence A that has or comprises
3 5 a certain 9'° amino acid sequence identity to, with, or against a
given amino acid sequence B) is calculated as follows:
9
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100 times the fraction XIY
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program ALIGN-2
in that program s alignment of A and B, and where Y is the total number of
amino acid residues in B. It will be appreciated
that where the length of amino acid sequence A is not equal to the length of
amino acid sequence B, the % amino acid
sequence identity of A to B will not equal the % amino acid sequence identity
of B to A. As examples of % amino acid
sequence identity calculations, Tables 2 and 3 below demonstrate how to
calculate the % amino acid sequence identity
of the amino acid sequence designated Comparison Protein to the amino acid
sequence designated PRO .
Unless specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as
described above using the ALIGN-2 sequence comparison computer program.
However, % amino acid sequence identity
may also be determined using the sequence comparison program NCBI-BLAST2
(Altschul et al., Nucleic Acids Res.
25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be
downloaded from
http:llwww.ncbi.nlm.nih.gov or otherwise obtained from the National Institute
of Health, Bethesda, MD. NCBI-BLAST2
uses several search parameters, wherein all of those search parameters are set
to default values including, for example,
unmask = yes, strand = all, expected occurrences = 10, minimum low complexity
length = 1515, multi-pass e-value =
0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25
and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for amino acid sequence
comparisons, the % amino acid sequence
identity of a given amino acid sequence A to, with, or against a given amino
acid sequence B (which can alternatively be
phrased as a given amino acid sequence A that has or comprises a certain %
amino acid sequence identity to, with, or
2 0 against a given amino acid sequence B) is calculated as follows:
100 times the fraction XIY
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program NCBI-
2 5 BLAST2 in that program s alignment of A and B, and where Y is the total
number of amino acid residues in B. It will be
appreciated that where the length of amino acid sequence A is not equal to the
length of amino acid sequence B, the °Yo
amino acid sequence identity of A to B will not equal the % amino acid
sequence identity of B to A.
"CHEPO variant polynucleotide" or CHEPO variant nucleic acid sequence means a
nucleic acid molecule which
encodes an active CHEPO polypeptide as defined below and which has at least
about 80% nucleic acid sequence identity
3 0 with either f a) a nucleic acid sequence which encodes residues 1 or about
28 to 193 of the CHEPO polypeptide shown in
Figure 3 (SED ID NOS:2 and 51, (b) a nucleic acid sequence which encodes
residues X to 193 of the CHEPO polypeptide
shown in Figure 3 (SEO ID NOS:2 and 5), wherein X is any amino acid residue
from 23 to 32 of Figure 3 (SED ID NOS:2
and 5) or (c) a nucleic acid sequence which encodes another.specifically
derived fragment of the amino acid sequence
shown in Figure 3 (SEO ID NOS:2 and 5). Ordinarily, a CHEPO variant
polynucleotide will have at least about 80% nucleic
3 5 acid sequence identity, alternatively at least about 81 % nucleic acid
sequence identity, alternatively at least about 82%
nucleic acid sequence identity, alternatively at least about 83% nucleic acid
sequence identity, alternatively at least about
CA 02369605 2001-10-O1
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849'° nucleic acid sequence identity, alternatively at least about 85%
nucleic acid sequence identity, alternatively at least
about 869'° nucleic acid sequence identity, alternatively at least
about 879'° nucleic acid sequence identity, alternatively
at least about 88% nucleic acid sequence identity, alternatively at least
about 89% nucleic acid sequence identity,
alternatively at least about 90% nucleic acid sequence identity, alternatively
at least about 91 % nucleic acid sequence
identity, alternatively at least about 92% nucleic acid sequence identity,
alternatively at least about 93% nucleic acid
sequence identity, alternatively at least about 94% nucleic acid sequence
identity, alternatively at least about 959'° nucleic
acid sequence identity, alternatively at least about 96% nucleic acid sequence
identity, alternatively at least about 97%
nucleic acid sequence identity, alternatively at least about 98% nucleic acid
sequence identity and alternatively at least
about 99% nucleic acid sequence identity with either (a) a nucleic acid
sequence which encodes residues 1 or about 28
to 193 of the CHEPO polypeptide shown in Figure 3 (SEO ID NOS:2 and 51, (b) a
nucleic acid sequence which encodes
residues_X to 193 of the CHEPO polypeptide shown in Figure 3 (SEO ID NOS:2 and
5), wherein X is any amino acid residue
from 23 to 32 of Figure 3 (SED ID NOS:2 and 5) or (c) a nucleic acid sequence
which encodes another specifically derived
fragment of the amino acid sequence shown in Figure 3 (SEO ID NOS:2 and 5).
CHEPO polynucleotide variants do not
encompass the native CHEPO nucleotide sequence.
Ordinarily, CHEPO variant polynucleotides are at least about 30 nucleotides in
length, alternatively at least about
60 nucleotides in length, alternatively at least about 90 nucleotides in
length, alternatively at least about 120 nucleotides
in length, alternatively at least about 150 nucleotides in length,
alternatively at least about 180 nucleotides in length,
alternatively at least about 210 nucleotides in length, alternatively at least
about 240 nucleotides in length, alternatively
at least about 270 nucleotides in length, alternatively at least about 300
nucleotides in length, alternatively at least about
2 0 450 nucleotides in length, alternatively at least about 600 nucleotides in
length, alternatively at least about 900
nucleotides in length, or more.
"Percent (%) nucleic acid sequence identity" with respect to the CHEPO
polypeptide-encoding nucleic acid
sequences identified herein is defined as the percentage of nucleotides in a
candidate sequence that are identical with the
nucleotides in a CHEPO polypeptide-encoding nucleic acid sequence, after
aligning the sequences and introducing gaps, if
2 5 necessary, to achieve the maximum percent sequence identity. Alignment for
purposes of determining percent nucleic acid
sequence identity can be achieved in various ways that are within the skill in
the art, for instance, using publicly available
computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)
software. Those skilled in the art
can determine appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal
alignment over the full-length of the sequences being compared. For purposes
herein, however, % nucleic acid sequence
3 0 identity values are obtained as described below by using the sequence
comparison computer program ALIGN-2, wherein
the complete source code for the ALIGN-2 program is provided in Table 1. The
ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc. and the source code shown in Table 1
has been filed with user documentation
in the U.S. Copyright Office, Washington D.C., 20559, where it is registered
under U.S. Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc.,
South San Francisco, California or may
3 5 be compiled from the source code provided in Table 1. The ALIGN-2 program
should be compiled for use on a UNIX
11
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operating system, preferably digital UNIX V4.OD. All sequence comparison
parameters are set by the ALIGN-2 program
and do not vary.
For purposes herein, the % nucleic acid sequence identity of a given nucleic
acid sequence C to, with, or against
a given nucleic acid sequence D (which can alternatively be phrased as a given
nucleic acid sequence C that has or
comprises a certain 9'° nucleic acid sequence identity to, with, or
against a given nucleic acid sequence D) is calculated as
follows:
100 times the fraction WIZ
where W is the number of nucleotides scored as identical matches by the
sequence alignment program ALIGN-2 in that
program s alignment of C and D, and where Z is the total number of nucleotides
in D. It will be appreciated that where
the length of nucleic acid sequence C is not equal to the length of nucleic
acid sequence D, the % nucleic acid sequence
identity of C to D will not equal the % nucleic acid sequence identity of D to
C. As examples of % nucleic acid sequence
identity calculations, Tables 4 and 5 below demonstrate how to calculate the %
nucleic acid sequence identity of the
nucleic acid sequence designated Comparison DNA to the nucleic acid sequence
designated PRO-DNA .
Unless specifically stated otherwise, all % nucleic acid sequence identity
values used herein are obtained as
described above using the ALIGN-2 sequence comparison computer program.
However, % nucleic acid sequence identity
may also be determined using the sequence comparison program NCBI-BLAST2
(Altschul et al., Nucleic Acids Res.
25:3389-3402 (19971). The NCBI-BLAST2 sequence comparison program may be
downloaded from
2 0 http:llwww.ncbi.nlm.nih.gov or otherwise obtained from the National
Institute of Health, Bethesda, MD. NCBI-BLAST2
uses several search parameters, wherein all of those search parameters are set
to default values including, for example,
unmask = yes, strand m all, expected occurrences = 10, minimum low complexity
length = 1515, multi-pass e-value =
0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25
and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for sequence comparisons, the %
nucleic acid sequence identity
2 5 of a given nucleic acid sequence C to, with, or against a given nucleic
acid sequence D (which can alternatively be phrased
as a given nucleic acid sequence C that has or comprises a certain ~°
nucleic acid sequence identity to, with, or against
a given nucleic acid sequence D) is calculated as follows:
100 times the fraction WIZ
where W is the number of nucleotides scored as identical matches by the
sequence alignment program NCBI-BLAST2 in
that program s alignment of C and D, and where Z is the total number of
nucleotides in D. It will be appreciated that where
the length of nucleic acid sequence C is not equal to the length of nucleic
acid sequence D, the % nucleic acid sequence
identity of C to D will not equal the % nucleic acid sequence identity of D to
C.
3 5 In other embodiments, CHEPO variant polynucleotides are nucleic acid
molecules that encode an active CHEPO
polypeptide and which are capable of hybridizing, preferably under stringent
hybridization and wash conditions, to
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nucleotide sequences encoding the full-length CHEPO polypeptide shown in
Figure 3 (SEO ID NOS:2 and 5). CHEPO variant
polypeptides may be those that are encoded by a CHEPO variant polynucleotide.
The term "positives", in the context of the amino acid sequence identity
comparisons performed as described
above, includes amino acid residues in the sequences compared that are not
only identical, but also those that have similar
properties. Amino acid residues that score a positive value to an amino acid
residue of interest are those that are either
identical to the amino acid residue of interest or are a preferred
substitution (as defined in Table 6 below) of the amino acid
residue of interest.
For purposes herein, the % value of positives of a given amino acid sequence A
to, with, or against a given amino
acid sequence B (which can alternatively be phrased as a given amino acid
sequence A that has or comprises a certain
positives to, with, or against a given amino acid sequence B1 is calculated as
follows:
100 times the fraction XIY
where X is the number of amino acid residues scoring a positive value as
defined above by the sequence alignment program
ALIGN-2 in that program s alignment of A and B, and where Y is the total
number of amino acid residues in B. It will be
appreciated that where the length of amino acid sequence A is not equal to the
length of amino acid sequence B, the °Y°
positives of A to B will not equal the % positives of B to A.
"Isolated," when used to describe the various polypeptides disclosed herein,
means polypeptide that has been
identified and separated andlor recovered from a component of its natural
environment. Preferably, the isolated polypeptide
2 0 is free of association with all components with which it is naturally
associated. Contaminant components of its natural
environment are materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and may
include enzymes, hormones, and other proteinaceous or non-proteinaceous
solutes. In preferred embodiments, the
polypeptide will be purified (1) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-
PAGE under non-reducing or reducing
2 5 conditions using Coomassie blue or, preferably, silver stain. Isolated
polypeptide includes polypeptide in situ within
recombinant cells, since at least one component of the CHEPO natural
environment will not be present. Ordinarily,
however, isolated polypeptide will be prepared by at least one purification
step.
An "isolated" nucleic acid molecule encoding a CHEPO polypeptide is a nucleic
acid molecule that is identified
and separated from at least one contaminant nucleic acid molecule with which
it is ordinarily associated in the natural
3 0 source of the CHEPO-encoding nucleic acid. Preferably, the isolated
nucleic is free of association with all components with
which it is naturally associated. An isolated CHEPO-encoding nucleic acid
molecule is other than in the form or setting in
which it is found in nature. Isolated nucleic acid molecules therefore are
distinguished from the CHEPO-encoding nucleic
acid molecule as it exists in natural cells. However, an isolated nucleic acid
molecule encoding a CHEPO polypeptide
includes CHEPO-encoding nucleic acid molecules contained in cells that
ordinarily express CHEPO where, for example, the
3 5 nucleic acid molecule is in a chromosomal location different from that of
natural cells.
13
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The term "control sequences" refers to DNA sequences necessary for the
expression of an operably linked coding
sequence in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a
promoter, optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters,
polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid
sequence. For example, DNA for a presequence or secretary leader is operably
linked to DNA for a polypeptide if it is
expressed as a preprotein that participates in the secretion of the
polypeptide; a promoter or enhancer is operably linked
to a coding sequence if it affects the transcription of the sequence; or a
ribosome binding site is operably linked to a coding
sequence if it is positioned so as to facilitate translation. Generally,
"operably linked" means that the DNA sequences being
linked are contiguous, and, in the case of a secretary leader, contiguous and
in reading phase. However, enhancers do not
have to be contiguous. linking is accomplished by ligation at convenient
restriction sites. If such sites do not exist, the
synthetic oligonucleotide adaptors or linkers are used in accordance with
conventional practice.
The term "antibody" is used in the broadest sense and specifically covers, for
example, single anti-CHEPO
monoclonal antibodies (including agonist, antagonist, and neutralizing
antibodies), anti-CHEPO antibody compositions with
polyepitopic specificity, single chain anti-CHEPO antibodies, and fragments of
anti-CHEPO antibodies (see below). The term
"monoclonal antibody" as used herein refers to an antibody obtained from a
population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the population are
identical except for possible naturally-occurring
mutations that may be present in minor amounts.
"Stringency" of hybridization reactions is readily determinable by one of
ordinary skill in the art, and generally
2 0 is an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer
probes require higher temperatures for proper annealing, while shorter probes
need lower temperatures. Hybridization
generally depends on the ability of denatured DNA to reanneal when
complementary strands are present in an environment
below their melting temperature. The higher the degree of desired homology
between the probe and hybridizable sequence,
the higher the relative temperature which can be used. As a result, it follows
that higher relative temperatures would tend
2 5 to make the reaction conditions more stringent, while lower temperatures
less so. For additional details and explanation
of stringency of hybridization reactions, see Ausubel et al., Current
Protocols in Molecular Biolo4y, Wiley Interscience
Publishers,11995).
"Stringent conditions" or "high stringency conditions", as defined herein, may
be identified by those that: (11
employ low ionic strength and high temperature for washing, for example 0.015
M sodium chloride10.0015 M sodium
3 0 citrate10.1 ~ sodium dodecyl sulfate at 50 C; (2) employ during
hybridization a denaturing agent, such as formamide, for
example, 50% (vlv) formamide with 0.1 % bovine serum albumin10.1 % Fico1110.1
% polyvinylpyrrolidone150mM sodium
phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate
at 42 C; or (3) employ 50% formamide,
5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH
6.8), 0.1 % sodium pyrophosphate, 5 x
Denhardt s solution, sonicated salmon sperm DNA (50 glmll, 0.1 % SDS, and 10%
dextran sulfate at 42 C, with washes
3 5 at 42 C in 0.2 x SSC (sodium chloridelsodium citrate) and 50% formamide at
55 C, followed by a high-stringency wash
consisting of 0.1 x SSC containing EDTA at 55 C.
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"Moderately stringent conditions" may be identified as described by Sambrook
et al., Molecular Cloning: A
L.aboratorv Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization
conditions (e.g., temperature, ionic strength and %SDS) less stringent that
those described above. An example of
moderately stringent conditions is overnight incubation at 37 C in a solution
comprising: 20% formamide, 5 x SSC (150
mM NaCI,15 mM trisodium citratel, 50 mM sodium phosphate (pH 7.61, 5 x
Denhardt s solution, 10% dextran sulfate, and
20 mglml denatured sheared salmon sperm DNA, followed by washing the filters
in 1 x SSC at about 37-50 C. The skilled
artisan will recognize how to adjust the temperature, ionic strength, etc. as
necessary to accommodate factors such as
probe length and the like.
The term "epitope tagged" when used herein refers to a chimeric polypeptide
comprising a CHEPO polypeptide
fused to a "tag polypeptide". The tag polypeptide has enough residues to
provide an epitope against which an antibody
can be made, yet is short enough such that it does not interfere with activity
of the polypeptide to which it is fused. The
tag polypeptide preferably also is fairly unique so that the antibody does not
substantially cross-react with other epitopes.
Suitable tag polypeptides generally have at least six amino acid residues and
usually between about 8 and 50 amino acid
residues (preferably, between about 10 and 20 amino acid residues).
As used herein, the term "immunoadhesin" designates antibody-like molecules
which combine the binding
specificity of a heterologous protein (an "adhesin") with the effector
functions of immunoglobulin constant domains.
Structurally, the immunoadhesins comprise a fusion of an amino acid sequence
with the desired binding specificity which
is other than the antigen recognition and binding site of an antibody (i.e.,
is "heterologous"1, and an immunoglobulin
constant domain sequence. The adhesin part of an immunoadhesin molecule
typically is a contiguous amino acid sequence
2 0 comprising at least the binding site of a receptor or a ligand. The
immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2,
IgG-3, or IgG-4 subtypes, IgA (including
IgA-1 and IgA-2/, IgE, IgD or IgM.
"Active" or "activity" for the purposes herein refers to formls) of CHEPO
which retain a biological andlor an
immunological activity of native or naturally-occurring CHEPO, wherein
biological activity refers to a biological function
(either inhibitory or stimulatory) caused by a native or naturally-occurring
CHEPO other than the ability to induce the
production of an antibody against an antigenic epitope possessed by a native
or naturally-occurring CHEPO and an
immunological activity refers to the ability to induce the production of an
antibody against an antigenic epitope possessed
by a native or naturally-occurring CHEPO. Preferred biological activities
includes, for example, the ability to regulate red
blood cell production, to bind to receptors on the surface of committed
progenitor cells of the bone marrow andlor other
3 0 hematopoietic tissues andlor to induce proliferation andlor terminal
maturation of erythroid cells.
The term "antagonist" is used in the broadest sense, and includes any molecule
that partially or fully blocks,
inhibits, or neutralizes a biological activity of a native CHEPO polypeptide
disclosed herein. In a similar manner, the term
"agonist" is used in the broadest sense and includes any molecule that mimics
a biological activity of a native CHEPO
polypeptide disclosed herein. Suitable agonist or antagonist molecules
specifically include agonist or antagonist antibodies
3 5 or antibody fragments, fragments or amino acid sequence variants of native
CHEPO polypeptides, peptides, small organic
molecules, etc. Methods for identifying agonists or antagonists of a CHEPO
polypeptide may comprise contacting a
CA 02369605 2001-10-O1
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CHEPO polypeptide with a candidate agonist or antagonist molecule and
measuring a detectable change in one or more
biological activities normally associated with the CHEPO polypeptide.
"Treatment" refers to both therapeutic treatment and prophylactic or
preventative measures, wherein the object
is to prevent or slow down (lessens the targeted pathologic condition or
disorder. Those in need of treatment include those
already with the disorder as well as those prone to have the disorder or those
in whom the disorder is to be prevented.
"Chronic" administration refers to administration of the agents) in a
continuous mode as opposed to an acute
mode, so as to maintain the initial therapeutic effect lactivity) for an
extended period of time. Intermittent administration
is treatment that is not consecutively done without interruption, but rather
is cyclic in nature.
"Mammal" for purposes of treatment refers to any animal classified as a
mammal, including humans, domestic
and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle,
horses, sheep, pigs, goats, rabbits, etc.
Preferably, the mammal is human.
Administration "in combination with" one or more further therapeutic agents
includes simultaneous (concurrent)
and consecutive administration in any order.
"Carriers" as used herein include pharmaceutically acceptable carriers,
excipients, or stabilizers which are
nontoxic to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the
physiologically acceptable carrier is an aqueous pH buffered solution.
Examples of physiologically acceptable carriers
include buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid; low molecular
weight (less than about 10 residues) polypeptide; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic
polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine;
2 0 monosaccharides, disaccharides, and other carbohydrates including glucose,
mannose, or dextrins; chelating agents such
as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions
such as sodium; andlor nonionic surfactants
such as TWEEN , polyethylene glycol (PEG), and PLURONICS .
"Antibody fragments" comprise a portion of an intact antibody, preferably the
antigen binding or variable region
of the intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab'IZ, and Fv fragments; diabodies; linear
2 5 antibodies IZapata et al., Protein Ena. 8110): 1057-1062 [1995]); single-
chain antibody molecules; and multispecific
antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antigen-binding
fragments, called "Fab" fragments, each
with a single antigen-binding site, and a residual "Fc" fragment, a
designation reflecting the ability to crystallize readily.
Pepsin treatment yields an Flab'Iz fragment that has two antigen-combining
sites and is still capable of cross-linking
3 0 antigen.
"Fv" is the minimum antibody fragment which contains a complete
antigemrecognition and -binding site. This
region consists of a dimer of one heavy- and one light-chain variable domain
in tight, non-covalent association. It is in this
configuration that the three CDRs of each variable domain interact to define
an antigen-binding site on the surface of the
VH-V~ dimer. Collectively, the six CDRs confer antigen-binding specificity to
the antibody. However, even a single variable
3 5 domain (or half of an Fv comprising only three CDRs specific for an
antigen) has the ability to recognize and bind antigen,
although at a lower affinity than the entire binding site.
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The Fab fragment also contains the constant domain of the light chain and the
first constant domain (CH1) of
the heavy chain. Fab fragments differ from Fab fragments by the addition of a
few residues at the carboxy terminus of
the heavy chain CH1 domain including one or more cysteines from the antibody
hinge region. Fab'-SH is the designation
herein for Fab' in which the cysteine residues! of the constant domains bear a
free thiol group. F(ab'IZ antibody fragments
originally were produced as pairs of Fab' fragments which have hinge cysteines
between them. Other chemical couplings
of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species
can be assigned to one of two
clearly distinct types, called kappa and lambda, based on the amino acid
sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of their heavy
chains, immunoglobulins can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several
of these may be further divided into subclasses (isotypes), e.g., IgGI, IgG2,
IgG3, IgG4, IgA, and IgA2.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains
of antibody, wherein these
domains are present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker
between the VH and VL domains which enables the sFv to form the desired
structure for antigen binding. For a review of
sFv, see Pluckthun in The Pharmacolony of Monoclonal Antibodies vol. 113,
Rosenburg and Moore eds., Springer-Verlag,
New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two antigen-
binding sites, which fragments
comprise a heavy-chain variable domain (VH) connected to a light-chain
variable domain (VL) in the same polypeptide chain
(VH - VL). By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains
2 0 are forced to pair with the complementary domains of another chain and
create two antigen-binding sites. Diabodies are
described more fully in, for example, EP 404,097; WO 93111161; and Hollinger
et al., Proc. Natl. Acad. Sci. USA, 90:6444-
6448 ( 1993).
An "isolated" antibody is one which has been identified and separated andlor
recovered from a component of its
natural environment. Contaminant components of its natural environment are
materials which would interfere with
2 5 diagnostic or therapeutic uses for the antibody, and may include enzymes,
hormones, and other proteinaceous or
nonproteinaceous solutes. In preferred embodiments, the antibody will be
purified (1 ) to greater than 95% by weight of
antibody as determined by the Lowry method, and most preferably more than 99%
by weight, (2) to a degree sufficient
to obtain at least 15 residues of N-terminal or internal amino acid sequence
by use of a spinning cup sequenator, or (3) to
homogeneity by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue ar, preferably, silver stain.
3 0 Isolated antibody includes the antibody in situ within recombinant cells
since at least one component of the antibody's
natural environment will not be present. Ordinarily, however, isolated
antibody will be prepared by at least one purification
step.
The word "label" when used herein refers to a detectable compound or
composition which is conjugated directly
or indirectly to the antibody so as to generate a "labeled" antibody. The
label may be detectable by itself (e.g. radioisotope
3 5 labels or fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound
or composition which is detectable.
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By "solid phase" is meant a non-aqueous matrix to which the antibody of the
present invention can adhere.
Examples of solid phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled pore glassh
polysaccharides (e.g., agaroseh polyacrylamides, polystyrene, polyvinyl
alcohol and silicones. In certain embodiments,
depending on the context, the solid phase can comprise the well of an assay
plate; in others it is a purification column (e.g.,
an affinity chromatography columnl. This term also includes a discontinuous
solid phase of discrete particles, such as
those described in U.S. Patent No. 4,275,149.
A "liposome" is a small vesicle composed of various types of lipids,
phospholipids andlor surfactant which is
useful for delivery of a drug (such as a CHEPO polypeptide or antibody
thereto) to a mammal. The components of the
liposome are commonly arranged in a bilayer formation, similar to the lipid
arrangement of biological membranes.
A small molecule is defined herein to have a molecular weight below about 500
Daltons.
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Table 1
I*
*
* C-C increased from 12 to 15
* Z is average of EO
* B is average of ND
* match with stop is M; stop-stop = 0; J (joker) match = 0
.I
#define _M -8 I* value of a match with a stop *I
int -day[26][261 = {
I* A B C D E F G H I J
K L M N 0 P 0 R S
T U V W X Y Z *I
I* A { 2, 0,-2, 0, 0.-4, M. 1, 0,-2, 1, 1, 0, 0,-6, 0,-3,
*I 1.-1,-1, 0,-1,-2,-1, 0},
0-
I* B { 0, 3,-4, 3, 2,-5, M,-1, 1, 0, 0, 0, 0,-2,-5, 0,-3,
*I 0, 1,-2, 0, 0,-3,-2, 1},
2_
I* {-2,-4,15,-5,-5,-4,-3,-3,-2,M,-3,-5,-4, 0,-2, 0,-2,-8, 0, 0,-5},
C *I 0,-5,-6,-5,-4,
I* D { 0, 3,-5, 4, 3,-6, M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4,
*I 1, 1,-2, 0, 0,-4,-3, 2},
2-
I* E { 0, 2,-5, 3, 4,-5, M,-1, 2,-1, 0, 0, 0,-2,-7, 0,-4,
*I 0, 1,-2, 0, 0,-3,-2, 3},
1 _
I* F {-4,-5,-4,-6,-5, 9,-5,-2,-M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5},
*I 1, 0,-5, 2, 0,-4,
I* G { 1, 0,-3, 1, 0,-5, _M,-1,-1,-3, 1, 0, 0,-1,-7, 0,-5,
*I 5,-2,-3, 0,-2,-4,-3, 0},
0,
I* {-1, 1,-3, 1, 1,-2,-2,-M, 0, 3, 2,-1,-1, 0,-2,-3, 0, 0,
H *I 6,-2, 0, 0,-2,-2, 2}.
2,
I* I {-1,-2,-2,-2,-2, 1,-3,-2,M,-2,-2,-2,-1, 0, 0, 4,-5, 0,-1,-2},
*I 5, 0,-2, 2, 2,-2 _
I*J*I {0,0,0,0,0,0,0,0,0,0,0,0,0,0-M,0,0,0,0,0,0,0,0,0,0,0},
I* K {-1, 0,-5, 0, 0,-5,-2,M,-1, 1, 3, 0, 0, 0,-2,-3, 0,-4,
*I 0,-2, 0, 5,-3, 0, 0},
1 -
I* L {-2,-3,-6,-4,-3, 2,-4,-2,M,-3,-2,-3,-3,-1, 0, 2,-2, 0,-1,-2},
*/ 2, 0,-3, 6, 4,-3 _
2 5 I* I {-1,-Z,-5.-3,-2, M,-2,-1. 0.-2,-1. 0, 2,-4, 0,-2,-1
M * 0,-3,-2, 2, 0, 0, },
4, 6,-2 _
I* N { 0, 2,-4, 2, 1,-4, M,-1, 1, 0, 1, 0, 0,-2,-4, 0,-2,
* I 0, 2,-2, 0, 1,-3,-2, 1 },
2 -
I* 0 { M _M _M -M _M _M M _M -M -M _M, 0 -M -M -M _M -M
*I -M _M -M - -M -M _M -M -M _M},
I* P { 1,-1,-3,-1,-1,-5,-1,_M, 6, 0, 0, 1, 0, 0,-1,-6, 0,-5,
*I 0,-2, 0,-1,-3,-2,-1, 0},
I* 0 { 0, 1,-5, 2, 2,-5,-1,_M, 0, 4, 1,-1,-1, 0,-2,-5, 0,-4,
*I 3,-2, 0, 1,-2,-1, 3},
1,
3 0 I* {-2, 0,-4,-1,-1,-4,-3,M, 0, 1, 6, 0,-1, 0,-2, 2, 0,-4,
R *I 2,-2, 0, 3,-3, 0, 0},
0 _
I* S { 1, 0, 0, 0, 0,-3, M, 1,-1, 0, 2, 1, 0,-1,-2, 0,-3,
"I 1,-1,-1, 0, 0,-3,-2, 0},
1 _
I* T { 1, 0,-2, 0, 0,-3, _M, 0,-1,-1, 1, 3, 0, 0,-5, 0,-3,
*I 0,-1, 0, 0, 0,-1,-1, 0},
0,
I*U*I {0,0,0,0,0,0,0,0,0,0,0,0,0,0-M,0,0,0,0,0,0,0,0,0,0,0},
I* ~ { 0~-2.-2.-2--2~-1--1--2--M--1~-2.-2--1- 0- 0 4--6. 0--2.-2},
*I 4. 0.-2. 2~ 2.-2
3 5 I* I {-6,-5,-8,-7,-7, _M,-6,-5, 2,-2,-5, 0,-6,17, 0, 0,-6},
W * 0,-7,-3,-5, 0,-3,-2,-4,-4,
I*X"I {0,0,0,0,0,0,0,0,0,0,0,0,0,0-M,0,0,0,0,0,0,0,0,0,0,0},
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I' Y'I {-3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-4,-1,-2,-2, M,-5,-4,-4,-3,-3, 0,-2, 0,
0,10,-4},
I" Z "I { 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1 _M, 0, 3, 0, 0, 0, 0,-2,-6,
0,-4, 4}
};
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Table t Icont )
I*
*I
#include < stdio.h >
#include < ctype.h >
#define MAXJMP 16 I* max jumps in a diag *I
#define MAXGAP24 I* continue to penalize gaps
don'tlarger than this *I
#define JMPS 1024 I* max jmps in an path *I
#defineMX 4 I* save if there's at least
MX-1 bases since last jmp
*I
#define DMAT 3 I* value of matching bases
*I
#define DMIS 0 I* penalty for mismatched
bases *I
#define DINSO 8 I* penalty for a gap *I
#defineDINS1 1 I* penalty per base *I
#define PINSO 8 I* penalty for a gap *I
#define PINS1 4 I* penalty per residue *I
struct jmp
2 0 short n[MAXJMP]; I* size of jmp (neg for dely] *I
unsigned short x[MAXJMP]; I* base no. of jmp in seq x *I
}; I* limits seq to 2" 16 -1 *I
struct diag ~
int score; I* score at last jmp *I
long offset; I* offset of prev block *I
short ijmp; I* current jmp index *I
struct jmp jp; I* list of jmps *I
};
struct path {
int spc; I* number of leading spaces *I
short n[JMPS]; I* size of jmp (gap) *I
int x(JMPS]; I* loc of jmp (last elem before gapl *I
3 5 };
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char *ofile; I* output file name
*I
char *namex[2]; I* seq names: getseqs()
*I
char *prog; I* prog name for
err msgs *I
char *seqx[2]; I* seqs: getseqsl)
*I
int dmax; I* best diag: nwp
*I
int dmax(); I* final diag *I
int dna; I* set if dna: maim)
*I
int endgaps; I" set if penalizing
end gaps *I
int gapx, gapy; I* total gaps in
seqs *I
int IenO, lenl; I* seq lens *I
int ngapx, ngapy;I* total size of
gaps *I
int smax; I* max score: nwl)
*I
int *xbm; I* bitmap for matching
*I
long offset; I* current offset
in jmp file *I
struct *dx; I* holds diagonals
diag *I
struct path pp(2]; I* holds path for
seqs *I
char *calloc(1, *mallocll, *index0, *strcpy();
char *getseq0, *g calloc0;
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Table 1 Icont )
Ix Needleman-Wunsch alignment program
x
x usage: progs filel filet
x where filel and filet are two dna or two protein sequences.
x The sequences can be in upper- or lower-case an may contain ambiguity
x Any lines beginning with ';', ' > ' or ' <' are ignored
x Max file length is 65535 (limited by unsigned short x in the jmp struct)
x A sequence with 113 or more of its elements ACGTU is assumed to be DNA
" Output is in the file "align.out"
x
x The program may create a tmp file in Itmp to hold info about traceback.
" Original version developed under BSD 4.3 on a vax 8650
xl
#include "nw.h"
#include "day.h"
static dbval(26] a {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
2 0 ~;
static pbval[26]
1, 2 ~ (1 < < ('D'-'A')1 ~ (1 < < ('N'-'A'11, 4, 8, 16, 32, 64,
128, 256, OxFFFFFFF, 1 « 10, 1 « 11, 1 « 12, 1 « 13, 1 « 14,
1«15,1«16,1«17,1«18,1«19,1«20,1«21,1«22,
1 < < 23, 1 < < 24, 1 < < 25 ~ (1 < < ('E'-'A'1) ~ (1 < < ('O'-'A'1)
};
mainlac, av] main
3 0 int ac;
char xav[];
prog - av[0];
if (ac ! = 3) {
3 5 fprintf(stderr,"usage: %s filel file2ln", prog];
fprintflstderr,"where filel and filet are two dna or two protein
sequences.ln");
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fprintf(stderr,"The sequences can be in upper- or lower-casein");
fprintf(stderr,"Any lines beginning with ';' or ' <' are ignoredln"1;
fprintflstderr,"Output is in the file I"align.outl"In"1;
exit111;
namex[0] - av(1];
namex[1] - av[2];
seqx(0] - getseq(namex[0], &Ien0l;
seqx[1] = getseqlnamex[1], &1en11;
xbm = (final? dbval : _pbval;
endgaps = 0; I* 1 to penalize endgaps *I
ofile = "align.out"; I* output file *I
nw0; I* fill in the matrix, get the possible jmps *I
readjmps(1; I* get the actual jmps *I
print0; I* print stats, alignment *I
cleanup(0); I* unlink any tmp files *I
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Table 1 (coot 1
I* do the alignment, return best score: main()
* dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro: PAM 250 values
* When scares are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y.
*I
nw0 nw
char *px, *py; I* seqs and ptrs
*I
int *ndely, I* keep track
*dely; of dely *I
int ndelx, delx;I* keep track
of deli *I
int *tmp; I* for swapping
row0, rowl *I
int mis; I* score for each
type *I
int ins0, inst;I* insertion penalties
*I
register id; I* diagonal index
*I
register ij; I* jmp index *I
register *col0, *coll;I* score for curr,
last row *I
2 0 register xx, yy; I* index into
seqs *I
dx = (struct diag *)g callocl"to get diags", IenO+lenl + 1, sizeoflstruct
diag)1;
ndely = (int *)g calloc("to get ndely", lenl +1, sizeoflint));
dely = lint *)g callocl"to get dely", lenl+1, sizeof(int)1;
col0 = (int *Ig callocl"to get col0", lenl +1, sizeof(int));
toll = (int *)g calloc("to get coil", lenl +1, sizeof(intll;
ins0 = Idna1? DINSO : PINSO;
insl = Idna)? DINS1 : PINS1;
smax = -10000;
if (endgaps) {
for (col0[0] = dely[0] _ -ins0, yy - 1; yy < = lenl; yy++) f
col0[yy1 = dely[yy] = col0[yy-1] - insl;
3 5 ndely[yy] - yy;
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col0(0] - 0; I" Waterman Bull Math Biol 84 "I
else
for (yy = 1; yy < = lenl; yy++)
dely[yy] - -ins0;
I" fill in match matrix
"I
for (px = seqx[0], xx = 1; xx < - IenO; px++, xx++) {
I" initialize first entry in col
"I
if (endgaps) {
if (xx - - 1)
coll[0] = delx = -/ins0+instl;
else
coll(0] = deli = col0[0] - insl;
ndelx = xx;
else {
2 0 col l (0] = 0;
delx - -ins0;
ndelx = 0;
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Table 1 Icont 1
...nw
for (py = seqx[1], yy = 1; yy < = lenl; py++, yy++) {
mis = col0[yy-1];
if (dna)
mis + _ (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS;
else
mis += day[*px-'A'][*py-'A'l;
I* update penalty for del in x seq;
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps
*I
if (endgaps ~ ~ ndely[yy] < MAXGAP) {
if (col0[yy] - ins0 > = dely(yy]) {
dely[yy] = col0[yy] - (ins0+insl );
ndely[yy] = 1;
} else {
dely[yy] -= insl;
2 0 ndely[yy]+ +;
} else {
if (col0[yy] - (ins0+insl) > = dely[yy]) {
dely[yy] = col0(yy] - (ins0+insl );
ndely[yy] = 1;
} else
ndely[yy]+ +;
3 0 I* update penalty for del in y seq;
* favor new del over ongong del
*I
if (endgaps ~ ~ ndelx < MAXGAP) {
if (coll[yy-1] - ins0 > = delx) {
delx = coil[yy-1]-(ins0+insl);
ndelx = 1;
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} else ~
delx -- insl;
ndelx++;
}
} else {
if (colt[yy-1] - (ins0+insl) > = delx) {
delx = coil[yy-1]-(ins0+ins1l;
ndelx = 1;
} else
ndelx++;
}
I" pick the maximum score; we're favoring
" mis over any del and delx over dely
"I
25
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Table 1 Icont 1
...nw
id = xx - yy + lenl - 1;
if (mis > = delx && mis > = dely[yy])
call[yy] = mis;
else if (delx > = dely[yy]) {
coil[yy] = delx;
ij = dx[id].ijmp;
if (dx(id].jp.n[0] && (!dna ~ ~ (ndelx > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) ~ ~ mis > dx[id].score+DINS011 {
dx[id].ijmp++;
if (++ij > = MAXJMP) {
writejmps(idl;
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset += sizeoflstruct jmp) + sizeofloffsetl;
dx[id].jp.n[ij] = ndelx;
2 0 dx[id].jp.x[ij] = xx;
dx[id].score = delx;
else {
coil[YV] = dely[YY];
2 5 ij = dx(id].ijmp;
if (dx[id].jp.n[0] && (!dna ~ ~ (ndely[yy] > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) ~ ~ mis > dx[id].score+DINSO)) {
dx[id].ijmp+ +;
if (++ij > = MAXJMP) {
3 0 writejmps(idl;
ij = dx(id].ijmp - 0;
dx[id].offset = offset;
offset + = sizeoflstruct jmp) + sizeofloffset);
dx[id].jp.n[ij] _ -ndely[yy];
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dx[id].jp.x[ij] - xx;
dx(id].score - dely[yy];
if Ixx = = IenO && yy < len 1 ) {
I" last cal
"I
if (endgaps)
coil[yy]-= ins0+insl"Ilen1-yy);
if (coil(yy] > smax) {
smax = cal t [yy];
dmax = id;
if (endgaps && xx < Ien0)
toll[yy-1]-= ins0+insl"pen0-xx);
if (toll[yy-1] > smax) {
smax = toll[yy-1];
dmax = id;
tmp = col0; col0 = call; coil = tmp;
(void) freellchar ")ndelyl;
(void) freellchar ")delyl;
2 5 (void) freellchar "Ico101;
paid) freellchar ")co111; }
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Table 1 Icont 1
I*
*
* print() -- only routine visible outside this module
* static:
* getmatl) -- trace back best path, count matches: print()
* pr align/) -- print alignment of described in array p[]: print/)
* dumpblockl) -- dump a block of lines with numbers, stars: pr_align()
* numsl) -- put out a number line: dumpblockp
* putlinel) -- put out a line (name, [num], seq, [num]I: dumpblock()
* stars/) - -put a line of stars: dumpblockQ
* stripnamel] -- strip any path and prefix from a seqname
*I
#include "nw.h"
#define SPC 3
#define P LINE 256 I* maximum output line *I
2 0 #define P SPC 3 I* space between name or num and seq *I
extern day[26][26];
int oleo; I* set output line length *I
FILE *fx; I" output file *I
print/)
print
f
int Ix, ly, firstgap, lastgap; I* overlap *I
3 0 if ((fx = fopenlofile, "w"1) - - 0) {
fprintflstderr,"%s: can't write %sln", prog, ofilel;
cleanup(11;
fprintflfx, " < first sequence: %s length - %dlln", namex[0], Ien0l;
3 5 fprintflfx, " < second sequence: %s (length = %dlln", namex[1 ], lenl ];
oleo - 60;
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Ix = IenO;
ly = lenl;
firstgap - lastgap = 0;
if (dmax < lenl - 1) { I* leading gap in x *I
pp[0].spc = firstgap = lenl - dmax - 1;
ly -- pp[0].spc;
else if fdmax > lenl - 1) { I* leading gap in y *I
pp[1].spc = firstgap = dmax - (lenl - 11;
Ix -= pp[1]-spc;
if (dmax0 < IenO - 1 ) { I* trailing gap in x *I
lastgap = IenO - dmax0 -1;
Ix -= lastgap;
else if (dmax0 > IenO - 1) { I* trailing gap in y *I
lastgap = dmax0 - (IenO - 1 );
ly -- lastgap;
2 0 getmat(Ix, ly, firstgap, lastgapl;
pr alignll;
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Table 1 Icont )
I"
" trace back the best path, count matches
"I
static
getmatllx, ly, firstgap, Iastgap) getmat
int Ix, ly; I" "core" (minus endgaps) "I
int firstgap, lastgap; I" leading trailing overlap "I
int nm, i0,
i1, siz0,
sizl;
char outx[321;
double pct;
register n0, n1;
register char "p0, "p1;
" get total matches, score
"I
i0=i1 =siz0=sitl =0;
p0 s seqx[0] + PP[1].spc;
p1 = seqx[1] + pp[0].spc;
n0 - pp[1].spc + 1;
n1 = pp(0].spc + 1;
nm=0;
while ( "p0 && "p1 ) ~
if (siz0) {
p1++;
nl++;
siz0--;
else if (sizl) ~
p0++;
n0++;
siz 1--;
else ~
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if (xbm["p0-'A']&xbm['p1-'A'])
nm++;
ifln0++ _=pp[0].x[i0])
siz0 = pp[0].n[i0++];
iflnl++ _=pp[1].x[il])
sizl = pp[1].n[i1++];
p0++;
pl ++;
I" pct homology:
" if penalizing endgaps, base is the shorter seq
" else, knock off overhangs and take shorter core
1 5 "~
if (endgaps)
Ix = (IenO < Ienl1? IenO : lenl;
else
Ix = (Ix < ly)? Ix : ly;
2 0 pct = 100."(double)nm1(double)Ix;
fprintf(fx, "In");
fprintf(fx, " < %d match%s in an overlap of %d: %.2f percent similarityln",
nm, (nm = = 11? "" : "es", Ix, pct);
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Table 1 Icont 1
fprintf(fx, " < gaps in first sequence: %d", gapx); ...getmat
if (gapx) {
(void) sprintf(outx, " (%d %s%s)",
ngapx, (dna)? "base":"residue", (ngapx = = 1 )? "":"s");
fprintflfx,"%s", outx);
fprintflfx, ", gaps in second sequence: %d", gapy);
if (gapy)
(void) sprintf(outx, " (%d %s%s)",
ngapy, (dual? "base":"residue", (ngapy = = 1 )? "":"s"];
fprintf(fx,"%s", outx);
if (dna)
fprintflfx,
"In < score: %d (match = %d, mismatch = %d, gap penalty = %d + %d per
baselln",
smax, DMAT, DMIS, DINSO, DINS1);
else
2 0 fprintf(fx,
"In < score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %d per
residue)In",
smax, PINSO, PINS1);
if (endgaps)
fprintf(fx,
2 5 " < endgaps penalized. left endgap: %d %s%s, right endgap: %d %s%sln",
firstgap, (dna)? "base" : "residue", (firstgap == 1)? "" : "s",
lastgap, (final? "base" : "residue", hastgap -- 1)? "" : "s");
else
fprintf(fx, " < endgaps not penalizedln"1;
static nm; I" matches in core --
for checking "I
static Imax; I' lengths of stripped
file names "I
static ij[2]; I' jmp index for a path
'I
3 5 static nc(2]; I' number at start of
current line "I
static ni[2]; I* current elem number
-- for gapping "I
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static siz[2];
static char *ps[2]; I* ptr to current element *I
static char *po[2]; I* ptr to next output char slat *I
static char out[2][P LINE]; I" output line *I
static char star[P LINE]; I* set by stars() *I
I*
* print alignment of described in struct path pp[]
*I
static
pr align/) pr align
int nn; I* char count *I
int more;
register i;
for (i = 0, Imax = 0; i < 2; i++) {
nn - stripname(namex[i]~;
if (nn > Imax)
2 0 Imax - nn;
nc[i] = 1;
ni[i] - 1;
siz[i] = ij[i] = 0;
2 5 ps[i] = seqx[i];
po[i] = out[i]; }
36
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Table t Icont 1
for Inn = nm = 0, more = 1; more; l f ...pr align
for (i = more = 0; i < 2; i++) f
I*
* do we have more of this sequence?
*I
if 1!*ps[i])
continue;
mare++;
if (pp[i].spc) { I* leading space *I
*po[i]++ _ ,
pp[i].spc--;
else if (siz[i1) { I* in a gap *I
*po[i)++ _ , ~;
siz[i]--;
else ~ I* we're putting a seq element
*I
*Po[il _ *Ps[il;
if (islower(*ps[i]))
*ps[i] = toupperl*ps[i]/;
po[i]++;
Ps[i1+ +;
I*
3 0 * are we at next gap for this seq?
*I
if (ni[i] _ = PP[il.x[ij[i]1) f
I*
* we need to merge all gaps
3 5 * at this location
*I
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siz[il - PPfil.n[ij[il++1;
while (ni[il -- pp[il.x[ij[ill)
siz[il +- PP[il.n[ij[il++1;
ni[i]+ +;
if (++nn -- oleo ~ ~ !more && nn) f
dumpblockll;
for (i = 0; i < 2; i++)
po[il - out[i];
nn-0;
I"
dump a block of lines, including numbers, stars: pr align()
"I
2 0 static
dumpblock() dumpblock
register i;
for (i - 0; i < 2; i++)
~po[il__ _ '10';
38
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Table 1 (coot )
...dumpblock
(void) putc('In', fx);
for (i = 0; i < 2; i++) {
if 1*out[i) && 1*out[i] !_ ~ ~ ~ ~ *(Po[ill !- ")) {
if (i - - 0)
numsli);
if (i -- 0 && "out[1])
stars0;
putline(i);
if fi =- 0 && *out[1])
fprintflfx, star);
if (i -= 1)
nums(i);
2 0 I*
* put out a number line: dumpblock()
static
nums(ix) nums
2 5 int ix; I* index in out[] holding seq line *I
char nline[P LINE];
register i, j;
register char *pn, *px, *py;
for (pn = nline, i = 0; i < Imax+P SPC; i++, pn++)
*pn = . .;
for (i = nc[ix], py = out[ix]; *py; py++, pn++) f
if (*py = _ " ~ ~ *PY =- '-')
*pn-";
else
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if (i% 10 - - 0 ~ ~ 6 - - 1 && nc[ix] ! - 11) {
j - li < 0)? -i : i;
for (px - pn; j; j I= 10, px--)
'"pX ' j%10 + '0';
if (i < 0)
"Px - ,
else
"Pn ° ,
i++;
.Pn = ,~0..
nc[ix] = i; /
for (pn = nline;'"pn; pn++)
(void) putcl"pn, fxl;
(void) putcl'In', fxl;
2 0 I"
" put out a line (name, [num], seq, [num]l: dumpblockl)
"I
static
putlinelix) putline
2 5 int ix;
CA 02369605 2001-10-O1
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Table 1 (coot )
...putline
int i;
register char *px;
for (px = namex[ix], i = 0; *px && *px ! _ '~'; px++, i++)
(void) putcl*px, fxl;
for (; i < Imax+P SPC; i++)
(void) putcl' ', fxl;
* these count from 1:
* ni[] is current element (from 1)
* ncp is number at start of current line
*I
for (px = out[ix]; *px; px++)
(void) putt(*px&Ox7F, fx);
(void) putcl'In', fx);
I*
* put a line of stars (seqs always in out[0], out[1]): dumpblockp
*I
2 5 static
stars() stars
f
int i;
register char *p0, *p1, cx, *px;
if 1! *out[O1 ~ ~ 1"out[0) a ~ ' && *(Po[O1) - _ ' ') ~ ~
!*out[11 ~ ~ 1*out[1] a = ' ' && *Ipo[1]) a a ' '1)
return;
px - star;
3 5 for (i ~ Imax+P SPC; i; i--)
*px++ , , ~;
41
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for (p0 = out[0], p1 = out[1]; "p0 && "p1; p0++, p1 ++) ~
if (isalpha("p0) && isalphal"p1)) {
if (xbm["p0-'A']&xbm["p1-'A']) ~
cx = '"';
nm++;
else if (!dna && day["p0-'A']["p1-'A'] > 0)
cx=";
else
cx = ' ';
else
1 5 cx = ,
"px++ ~ cx;
"Px++ ° ~~n';
"Px ° 't0';
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Table 1 (coot I
I*
* strip path or prefix from pn, return len: pr align()
*I
static
stripnamelpn) stripname
char *pn; I* file name (may be path) *I
register char *px, *py;
PY ' ~;
for (px = pn; *px; px++)
if (*px =_ 'I')
PY°Px+1;
if (py)
(void) strcpy(pn, py);
return(strlen(pn));
25
35
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Table 1 (coot )
I*
* cleanup() -- cleanup any tmp file
* getseq(1-- read in seq, set dna, len, maxlen
* g calloc() -- callocp with error checkin
* readjmpsU -- get the good jmps, from tmp file if necessary
* writejmps() -- write a filled array of jmps to a tmp file: nw()
*I
#include "nw.h"
#include < sysl file.h >
char *jname = "ItmplhomgXXXXXX"; I* tmp file for jmps *I
FILE *tj;
int cleanup0; I* cleanup tmp file *I
long Iseekll;
I*
* remove any tmp file if we blow
2 0 *I
cleanup(i) cleanup
int i;
if (fj)
2 5 (void) unlink(jnamel;
exit(il;
I*
3 0 * read, return ptr to seq, set dna, len, maxlen
* skip lines starting with ';', ' < ', or ' > '
* seq in upper or lower case
*I
char *
3 5 getseqlfile, len) getseq
char *file; I* file name *I
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int *len; I* seq ten *I
f
char line[1024], *pseq;
register char *px, *py;
int natgc, tlen;
FILE *fp;
if ((fp = fopenlfile,"r"11 == 0) {
fprintf(stderr,"%s: can't read %sln", prog, filet;
exit(11;
tlen = natgc = 0;
while (fgets(line, 1024, fpl) {
if ("line = - ,' ~ ~ "line = _ ' <' ~ ~ "line = - ' > ')
continue;
for (px = line; *px ! _ 'In'; px++)
if (isupperl*px) ~ ~ islowerl*px))
tlen++;
2 0 if (Ipseq = mallocl(unsignedlltlen+6))) _ = 01 {
fprintf(stderr,"%s: mallocl) failed to get %d bytes for %sln", prog, tlen+(i,
filet;
exit111;
pseq[0] = pseq[1] = pseq[2] = pseq[3) _ '10';
45
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Table 1 (coot 1
...getseq
PY - Pseq + 4;
*len - tlen;
rewind(fpl;
while (fgetslline, 1024, fpl) f
if (*line -- ';' ~ ~ *line -- ' <' ~ ~ *line - _ ' >')
continue;
for (px - line; *px !_ 'In'; px++)
if (isupper(*px))
*PY++ - *Px;
else if (islowerl*px))
*py++ = toupperl*px);
if (index)"ATGCU",*(py-1)))
natgc+ +;
*pY++ a ~~0~.
*PY -'10'; I
(void) fclose(fpl;
dna - natgc > (tlenl3l;
return(pseq+41;
char *
g calloclmsg, nx, sz) g calloc
char *msg; I* program, calling routine *I
int nx, sz; I* number and size of elements *I
char *px, *callocll;
if ((px = callocl(unsigned)nx, (unsignedlszl) _ - 0) {
if (*msg) {
3 5 fprintf(stderr, "%s: g callocU failed %s fn-%d, sz-%d)In", prog, msg, nx,
szl;
exit(11;
46
CA 02369605 2001-10-O1
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returnlpx);
I"'
" get final jmps from dx[] or tmp file, set pp[], reset dmax: maim)
"I
readjmpsl) readjmps
int fd = -1;
int siz, i0, i1;
register i, j, xx;
if (fj) {
(void) fcloselfjl;
if ((fd = open(jname, 0 RDONLY, 0)) < 0) {
fprintf(stderr, "%s: can't open() %sln", prog, jname);
cleanup(1);
for (i = i0 = i1 = 0, dmax0 = dmax, xx = IenO; ; i++) ~
while (1) {
for (j = dx[dmax].ijmp; j > = 0 && dx[dmax].jp.x[j] > = xx; j--)
,
47
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Table 1 (coot 1
...readjmps
if (j < 0 && dx[dmax].offset && fj) {
(void) Iseek(fd, dx[dmax].offset, 0);
(void) read(fd, (char *)&dx[dmax].jp, sizeoflstruct jmp));
(void) readlfd, (char *)&dx(dmax].offset, sizeof(dx[dmax].offset));
dx[dmax].ijmp = MAXJMP-1;
else
break;
if (i > = JMPS) {
fprintflstderr, "%s: too many gaps in alignmentln", progl;
cleanupll );
if (j >=0){
siz = dx(dmax].jp.n[j];
xx = dx[dmax].jp.x[j];
dmax += siz;
if (siz < 0) { I* gap in second seq *I
pp(1].n[i1] _ -siz;
XX + = SIZ;
I*id=xx-yy+lenl-1
*I
pp[1].x[i1] = xx - dmax + lenl - 1;
9aPY+ +.
ngapy -= siz;
I* ignore MAXGAP when doing endgaps *I
siz = (-siz < MAXGAP ~ ~ endgaps)? -siz : MAXGAP;
il++;
else if (siz > 0) { I* gap in first seq *I
pp(0].n(i0] = siz;
pp[0].x[i0] = xx;
gapx++;
ngapx + - siz;
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I* ignore MAXGAP when doing endgaps 'I
siz - (siz < MAXGAP ~ ~ endgapsl? siz : MAXGAP;
i0++;
else
break;
I' reverse the order of jmps
'I
for (j = 0, i0--; j < i0; j++, i0--) {
i ° PP[01.n[jl; PP[01.n[!1 - PP[01.n[i0]; PP[01.n[i0] = i;
i - PPf0l.x[jl; PP[01.x[ll - PP[01.x[i01; PPfOI.x[i0] = i;
for (j - 0, i1--; j < i1; j++, i1--1 {
i - PP[11.n[jl; PP[ll.n[jl ° PP[ll.n[i11; PP[11.n[i1] = i;
i - PP[11.x[jl; PP[11.x[jl ° PP[11.x[i11; PP[11.x[i1] - i;
2 0 if (fd > = 0)
(void) closelfd);
if (fjl ~
(void) unlinkljnamel;
fj-0;
2 5 offset = 0;
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Table 1 (coot 1
" write a filled jmp struct offset of the prev one (if anyl: nw()
"I
writejmps(ix) writejmps
int ix;
{
char "mktemp(];
if (!fj) {
if (mktempljname) < 0) {
fprintflstderr, "%s: can't mktemp() %sln", prog, jnamel;
cleanupll );
if ((fj = fopen(jname, "w")) ~ = 0) {
fprintflstderr, "%s: can't write %sln", prog, jnamel;
exit111;
(void) fwritel(char ")&dx[ix].jp, sizeof(struct jmpl, 1, fj);
(void) fwritel(char ~1&dx[ix].offset, sizeofldx[ix].offsetl, 1, fj);
30
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Table 2
PRO XXXXXXXXXXXXXXX (Length ~ 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acidsl
amino acid sequence identity =
(the number of identically matching amino acid residues between the two
polypeptide sequences as determined by ALIGN-2)
divided by (the total number of amino acid residues of the PRO polypeptide) _
5 divided by 15 = 33.3%
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Table 3
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
amino acid sequence identity =
(the number of identically matching amino acid residues between the two
polypeptide sequences as determined by ALIGN~2)
divided by (the total number of amino acid residues of the PRO polypeptide) _
5 divided by 10 = 50%
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Table 4
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by ALIGN-2) divided
by (the total number of nucleotides of the PRO-DNA nucleic acid sequence)
6 divided by 14 = 42.9%
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Table 5
PRO-DNA NNNNNNNNNNNN (Length - 12 nucleotides)
Comparison DNA NNNNLLLIIU (Length = 9 nucleotides)
nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by ALIGN-2) divided
by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) _
4 divided by 12 = 33.3%
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II. Comuositions and Methods of the Invention
A. Full-len4th CHEPO Polyoeptide
The present invention provides newly identified and isolated nucleotide
sequences encoding polypeptides referred
to in the present application as CHEPO. In particular, DNA encoding a CHEPO
polypeptide has been identified and isolated,
as disclosed in further detail in the Examples below.
B. CHEPO Variants
In addition to the full-length native sequence CHEPO polypeptides described
herein, it is contemplated that
CHEPO variants can be prepared. CHEPO variants can be prepared by introducing
appropriate nucleotide changes into the
CHEPO DNA, andlar by synthesis of the desired CHEPO polypeptide. Those skilled
in the art will appreciate that amino
acid changes may alter post-translational processes of the CHEPO, such as
changing the number or position of
glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence CHEPO or in various domains of
the CHEPO described herein, can
be made, for example, using any of the techniques and guidelines for
conservative and non-conservative mutations set
forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a
substitution, deletion or insertion of one or more
colons encoding the CHEPO that results in a change in the amino acid sequence
of the CHEPO as compared with the native
sequence CHEPO. Optionally the variation is by substitution of at least one
amino acid with any other amino acid in one
or more of the domains of the CHEPO. Guidance in determining which amino acid
residue may be inserted, substituted or
deleted without adversely affecting the desired activity may be found by
comparing the sequence of the CHEPO with that
2 0 of homologous known protein molecules and minimizing the number of amino
acid sequence changes made in regions of
high homology. Amino acid substitutions can be the result of replacing one
amino acid with another amino acid having
similar structural andlor chemical properties, such as the replacement of a
leucine with a serine, i.e., conservative amino
acid replacements. Insertions or deletions may optionally be in the range of
about 1 to 5 amino acids. The variation
allowed may be determined by systematically making insertions, deletions or
substitutions of amino acids in the sequence
2 5 and testing the resulting variants for activity exhibited by the full-
length or mature native sequence.
CHEPO polypeptide fragments are provided herein. Such fragments may be
truncated at the N-terminus or C-
terminus, or may lack internal residues, for example, when compared with a
full length native protein. Certain fragments
lack amino acid residues that are not essential for a desired biological
activity of the CHEPO polypeptide.
CHEPO fragments may be prepared by any of a number of conventional techniques.
Desired peptide fragments
3 0 may be chemically synthesized. An alternative approach involves generating
CHEPO fragments by enzymatic digestion,
e.g., by treating the protein with an enzyme known to cleave proteins at sites
defined by particular amino acid residues,
or by digesting the DNA with suitable restriction enzymes and isolating the
desired fragment. Yet another suitable
technique involves isolating and amplifying a DNA fragment encoding a desired
polypeptide fragment, by polymerase chain
reaction (PCR~. Oligonucleotides that define the desired termini of the DNA
fragment are employed at the 5' and 3' primers
3 5 in the PCR. Preferably, CHEPO polypeptide fragments share at least one
biological andlor immunological activity with the
native CHEPO polypeptides shown in Figure 3 (SEO ID NOS:2 and 51.
CA 02369605 2001-10-O1
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In particular embodiments, conservative substitutions of interest are shown in
Table 6 under the heading of
preferred substitutions. If such substitutions result in a change in
biological activity, then more substantial changes,
denominated exemplary substitutions in Table 6, or as further described below
in reference to amino acid classes, are
introduced and the products screened.
Table 6
OriginalExemplary Preferred
Residue Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gln; asn lys
Asn (N) gln; his; lys; arg gln
Asp (D) glu glu
Cys (C) ser ser
Gln (Q) asn asn
Glu (E) asp asp
Gly (G) pro; ala ala
His /H) asn; gln; lys; arg arg
Ile (I) leu; val; met; ala;
phe;
norleucine leu
2 Leu IL) norleucine; ile; val;
0
met; ala; phe ile
Lys (K) arg; gln; asn arg
Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; leu
tyr
2 Pro (P) ala ala
5
Ser(S) thr thr
Thr (T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) trp; phe; thr; ser phe
3 Val (V) ile; leu; met; phe;
0
ala; norleucine leu
Substantial modifications in function or immunological identity of the CHEPO
polypeptide are accomplished by
selecting substitutions that differ significantly in their effect on
maintaining (a) the structure of the polypeptide backbone
3 5 in the area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the
56
CA 02369605 2001-10-O1
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molecule at the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on
common 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.
Non-conservative substitutions will entail exchanging a member of one of these
classes for another class. Such
substituted residues also may be introduced into the conservative substitution
sites or, more preferably, into the remaining
(non-conserved) sites.
The variations can be made using methods known in the art such as
oligonucleotide-mediated (site-directed)
mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis
[Carter et al., Nucl. Acids Res.. 13:4331
11986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis
[Wells et al., Gene, 34:315 (1985)],
restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London
SerA, 317:415 (1986)] or other known
techniques can be performed on the cloned DNA to produce the CHEPO variant
DNA.
Scanning amino acid analysis can also be employed to identify one or more
amino acids along a contiguous
sequence. Among the preferred scanning amino acids are relatively small,
neutral amino acids. Such amino acids include
2 0 alanine, glycine, serine, and cysteine. Alanine is typically a preferred
scanning amino acid among this group because it
eliminates the side-chain beyond the beta-carbon and is less likely to alter
the main-chain conformation of the variant
[Cunningham and Wells, Science, 244: 1081-1085 (19891]. Alanine is also
typically preferred because it is the most
common amino acid. Further, it is frequently found in both buried and exposed
positions [Creighton, The Proteins, (W.H.
Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine
substitution does not yield adequate amounts of
2 5 variant, an isoteric amino acid can be used.
C. Modifications of CHEPO
Covalent modifications of CHEPO are included within the scope of this
invention. One type of covalent
modification includes reacting targeted amino acid residues of a CHEPO
polypeptide with an organic derivatizing agent that
3 0 is capable of reacting with selected side chains or the N- or C- terminal
residues of the CHEPO. Derivatization with
bifunctional agents is useful, for instance, for crosslinking CHEPO to a water-
insoluble support matrix ar surface for use
in the method for purifying anti-CHEPO antibodies, and vice-versa. Commonly
used crosslinking agents include, e.g., 1,1-
bisldiazoacetyll-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters,
for example, esters with 4-azidosalicylic acid,
homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-
dithiobislsuccinimidylpropionate), bifunctional
3 5 maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-
[Ip-azidophenyl)dithio]propioimidate.
57
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Other modifications include deamidation of glutaminyl and asparaginyl residues
to the corresponding glutamyl
and aspartyl residues, respectively, hydroxylation of praline and lysine,
phosphorylation of hydroxyl groups of seryl or
threonyl residues, methylation of the -amino groups of lysine, arginine, and
histidine side chains [T.E. Creighton, Proteins:
Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-
86 (1983~], acetylation of the N-terminal
amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of the CHEPO polypeptide included within
the scope of this invention
comprises altering the native glycosylation pattern of the polypeptide.
"Altering the native glycosylation pattern" is
intended for purposes herein to mean deleting one or more carbohydrate
moieties found in native sequence CHEPO (either
by removing the underlying glycasylation site or by deleting the glycosylation
by chemical andlor enzymatic meansl, andlor
adding one or more glycosylation sites that are not present in the native
sequence CHEPO. In addition, the phrase includes
qualitative changes in the glycosylation of the native proteins, involving a
change in the nature and proportions of the
various carbohydrate moieties present.
Addition of glycosylation sites to the CHEPO polypeptide may be accomplished
by altering the amino acid
sequence. The alteration may be made, for example, by the addition of, or
substitution by, one or more serine or threonine
residues to the native sequence CHEPO (for 0-linked glycosylation sited. The
CHEPO amino acid sequence may optionally
be altered through changes at the DNA level, particularly by mutating the DNA
encoding the CHEPO polypeptide at
preselected bases such that codons are generated that will translate into the
desired amino acids.
Another means of increasing the number of carbohydrate moieties on the CHEPO
polypeptide is by chemical or
enzymatic coupling of glycosides to the polypeptide. Such methods are
described in the art, e.g., in WO 87105330
published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev.
Biochem., pp. 259-306 (19811.
Removal of carbohydrate moieties present on the CHEPO polypeptide may be
accomplished chemically or
enzymatically or by mutational substitution of codons encoding for amino acid
residues that serve as targets for
glycosylation. Ghemical deglycosylation techniques are known in the art and
described, for instance, by Hakimuddin, et
al., Arch. Biochem. Biouhvs., 259:52 (1987) and by Edge et al., Anal.
Biochem., 118:131 (19811. Enzymatic cleavage of
2 5 carbohydrate moieties on polypeptides can be achieved by the use of a
variety of endo- and exo-glycosidases as described
by Thotakura et al., Meth. Enzvmol., 138:350 (19871.
Another type of covalent modification of CHEPO comprises linking the CHEPO
polypeptide to one of a variety
of nonproteinaceous polymers, e.g., polyethylene glycol (PEGI, polypropylene
glycol, or polyoxyalkylenes, in the manner
set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337.
3 0 The CHEPO of the present invention may also be modified in a way to form a
chimeric molecule comprising
CHEPO fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the CHEPO
with a tag polypeptide which
provides an epitope to which an anti-tag antibody can selectively bind. The
epitope tag is generally placed at the amino-
or carboxyl- terminus of the CHEPO. The presence of such epitope-tagged forms
of the CHEPO can be detected using an
3 5 antibody against the tag polypeptide. Also, provision of the epitope tag
enables the CHEPO to be readily purified by affinity
purification using an anti-tag antibody or another type of affinity matrix
that binds to the epitope tag. Various tag
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polypeptides and their respective antibodies are well known in the art.
Examples include poly-histidine (poly-his) or poly-
histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its
antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-
2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies
thereto [Evan et al., Molecular and
Cellular Biolo4y, 5:3610-3616 (1985)]; and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody [Paborsky
et al., Protein Engineerinp, 3(6):547-553 (1990)]. Other tag polypeptides
include the Flag-peptide (Hopp et al.,
BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide (Martin et al.,
Science, 255:192-194 (1992)]; an -tubulin
epitope peptide [Skinner et al., J. Biol. Chem.. 266:15163-15166 (19911]; and
the T7 gene 10 protein peptide tag [Lutz-
Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of
the CHEPO with an immunoglobulin
or a particular region of an immunoglobulin. Far a bivalent form of the
chimeric molecule (also referred to as an
immunoadhesin 1, such a fusion could be to the Fc region of an IgG molecule.
The Ig fusions preferably include the
substitution of a soluble (transmembrane domain deleted or inactivated) form
of a CHEPO polypeptide in place of at least
one variable region within an Ig molecule. In a particularly preferred
embodiment, the immunoglobulin fusion includes the
hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1
molecule. For the production of immunoglobulin
fusions see also US Patent No. 5,428,130 issued June 27, 1995.
D. Preparation of CHEPO
The description below relates primarily to production of CHEPO by culturing
cells transformed or transfected with
a vector containing CHEPO nucleic acid. It is, of course, contemplated that
alternative methods, which are well known
2 0 in the art, may be employed to prepare CHEPO. For instance, the CHEPO
sequence, or portions thereof, may be produced
by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart
et al., Solid-Phase Peptide Synthesis, W.H.
Freeman Co., San Francisco, CA (19691; Merrifield, J. Am. Chem. Soc., 85:2149-
2154 (1963)]. In vitro protein synthesis
may be performed using manual techniques or by automation. Automated synthesis
may be accomplished, for instance,
using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using
manufacturer's instructions. Various portions of
the CHEPO may be chemically synthesized separately and combined using chemical
or enzymatic methods to produce the
full-length CHEPO.
Isolation of ONA Encoding CHEPO
DNA encoding CHEPO may be obtained from a cDNA library prepared from tissue
believed to possess the CHEPO
3 0 mRNA and to express it at a detectable level. Accordingly, human CHEPO DNA
can be conveniently obtained from a cDNA
library prepared from human tissue, such as described in the Examples. The
CHEPO-encoding gene may also be obtained
from a genomic library or by known synthetic procedures (e.g., automated
nucleic acid synthesis).
libraries can be screened with probes (such as antibodies to the CHEPO or
oligonucleotides of at least about 20-
80 bases) designed to identify the gene of interest or the protein encoded by
it. Screening the cDNA or genomic library
3 5 with the selected probe may be conducted using standard procedures, such
as described in Sambrook et al., Molecular
Cloninn: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,
19891. An alternative means to isolate
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the gene encoding CHEPO is to use PCR methodology [Sambrook et al., supra;
Dieffenbach et al., PCR Primer: A Laboratory
Manual (Cold Spring Harbor Laboratory Press, 19951].
The Examples below describe techniques for screening a cDNA library. The
oligonucleotide sequences selected
as probes should be of sufficient length and sufficiently unambiguous that
false positives are minimized. The
oligonucleotide is preferably labeled such that it can be detected upon
hybridization to DNA in the library being screened.
Methods of labeling are well known in the art, and include the use of
radiolabels like'ZP-labeled ATP, biotinylation or
enzyme labeling. Hybridization conditions, including moderate stringency and
high stringency, are provided in Sambrook
et al., su ra.
Sequences identified in such library screening methods can be compared and
aligned to other known sequences
deposited and available in public databases such as GenBank or other private
sequence databases. Sequence identity Iat
either the amino acid or nucleotide level) within defined regions of the
molecule or across the full-length sequence can be
determined using methods known in the art and as described herein.
Nucleic acid having protein coding sequence may be obtained by screening
selected cDNA or genomic libraries
using the deduced amino acid sequence disclosed herein for the first time,
and, if necessary, using conventional primer
extension procedures as described in Sambrook et al., supra, to detect
precursors and processing intermediates of mRNA
that may not have been reverse-transcribed into cDNA.
2. Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors
described herein for CHEPO
2 0 production and cultured in conventional nutrient media modified as
appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired sequences. The
culture conditions, such as media,
temperature, pH and the like, can be selected by the skilled artisan without
undue experimentation. In general, principles,
protocols, and practical techniques for maximizing the productivity of cell
cultures can be found in Mammalian Cell
Biotechnolouy: a Practical Approach, M. Butler, ed. pRL Press, 1991 ) and
Sambrook et al., supra.
Methods of eukaryotic cell transfection and prokaryotic cell transformation
are known to the ordinarily skilled
artisan, for example, CaClz, CaP04, liposome-mediated and electroporation.
Depending on the host cell used,
transformation is performed using standard techniques appropriate to such
cells. The calcium treatment employing calcium
chloride, as described in Sambrook et al., su ra, or electroporation is
generally used for prokaryotes. Infection with
Agrnbacterium tumefaciens is used far transformation of certain plant cells,
as described by Shaw et al., Gene, 23:315
3 0 (1983) and WO 89105859 published 29 June 1989. For mammalian cells without
such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virolo4y, 52:456-457 (1978) can
be employed. General aspects of
mammalian cell host system transfections have been described in U.S. Patent
No. 4,399,216. Transformations into yeast
are typically carried out according to the method of Van Solingen et al., J.
Bact., 130:946 (1977) and Hsiao et al., Proc.
Natl. Acad. Sci. (USAI, 76:3829119791. However, other methods for introducing
DNA into cells, such as by nuclear
3 5 microinjection, electroporation, bacterial protoplast fusion with intact
cells, or polycations, e.g., polybrene, polyornithine,
CA 02369605 2001-10-O1
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may also be used. For various techniques for transforming mammalian cells, see
Keown et al., Methods in Enzymolooy,
185:527-537 (1990) and Mansour et al., Nature. 336:348-352 (19881.
Suitable host cells for cloning or expressing the DNA in the vectors herein
include prokaryote, yeast, or higher
eukaryote cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive
organisms, for example, Enterobacteriaceae such as E. coli. Various E. cvli
strains are publicly available, such as E. coli
K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E, coli strain
W3110 (ATCC 27,325) and K5 772 (ATCC
53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such
as Escherichia, eg., E. cvli, Entervbacter,
Erwinia, Klebsiella, Proteus, Salmonella, eg., Salmonella typhimurium,
Serratia, e.g., Serratia marcescans, and Shigella,
as well as Bacilli such as B. subtilis and B. lichenifvrmis (e.g., B.
lichenifvrmis 41 P disclosed in DD 266,710 published 12
April 19891, Pseudomvnas such as P, aeruginosa, and Streptomyces. These
examples are illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host because it is a
common host strain for recombinant DNA
product fermentations. Preferably, the host cell secretes minimal amounts of
proteolytic enzymes. For example, strain
W3110 may be modified to effect a genetic mutation in the genes encoding
proteins endogenous to the host, with examples
of such hosts including E. coli W3110 strain 1 A2, which has the complete
genotype tonA ; E. cvli W3110 strain 9E4, which
has the complete genotype tonA ptr3; E. cvli W3110 strain 27C7 (ATCC 55,2441,
which has the complete genotype tvnA
ptr3 phoA E15 (argf lac1169 degP vmpT kad; E. cvli W3110 strain 37D6, which
has the complete genotype tonA ptr3
phoA E15 (argf lac1169 degP vmpT rbs7 ilvG kanr; E. coli W3110 strain 4084,
which is strain 37D6 with a non-
kanamycin resistant degP deletion mutation; and an E. cvli strain having
mutant periplasmic protease disclosed in U.S.
Patent No. 4,946,783 issued 7 August 1990. Alternatively, in vitro methods of
cloning, e.g., PCR or other nucleic acid
2 0 polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or
yeast are suitable cloning or
expression hosts for CHEPO-encoding vectors. Saccharomyces cerevisiae is a
commonly used lower eukaryotic host
microorganism. Others include Schizosaccharvmyces pvmbe (Beach and Nurse,
Nature, 290: 140 [1981]; EP 139,383
published 2 May 1985); Kluyvervmyces hosts (U.S. Patent No. 4,943,529; Fleer
et al., BioITechnology. 9: 968-975 (1991 ))
such as, eg., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.
Bacteriol., 737 [1983]I, K. fragilis (ATCC
12,424 K. bulgaricus (ATCC 16,0451, K. wickeramii (ATCC 24,1781, K. waltii
(ATCC 56,5001, K, drosophilarum (ATCC
36,906; Uan den Berg etal., BioITechnolo4y, 8: 135 (1990)), K. thermvtvlerans,
and K. marxianus,~ yarrvwia (EP 402,226);
Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.. 28: 265-
278 [1988]); Candida,~ Trichvderma reesia (EP
244,234); Neurvspora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76: 5259-
5263 [1979]h Schwanniomyces such as
3 0 Schwanniomyces occidentalis (EP 394,538 published 31 October 19901; and
filamentous fungi such as, e.g., Neurospora,
Penicillium, Tolypocladium (WO 91100357 published 10 January 1991 ), and
Aspergillus hosts such as A- nidulans (Ballance
et al., Biochem. Bioohys. Res. Commun., 112: 284-289 [1983]; Tilburn et al.,
Gene, 26: 205-221 [1983]; Yelton et al.,
Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and A. niger (Kelly and
Hynes, EMBO J., 4: 475-479 [1985]).
Methylotropic yeasts are suitable herein and include, but are not limited to,
yeast capable of growth on methanol selected
3 5 from the genera consisting of Hansenula, Candida, Kloeckera, Pichia,
Saccharomyces, Torulopsis, and Rhodotorula. A list
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of specific species that are exemplary of this class of yeasts may be found in
C. Anthony, The Biochemistry of
Methvlotrouhs, 269 (1982).
Suitable host cells for the expression of glycosylated CHEPO are derived from
multicellular organisms. Examples
of invertebrate cells include insect cells such as Drosophila S2 and
Spodoptera Sf9, as well as plant cells. Examples of
useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS
cells. More specific examples include
monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 16511; human
embryonic kidney line 1293 or 293 cells
subcloned for growth in suspension culture, Graham et al., J. Gen Virol.,
36:59 (19771); Chinese hamster ovary cellsl-DHFR
(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA. 77:4216 (1980)1; mouse
sertoli cells (TM4, Mother, Biol. Reorod.,
23:243-251 (1980)1; human lung cells (W138, ATCC CCL 75); human liver cells
IHep G2, HB 8065); and mouse mammary
tumor (MMT 060562, ATCC CCL51 ). The selection of the appropriate host cell is
deemed to be within the skill in the art.
3. Selection and Use of a Reolicable Vector
The nucleic acid (e.g., cDNA or genomic DNA) encoding CHEPO may be inserted
into a replicable vector for
cloning (amplification of the DNA) or for expression. Various vectors are
publicly available. The vector may, for example,
be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate
nucleic acid sequence may be inserted into
the vector by a variety of procedures. In general, DNA is inserted into an
appropriate restriction endonuclease sitels) using
techniques known in the art. Vector components generally include, but are not
limited to, one or more of a signal sequence,
an origin of replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination
sequence. Construction of suitable vectors containing one or more of these
components employs standard ligation
2 0 techniques which are known to the skilled artisan.
The CHEPO may be produced recombinantly not only directly, but also as a
fusion polypeptide with a
heterologous polypeptide, which may be a signal sequence or other polypeptide
having a specific cleavage site at the N-
terminus of the mature protein or polypeptide. In general, the signal sequence
may be a component of the vector, or it may
be a part of the CHEPO-encoding DNA that is inserted into the vector. The
signal sequence may be a prokaryotic signal
2 5 sequence selected, for example, from the group of the alkaline
phosphatase, penicillinase, Ipp, or heat-stable enterotoxin
II leaders. For yeast secretion the signal sequence may be, eg., the yeast
invertase leader, alpha factor leader (including
Saccharomyces and Kluyveromyces -factor leaders, the latter described in U.S.
Patent No. 5,010,1821, or acid
phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published
4 April 1990), or the signal described in
WO 90113646 published 15 November 1990. In mammalian cell expression,
mammalian signal sequences may be used
3 0 to direct secretion of the protein, such as signal sequences from secreted
polypeptides of the same or related species, as
well as viral secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that
enables the vector to replicate in one
or more selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for mast Gram-negative
bacteria, the 2 plasmid origin is suitable for yeast,
3 5 and various viral origins (SV40, polyoma, adenovirus, VSV or BPVI are
useful for cloning vectors in mammalian cells.
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Expression and cloning vectors will typically contain a selection gene, also
termed a selectable marker. Typical
selection genes encode proteins that (a) confer resistance to antibiotics or
other toxins, eg., ampicillin, neomycin,
methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or
supply critical nutrients not available from
complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
An example of suitable selectable markers for mammalian cells are those that
enable the identification of cells
competent to take up the CHEPO-encoding nucleic acid, such as DHFR or
thymidine kinase. An appropriate host cell when
wild-type DHFR is employed is the CHO cell line deficient in DHFR activity,
prepared and propagated as described by Urlaub
et al., Proc. Natl. Acad. Sci. USA, 77:4216 (19801. A suitable selection gene
for use in yeast is the trill gene present in
the yeast plasmid YRp7 (Stinchcomb et al., Nature. 282:39 (1979); Kingsman et
al., Gene, 7:141 (1979); Tschemper et
al., Gene, 10:157 (19801]. The trill gene provides a selection marker for a
mutant strain of yeast lacking the ability to
grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics,
85:12 (1977)].
Expression and cloning vectors usually contain a promoter operably linked to
the CHEPO-encoding nucleic acid
sequence to direct mRNA synthesis. Promoters recognized by a variety of
potential host cells are well known. Promoters
suitable for use with prokaryotic hosts include the -lactamase and lactose
promoter systems [Chang et al., Nature,
275:615 (19781; Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase,
a tryptophan (trill promoter system
(Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters
such as the tac promoter [deBoer et al.,
Proc. Natl. Acad. Sci. USA. 80:21-25 (1983)]. Promoters for use in bacterial
systems also will contain a Shine-Dalgarno
(S.D.) sequence operably linked to the DNA encoding CHEPO.
Examples of suitable promoting sequences for use with yeast hosts include the
promoters for 3-phosphoglycerate
2 0 kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (19801] or other
glycolytic enzymes [Hess et al., J. Adv. Enzyme Ren.,
7:149 (19681; Holland, Biochemistry, 17:4900 (19781], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate
isomerase, 3-phosphoglycerate mutase,
pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase.
Other yeast promoters, which are inducible promoters having the additional
advantage of transcription controlled
by growth conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase,
degradative enzymes associated with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase,
and enzymes responsible for maltose and galactose utilization. Suitable
vectors and promoters for use in yeast expression
are further described in EP 73,657.
CHEPO transcription from vectors in mammalian host cells is controlled, for
example, by promoters obtained from
3 0 the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504
published 5 July 19891, adenovirus (such as
Adenovirus 21, bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus
40 (S1140), from heterologous mammalian promoters, e.g., the actin promoter or
an immunoglobulin promoter, and from
heat-shock promoters, provided such promoters are compatible with the host
cell systems.
Transcription of a DNA encoding the CHEPO by higher eukaryotes may be
increased by inserting an enhancer
3 5 sequence into the vector. Enhancers are cis-acting elements of DNA,
usually about from 10 to 300 bp, that act on a
promoter to increase its transcription. Many enhancer sequences are now known
from mammalian genes (globin, elastase,
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albumin, -fetoprotein, and insulin). Typically, however, one will use an
enhancer from a eukaryotic cell virus. Examples
include the SU40 enhancer on the late side of the replication origin (bp 100-
270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication origin, and
adenovirus enhancers. The enhancer may
be spliced into the vector at a position 5' or 3' to the CHEPO coding
sequence, but is preferably located at a site 5' from
the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant,
animal, human, or nucleated cells
from other multicellular organisms) will also contain sequences necessary for
the termination of transcription and for
stabilizing the mRNA. Such sequences are commonly available from the 5' and,
occasionally 3', untranslated regions of
eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments
transcribed as polyadenylated fragments
in the untranslated portion of the mRNA encoding CHEPO.
Still other methods, vectors, and host cells suitable for adaptation to the
synthesis of CHEPO in recombinant
vertebrate cell culture are described in Gething et al., Nature. 293:620-625
(1981); Mantei et al., Nature, 281:40-46
(1979); EP 117,060; and EP 117,058.
4. Detectinu Gene AmplificationlExnression
Gene amplification andlor expression may be measured in a sample directly, for
example, by conventional
Southern blotting, Northern blotting to quantitate the transcription of mRNA
(Thomas, Proc. Natl. Acad. Sci. USA,
77:5201-5205 (19801], dot blotting (DNA analysis), or in situ hybridization,
using an appropriately labeled probe, based
on the sequences provided herein. Alternatively, antibodies may be employed
that can recognize specific duplexes, including
2 0 DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein
duplexes. The antibodies in turn may be
labeled and the assay may be carried out where the duplex is bound to a
surface, so that upon the formation of duplex on
the surface, the presence of antibody bound to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological methods, such
as immunohistochemical
staining of cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene
2 5 product. Antibodies useful for immunohistochemical staining andlor assay
of sample fluids may be either monoclonal or
polyclonal, and may be prepared in any mammal. Conveniently, the antibodies
may be prepared against a native sequence
CHEPO polypeptide or against a synthetic peptide based on the DNA sequences
provided herein or against exogenous
sequence fused to CHEPO DNA and encoding a specific antibody epitope.
3 0 5. Purification of Polvoeutide
Forms of CHEPO may be recovered from culture medium or from host cell lysates.
If membrane-bound, it can
be released from the membrane using a suitable detergent solution (e.g. Triton-
X 100) or by enzymatic cleavage. Cells
employed in expression of CHEPO can be disrupted by various physical or
chemical means, such as freeze-thaw cycling,
sonication, mechanical disruption, or cell lysing agents.
3 5 It may be desired to purify CHEPO from recombinant cell proteins or
polypeptides. The following procedures are
exemplary of suitable purification procedures: by fractionation on an ion-
exchange column; ethanol precipitation; reverse
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phase HPLC; chromatography on silica or on a cation-exchange resin such as
DEAE; chromatofocusing; SDS-PAGE;
ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-
75; protein A Sepharose columns to remove
contaminants such as IgG; and metal chelating columns to bind epitope-tagged
forms of the CHEPO. Various methods of
protein purification may be employed and such methods are known in the art and
described for example in Deutscher,
Methods in Enzvmologv, 182 (19901; Scopes, Protein Purification: Princiules
and Practice, Springer-Uerlag, New York
(19821. The purification steps) selected will depend, for example, on the
nature of the production process used and the
particular CHEPO produced.
E. Uses for CHEPO
Nucleotide sequences (or their complement) encoding CHEPO have various
applications in the art of molecular
biology, including uses as hybridization probes, in chromosome and gene
mapping and in the generation of anti-sense RNA
and DNA. CHEPO nucleic acid will also be useful for the preparation of CHEPO
polypeptides by the recombinant techniques
described herein.
The full-length native sequence CHEPO cDNA (SED ID N0:31, or portions thereof,
may be used as hybridization
probes for a cDNA library to isolate the fulhlength CHEPO cDNA or to isolate
still other cDNAs (for instance, those
encoding naturally-occurring variants of CHEPO or CHEPO from other species)
which have a desired sequence identity to
the CHEPO sequence disclosed in Figure 2 (SEO ID N0:31. Optionally, the length
of the probes will be about 20 to about
50 bases. The hybridization probes may be derived from at least partially
novel regions of the nucleotide sequence of SED
ID N0:3 wherein those regions may be determined without undue experimentation
or from genomic sequences including
2 0 promoters, enhancer elements and introns of native sequence CHEPO. By way
of example, a screening method will
comprise isolating the coding region of the CHEPO gene using the known DNA
sequence to synthesize a selected probe of
about 40 bases. Hybridization probes may be labeled by a variety of labels,
including radionucleotides such as 32P or'SS,
or enzymatic labels such as alkaline phosphatase coupled to the probe via
avidinlbiotin coupling systems. Labeled probes
having a sequence complementary to that of the CHEPO gene of the present
invention can be used to screen libraries of
2 5 human cDNA, genomic DNA or mRNA to determine which members of such
libraries the probe hybridizes to. Hybridization
techniques are described in further detail in the Examples below.
Any EST sequences disclosed in the present application may similarly be
employed as probes, using the methods
disclosed herein.
Other useful fragments of the CHEPO nucleic acids include antisense or sense
oligonucleotides comprising a
3 0 singe-stranded nucleic acid sequence (either RNA or DNA) capable of
binding to target CHEPO mRNA (sense) or CHEPO
DNA Iantisense) sequences. Antisense or sense oligonucleotides, according to
the present invention, comprise a fragment
of the coding region of CHEPO DNA. Such a fragment generally comprises at
least about 14 nucleotides, preferably from
about 14 to 30 nucleotides. The ability to derive an antisense or a sense
oligonucleotide, based upon a cDNA sequence
encoding a given protein is described in, for example, Stein and Cohen
(CancerRes. 48:2659, 1988) and van der Krol et
3 5 al. (BioTechniques 6:958, 19881.
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Binding of antisense or sense oligonucleotides to target nucleic acid
sequences results in the formation of
duplexes that block transcription or translation of the target sequence by one
of several means, including enhanced
degradation of the duplexes, premature termination of transcription or
translation, or by other means. The antisense
oligonucleotides thus may be used to block expression of CHEPO proteins.
Antisense or sense oligonucleotides further
comprise oligonucleotides having modified sugar-phosphodiester backbones (or
other sugar linkages, such as those
described in WO 91106629) and wherein such sugar linkages are resistant to
endogenous nucleases. Such oligonucleotides
with resistant sugar linkages are stable in vivo (i.e., capable of resisting
enzymatic degradation) but retain sequence
specificity to be able to bind to target nucleotide sequences.
Other examples of sense or antisense oligonucleotides include those
oligonucleotides which are covalently linked
to organic moieties, such as those described in WO 90110048, and other
moieties that increases affinity of the
oligonucleotide for a target nucleic acid sequence, such as poly-IL-lysinel.
Further still, intercalating agents, such as
ellipticine, and alkylating agents or metal complexes may be attached to sense
or antisense oligonucleotides to modify
binding specificities of the antisense or sense oligonucleotide for the target
nucleotide sequence.
Antisense or sense oligonucleotides may be introduced into a cell containing
the target nucleic acid sequence by
any gene transfer method, including, for example, CaP04-mediated DNA
transfection, electraporation, or by using gene
transfer vectors such as Epstein-Barr virus. In a preferred procedure, an
antisense or sense oligonucleotide is inserted into
a suitable retroviral vector. A cell containing the target nucleic acid
sequence is contacted with the recombinant retroviral
vector, either in vivo or ex viva. Suitable retroviral vectors include, but
are not limited to, those derived from the murine
retrovirus M-MuLU, N2 (a retrovirus derived from M-MuLU), or the double copy
vectors designated DCTSA, DCTSB and
2 0 DCT5C (see WO 90113641 ).
Sense or antisense oligonucleotides also may be introduced into a cell
containing the target nucleotide sequence
by formation of a conjugate with a ligand binding molecule, as described in WO
91104753. Suitable ligand binding
molecules include, but are not limited ta, cell surface receptors, growth
factors, other cytokines, or other ligands that bind
to cell surface receptors. Preferably, conjugation of the ligand binding
molecule does not substantially interfere with the
2 5 ability of the ligand binding molecule to bind to its corresponding
molecule or receptor, or block entry of the sense or
antisense oligonucleotide or its conjugated version into the cell.
Alternatively, a sense or an antisense oligonucleotide may be introduced into
a cell containing the target nucleic
acid sequence by formation of an oligonucleotide-lipid complex, as described
in WO 90110448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the cell by an
endogenous lipase.
3 0 The probes may also be employed in PCR techniques to generate a pool of
sequences for identification of closely
related CHEPO coding sequences.
Nucleotide sequences encoding a CHEPO can also be used to construct
hybridization probes for mapping the gene
which encodes that CHEPO and for the genetic analysis of individuals with
genetic disorders. The nucleotide sequences
provided herein may be mapped to a chromosome and specific regions of a
chromosome using known techniques, such as
3 5 in situ hybridization, linkage analysis against known chromosomal markers,
and hybridization screening with libraries.
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When the coding sequences for CHEPO encode a protein which binds to another
protein (example, where the
CHEPO is a receptorl, the CHEPO can be used in assays to identify the other
proteins or molecules involved in the binding
interaction. By such methods, inhibitors of the receptorhigand binding
interaction can be identified. Proteins involved in
such binding interactions can also be used to screen for peptide or small
molecule inhibitors or agonists of the binding
interaction. Also, the receptor CHEPO can be used to isolate correlative
ligand(s). Screening assays can be designed to
find lead compounds that mimic the biological activity of a native CHEPO or a
receptor for CHEPO. Such screening assays
will include assays amenable to high-throughput screening of chemical
libraries, making them particularly suitable for
identifying small molecule drug candidates. Small molecules contemplated
include synthetic organic or inorganic
compounds. The assays can be performed in a variety of formats, including
protein-protein binding assays, biochemical
screening assays, immunoassays and cell based assays, which are well
characterized in the art.
Nucleic acids which encode CHEPO or its modified forms can also be used to
generate either transgenic animals
or "knock out" animals which, in turn, are useful in the development and
screening of therapeutically useful reagents. A
transgenic animal (e.g., a mouse or rat) is an animal having cells that
contain a transgene, which transgene was introduced
into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic
stage. A transgene is a DNA which is
integrated into the genome of a cell from which a transgenic animal develops.
In one embodiment, cDNA encoding CHEPD
can be used to clone genomic DNA encoding CHEPO in accordance with established
techniques and the genomic sequences
used to generate transgenic animals that contain cells which express DNA
encoding CHEPO. Methods for generating
transgenic animals, particularly animals such as mice or rats, have become
conventional in the art and are described, for
example, in U.S. Patent Nos. 4,736,866 and 4,870,009. Typically, particular
cells would be targeted for CHEPO transgene
2 0 incorporation with tissue-specific enhancers. Transgenic animals that
include a copy of a transgene encoding CHEPO
introduced into the germ line of the animal at an embryonic stage can be used
to examine the effect of increased expression
of DNA encoding CHEPO. Such animals can be used as tester animals for reagents
thought to confer protection from, for
example, pathological conditions associated with its overexpression. In
accordance with this facet of the invention, an
animal is treated with the reagent and a reduced incidence of the pathological
condition, compared to untreated animals
2 5 bearing the transgene, would indicate a potential therapeutic intervention
for the pathological condition.
Alternatively, non-human homologues of CHEPO can be used to construct a CHEPO
"knock out" animal which
has a defective or altered gene encoding CHEPO as a result of homologous
recombination between the endogenous gene
encoding CHEPO and altered genomic DNA encoding CHEPO introduced into an
embryonic stem cell of the animal. For
example, cDNA encoding CHEPO can be used to clone genomic DNA encoding CHEPO
in accordance with established
3 0 techniques. A portion of the genomic DNA encoding CHEPO can be deleted or
replaced with another gene, such as a gene
encoding a selectable marker which can be used to monitor integration.
Typically, several kilobases of unaltered flanking
DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomas
and Capecchi, Cell, 51:503 (1987) for a
description of homologous recombination vectors]. The vector is introduced
into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced DNA has homologously
recombined with the endogenous DNA are
3 5 selected [see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells
are then injected into a blastocyst of an animal (e.g.,
a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in
Teratocarcinomas and Embryonic Stem Cells: A Practical
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Approach, E. J. Robertson, ed. (IRL, Oxford, 19871, pp. 113-152]. A chimeric
embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term to create a
"knock out" animal. Progeny harboring
the homologously recombined DNA in their germ cells can be identified by
standard techniques and used to breed animals
in which all cells of the animal contain the homologously recombined DNA.
Knockout animals can be characterized for
instance, for their ability to defend against certain pathological conditions
and for their development of pathological
conditions due to absence of the CHEPO polypeptide.
Nucleic acid encoding the CHEPO polypeptides may also be used in gene therapy.
In gene therapy applications,
genes are introduced into cells in order to achieve in vivo synthesis of a
therapeutically effective genetic product, for
example for replacement of a defective gene. "Gene therapy" includes both
conventional gene therapy where a lasting
effect is achieved by a single treatment, and the administration of gene
therapeutic agents, which involves the one time
or repeated administration of a therapeutically effective DNA or mRNA.
Antisense RNAs and DNAs can be used as
therapeutic agents for blocking the expression of certain genes in vivo. It
has already been shown that short antisense
oligonucleotides can be imported into cells where they act as inhibitors,
despite their low intracellular concentrations
caused by their restricted uptake by the cell membrane. (Zamecnik et al.,
Proc. Natl. Acad. Sci. USA 83, 4143-4146
[1986]I. The oligonucleotides can be modified to enhance their uptake, e.g. by
substituting their negatively charged
phosphodiester groups by uncharged groups.
There are a variety of techniques available for introducing nucleic acids into
viable cells. The techniques vary
depending upon whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host.
Techniques suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes,
2 0 electroporation, microinjection, cell fusion, DEAE-dextran, the calcium
phosphate precipitation method, etc. The currently
preferred in vivo gene transfer techniques include transfection with viral
(typically retroviral) vectors and viral coat protein-
liposome mediated transfection (Dzau et al., Trends in Biotechnolo4y 11, 205-
210 [1993]I. In some situations it is
desirable to provide the nucleic acid source with an agent that targets the
target cells, such as an antibody specific for
a cell surface membrane protein or the target cell, a ligand for a receptor on
the target cell, etc. Where liposomes are
2 5 employed, proteins which bind to a cell surface membrane protein
associated with endocytosis may be used for targeting
andlor to facilitate uptake, e.g. capsid proteins or fragments thereof tropic
for a particular cell type, antibodies for proteins
which undergo internalization in cycling, proteins that target intracellular
localization and enhance intracellular half-life.
The technique of receptor-mediated endocytosis is described, for example, by
Wu et al., J. Biol. Chem. 262, 4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (19901.
For review of gene marking and gene
3 0 therapy protocols see Anderson et al., Science 256, 808-813 (19921.
The CHEPO polypeptides described herein may also be employed as molecular
weight markers for protein
electrophoresis purposes.
The nucleic acid molecules encoding the CHEPO polypeptides or fragments
thereof described herein are useful
for chromosome identification. In this regard, there exists an ongoing need to
identify new chromosome markers, since
3 5 relatively few chromosome marking reagents, based upon actual sequence
data are presently available. Each CHEPO
nucleic acid molecule of the present invention can be used as a chromosome
marker.
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The CHEPO polypeptides and nucleic acid molecules of the present invention may
also be used for tissue typing,
wherein the CHEPO polypeptides of the present invention may be differentially
expressed in one tissue as compared to
another. CHEPO nucleic acid molecules will find use for generating probes for
PCR, Northern analysis, Southern analysis
and Western analysis.
The CHEPO polypeptides described herein may also be employed as therapeutic
agents. The CHEPO polypeptides
of the present invention can be formulated according to known methods to
prepare pharmaceutically useful compositions,
whereby the CHEPO product hereof is combined in admixture with a
pharmaceutically acceptable carrier vehicle.
Therapeutic formulations are prepared for storage by mixing the active
ingredient having the desired degree of purity with
optional physiologically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (198011, in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients or
stabilizers are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate,
citrate and other organic acids; antioxidants including ascorbic acid; low
molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins;
hydrophilic polymers such as
polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine,
arginine or lysine; monosaccharides, disaccharides
1 S and other carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; salt-forming counterions such as sodium; andlor nonionic
surfactants such as TWEEN'"", PLURONICSr""
or PEG.
The formulations to be used for in vivo administration must be sterile. This
is readily accomplished by filtration
through sterile filtration membranes, prior to or following lyophilization and
reconstitution.
2 0 Therapeutic compositions herein 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.
The route of administration is in accord with known methods, e.g. injection or
infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or
intralesional routes, topical administration, or by
sustained release systems.
2 5 Dosages and desired drug concentrations of pharmaceutical compositions of
the present invention may vary
depending on the particular use envisioned. The determination of the
appropriate dosage or route of administration is well
within the skill of an ordinary physician. Animal experiments provide reliable
guidance for the determination of effective
doses for human therapy. Interspecies scaling of effective doses can be
performed following the principles laid down by
Mordenti, J. and Chappell, W. "The use of interspecies scaling in
toxicokinetics" In Toxicokinetics and New Drug
3 0 Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 4296.
When in vivo administration of a CHEPO polypeptide or agonist or antagonist
thereof is employed, normal dosage
amounts may vary from about 10 nglkg to up to 100 mglkg of mammal body weight
or more per day, preferably about 1
glkglday to 10 mglkglday, depending upon the route of administration. Guidance
as to particular dosages and methods
of delivery is provided in the literature; see, for example, U.S. Pat. Nos.
4,657,760; 5,206,344; or 5,225,212. It is
3 5 anticipated that different formulations will be effective for different
treatment compounds and different disorders, that
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CA 02369605 2001-10-O1
WO 00/68376 PCT/US00/12370
administration targeting one organ or tissue. for example, may necessitate
delivery in a manner different from that to
another organ or tissue.
Where sustained-release administration of a CHEPO potypeptide is desired in a
formulation with release
characteristics suitable for the treatment of any disease or disorder
requiring administration of the CHEPO polypeptide,
microencapsulation of the CHEPO polypeptide is contemplated.
Microencapsulation of recombinant proteins for sustained
release has been successfully performed with human growth hormone (rhGH),
interferon- (rhIFN-1, interleukin-2, and MN
rgp120. Johnson et al., Nat. Med.. 2: 795-799 (19961; Yasuda. Biomed. Ther.,
27: 1221-1223 (19931; Hora et al.,
BioITechnoloay. 8: 755-758 (19901; Cleland, "Design and Production of Single
Immunization Vaccines Using Polylactide
Polyglycolide Microsphere Systems," in Vaccine Design: The Subunit and
Adiuvant Auproach, Powell and Newman, eds,
(Plenum Press: New York,19951, pp. 439-462; WO 97103692, WO 96140072, WO
96107399; and U.S Pat. No. 5,654,010.
The sustained-release formulations of these proteins were developed using poly-
lactic-coglycolic acid (PLGA)
polymer due to its biocompatibility and wide range of biodegradable
properties. The degradation products of PLGA, lactic
and glycolic acids, can be cleared quickly within the human body. Moreover,
the degradability of this polymer can be
adjusted from months to years depending on its molecular weight and
composition. Lewis, "Controlled release of bioactive
agents from lactidelglycolide polymer," in: M. Chasin and R. Langer (Eds.l,
Biode4radable Polymers as Drug Delivery
S sv tams (Marcel Dekker: New York, 1990), pp. 1-41.
This invention encompasses methods of screening compounds to identify those
that mimic the CHEPO
polypeptide (agonistsl or prevent the effect of the CHEPO polypeptide
(antagonists). Screening assays for antagonist drug
candidates are designed to identify compounds that bind or complex with the
CHEPO polypeptides encoded by the genes
2 0 identified herein, or otherwise interfere with the interaction of the
encoded polypeptides with other cellular proteins. Such
screening assays will include assays amenable to high-throughput screening of
chemical libraries, making them particularly
suitable for identifying small molecule drug candidates.
The assays can be performed in a variety of formats, including protein-protein
binding assays, biochemical
screening assays, immunoassays, and cell-based assays, which are well
characterized in the art. All assays for
2 5 antagonists are common in that they call for contacting the drug candidate
with a CHEPO polypeptide encoded by a nucleic
acid identified herein under conditions and for a time sufficient to allow
these two components to interact.
In binding assays, the interaction is binding and the complex formed can be
isolated or detected in the reaction
mixture. In a particular embodiment, the CHEPO polypeptide encoded by the gene
identified herein or the drug candidate
is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or
non-covalent attachments. Non-covalent
3 0 attachment generally is accomplished by coating the solid surface with a
solution of the CHEPO polypeptide and drying.
Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific
for the CHEPO polypeptide to be immobilized
can be used to anchor it to a solid surface. The assay is performed by adding
the non-immobilized component, which may
be labeled by a detectable label, to the immobilized component, e.g., the
coated surface containing the anchored
component. When the reaction is complete, the non-reacted components are
removed, e.g., by washing, and complexes
3 5 anchored on the solid surface are detected. When the originally non-
immobilized component carries a detectable label, the
detection of label immobilized on the surface indicates that complexing
occurred. Where the originally non-immobilized
CA 02369605 2001-10-O1
WO 00/68376 PCT/US00/12370
component does not carry a label. complexing can be detected, for example, by
using a labeled antibody specifically binding
the immobilized complex.
If the candidate compound interacts with but does not bind to a particular
CHEPO polypeptide encoded by a gene
identified herein, its interaction with that polypeptide can be assayed by
methods well known for detecting protein-protein
interactions. Such assays include traditional approaches, such as, e.g., cross-
linking, co-immunoprecipitation, and co
purification through gradients or chromatographic columns. In addition,
protein-protein interactions can be monitored by
using a yeast-based genetic system described by Fields and co-workers (Fields
and Song, Nature (Londonh 340: 245-246
(1989); Chien etal., Proc. Natl. Acad. Sci. USA. 88: 9578-9582 (19911) as
disclosed by Chevray and Nathans, Proc. Natl.
Acad. Sci. USA, 89: 5789-5793 (19911. Many transcriptional activators, such as
yeast GAL4, consist of two physically
discrete modular domains, one acting as the DNA-binding domain, the other one
functioning as the transcription-activation
domain. The yeast expression system described in the foregoing publications
(generally referred to as the "two-hybrid
system") takes advantage of this property, and employs two hybrid proteins,
one in which the target protein is fused to
the DNA-binding domain of GAL4, and another, in which candidate activating
proteins are fused to the activation domain.
The expression of a GAL1-/acZ reporter gene under control of a GAL4-activated
promoter depends on reconstitution of
GAL4 activity via protein-protein interaction. Colonies containing interacting
polypeptides are detected with a chromogenic
substrate for -galactosidase. A complete kit (MATCHMAKERT"') for identifying
protein-protein interactions between two
specific proteins using the two-hybrid technique is commercially available
from Clontech. This system can also be extended
to map protein domains involved in specific protein interactions as well as to
pinpoint amino acid residues that are crucial
for these interactions.
2 0 Compounds that interfere with the interaction of a gene encoding a CHEPO
polypeptide identified herein and
other intro- or extracellular components can be tested as follows: usually a
reaction mixture is prepared containing the
product of the gene and the intro- or extracellular component under conditions
and for a time allowing for the interaction
and binding of the two products. To test the ability of a candidate compound
to inhibit binding, the reaction is run in the
absence and in the presence of the test compound. In addition, a placebo may
be added to a third reaction mixture, to serve
2 5 as positive control. The binding (complex formation) between the test
compound and the intro- or extracellular component
present in the mixture is monitored as described hereinabove. The formation of
a complex in the control reactionls) but
not in the reaction mixture containing the test compound indicates that the
test compound interferes with the interaction
of the test compound and its reaction partner.
To assay for antagonists, the CHEPO polypeptide may be added to a cell along
with the compound to be screened
3 0 for a particular activity and the ability of the compound to inhibit the
activity of interest in the presence of the CHEPO
polypeptide indicates that the compound is an antagonist to the CHEPO
polypeptide. Alternatively, antagonists may be
detected by combining the CHEPO polypeptide and a potential antagonist with
membrane-bound CHEPO polypeptide
receptors or recombinant receptors under appropriate conditions for a
competitive inhibition assay. The CHEPO polypeptide
can be labeled, such as by radioactivity, such that the number of CHEPO
polypeptide molecules bound to the receptor can
3 5 be used to determine the effectiveness of the potential antagonist. The
gene encoding the receptor can be identified by
numerous methods known to those of skill in the art, for example, ligand
panning and FACS sorting. Coligan etal., Current
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Protocols in Immun., 1(21: Chapter 5 (19911. Preferably, expression cloning is
employed wherein polyadenylated RNA is
prepared from a cell responsive to the CHEPO palypeptide and a cDNA library
created from this RNA is divided into pools
and used to transfect COS cells or other cells that are not responsive to the
CHEPO polypeptide. Transfected cells that
are grown on glass slides are exposed to labeled CHEPO polypeptide. The CHEPO
polypeptide can be labeled by a variety
of means including iodination or inclusion'of a recognition site for a site-
specific protein kinase. Following fixation and
incubation, the slides are subjected to autoradiographic analysis. Positive
pools are identified and sub-pools are prepared
and re-transfected using an interactive sub-pooling and re-screening process,
eventually yielding a single clone that encodes
the putative receptor.
As an alternative approach for receptor identification, labeled CHEPO
polypeptide can be photoaffinity-linked with
cell membrane or extract preparations that express the receptor molecule.
Cross-linked material is resolved by PAGE and
exposed to X-ray film. The labeled complex containing the receptor can be
excised, resolved into peptide fragments, and
subjected to protein micro-sequencing. The amino acid sequence obtained from
micro- sequencing would be used to design
a set of degenerate oligonucleotide probes to screen a cDNA library to
identify the gene encoding the putative receptor.
In another assay for antagonists, mammalian cells or a membrane preparation
expressing the receptor would be
incubated with labeled CHEPO polypeptide in the presence of the candidate
compound. The ability of the compound to
enhance or block this interaction could then be measured.
More specific examples of potential antagonists include an oligonucleotide
that binds to the tusions of
immunoglobulin with CHEPO polypeptide, and, in particular, antibodies
including, without limitation, poly- and monoclonal
antibodies and antibody fragments, single-chain antibodies, anti-idiotypic
antibodies, and chimeric or humanized versions
2 0 of such antibodies or fragments, as well as human antibodies and antibody
fragments. Alternatively, a potential antagonist
may be a closely related protein, for example, a mutated form of the CHEPO
polypeptide that recognizes the receptor but
imparts no effect, thereby competitively inhibiting the action of the CHEPO
polypeptide.
Another potential CHEPO polypeptide antagonist is an antisense RNA or DNA
construct prepared using antisense
technology, where, e.g., an antisense RNA or DNA molecule acts to block
directly the translation of mRNA by hybridizing
2 5 to targeted mRNA and preventing protein translation. Antisense technology
can be used to control gene expression through
triple-helix formation or antisense DNA or RNA, both of which methods are
based on binding of a polynucleotide to DNA
or RNA. For example, the 5' coding portion of the polynucleotide sequence,
which encodes the mature CHEPO polypeptides
herein, is used to design an antisense RNA oligonucleotide of from about 10 to
40 base pairs in length. A DNA
oligonucleotide is designed to be complementary to a region of the gene
involved in transcription (triple helix - see Lee et
3 0 a/., Nucl. Acids Res., 6: 3073 (19791; Cooney et al., Science, 241: 456
(1988); Dervan et al., Science, 251:1360 (199111,
thereby preventing transcription and the production of the CHEPO polypeptide.
The antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule
into the CHEPO polypeptide (antisense -
Okano, Neurochem., 56: 560 (19911; Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression (CRC Press: Boca
Baton, FL, 19881. The oligonucleotides described above can also be delivered
to cells such that the antisense RNA or DNA
3 5 may be expressed in vivo to inhibit production of the CHEPO polypeptide.
When antisense DNA is used,
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oligodeoxyribonucleotides derived from the translation-initiation site, e.g.,
between about -10 and + 10 positions of the
target gene nucleotide sequence, are preferred.
Potential antagonists include small molecules that bind to the active site,
the receptor binding site, or growth
factor or other relevant binding site of the CHEPO polypeptide, thereby
blocking the normal biological activity of the CHEPO
polypeptide. Examples of small molecules include, but are not limited to,
small peptides or peptide-like molecules,
preferably soluble peptides, and synthetic non-peptidyl organic or inorganic
compounds.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific
cleavage of RNA. Ribozymes act by
sequence-specific hybridization to the complementary target RNA, followed by
endonucleolytic cleavage. Specific ribozyme
cleavage sites within a potential RNA target can be identified by known
techniques. For further details see, e.g., Rossi,
Current Bioloay, 4: 469-471 (19941, and PCT publication No. WO 97133551
(published September 18, 19971.
Nucleic acid molecules in triple-helix formation used to inhibit transcription
should be single-stranded and
composed of deoxynucleotides. The base composition of these oligonucleotides
is designed such that it promotes triple-
helix formation via Hoogsteen base-pairing rules, which generally require
sizeable stretches of purines or pyrimidines on
one strand of a duplex. For further details see, eg., PCT publication Na. WO
97133551, supra.
These small molecules can be identified by any one or more of the screening
assays discussed hereinabove andlor
by any other screening techniques well known for those skilled in the art.
F. Anti-CHEPO Antibodies
The present invention further provides anti-CHEPO antibodies. Exemplary
antibodies include polyclonal,
2 0 monoclonal, humanized, bispecific, and heteroconjugate antibodies.
Polvclonal Antibodies
The anti-CHEPO antibodies may comprise polyclonal antibodies. Methods of
preparing polyclonal antibodies are
known to the skilled artisan. Polyclonal antibodies can be raised in a mammal,
for example, by one or more injections of
2 5 an immunizing agent and, if desired, an adjuvant. Typically, the
immunizing agent andlor adjuvant will be injected in the
mammal by multiple subcutaneous or intraperitoneal injections. The immunizing
agent may include the CHEPO polypeptide
or a fusion protein thereof. It may be useful to conjugate the immunizing
agent to a protein known to be immunogenic in
the mammal being immunized. Examples of such immunogenic proteins include but
are not limited to keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin
inhibitor. Examples of adjuvants which may be
3 0 employed include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose
dicorynomycolatel. The immunization protocol may be selected by one skilled in
the art without undue experimentation.
2. Monoclonal Antibodies
The anti-CHEPO antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared
3 5 using hybridoma methods, such as those described by Kohler and Milstein,
Nature, 256:495 (19751. In a hybridoma
method, a mouse, hamster, or other appropriate host animal, is typically
immunized with an immunizing agent to elicit
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lymphocytes that produce or are capable of producing antibodies that will
specifically bind to the immunizing agent.
Alternatively, the lymphocytes may be immunized in vitro.
The immunizing agent will typically include the CHEPO polypeptide or a fusion
protein thereof. Generally, either
peripheral blood lymphocytes ["PBLs") are used if cells of human origin are
desired, or spleen cells or lymph node cells are
used if non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using
a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell
[coding, Monoclonal Antibodies: Principles
and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are
usually transformed mammalian cells,
particularly myeloma cells of rodent, bovine and human origin. Usually, rat or
mouse myeloma cell lines are employed. The
hybridoma cells may be cultured in a suitable culture medium that preferably
contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For example, if the
parental cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRTI, the culture medium for the
hybridomas typically will include
hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances
prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high level expression of antibody
by the selected antibody-producing cells, and are sensitive to a medium such
as HAT medium. More preferred immortalized
cell lines are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center,
San Diego, California and the American Type Culture Collection, Manassas,
Virginia. Human myeloma and mouse-human
heteromyeloma cell lines also have been described for the production of human
monoclonal antibodies [Kozbor, J. Immunol..
133:3001 (19841; Brodeur et al., Monoclonal Antibody Production TechniQUes and
Annlications Marcel Dekker, Inc., New
York, (1987) pp. 51-63].
2 0 The culture medium in which the hybridoma cells are cultured can then be
assayed for the presence of monoclonal
antibodies directed against CHEPO. Preferably, the binding specificity of
monoclonal antibodies produced by the hybridoma
cells is determined by immunoprecipitation or by an in vitro binding assay,
such as radioimmunoassay (RIA) or enzyme
linked immunoabsorbent assay (ELISAI. Such techniques and assays are known in
the art. The binding affinity of the
monoclonal antibody can, for example, be determined by the Scatchard analysis
of Munson and Pollard, Anal. Biochem., .
107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned
by limiting dilution procedures and
grown by standard methods [coding, suura]. Suitable culture media for this
purpose include, for example, Dulbecco's
Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma
cells may be grown in vivo as ascites in
a mammal.
3 0 The monoclonal antibodies secreted by the subclones may be isolated or
purified from the culture medium or
ascites fluid by conventional immunoglobulin purification procedures such as,
for example, protein A-Sepharose,
hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity
chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as
those described in U.S.
Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention
can be readily isolated and sequenced
3 5 using conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes
encoding the heavy and light chains of murine antibodies). The hybridoma cells
of the invention serve as a preferred source
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WO 00/68376 PCT/US00/12370
of such DNA. Once isolated, the DNA may be placed into expression vectors,
which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CH0) cells, or myeloma cells
that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in
the recombinant host cells. The DNA also may
be modified, for example, by substituting the coding sequence for human heavy
and light chain constant domains in place
of the homologous murine sequences [U.S. Patent No. 4,816,567; Morrison et
al., supra] or by covalently joining to the
immunoglobulin coding sequence all or part of the coding sequence for a non-
immunoglobulin polypeptide. Such a non-
immunoglobulin polypeptide can be substituted for the constant domains of an
antibody of the invention, or can be
substituted for the variable domains of one antigen-combining site of an
antibody of the invention to create a chimeric
bivalent antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent
antibodies are well known in
the art. For example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain.
The heavy chain is truncated generally at any point in the Fc region so as to
prevent heavy chain crosslinking.
Alternatively, the relevant cysteine residues are substituted with another
amino acid residue or are deleted so as to prevent
crosslinking.
/n vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce
fragments thereof, particularly, Fab fragments, can be accomplished using
routine techniques known in the art.
3. Human and Humanized Antibodies
The anti-CHEPO antibodies of the invention may further comprise humanized
antibodies or human antibodies.
2 0 Humanized forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', Flab'IZ or other antigen-binding subsequences
of antibodies) which contain minimal sequence
derived from non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in
which residues from a complementary determining region (CDR) of the recipient
are replaced by residues from a CDR of
a non-human species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity.
2 5 In some instances, Fv framework residues of the human immunoglobulin are
replaced by corresponding non-human
residues. Humanized antibodies may also comprise residues which are found
neither in the recipient antibody nor in the
imported CDR or framework sequences. In general, the humanized antibody will
comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially all of the
CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are those of a
human immunoglobulin consensus sequence.
3 0 The humanized antibody optimally also will comprise at least a portion of
an immunoglobulin constant region [Fc], typically
that of a human immunoglobulin [Jones et al., Nature. 321:522-525 (1986);
Riechmann et al., Nature. 332:323-329
(19881; and Presto, Curr. Oa. Struct. Biol., 2:593-596 (19921].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized antibody has
one or more amino acid residues introduced into it from a source which is non-
human. These non-human amino acid
3 5 residues are often referred to as "import" residues, which are typically
taken from an "import" variable domain.
Humanization can be essentially performed following the method of Winter and
co-workers [Jones et al., Nature. 321:522-
CA 02369605 2001-10-O1
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525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Uerhoeyen et al.,
Science, 239:1534-1536 (1988)], by
substituting rodent CDRs or CDR sequences for the corresponding sequences of a
human antibody. Accordingly, such
"humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567),
wherein substantially less than an intact
human variable domain has been substituted by the corresponding sequence from
a non-human species. In practice,
humanized antibodies are typically human antibodies in which some CDR residues
and possibly some FR residues are
substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the
art, including phage display
libraries [Hoogenboam and Winter, J. Mal. Biol., 227:381 (19911; Marks et al.,
J. Mol. Biol., 222:581 (1991)]. The
techniques of Cole et al. and Boerner et al. are also available for the
preparation of human monoclonal antibodies (Cole et
al., Monoclonal Antibodies and Cancer Therauv, Alan R. Liss, p. 77 (1985) and
Boerner et al., J. Immunol., 14711):86-95
(1991)]. Similarly, human antibodies can be made by introducing of human
immunoglobulin loci into transgenic animals,
e.g., mice in which the endogenous immunoglobulin genes have been partially or
completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that seen in
humans in all respects, including gene
rearrangement, assembly, and antibody repertoire. This approach is described,
for example, in U.S. Patent Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following
scientific publications: Marks et al.,
BiolTechnolony 0 779-78311992); Lonberg et al., Nature 368 856-859 (19941;
Morrison, Nature 368, 812-13 (1994);
Fishwild et al., Nature Biotechnolouy 4 845-51 (1996); Neuberger, Nature
Biotechnoloay 4 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
2 0 4. Bisoecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that have binding specificities
for at least two different antigens. In the present case, one of the binding
specificities is for the CHEPO, the other one
is for any other antigen, and preferably for a cell-surface protein or
receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the recombinant production of
2 5 bispecific antibodies is based on the co-expression of two immunoglobulin
heavy-chainhight-chain pairs, where the two
heavy chains have different specificities [Milstein and Cuello, Nature,
305:537-539 (1983)]. Because of the random
assortment of immunoglobulin heavy and light chains. these hybridomas
(quadromas) produce a potential mixture of ten
different antibody molecules, of which only one has the correct bispecific
structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps. Similar
procedures are disclosed in WO 93108829,
3 0 published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659
(1991 ).
Antibody variable domains with the desired binding specificities (antibody-
antigen combining sites) can be fused
to immunoglobulin constant domain sequences. The fusion preferably is with an
immunoglobulin heavy-chain constant
domain, comprising at least part of the hinge, CH2, and CH3 regions. It is
preferred to have the first heavy-chain constant
region (CH1) containing the site necessary for light-chain binding present in
at least one of the fusions. DNAs encoding
3 5 the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin
light chain, are inserted into separate
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expression vectors, and are co-transfected into a suitable host organism. For
further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymolo4y 121:210
(19861.
According to another approach described in WO 96127011, the interface between
a pair of antibody molecules
can be engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The
preferred interface comprises at least a part of the CH3 region of an antibody
constant domain. In this method, one or
more small amino acid side chains from the interface of the first antibody
molecule are replaced with larger side chains
(e.g. tyrosine or tryptophanl. Compensatory cavities of identical or similar
size to the large side chains) are created on
the interface of the second antibody molecule by replacing large amino acid
side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such
as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments (e.g. F(ab )z bispecific
antibodies). Techniques for generating bispecific antibodies from antibody
fragments have been described in the literature.
For example, bispecific antibodies can be prepared can be prepared using
chemical linkage. Brennan et ah, Science 229:81
(1985) describe a procedure wherein intact antibodies are proteolytically
cleaved to generate Flab )Z fragments. These
fragments are reduced in the presence of the dithiol complexing agent sodium
arsenite to stabilize vicinal dithiols and
prevent intermolecular disulfide formation. The Fab fragments generated are
then converted to thionitrobenzoate (TNB)
derivatives. One of the Fab -TNB derivatives is then reconverted to the Fab -
thiol by reduction with mercaptoethylamine
and is mixed with an equimolar amount of the other Fab -TNB derivative to form
the bispecific antibody. The bispecific
antibodies produced can be used as agents for the selective immobilization of
enzymes.
2 0 Fab fragments may be directly recovered from E. coii and chemically
coupled to form bispecific antibodies.
Shalaby et al., J. Exn. Med. 175:217-225 (1992) describe the production of a
fully humanized bispecific antibody Flab Iz
molecule. Each Fab fragment was separately secreted from E. cvii and subjected
to directed chemical coupling in vitro
to form the bispecific antibody. The bispecific antibody thus formed was able
to bind to cells overexpressing the ErbB2
receptor and normal human T cells, as well as trigger the lytic activity of
human cytotoxic lymphocytes against human
2 5 breast tumor targets.
Various technique for making and isolating bispecific antibody fragments
directly from recombinant cell culture
have also been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al.,
J. Immunol. 148(5):1547-1553 (19921. The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab
portions of two different antibodies by gene fusion. The antibody homodimers
were reduced at the hinge region to form
3 0 monomers and then re-oxidized to form the antibody heterodimers. This
method can also be utilized for the production of
antibody homodimers. The diabody technology described by Hollinger et al.,
Proc. Natl. Acad. Sci. USA 90:6444-6448
(1993) has provided an alternative mechanism for making bispecific antibody
fragments. The fragments comprise a
heavy-chain variable domain (VH) connected to a light-chain variable domain
(V~) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the VH and V~
domains of one fragment are forced to
3 5 pair with the complementary V~ and VH domains of another fragment, thereby
forming two antigen-binding sites. Another
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strategy for making bispecific antibody fragments by the use of single-chain
Fv (sFv) dimers has also been reported. See,
Gruber et al., J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can be prepared. Tutt et
a/., J. Immunol. 147:60 (19911.
Exemplary bispecific antibodies may bind to two different epitopes on a given
CHEPO polypeptide herein.
Alternatively, an anti-CHEPO polypeptide arm may be combined with an arm which
binds to a triggering molecule on a
leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or
Fc receptors for IgG (Fc Rl, such as Fc RI
(CD641, Fc RII (C032) and Fc RIII (CD161 so as to focus cellular defense
mechanisms to the cell expressing the particular
CHEPO polypeptide. Bispecific antibodies may also be used to localize
cytotoxic agents to cells which express a particular
CHEPO polypeptide. These antibodies possess a CHEPO-binding arm and an arm
which binds a cytotoxic agent or a
radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific
antibody of interest binds the CHEPO
polypeptide and further binds tissue factor (TF).
5. Heteroconiu4ate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are
composed of two covalently joined antibodies. Such antibodies have, for
example, been proposed to target immune system
cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV
infection [WO 91100360; WO 921200373;
EP 03089]. It is contemplated that the antibodies may be prepared in vitro
using known methods in synthetic protein
chemistry, including those involving crosslinking agents. For example,
immunotoxins may be constructed using a disulfide
2 0 exchange reaction or by forming a thioether bond. Examples of suitable
reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent
No. 4,676,980.
6. Effector Function EnQineerinQ
It may be desirable to modify the antibody of the invention with respect to
effector function, so as to enhance,
2 5 e.g., the effectiveness of the antibody in treating cancer. For example,
cysteine residues) may be introduced into the Fc
region, thereby allowing interchain disulfide bond formation in this region.
The homodimeric antibody thus generated may
have improved internalization capability andlor increased complement-mediated
cell killing and antibody-dependent cellular
cytotoxicity (ADCCI. See Caron et al., J. Exu Med., 176: 1191-1195 (1992) and
Shopes, J. Immunol., 148: 2918-2922
(1992). Homodimeric antibodies with enhanced anti-tumor activity may also be
prepared using heterobifunctional cross-
3 0 linkers as described in Wolff et al. Cancer Research. 53: 2560-2565
(1993). Alternatively, an antibody can be engineered
that has dual Fc regions and may thereby have enhanced complement lysis and
ADCC capabilities. See Stevenson etal.,
Anti-Cancer Drun Design, 3: 219-230 (1989).
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Immunoconiu4ates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent such
as a chemotherapeutic agent, toxin (e,g., an enzymatically active toxin of
bacterial, fungal, plant, or animal origin, or
fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described above.
Enzymatically active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active fragments
of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosal, ricin A
chain, abrin A chain, modeccin A chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca
americana proteins (PAPI, PAPII, and PAP-S), momordica
charantia inhibitor, curcin, crotin, sapaanaria officinalis inhibitor,
gelonin, mitogellin, restrictocin, phenomycin, enomycin,
and the tricothecenes. A variety of radionuclides are available for the
production of radioconjugated antibodies. Examples
include z'ZBi, "'I,'3'In, °°Y, and'B6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein-coupling agents
such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane
(IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediaminel, bis-
diazonium derivatives (such as bis-(p-
diazoniumbenzoyl)-ethylenediaminel, diisocyanates (such as tolyene 2,6-
diisocyanate), and bis-active fluorine compounds
(such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin
can be prepared as described in Uitetta et al.,
Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-
methyldiethylene triaminepentaacetic acid (MX-
DTPA) is an exemplary chelating agent tar conjugation of radionucleotide to
the antibody. See W094111026.
2 0 In another embodiment, the antibody may be conjugated to a "receptor"
(such streptavidin) far utilization in tumor
pretargeting wherein the antibody-receptor conjugate is administered to the
patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then administration
of a "ligand" (e.g., avidin) that is conjugated
to a cytotoxic agent le.g., a radionucleotidel.
2 5 8. Immunoliposomes
The antibodies disclosed herein may also be formulated as immunoliposomes.
Liposomes containing the antibody
are prepared by methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA. 82: 3688 (1985);
Hwang etal., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos.
4,485,045 and 4,544,545. Liposomes with
enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
3 0 Particularly useful liposomes can be generated by the reverse-phase
evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are
extruded through fitters of defined pore size to yield liposomes with the
desired diameter. Fab' fragments of the antibody
of the present invention can be conjugated to the liposomes as described in
Martin et al., J. Biol. Chem.. 257: 286-288
(1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as
Doxorubicin) is optionally contained within
3 5 the liposome. See Gabizon et al., J. National Cancer Inst.. 81 (191: 1484
(19891.
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9. Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a CHEPO polypeptide identified herein, as well
as other molecules identified by
the screening assays disclosed hereinbefore, can be administered for the
treatment of various disorders in the form of
pharmaceutical compositions.
If the CHEPO polypeptide is intracellular and whole antibodies are used as
inhibitors, internalizing antibodies are
preferred. However, lipofections or liposomes can also be used to deliver the
antibody, or an antibody fragment, into cells.
Where antibody fragments are used, the smallest inhibitory fragment that
specifically binds to the binding domain of the
target protein is preferred. For example, based upon the variable-region
sequences of an antibody, peptide molecules can
be designed that retain the ability to bind the target protein sequence. Such
peptides can be synthesized chemically andlor
produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl.
Acad. Sci. USA, 90: 7889-7893
(1993). The formulation herein may also contain more than one active compound
as necessary far the particular indication
being treated, preferably those with complementary activities that do not
adversely affect each other. Alternatively, or
in addition, the composition may comprise an agent that enhances its function,
such as, for example, a cytotoxic agent,
cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules
are suitably present in combination in
amounts that are effective for the purpose intended.
The active ingredients may also be entrapped in microcapsules prepared, for
example, by coacervation techniques
2 0 or by interfacial polymerization, for example, hydroxymethylcellulose ar
gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres,
microemulsions, nano-particles, and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's
Pharmaceutical Sciences, supra.
The formulations to be used for in viva administration must be sterile. This
is readily accomplished by filtration
2 5 through sterile filtration membranes.
Sustained-release preparations may be prepared. Suitable examples of sustained-
release preparations include
semipermeable matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped
articles, e.g., films, or microcapsules. Examples of sustained-release
matrices include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylatel, or poly(vinylalcohol)), polylactides (U.S.
Pat. No. 3,773,919), copolymers of L-glutamic
3 0 acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such
as the LUPRON DEPOT ~" (injectable microspheres composed of lactic acid-
glycolic acid copolymer and leuprolide acetateh
and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl
acetate and lactic acid-glycolic acid enable
release of molecules for over 100 days, certain hydrogels release proteins for
shorter time periods. When encapsulated
antibodies remain in the body for a long time, they may denature or aggregate
as a result of exposure to moisture at 37 C,
3 5 resulting in a loss of biological activity and possible changes in
immunogenicity. Rational strategies can be devised for
stabilization depending on the mechanism involved. For example, if the
aggregation mechanism is discovered to be
CA 02369605 2001-10-O1
WO 00/68376 PCT/US00/12370
intermolecular S-S bond formation through thio-disulfide interchange,
stabilization may be achieved by modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture content,
using appropriate additives, and developing specific
polymer matrix compositions.
G. Uses for anti-CHEPO Antibodies
The anti-CHEPO antibodies of the invention have various utilities. For
example, anti-CHEPO antibodies may be
used in diagnostic assays for CHEPO, eg., detecting its expression in specific
cells, tissues, or serum. Various diagnostic
assay techniques known in the art may be used, such as competitive binding
assays, direct or indirect sandwich assays
and immunaprecipitation assays conducted in either heterogeneous or
homogeneous phases (Zola, Monoclonal Antibodies:
A Manual of Techniaues, CRC Press, Inc. (1987) pp.147-158]. The antibodies
used in the diagnostic assays can be labeled
with a detectable moiety. The detectable moiety should be capable of
producing, either directly or indirectly, a detectable
signal. For example, the detectable moiety may be a radioisotope, such as
'H,'4C,'zp, 355, or'z51, a fluorescent or
chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or
luciferin, or an enzyme, such as alkaline
phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in
the art for conjugating the antibody
to the detectable moiety may be employed, including those methods described by
Hunter et al., Nature, 144:945 (19621;
David et al., Biochemistry, 13:1014 (1974/; Pain et al., J. Immunol. Meth.,
40:219 (19811; and Nygren, J. Histochem. and
Cytochem., 30:407 (1982).
Anti-CHEPO antibodies also are useful for the affinity purification of CHEPO
from recombinant cell culture or
natural sources. In this process, the antibodies against CHEPO are immobilized
on a suitable support, such a Sephadex
resin or filter paper, using methods well known in the art. The immobilized
antibody then is contacted with a sample
2 0 containing the CHEPO to be purified, and thereafter the support is washed
with a suitable solvent that will remove
substantially all the material in the sample except the CHEPO, which is bound
to the immobilized antibody. Finally, the
support is washed with another suitable solvent that will release the CHEPO
from the antibody.
The following examples are offered for illustrative purposes only, and are not
intended to limit the scope of the
present invention in any way.
2 5 All patent and literature references cited in the present specification
are hereby incorporated by reference in their
entirety.
EXAMPLES
Commercially available reagents referred to in the examples were used
according to manufacturer's instructions
3 0 unless otherwise indicated. The source of those cells identified in the
following examples, and throughout the specification,
by ATCC accession numbers is the American Type Culture Collection, Manassas,
VA.
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EXAMPLE 1
Isolation of Nucleic Acid Encodinu CHEPO
Genomic DNA was isolated from two chimpanzee cell lines (ATCC CRL1609 and
CRL1857) using a Diagen kit
(cat#10262) as recommended by the manufacturer's instructions. The chimp Epo
gene was then obtained on 3 separate
fragments by PCR using 1 g of genomic DNA as template and the following primer
pairs:
EPO.F: 5'-ACCGCGCCCCCTGGACAG-3' (SED ID N0:12)
EPO.INT1.R: 5'-CATCCACTTCTCCGGCCAAACTTCA-3' (SED ID N0:13)
EPO.INT1F:5'-TTTGGCCGGAGAAGTGGATGC-3' (SEDIDN0:14)
EPO.INT4R: 5'-TCACTCACTCACTCATTCATTCATTCATTCA-3' (SEO ID N0:15)
EPO.INT4F: 5'-GTTGAATGAATGATTGAATGAATGAGTGA-3' (SED ID N0:16)
EPO.R: 5'-GCACTGGAGTGTCCATGGGACAG-3' (SED ID N0:17)
Each PCR reaction contained 5 I of 10x PCR Buffer (Perkin Elmerl, 1 I dNTP
(20mM1, 1 g genomic DNA, 1 I of
each primer, 1 I of Taq polymerase (Clontech) and HZO to bring the total
volume to 50 I. The reaction was first denatured
for 4 min. at 94 C then amplified for 40 cycles of 1 min. at 94 C, 1 min. at
65 C or 66 C then extended for 1 min. at 72 C.
A last extension step of 5 min. at 72 C was performed. The reaction was then
analyzed on agarose gel. PCR product of
500bp, 1200bp and 750bp were observed for each PCR product, respectively. The
PCR reactions were then purified using
2 0 a Wizard kit (Promega cat # A7170) then directly sequenced. DNA sequencing
of the PCR products was done using an
Applied Biosystems 377 DNA Sequencer (PEIApplied Biosystems, Foster City, CA).
The chemistry used was DYE
Terminator Cycle Sequencing with dRhodamine and BIG DYE terminators
(PEIApplied Biosystems, Foster City, CAI.
Sequence assembly and editing done with Sequencher software (Gene Codes, Ann
Arbor, MI).
The 5 coding exons were identified by homology with the human erythropoietin
sequence and assembled into a
2 5 predicted full length cDNA. The coding region of CHEPO cDNA is 579
nucleotides long (Figure 1 ) and encodes a predicted
protein of 193 amino acids (Figure 3). There are 3 putative signal cleavage
sites predicted after amino acid residue 22, 24
and 27. In accordance with the N-terminus of human Epo, the latest one is
likely to correspond to the cleavage site for
chimp Epo. The hydrophobic 27 amino acid signal peptide is followed by a 166
amino acid long mature protein containing
3 potential N-glycosylation sites. A single nucleotide polymorphism is present
in the predicted sequence obtained from
3 0 CRL1609 and changes the protein sequence at amino acid position 142 from a
D to a K. Alignment of the chimp Epo protein
with the human sequence indicates a single change at amino acid position 84
(Figure 3).
EXAMPLE 2
Use of CHEPO cDNA as a hybridization urobe
35 The following method describes use of a nucleotide sequence encoding CHEPO
as a hybridization probe.
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DNA comprising the coding sequence of full-length or mature CHEPO (as shown in
Figure 2, SEO ID N0:3) is
employed as a probe to screen for homologous DNAs (such as those encoding
naturally-occurring variants of CHEP01 in
human tissue cDNA libraries or human tissue genomic libraries.
Hybridization and washing of filters containing either library DNAs is
performed under the following high
stringency conditions. Hybridization of radiolabeled CHEPO-derived probe to
the filters is performed in a solution of 50%
formamide, 5x SSC, 0.1 % SDS, 0.1 % sodium pyrophosphate, 50 mM sodium
phosphate, pH 6.8, 2x Denhardt's solution,
and 10% dextran sulfate at 42°C for 20 hours. Washing of the filters is
performed in an aqueous solution of 0.1 x SSC and
0.19'o SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length
native sequence CHEPO can then be
identified using standard techniques known in the art.
EXAMPLE 3
Expression of CHEPO in E. coli
This example illustrates preparation of an unglycosylated form of CHEPO by
recombinant expression in E. coli.
The DNA sequence encoding CHEPO (SEO ID N0:3) is initially amplified using
selected PCR primers. The primers
should contain restriction enzyme sites which correspond to the restriction
enzyme sites on the selected expression vector.
A variety of expression vectors may be employed. An example of a suitable
vector is pBR322 (derived from E. coli; see
Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin and
tetracycline resistance. The vector is digested
with restriction enzyme and dephosphorylated. The PCR amplified sequences are
then ligated into the vector. The vector
2 0 will preferably include sequences which encode for an antibiotic
resistance gene, a trp promoter, a polyhis leader (including
the first six STII codons, polyhis sequence, and enterokinase cleavage sitel,
the CHEPO coding region, lambda
transcriptional terminator, and an argU gene.
The ligation mixture is then used to transform a selected E. cvli strain using
the methods described in Sambrook
et al., supra. Transformants are identified by their ability to grow on LB
plates and antibiotic resistant colonies are then
2 5 selected. Plasmid DNA can be isolated and confirmed by restriction
analysis and DNA sequencing.
Selected clones can be grown overnight in liquid culture medium such as LB
broth supplemented with antibiotics.
The overnight culture may subsequently be used to inoculate a larger scale
culture. The cells are then grown to a desired
optical density, during which the expression promoter is turned on.
After culturing the cells for several more hours, the cells can be harvested
by centrifugation. The cell pellet
3 0 obtained by the centrifugation can be solubilized using various agents
known in the art, and the solubilized CHEPO protein
can then be purified using a metal chelating column under conditions that
allow tight binding of the protein.
CHEPO may be expressed in E. coli in a poly-His tagged form, using the
following procedure. The DNA encoding
CHEPO is initially amplified using selected PCR primers. The primers will
contain restriction enzyme sites which correspond
to the restriction enzyme sites on the selected expression vector, and other
useful sequences providing for efficient and
3 5 reliable translation initiation, rapid purification on a metal chelation
column, and proteolytic removal with enterokinase.
The PCR-amplified, poly-His tagged sequences are then ligated into an
expression vector, which is used to transform an
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E. coli host based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHtsIhtpRts)
clpP(laclql. Transformants are first grown in
LB containing 50 mglml carbenicillin at 30 C with shaking until an O.D.600 of
3-5 is reached. Cultures are then diluted
50-100 fold into CRAP media (prepared by mixing 3.57 g (NH4)ZS04, 0.71 g
sodium citrate 2H20, 1.07 g KCI, 5.36 g Difco
yeast extract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 110 mM
MPOS, pH 7.3, 0.55% Iwlv) glucose and
7 mM MgS04) and grown for approximately 20-30 hours at 30 C with shaking.
Samples are removed to verify expression
by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells.
Cell pellets are frozen until purification and
refolding.
E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in
10 volumes (wlv) in 7 M guanidine,
20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is
added to make final concentrations of 0.1 M
and 0.02 M, respectively, and the solution is stirred overnight at 4 C. This
step results in a denatured protein with all
cysteine residues blocked by sulfitolization. The solution is centrifuged at
40,000 rpm in a Beckman Ultracentifuge for 30
min. The supernatant is diluted with 3-5 volumes of metal chelate column
buffer (6 M guanidine, 20 mM Tris, pH 7.41 and
filtered through 0.22 micron filters to clarify. The clarified extract is
loaded onto a 5 ml Qiagen Ni-NTA metal chelate
column equilibrated in the metal chelate column buffer. The column is washed
with additional buffer containing 50 mM
imidazole (Calbiochem, Utrol gradeL pH 7.4. The protein is eluted with buffer
containing 250 mM imidazole. Fractions
containing the desired protein are pooled and stored at 4 C. Protein
concentration is estimated by its absorbance at 280
nm using the calculated extinction coefficient based on its amino acid
sequence.
The proteins are refolded by diluting the sample slowly into freshly prepared
refolding buffer consisting of: 20
mM Tris, pH 8.6, 0.3 M NaCI, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM
EDTA. Refolding volumes are chosen
2 0 so that the final protein concentration is between 50 to 100
microgramslml. The refolding solution is stirred gently at 4 C
for 12-36 hours. The refolding reaction is quenched by the addition of TFA to
a final concentration of 0.4% (pH of
approximately 31. Before further purification of the protein, the solution is
filtered through a 0.22 micron filter and
acetonitrile is added to 2-10% final concentration. The refolded protein is
chromatographed on a Poros R11H reversed
phase column using a mobile buffer of 0.1 % TFA with elution with a gradient
of acetonitrile from 10 to 80%. Aliquots
2 5 of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels
and fractions containing homogeneous
refolded protein are pooled. Generally, the properly refolded species of most
proteins are eluted at the lowest
concentrations of acetonitrile since those species are the most compact with
their hydrophobic interiors shielded from
interaction with the reversed phase resin. Aggregated species are usually
eluted at higher acetonitrile concentrations. In
addition to resolving misfolded forms of proteins from the desired form, the
reversed phase step also removes endotoxin
3 0 from the samples.
Fractions containing the desired folded CHEPO polypeptide are pooled and the
acetonitrile removed using a gentle
stream of nitrogen directed at the solution. Proteins are formulated into 20
mM Hepes, pH 6.8 with 0.14 M sodium
chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine
(Pharmacia) resins equilibrated in the
formulation buffer and sterile filtered.
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EXAMPLE 4
Expression of CHEPO in mammalian cells
This example illustrates preparation of a potentially glycosylated form of
CHEPO by recombinant expression in
mammalian cells.
The vector, pRK5 (see EP 307,247, published March 15, 1989], is employed as
the expression vector.
Optionally, the CHEPO DNA is ligated into pRK5 with selected restriction
enzymes to allow insertion of the CHEPO DNA
using ligation methods such as described in Sambrook et al., suura. The
resulting vector is called pRKS-CHEPO.
In one embodiment, the selected host cells may be 293 cells. Human 293 cells
(ATCC CCL 1573) are grown to
confluence in tissue culture plates in medium such as DMEM supplemented with
fetal calf serum and optionally, nutrient
components andlor antibiotics. About 10 g pRKS-CHEPO DNA is mixed with about 1
g DNA encoding the VA RNA gene
[Thimmappaya et al., Cell, 31:543 (19821] and dissolved in 500 I of 1 mM Tris-
HCI, 0.1 mM EDTA, 0.227 M CaClz. To
this mixture is added, dropwise, 500 I of 50 mM HEPES (pH 7.35], 280 mM NaCI,
1.5 mM NaPO,, and a precipitate is
allowed to form for 10 minutes at 25°C. The precipitate is suspended
and added to the 293 cells and allowed to settle
for about four hours at 37°C. The culture medium is aspirated off and 2
ml of 20~'o glycerol in PBS is added for 30
seconds. The 293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for
about 5 days.
Approximately 24 hours after the transfections, the culture medium is removed
and replaced with culture medium
(alone) or culture medium containing 200 Cilml'SS-cysteine and 200 Cilml 35S-
methionine. After a 12 hour incubation,
the conditioned medium is collected, concentrated on a spin filter, and loaded
onto a 15% SDS gel. The processed gel may
2 0 be dried and exposed to film for a selected period of time to reveal the
presence of CHEPO polypeptide. The cultures
containing transfected cells may undergo further incubation (in serum free
medium) and the medium is tested in selected
bioassays.
In an alternative technique, CHEPO may be introduced into 293 cells
transiently using the dextran sulfate method
described by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (19811. 293
cells are grown to maximal density in a
spinner flask and 700 g pRKS-CHEPO DNA is added. The cells are first
concentrated from the spinner flask by
centrifugation and washed with PBS. The DNA-dextran precipitate is incubated
on the cell pellet for four hours. The cells
are treated with 20% glycerol for 90 seconds, washed with tissue culture
medium, and re-introduced into the spinner flask
containing tissue culture medium, 5 glml bovine insulin and 0.1 glml bovine
transferrin. After about four days, the
conditioned media is centrifuged and filtered to remove cells and debris. The
sample containing expressed CHEPO can then
3 0 be concentrated and purified by any selected method, such as dialysis
andlor column chromatography.
In another embodiment, CHEPO can be expressed in CHO cells. The pRKS-CHEPO can
be transfected into CHO
cells using known reagents such as CaP04 or DEAE-dextran. As described above,
the cell cultures can be incubated, and
the medium replaced with culture medium (alonel or medium containing a
radiolabel such as 35S-methionine. After
determining the presence of CHEPO polypeptide, the culture medium may be
replaced with serum free medium. Preferably,
3 5 the cultures are incubated for about 6 days, and then the conditioned
medium is harvested. The medium containing the
expressed CHEPO can then be concentrated and purified by any selected method.
CA 02369605 2001-10-O1
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Epitope-tagged CHEPO may also be expressed in host CHO cells. The CHEPO may be
subcloned out of the pRK5
vector. The subclone insert can undergo PCR to fuse in frame with a selected
epitope tag such as a poly-his tag into a
Baculovirus expression vector. The poly-his tagged CHEPO insert can then be
subcloned into a SV40 driven vector
containing a selection marker such as DHFR for selection of stable clones.
Finally, the CHO cells can be transfected (as
described above) with the SV40 driven vector. Labeling may be performed, as
described above, to verify expression. The
culture medium containing the expressed poly-His tagged CHEPO can then be
concentrated and purified by any selected
method, such as by Niz'-chelate affinity chromatography.
CHEPO may also be expressed in CHO andlor COS cells by a transient expression
procedure or in CHO cells by
another stable expression procedure.
Stable expression in CHO cells is performed using the following procedure. The
proteins are expressed as an IgG
construct (immunoadhesin), in which the coding sequences for the soluble forms
(e.g. extracellular domains) of the
respective proteins are fused to an IgG1 constant region sequence containing
the hinge, CH2 and CH2 domains andlor is
a poly-His tagged form.
Following PCR amplification, the respective DNAs are subcloned in a CHO
expression vector using standard
techniques as described in Ausubel et al., Current Protocols of Molecular
Biology Unit 3.16, John Wiley and Sons (1997).
CHO expression vectors are constructed to have compatible restriction sites 5
and 3 of the DNA of interest to allow the
convenient shuttling of cDNA s. The vector used expression in CHO cells is as
described in Lucas et al., Nucl. Acids Res.
24:9 (1774-1779 (1996), and uses the SV40 early promoterlenhancer to drive
expression of the cDNA of interest and
dihydrofolate reductase (DHFR1. DHFR expression permits selection for stable
maintenance of the plasmid following
2 0 transfection.
Twelve micrograms of the desired plasmid DNA is introduced into approximately
10 million CHO cells using
commercially available transfection reagents Superfect (Quiagen), Dosper or
Fugene (Boehringer Mannheim). The cells
are grown as described in Lucas et al., supra. Approximately 3 x 10'' cells
are frozen in an ampule for further growth and
production as described below.
2 5 The ampules containing the plasmid DNA are thawed by placement into water
bath and mixed by vortexing. The
contents are pipetted into a centrifuge tube containing 10 mls of media and
centrifuged at 1000 rpm for 5 minutes. The
supernatant is aspirated and the cells are resuspended in 10 mL of selective
media (0.2 m filtered PS20 with 5% 0.2 m
diafiltered fetal bovine serum). The cells are then aliquoted into a 100 mL
spinner containing 90 mL of selective media.
After 1-2 days. the cells are transferred into a 250 mL spinner filled with
150 mL selective growth medium and incubated
3 0 at 37°C. After another 2-3 days, 250 mL, 500 mL and 2000 ml
spinners are seeded with 3 x 105 ceIIsImL. The cell media
is exchanged with fresh media by centrifugation and resuspension in production
medium. Although any suitable CHO media
may be employed, a production medium described in U.S. Patent No. 5,122,469,
issued June 16, 1992 may actually be
used. A 3L production spinner is seeded at 1.2 x 106 ceIIsImL. On day 0, the
cell number pH ie determined. On day 1, the
spinner is sampled and sparging with filtered air is commenced. On day 2, the
spinner is sampled, the temperature shifted
3 5 to 33°C, and 30 mL of 500 gIL glucose and 0.6 mL of 10% antifoam
(e.g., 35% polydimethylsiloxane emulsion, Dow
Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH
is adjusted as necessary to keep it at
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around 7.2. After 10 days, or until the viability dropped below 709'0, the
cell culture is harvested by centrifugation and
filtering through a 0.22 m filter. The filtrate was either stored at
4°C or immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins are purified using a Ni-NTA
column (Qiagenl. Before purification,
imidazole is added to the conditioned media to a concentration of 5 mM. The
conditioned media is pumped onto a 6 ml Ni-
NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCI
and 5 mM imidazole at a flow rate of 4-5
mllmin. at 4°C. After loading, the column is washed with additional
equilibration buffer and the protein eluted with
equilibration buffer containing 0.25 M imidazole. The highly purified protein
is subsequently desalted into a storage buffer
containing 10 mM Hepes, 0.14 M NaCI and 4% mannitol, pH 6.8, with a 25 ml G25
Superfine (Pharmacia) column and
stored at -80°C.
Immunoadhesin IFc-containing) constructs are purified from the conditioned
media as follows. The conditioned
medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been
equilibrated in 20 mM Na phosphate buffer,
pH 6.8. After loading, the column is washed extensively with equilibration
buffer before elution with 100 mM citric acid,
pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml
fractions into tubes containing 275 L of 1 M Tris
buffer, pH 9. The highly purified protein is subsequently desalted into
storage buffer as described above far the poly-His
tagged proteins. The homogeneity is assessed by SDS polyacrylamide gels and by
N-terminal amino acid sequencing by
Edman degradation.
EXAMPLE 5
Expression of CHEPO in Yeast
2 0 The following method describes recombinant expression of CHEPO in yeast.
First, yeast expression vectors are constructed for intracellular production
or secretion of CHEPO from the
ADH21GAPDH promoter. DNA encoding CHEPO and the promoter is inserted into
suitable restriction enzyme sites in the
selected plasmid to direct intracellular expression of CHEPO. For secretion,
DNA encoding CHEPO can be cloned into the
selected plasmid, together with DNA encoding the ADH21GAPDH promoter, a native
CHEPO signal peptide or other
2 5 mammalian signal peptide, or, for example, a yeast alpha-factor or
invertase secretory signalheader sequence, and linker
sequences (if needed) for expression of CHEPO.
Yeast cells, such as yeast strain AB110, can then be transformed with the
expression plasmids described above
and cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with
10~ trichloroacetic acid and separation by SDS-PAGE, followed by staining of
the gels with Coomassie Blue stain.
3 0 Recombinant CHEPO can subsequently be isolated and purified by removing
the yeast cells from the fermentation
medium by centrifugation and then concentrating the medium using selected
cartridge filters. The concentrate containing
CHEPO may further be purified using selected column chromatography resins.
EXAMPLE 6
3 5 Expression of CHEPO in Baculovirus-Infected Insect Cells
The following method describes recombinant expression of CHEPO in Baculovirus-
infected insect cells.
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The sequence coding for CHEPO is fused upstream of an epitope tag contained
within a baculovirus expression
vector. Such epitope tags include poly-his tags and immunoglobulin tags hike
Fc regions of IgGI. A variety of plasmids may
be employed, including plasmids derived from commercially available plasmids
such as pVL1393 (Novagen). Briefly, the
sequence encoding CHEPO or the desired portion of the coding sequence of CHEPO
such as the sequence encoding the
extracellular domain of a transmembrane protein or the sequence encoding the
mature protein if the protein is extracellular
is amplified by PCR with primers complementary to the 5' and 3' regions. The
5' primer may incorporate flanking (selected)
restriction enzyme sites. The product is then digested with those selected
restriction enzymes and subcloned into the
expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and
BaculoGoIdT"' virus DNA
(Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from
GIBCO-BRL). After 4 - 5 days of incubation at 28°C, the released
viruses are harvested and used for further amplifications.
Viral infection and protein expression are performed as described by 0'Reilley
et al., Baculovirus expression vectors: A
Laboratory Manual, Oxford: Oxford University Press (19941.
Expressed poly-his tagged CHEPO can then be purified, for example, by Nip'-
chelate affinity chromatography as
follows. Extracts are prepared from recombinant virus-infected Sf9 cells as
described by Rupert et al., Nature, 362:175-
179 (19931. Briefly, Sf9 cells are washed, resuspended in sonication buffer
(25 mL Hepes, pH 7.9; 12.5 mM MgCl2; 0.1
mM EDTA; 10% glycerol; 0.1 % NP-40; 0.4 M KCI), and sonicated twice for 20
seconds on ice. The sonicates are cleared
by centrifugation, and the supernatant is diluted 50-fold in loading buffer
(50 mM phosphate, 300 mM NaCI, 10~ glycerol,
pH 7.8) and filtered through a 0.45 m filter. A Niz'-NTA agarose column
(commercially available from Qiagen) is prepared
2 0 with a bed volume of 5 mL, washed with 25 mL of water and equilibrated
with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column at 0.5 mL per minute. The column is washed
to baseline AZeo with loading buffer, at
which point fraction collection is started. Next, the column is washed with a
secondary wash buffer 150 mM phosphate;
300 mM NaCI, 109'o glycerol, pH 6.0), which elutes nonspecifically bound
protein. After reaching AZeo baseline again, the
column is developed with a 0 to 500 mM Imidazoie gradient in the secondary
wash buffer. One mL fractions are collected
2 5 and analyzed by SDS-PAGE and silver staining or Western blot with Nip'-NTA-
conjugated to alkaline phosphatase (Qiagen).
Fractions containing the eluted His,o-tagged CHEPO are pooled and dialyzed
against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) CHEPO can be
performed using known chromatography
techniques, including for instance, Protein A or protein G column
chromatography.
3 0 EXAMPLE 7
Preparation of Antibodies that Bind CHEPO
This example illustrates preparation of monoclonal antibodies which can
specifically bind CHEPO.
Techniques for producing the monoclonal antibodies are known in the art and
are described, for instance, in
Goding, su ra. Immunogens that may be employed include purified CHEPO, fusion
proteins containing CHEPO, and cells
3 5 expressing recombinant CHEPO on the cell surface. Selection of the
immunogen can be made by the skilled artisan without
undue experimentation.
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Mice, such as Balblc, are immunized with the CHEPO immunogen emulsified in
complete Freund's adjuvant and
injected subcutaneously or intraperitoneally in an amount from 1-100
micrograms. Alternatively, the immunogen is
emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT)
and injected into the animal's hind foot
pads. The immunized mice are then boosted 10 to 12 days later with additional
immunogen emulsified in the selected
adjuvant. Thereafter, for several weeks, the mice may also be boosted with
additional immunization injections. Serum
samples may be periodically obtained from the mice by retro-orbital bleeding
for testing in ELISA assays to detect anti-
CHEPO antibodies.
After a suitable antibody titer has been detected, the animals "positive" for
antibodies can be injected with a
final intravenous injection of CHEPO. Three to four days later, the mice are
sacrificed and the spleen cells are harvested.
The spleen cells are then fused (using 359'o polyethylene glycol) to a
selected murine myeloma cell line such as
P3X63AgU.l, available from ATCC, No. CRL 1597. The fusions generate hybridoma
cells which can then be plated in 96
well tissue culture plates containing HAT (hypoxanthine, aminopterin, and
thymidine) medium to inhibit proliferation of non-
fused cells, myeloma hybrids, and spleen cell hybrids.
The hybridoma cells will be screened in an ELISA for reactivity against CHEPO.
Determination of "positive"
hybridoma cells secreting the desired monoclonal antibodies against CHEPO is
within the skill in the art.
The positive hybridoma cells can be injected intraperitoneally into syngeneic
Balblc mice to produce ascites
containing the anti-CHEPO monoclonal antibodies. Alternatively, the hybridoma
cells can be grown in tissue culture flasks
or roller bottles. Purification of the monoclonal antibodies produced in the
ascites can be accomplished using ammonium
sulfate precipitation, followed by gel exclusion chromatography.
Alternatively, affinity chromatography based upon binding
2 0 of antibody to protein A or protein G can be employed.
EXAMPLE 8
Purification of CHEPO Polynentides Usina Specific Antibodies
Native or recombinant CHEPO polypeptides may be purified by a variety of
standard techniques in the art of
2 5 protein purification. For example, pro-CHEPO polypeptide, mature CHEPO
polypeptide, or pre-CHEPO polypeptide is purified
by immunoaffinity chromatography using antibodies specific for the CHEPO
polypeptide of interest. In general, an
immunoaffinity column is constructed by covalently coupling the anti-CHEPO
polypeptide antibody to an activated
chromatographic resin.
Polyclonal immunoglobulins are prepared from immune sera either by
precipitation with ammonium sulfate or by
3 0 purification on immobilized Protein A (Pharmacia LKB Biotechnology,
Piscataway, N.J.). Likewise, monoclonal antibodies
are prepared from mouse ascites fluid by ammonium sulfate precipitation or
chromatography on immobilized Protein A.
Partially purified immunoglobulin is covalently attached to a chromatographic
resin such as CnBr-activated SEPHAROSE'""
(Pharmacia LKB Biotechnologyl. The antibody is coupled to the resin, the resin
is blocked, and the derivative resin is
washed according to the manufacturer's instructions.
3 5 Such an immunoaffinity column is utilized in the purification of CHEPO
polypeptide by preparing a fraction from
cells containing CHEPO polypeptide in a soluble form. This preparation is
derived by solubilization of the whole cell or of
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a subcellular fraction obtained via differential centrifugation by the
addition of detergent or by other methods well known
in the art. Alternatively, soluble CHEPO polypeptide containing a signal
sequence may be secreted in useful quantity into
the medium in which the cells are grown.
A soluble CHEPO polypeptide-containing preparation is passed over the
immunoaffinity column, and the column
is washed under conditions that allow the preferential absorbance of CHEPO
polypeptide (eg., high ionic strength buffers
in the presence of detergentl. Then, the column is eluted under conditions
that disrupt antibodyICHEPO polypeptide binding
(e.g., a low pH buffer such as approximately pH 2-3, or a high concentration
of a chaotrope such as urea or thiocyanate
ionl, and CHEPO polypeptide is collected.
EXAMPLE 9
Drug Screening
This invention is particularly useful for screening compounds by using CHEPO
polypeptides or binding fragment
thereof in any of a variety of drug screening techniques. The CHEPO
polypeptide or fragment employed in such a test may
either be free in solution, affixed to a solid support, borne on a cell
surface, or located intracellularly. One method of drug
screening utilizes eukaryotic or prokaryotic host cells which are stably
transformed with recombinant nucleic acids
expressing the CHEPO polypeptide or fragment. Drugs are screened against such
transformed cells in competitive binding
assays. Such cells, either in viable or fixed form, can be used for standard
binding assays. One may measure, for example,
the formation of complexes between CHEPO polypeptide or a fragment and the
agent being tested. Alternatively, one can
examine the diminution in complex formation between the CHEPO polypeptide and
its target cell or target receptors caused
2 0 by the agent being tested.
Thus, the present invention provides methods of screening for drugs or any
other agents which can affect a
CHEPO polypeptide-associated disease or disorder. These methods comprise
contacting such an agent with an CHEPO
polypeptide or fragment thereof and assaying (1) for the presence of a complex
between the agent and the CHEPO
polypeptide or fragment, or (ii) for the presence of a complex between the
CHEPO polypeptide or fragment and the cell,
by methods well known in the art. In such competitive binding assays, the
CHEPO polypeptide or fragment is typically
labeled. After suitable incubation, free CHEPO polypeptide or fragment is
separated from that present in bound form, and
the amount of free or uncomplexed label is a measure of the ability of the
particular agent to bind to CHEPO polypeptide
or to interfere with the CHEPO polypeptidelcell complex.
Another technique for drug screening provides high throughput screening for
compounds having suitable binding
3 0 affinity to a polypeptide and is described in detail in WO 84103564,
published on September 13, 1984. Briefly stated, large
numbers of different small peptide test compounds are synthesized on a solid
substrate, such as plastic pins or some other
surface. As applied to a CHEPO polypeptide, the peptide test compounds are
reacted with CHEPO polypeptide and washed.
Bound CHEPO polypeptide is detected by methods well known in the art. Purified
CHEPO polypeptide can also be coated
directly onto plates for use in the aforementioned drug screening techniques.
In addition, non-neutralizing antibodies can
3 5 be used to capture the peptide and immobilize it on the solid support.
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This invention also contemplates the use of competitive drug screening assays
in which neutralizing antibodies
capable of binding CHEPO polypeptide specifically compete with a test compound
for binding to CHEPO polypeptide or
fragments thereof. In this manner, the antibodies can be used to detect the
presence of any peptide which shares one ar
more antigenic determinants with CHEPO polypeptide.
EXAMPLE 10
Rational Dru4 Design
The goal of rational drug design is to produce structural analogs of
biologically active polypeptide of interest (ie.,
a CHEPO polypeptide) or of small molecules with which they interact, e.g.,
agonists, antagonists, or inhibitors. Any of
these examples can be used to fashion drugs which are more active or stable
forms of the CHEPO polypeptide or which
enhance or interfere with the function of the CHEPO polypeptide in vivo (c.f.,
Hodgson, BioITechnoloay, 9: 19-21 (1991)1.
In one approach, the three-dimensional structure of the CHEPO polypeptide, or
of an CHEPO polypeptide-inhibitor
complex, is determined by x-ray crystallography, by computer modeling or, most
typically, by a combination of the two
approaches. Both the shape and charges of the CHEPO polypeptide must be
ascertained to elucidate the structure and
to determine active sitels) of the molecule. Less often, useful information
regarding the structure of the CHEPO polypeptide
may be gained by modeling based on the structure of homologous proteins. In
both cases, relevant structural information
is used to design analogous CHEPO polypeptide-like molecules or to identify
efficient inhibitors. Useful examples of rational
drug design may include molecules which have improved activity or stability as
shown by Braxton and Wells, Biochemistry,
31:7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of
native peptides as shown by Athauda et al.,
2 0 J. Biochem., 113:742-746 (1993).
It is also possible to isolate a target-specific antibody, selected by
functional assay, as described above, and then
to solve its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent drug design can be
based. It is possible to bypass protein crystallography altogether by
generating anti-idiotypic antibodies (anti-ids) to a
functional, pharmacologically active antibody. As a mirror image of a mirror
image, the binding site of the anti-ids would
2 5 be expected to be an analog of the original receptor. The anti-id could
then be used to identify and isolate peptides from
banks of chemically or biologically produced peptides. The isolated peptides
would then act as the pharmacore.
By virtue of the present invention, sufficient amounts of the CHEPO
polypeptide may be made available to
perform such analytical studies as X-ray crystallography. In addition,
knowledge of the CHEPO polypeptide amino acid
sequence provided herein will provide guidance to those employing computer
modeling techniques in place of or in addition
3 0 to x-ray crystallography.
The foregoing written specification is considered to be sufficient to enable
one skilled in the art to practice the
invention as claimed. Various modifications of the invention in addition to
those shown and described herein will become
apparent to those skilled in the art from the foregoing description and fall
within the scope of the appended claims.
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