U.S. patent application number 09/983000 was filed with the patent office on 2003-06-26 for use of protein biomolecular targets in the treatment and visualization of brain tumors.
This patent application is currently assigned to AGY Therapeutics. Invention is credited to Chin, Daniel, Melcher, Thorsten, Muller, Sabine.
Application Number | 20030118585 09/983000 |
Document ID | / |
Family ID | 25529729 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030118585 |
Kind Code |
A1 |
Muller, Sabine ; et
al. |
June 26, 2003 |
Use of protein biomolecular targets in the treatment and
visualization of brain tumors
Abstract
The present invention relates to the use of proteins which are
differentially expressed in primary brain tumor tissues, as
compared to normal brain tissues, as biomolecular targets for brain
tumor treatment therapies. Specifically, the present invention
relates to the use of immunotherapeutic and immunoimaging agents
which specifically bind to one or more of human proteins
angiopoietin related protein 2 (ARP-2,) secreted protein acidic,
rich in cysteine (SPARC,) c-met proto-oncogene (C-MET,) brevican
(BEHAB,) CD-44 antigen (CD-44,) tetraspanin 3 (TSPN3,) pleiotrophin
(PTN,) osteopontin (OPN,) vasoactive intestinal peptide receptor-2
(VIPR-2,) and receptor protein tyrosine phosphatase zeta
(PTP.zeta.) for the treatment and visualization of brain tumors in
patients. The present invention also provides compounds and
pharmaceutically acceptable compositions for administration in the
methods of the invention. The present invention also provides novel
splice variants of protein PTP.zeta., PTP.zeta. SM1 and PTP.zeta.
SM2. Nucleic acid probes specific for the spliced mRNA encoding
these variants and affinity reagents specific for the novel
proteins are also provided.
Inventors: |
Muller, Sabine; (San
Francisco, CA) ; Melcher, Thorsten; (San Francisco,
CA) ; Chin, Daniel; (Foster City, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Assignee: |
AGY Therapeutics
|
Family ID: |
25529729 |
Appl. No.: |
09/983000 |
Filed: |
October 17, 2001 |
Current U.S.
Class: |
424/143.1 |
Current CPC
Class: |
A61K 49/0002
20130101 |
Class at
Publication: |
424/143.1 |
International
Class: |
A61K 039/395 |
Claims
We claim:
1. A method to treat a brain tumor comprising administering a
therapeutic amount of a composition comprising: a compound of the
general formula .alpha.(VIPR2), wherein .alpha.(VIPR2) is one or
more moieties which specifically binds to a human vasoactive
intestinal peptide receptor-2, wherein the binding of
.alpha.(VIPR2) alters the function of the vasoactive intestinal
peptide receptor-2, and a pharmaceutically acceptable carrier.
2. The method of claim 1 wherein the therapeutic composition is
administered by intrathecal administration.
3. The method of claim 1 wherein the therapeutic composition is
administered by intravascular administration.
4. The method of claim 1 wherein the brain tumor is a
glioblastoma.
5. The method of claim 1 wherein .alpha.(VIPR2) is selected from
the group consisting of an antibody and an antibody fragment.
6. The method of claim 1 wherein the composition is administered as
a single dose.
7. The method of claim 1 wherein the composition is administered as
a series of doses over a period of time.
8. The method of claim 1 wherein .alpha.(VIPR2) further comprises
attached thereto one or more cytotoxic moieties C.
9. The method of claim 8 wherein the therapeutic composition is
administered by intrathecal administration.
10. The method of claim 8 wherein the therapeutic composition is
administered by intravascular administration.
11. The method of claim 8 wherein the brain tumor is a
glioblastoma.
12. The method of claim 8 wherein .alpha.(VIPR2) is selected from
the group consisting of an antibody and an antibody fragment.
13. The method of claim 8 wherein C is a radioactive moiety.
14. The method of claim 8 wherein C is a chemotoxic moiety.
15. The method of claim 8 wherein C is a toxin protein moiety.
16. The method of claim 8 wherein the composition is administered
as a single dose.
17. The method of claim 8 wherein the composition is administered
as a series of doses over a period of time.
Description
FIELD OF USE
[0001] The present invention relates to the use of proteins which
are differentially expressed in primary brain tumor tissues, as
compared to normal brain tissues, as biomolecular targets for brain
tumor treatment therapies. Specifically, the present invention
relates to the use of immunotherapeutic and immunoimaging agents
which specifically bind to one or more of angiopoietin related
protein 2 (ARP-2,) secreted protein acidic, rich in cysteine
(SPARC,) c-met proto-oncogene (C-MET,) brevican (BEHAB,) CD-44
antigen (CD-44,) tetraspanin 3 (TSPN3,) pleiotrophin (PTN,)
osteopontin (OPN,) vasoactive intestinal peptide receptor-2
(VIPR-2,) and receptor protein tyrosine phosphatase zeta
(PTP.zeta.) for the treatment and visualization of brain tumors in
patients. The present invention also provides compounds and
pharmaceutically acceptable compositions for administration.
BACKGROUND OF THE INVENTION
[0002] Brain Tumor Biology and Etiology
[0003] Brain tumors are considered to have one of the least
favorable prognoses for long term survival: the average life
expectancy of an individual diagnosed with a central nervous system
(CNS) tumor is just eight to twelve months. Several unique
characteristics of both the brain and its particular types of
neoplastic cells create daunting challenges for the complete
treatment and management of brain tumors. Among these are 1) the
physical characteristics of the intracranial space, 2) the relative
biological isolation of the brain from the rest of the body, 3) the
relatively essential and irreplaceable nature of the organ mass,
and 4) the unique nature of brain tumor cells.
[0004] First and foremost, the intracranial space and physical
layout of the brain create significant obstacles to treatment and
recovery. The brain is made of, primarily, astrocytes (which make
up the majority of the brain mass, and serve as a scaffold and
support for the neurons), neurons (which carry the actual
electrical impulses of the nervous system), and a minor contingent
of other cells such as insulating oligodendrocytes (which produce
myelin). These cell types give rise to primary brain tumors (e.g.,
astrocytomas, neuroblastomas, glioblastomas, oligodendrogliomas,
etc.) Although the World Health Organization has recently
established standard guidelines, the nomenclature for brain tumors
is somewhat imprecise, and the terms astrocytoma and glioblastoma
are often used broadly. The brain is encased in the relatively
rigid shell of the skull, and is cushioned by the cerebrospinal
fluid, much like a fetus in the womb. Because of the relatively
small volume of the skull cavity, minor changes in the volume of
tissue in the brain can dramatically increase intracranial
pressure, causing damage to the entire organ (i.e., "water on the
brain"). Thus, even small tumors can have a profound and adverse
affect on the brain's function. In contrast, tumors in the
relatively distensible abdomen may reach several pounds in size
before the patient experiences adverse symptoms. The cramped
physical location of the cranium also makes surgery and treatment
of the brain a difficult and delicate procedure. However, because
of the dangers of increased intracranial pressure from the tumor,
surgery is often the first strategy of attack in treating brain
tumors.
[0005] In addition to its physical isolation, the brain is
chemically and biologically isolated from the rest of the body by
the so-called "Blood-Brain-Barrier" (or BBB). This physiological
phenomenon arises because of the "tightness" of the epithelial cell
junctions in the lining of the blood vessels in the brain. Although
nutrients, which are actively transported across the cell lining,
may reach the brain, other molecules from the bloodstream are
excluded. This prevents toxins, viruses, and other potentially
dangerous molecules from entering the brain cavity. However, it
also prevents therapeutic molecules, including many
chemotherapeutic agents that are useful in other types of tumors,
from crossing into the brain. Thus, many therapies directed at the
brain must be delivered directly into the brain cavity (e.g., by an
Ommaya reservoir), or administered in elevated dosages to ensure
the diffusion of an effective amount across the BBB.
[0006] With the difficulties of administering chemotherapies to the
brain, radiotherapy approaches have also been attempted. However,
the amount of radiation necessary to completely destroy potential
tumor-producing cells also produce unacceptable losses of healthy
brain tissue. The retention of patient cognitive function while
eliminating the tumor mass is another challenge to brain tumor
treatment. Neoplastic brain cells are often pervasive, and travel
throughout the entire brain mass. Thus, it is impossible to define
a true "tumor margin," unlike, for example, in lung or bladder
cancers. Unlike reproductive (ovarian, uterine, testicular,
prostate, etc.), breast, kidney, or lung cancers, the entire organ,
or even significant portions, cannot be removed to prevent the
growth of new tumors. In addition, brain tumors are very
heterogeneous, with different cell doubling times, treatment
resistances, and other biochemical idiosyncrasies between the
various cell populations that make up the tumor. This pervasive and
variable nature greatly adds to the difficulty of treating brain
tumors while preserving the health and function of normal brain
tissue.
[0007] Although current surgical methods offer considerably better
post-operative life for patients, the current combination therapy
methods (surgery, low-dosage radiation, and chemotherapy) have only
improved the life expectancy of patients by one month, as compared
to the methods of 30 years ago. Without effective agents to prevent
the growth of brain tumor cells that are present outside the main
tumor mass, the prognosis for these patients cannot be
significantly improved. Although some immuno-affinity agents have
been proposed and tested for the treatment of brain tumors, see,
e.g., the tenascin-targeting agents described in U.S. Pat. No.
5,624,659, these agents have not proven sufficient for the
treatment of brain tumors. Thus, therapeutic agents which are
directed towards new molecular targets, and are capable of
specifically targeting and killing brain tumor cells, are urgently
needed for the treatment of brain tumors.
[0008] ARP-2 (Angiopoeitin Related Protein-2, Angiopoeitin Like-2
[ANGPTL-2])
[0009] Angiopoeitin related protein-2 (ARP-2), is related to the
angiopoeitin family of proteins, that includes Ang-1 and Ang-2.
Like members of the angiopoeitin family, ARP-2 contains a
coiled-coil domain in the amino terminal portion and a
fibrinogen-like domain in the carboxyl terminal portion. However,
ARP-2 has a low homology with Ang-1 and Ang-2 and unlike Ang-1 and
Ang-2, ARP-2 does not bind to the Tie-2 receptor, nor does ARP-2
bind to the closely related Tie-1 receptor. Hence, ARP-2 is
believed to be part of a newly identified family of proteins termed
angiopocitin related proteins. Like the angiopoeitins, ARP-2 is a
member of the fibrinogen superfamily, which also includes the
fibrinogens and lectins.
[0010] ARP-2 is a glycosylated, secretory protein that induces
sprouting in endothelial cells, most likely through autocrine or
paracrine signaling, and it is preferentially expressed in the
blood vessels and muscle cells. Hence, ARP-2 mediates the
differentiated state of endothelial cells or for vascular
remodeling and development. ARP-2 has not heretofore been
associated with brain tumors.
[0011] SPARC (Secreted Protein, Aacidic, Cysteine-Rich;
Osteonectin; Basement Membrane Protein (bm) 40)
[0012] Secreted protein acidic and rich in cysteine, SPARC or
BM-40, is a member of the counter-adhesive family of proteins. It
is a developmentally regulated, secreted glycoprotein expressed in
fetal astrocytes, particularity during tissue remodeling, vessel
morphogenesis, and in response to stress. It has been hypothesized
that SPARC may affect cell migration and vascular morphogenesis
either by directly interacting with extracellular matrix (ECM)
proteins (such as collagens I, III, IV and V) or by initiating a
receptor mediated signaling event that induces changes in
cytoplasmic components associated with focal adhesions. SPARC has
been found to bind directly to vitronectin, a multifunctional
adhesive protein that is a component of the brain vascular basement
membranes.
[0013] SPARC may indirectly affect cell migration and motility by
regulating the expression of matrix metallo-proteases and by
modulating the expression of other proteolytic enzymes (such as
collagenase) that degrade the ECM. Increased SPARC expression has
also been observed in two forms of low-grade malignant gliomas, in
all grades of human astrocytic tumors, and in tumor cells invading
adjacent brain at the tumor/brain interface. Hence, SPARC may be an
astrocytoma invasion related gene that functions in connection with
vitronectin to balance the modulation of cellular adhesion to the
ECM and it may promote diffuse tumor cell infiltration into
adjacent brain by affecting both tumor and endothelial cell-ECM
interactions.
[0014] Because SPARC is also found in bone, dentine, and many
normal and neoplastic human soft tissues it may also play a
regulatory function in the control of such diverse processes as
bone mineralization, cell shape, tissue remodeling or repair, cell
migration, proliferation, and differentiation. SPARC is also
synthesized, stored, and secreted by human blood platelets, binds
to plasminogen, and enhances tissue plasminogen activator
conversion of plasminogen to plasmin.
[0015] c-MET (Met Proto-Oncogene Tyrosine Kinase, Hepatocyte Growth
Factor Receptor [HGFR])
[0016] c-MET is a member of the Hepatocyte Growth Factor Receptor
(HGFR) family and a heterodimeric cellular receptor for Hepatocyte
Growth Factor (HGF). c-MET contains a disulfide-linked
.alpha.-chain of 50-kDa (which is located in the extracellular
domain,) a 145-kDa .beta.-chain (which includes an extracellular
region,) a transmembrane spanning domain, and an intracellular
tyrosine kinase domain that can be activated by
autophosphorylation. Hence, HGFR is a subset of the protein
tyrosine-kinase family of membrane-spanning, cell surface
receptors.
[0017] The receptor-ligand pair, c-MET and HGF, function as a
growth factor, regulating cell growth, migration, and
morphogenesis, and hence, may play a role in neoplastic formation
and metastasis. Upon HGF or macrophage stimulating protein (MSP)
binding, the c-MET protein receptor goes through a conformational
change wherein the intracellular tyrosine residues of the .beta.
subunit become phosphorylated at residue 1235, and a second
messenger signal cascade is induced. This change activates c-MET's
intracellular receptor kinase activity, which is important to the
growth and differentiation of epithelial cells in normal and
malignant tissues. c-MET has been identified in both normal brain
and on glial tumors, and is thought to be determinant in the
pathological processes of various malignancies. For instance,
detailed studies have shown that glioblastoma multiforme (GBM), a
highly malignant brain tumor of astrocytic origin, expresses c-MET,
and this research suggests a role in tumor progression.
[0018] BEHAB (Brain-Enriched Hyaluronan Binding Protein,
Brevican)
[0019] BEHAB is a brain-specific, extracellular matrix protein,
that is a member of the chondroitin sulfate proteoglycan (CSPG)
family. BEHAB is expressed only in the CNS. Although its function
is unclear, BEHAB is reported to bind to HA at the N-terminus,
lectins at the C-terminus, and may mediate binding of other ECM
components like tenascin. This suggests that BEHAB may play a role
in cell-cell and cell-matrix interactions thereby maintaining the
extracellular environment of the brain. It has been reported that
the highest levels of expression of BEHAB is during brain
development and at times and places where glial cells are highly
motile, as in cases of brain injury or trauma. BEHAB expression is
also unregulated in primary gliomas of the central nervous system,
but not in tumors of non-glial origin. In surgical samples of human
gliomas (including astrocytoma, oligodendroglioma, and glioblastoma
tumors), BEHAB expression is consistently and dramatically
increased over the level of expression in the normal brain. Hence,
BEHAB expression correlates with an invasive phenotype that
promotes gliogenesis by contributing to cell movement through the
ECM.
[0020] CD-44 Antigen
[0021] CD-44 is a single-path, type I transmembrane protein with
extracellular domains that are flexibly linked to the transmembrane
segment. CD-44 is a member of the cartilage link protein family and
belongs to the hyaloadherin or link protein superfamily (LPSF). As
other members of the LPS family, CD-44 can be extensively
glycosylated and is typically decorated with glycosaminoglycans
(e.g., chondroitin, heparin, and keratin sulfate). The genomic
structure of CD-44 consists of 21 exons, at least 11 of which can
be variably spliced (v1 -v10), that are located in the
membrane-proximal extracellular region. Alternative splicing of
these exons give rise to a variety of CD-44 isoforms (at least 30
different isoforms have been characterized to date) that are widely
distributed and expressed in a cell-specific manner. Among the most
frequently occurring isoforms are CD-44H, expressed on
hematopoietic cells, and CD-44E, expressed in epithelial cells.
CD-44(H) has also been found to be expressed in lymphocytes,
macrophages, erythrocytes, fibroblasts, epithelial and endothelial
cells, and neurons. It is the predominant isoform in normal brain
and neuroectoderm-derived tumors and is expressed on both normal
astrocytes and oligodendrocytes as well on neoplastic astrocytes
and glioblastomas.
[0022] The family of CD-44 proteins has been implicated in
lymphocyte activation and homing, endothelial migration, and tumor
cell metastasis. CD-44 is believed to be the major receptor for
Hyaluronic acid (HA). CD-44/HA interactions underlie a wide
spectrum of functions in embryonic morphogenesis and organogenesis,
hematopoeisis, lymphocyte homing. CD-44 also mediates the
attachment of glioma cells to chondroitin sulfate, types I and IV
collagen, fibronectin laminin, vitronectin and Martrigel. This
suggest that CD-44 may play a role in cell-cell and cell-matrix
interactions, affecting the extracellular environment of the brain.
Because HA is a major component of the brain ECM, and CD-44 is one
of the principal cellular receptors of HA, CD-44 expression
coincides with brain tumor growth and invasiveness.
[0023] PTN (Pleiotrophin, Heparin Binding Growth Factor 8, Neurite
Growth-Promoting Factor 1)
[0024] Pleiotrophin or PTN, is a platelet-derived, growth factor
inducible, member of the pleiotrophin family of proteins that
includes midkine and retinoic acid-induced heparin-binding protein.
It is a developmentally regulated, secreted cytokine that
stimulates mitogenesis, angiogenesis, and neurite and glial process
outgrowth guidance activities. During development PTN is expressed
in the brain, intestine, muscle, skin, heart, lung and kidney. In
the adult, PTN is found primarily in the brain in association with
axonal tracts during active mitogenesis and may therefore play an
important role in the development and maintenance of the nervous
system. It has been found to bind heparin, heparin sulfate
proteoglycans, the extracellular matrix, and is also a natural
ligand for receptor protein tyrosine phosphatase (RPTP), signaling
through ligand dependant receptor inactivation of RPTP. Receptor
mediated endocytosis occurs following PTN binding and may be
disrupted by heparin.
[0025] PTN has also been found to have oncogenic properties,
inducing malignant transformation and tumor growth and progression.
It has been described as a proto-oncogene that is expressed in many
human tumors and cell lines derived from human tumors. PTN is a
mitogen for fibroblasts, epithelial and endothelial cells,
stimulates plasminogen-activator production, can induce tube
formation, and therefore can serve as a tumor angiogenesis
factor.
[0026] OPN (Osteopontin, Secreted Phosphoprotein 1, Bone
Sialoprotein-1)
[0027] Osteopontin or OPN, is a member of the osteopontin family.
It is a glycosylated sialoprotein that is heavily phosphorylated
and expressed in a variety of cells including bone, kidney,
placenta, nerve cells and macrophages, as well as T lymphocytes,
epidermal and bone cells. OPN is a part of the mineralized bone
matrix and may play a role in bone resorption, by facilitating the
attachment of osteoclasts to the bone surface, and may be
functionally important as an adhesive and chemotactic molecule for
vascular cells. OPN is a secreted protein that binds tightly to
hydroxyapatite, and hence, is important to cell matrix
interactions. It has been observed to interact with the CD-44
homing receptor to physiologically induce macrophage chemotaxis,
which may be a mechanism utilized by metastatic brain tumors in the
process of dissemination.
[0028] OPN has been observed in the microvasculature of
glioblastomas associated with VEGF expression and OPN mRNA has been
found to be overexpressed in high grade and metastatic brain
tumors. Hence, OPN expression correlates with the malignancy grade
of gliomas.
[0029] VIPR-2 (Vasoactive Intestinal Peptide Receptor-2)
[0030] Vasoactive intestinal polypeptide receptor II (VIPR-2),
VPAC-2, is a member of the G-protein receptor family, which
includes such members as the calcitonin, parathyroid hormone,
secretin, glucagon and VIP-1 receptors. VIPR-2 is a
seven-transmembrane spanning G protein-coupled receptor that
responds to VIP by stimulating cAMP production. VIPR-2 is found in
the brain as well as peripheral tissues such as the pancreas,
skeletal muscle, heart, lung, kidneys, stomach, adipocytes and the
liver, and in various cells of the immune system. In the brain,
VIPR-2 functions as a neuroendocrine hormone and neurotransmitter
receptor, and is found in the thalamus, hippocampus,
suprachiasmatic nucleus and hypothalamus.
[0031] VIPR-2 is encoded by a nucleotide sequence of approximately
2.8 kb, which codes for a 438 amino acid sequence of approximately
48-64 kDa. The receptor-ligand pair, VIPR-2 and VIP, have various
functions dependent upon the tissue where in they are located. VIP
is a late-developing, 28 amino acid peptide that, along with its
receptor, is widely distributed throughout the peripheral body, and
plays a role in cardiovascular, reproductive, pulmonary, immune and
gastrointestinal systems, to effect vasodilatation,
bronchodilation, immunosuppression, hormonal secretion, and
increased gastric motility. However, the cerebral cortex has one of
the highest reported concentrations of VIP, localized to intrinsic
neurons throughout all neocortical regions. In the brain, VIP and
its receptor, have behavioral, electrophysiological, secretory,
metabolic, vascular, and mitogenic effects. For instance, the
receptor-ligand pair play a role in cortical differentiation, the
relaying of sensory information to the cortex, and the regulation
of morphogenic events by the release of diffusible signals from
glial cells. VIPR-2 and VIP also play a role in the growth and
differentiation of neuroblastomas.
[0032] TSPAN3 (Tetraspanin 3, Tetraspanin TM-4A)
[0033] The Tetraspanin superfamily, is a family of approximately 20
integral membrane proteins that are broadly expressed in most human
tissues including neural and bone marrow derived tissues. The
family shares a common motif that includes four putative
transmembrane domains (TM1-4), a small extracellular domain (EC1)
of 20-27 amino acids, and a larger extracellular domain (EC2)
between TMS3 and TMS4 of 70-130 amino acids. Two conserved features
of tetraspanins are critical to their structure and function.
First, charged residues are present in or near the TM domains,
second, a cluster of cysteine residues is in the putative EC2
domain. Most of the tetraspanins are modified by
N-glycosylation.
[0034] Many Tetraspanin proteins affect the regulation of cellular
proliferation, motility, differentiation, development. In some
cells, Tetraspanins may act as adapters in ultimeric complexes that
link plasma membrane proteins, like integrins, into signaling
complexes with other signaling molecules (e.g.,
phosphatidylinositol 4-kinase) at the plasma membrane and play a
role in integrin-mediated cell migration, metastasis and tumor cell
invasion. A number of tetraspanins have also been discovered as
tumor-associated proteins, including C-029, PETA-3/SFA-1, and SAS,
which is amplified in a subset of sarcomas. Of the various TM4SF
proteins, CD9, CD63, CD81, CD82, and CD151 are the most widely
distributed. CD9 is expressed on 90% of non-T cell acute
lymphoblastic leukemia cells and on 50% of chronic lymphocytic and
acute myeloblastic leukemias. CD63 is also expressed in early stage
melanomas.
[0035] Protein Tyrosine Phosphatase Receptor Zeta (PTP.zeta.)
[0036] Vital cellular functions, such as cell proliferation and
signal transduction, are regulated in part by the balance between
the activities of protein kinases and protein phosphatases. These
protein-modifying enzymes add or remove a phosphate group from
serine, threonine, or tyrosine residues in specific proteins. Some
tyrosine kinases (PTK's) and phosphatases (PTPase's) have been
theorized to have a role in some types of oncogenesis, which is
thought to result from an imbalance in their activities. There are
two classes of PTPase molecules: low molecular weight proteins with
a single conserved phosphatase domain such as T-cell
protein-tyrosine phosphatase (PTPT; MIM 176887), and high molecular
weight receptor-linked PTPases with two tandemly repeated and
conserved phosphatase domains separated by 56 to 57 amino acids.
Examples of this latter group of receptor proteins include:
leukocyte-common antigen (PTPRC; MIM 151460) and leukocyte antigen
related tyrosine phosphatase (PTPRF; MIM 179590).
[0037] Protein tyrosine phosphatase zeta (PTP.zeta.) [also known as
PTPRZ, HPTP-ZETA, HPTPZ, RPTP-BETA(.beta.), or RPTPB] was isolated
as a cDNA sequence by two groups in the early nineties. The
complete cDNA sequence of the protein is provided in SEQ ID NO. 1,
and the complete deduced amino acid sequence is provided in SEQ ID
NO. 2. Splicing variants and features are indicated in the
sequences. Levy et al. ("The cloning of a receptor-type protein
tyrosine phosphatase expressed in the central nervous system" J.
Biol. Chem. 268: 10573-10581, (1993)) isolated cDNA clones from a
human infant brain step mRNA expression library, and deduced the
complete amino acid sequence of a large receptor-type protein
tyrosine phosphatase containing 2,307 amino acids.
[0038] Levy found that the protein, which they designated
PTP-.beta. (PTP.zeta.), is a transmembrane protein with 2
cytoplasmic PTPase domains and a 1,616-amino acid extracellular
domain. As in PTP-.gamma. (MIM 176886), the 266 N-terminal residues
of the extracellular domain are have a high degree of similarity to
carbonic anhydrases (see MIM 114880). The human gene encoding
PTP.zeta. has been mapped to chromosome 7q31.3-q32 by chromosomal
in situ hybridization (Ariyama et al., "Assignment of the human
protein tyrosine phosphatase, receptor-type, zeta (PTPRZ) gene to
chromosome band 7q31.3" Cytogenet. Cell Genet. 70: 52-54 (1995)).
Northern blot analysis has shown that showed that PTP-zeta is
expressed only in the human central nervous system. By in situ
hybridization, Levy et al. (1993) localized the expression to
different regions of the adult human brain, including the Purkinje
cell layer of the cerebellum, the dentate gyrus, and the
subependymal layer of the anterior horn of the lateral ventricle.
Levy stated that this was the first mammalian tyrosine phosphatase
whose expression is restricted to the nervous system. In addition,
high levels of expression in the murine embryonic brain suggest an
important role in CNS development.
[0039] Northern analysis has shown three splice variants: the
extracellular proteoglycan phosphacan, which contains the full
extracellular region of the protein, and the long (.alpha.) and
short (.beta.) forms of the transmembrane phosphatase. The .beta.
form lacks the extracellular 860 aa long insert domain of the
protein, therefore it is not glycosylated. PCR studies of the gene
in rat genomic DNA indicated that there are no introns at the
putative 5' and 3' splice sites or in the 2.6 kb segment which is
deleted in the short transmembrane protein. The phosphatases and
the extracellular proteoglycan have different 3'-untranslated
regions. Additional alternative mRNA splicing is likely to result
in the deletion of a 7 amino acid insert from the intracellular
juxtamembrane region of both long and short phosphatase isoforms.
Simultaneous quantitation of the three major isoforms indicated
that the mRNA encoding phosphacan had the highest relative
abundance in the CNS while that encoding the short phosphatase
isoform was most abundant relative to the other PTP.zeta. variants
in the PNS.
[0040] PTP.zeta. has only been found to be expressed in the nervous
system. By in situ hybridization, it has been localized to
different regions of the adult brain, including the Purkinje cell
layer of the cerebellum, the dentate gyrus, and the subependymal
layer of the anterior horn of the lateral ventricle. High levels of
PTP.zeta. have been seen in regions of the brain where there is
continued neurogenesis and neurite outgrowth, and it seems to play
a role in morphogenesis and plasticity of the nervous system.
Phosphacan immunoreactivity has been associated with perineuronal
nets around parvalbumin-expressing neurons in adult rat cerebral
cortex. Neurons as well as astrocytes have been shown to express
phosphacan.
[0041] The transmembrane forms of PTP.zeta. are expressed on the
migrating neurons especially at the lamellipodia along the leading
processes. PTP.zeta. is postulated to be involved in the neuronal
migration as a neuronal receptor of pleiotrophin distributed along
radial glial fibers. PTP.zeta. has been shown to be highly
expressed in radial glia and other forms of glial cells that play
an important role during development. The anti-PTP.zeta. staining
localizes to the radial processes of these cells, which act as
guides during neuronal migration and axonal elongation. The pattern
of RPTP-zeta expression has also been shown to change with the
progression of glial cell differentiation.
[0042] The three splicing variants of RPTP-zeta have been shown to
have different spatial and temporal patterns of expression in the
developing brain. The 9.5-kb and 6.4-kb transcripts, which encode
the .alpha. and .beta. transmembrane protein tyrosine phosphatases,
were predominantly expressed in glial progenitors located in the
subventricular zone. The 8.4-kb transcript, which encodes the
secreted chondroitin sulfate proteoglycan phosphacan, was expressed
at high levels by more mature glia that have migrated out of the
subventricular zone. The three transcripts have also been shown to
be differentially expressed in glial cell cultures.
[0043] In knockout studies, PTP.zeta.-deficient mice were viable,
fertile, and showed no gross anatomical alterations in the nervous
system or other organs. Therefore, it was deduced that PTP.zeta. is
not essential for neurite outgrowth and node formation in mice. The
ultrastructure of nerves of the central nervous system in
PTP.zeta.-deficient mice suggests a fragility of myelin. However,
conduction velocity was not altered. The normal development of
neurons and glia in was thought to indicate that PTP.zeta. function
is not necessary for these processes in vivo, or that a loss of
PTP.zeta. can be compensated for by other protein tyrosine
phosphatases expressed in the nervous system.
[0044] Following CNS injury, robust induction of phosphatase forms
of PTP.zeta. mRNA has been observed in areas of axonal sprouting,
and of both phosphatases and phosphacan mRNAs in areas of glial
scarring. This is thought to imply that the encoded proteins and
the cell adhesion molecules and extracellular matrix proteins to
which they bind may contribute to recovery from injury and perhaps
also to the regulation of axonal regrowth in the nervous system.
Following peripheral nerve crush, all PTP.zeta. mRNAs, including
phosphacan and the phosphatase variants with and without the 21
base insert, were observed to be significantly induced in the
distal segments of the sciatic nerve with a time course that
correlated well with the response of Schwann cells to this
injury.
[0045] The extracellular domains of PTP.zeta. have been shown to be
capable of binding to several cell adhesion molecules. Phosphacan,
which is the shortest, secreted form of PTP.zeta., containing the
full extracellular region, previously was designated 3F8 and 6B4
chondroitin sulfate proteoglycan or 3H1 keratin sulfate
proteoglycan depending on the glycosylation status. It is
synthesized mainly by glia and binds to neurons and to the neural
cell adhesion molecules Ng-CAM/L1, NCAM, TAG-1/axonin-1, to
tenascin-C and R, to amphoterin and pleiotrophin/heparin-binding
growth-associated molecule (HB-GAM) (amphoterin and pleiotrophin
are heparin-binding proteins that are developmentally regulated in
brain and functionally involved in neurite outgrowth). Binding of
phosphacan to Ng-CAM/L1, NCAM, and tenascin-C (FNIII domain) is
mediated by complex-type N-linked oligosaccharides on the
proteoglycan. Phosphacan, shows saturable, reversible,
high-affinity binding to fibroblast growth factor-2 (FGF-2). The
interaction is mediated primarily through the core protein.
Immunocytochemical studies have also shown an overlapping
localization of FGF-2 and phosphacan in the developing central
nervous system. The core protein of phosphacan may also regulate
the access of FGF-2 to cell surface signaling receptors in nervous
tissue.
[0046] The carbonic anhydrase (CAH) domain of PTP.zeta. has been
shown to bind specifically to contactin. Contactin is a 140 kDa GPI
membrane-anchored neuronal cell recognition protein expressed on
the surface of neuronal cells. The CAH domain of RPTP zeta was
shown to induce cell adhesion and neurite growth of primary tectal
neurons, and differentiation of neuroblastoma cells. These
responses were blocked by antibodies against contactin,
demonstrating that contactin is a neuronal receptor for RPTP zeta.
Caspr ((p190/Caspr, a contactin-associated transmembrane receptor)
and contactin exist as a complex in rat brain and are bound to each
other by means of lateral (cis) interactions in the plasma
membrane. The extracellular domain of Caspr contains a
neurophilin/coagulation factor homology domain, a region related to
fibrinogen beta/gamma, epidermal growth factor-like repeats,
neurexin motifs as well as unique PGY repeats found in a molluscan
adhesive protein. The cytoplasmic domain of Caspr contains a
proline-rich sequence capable of binding to a subclass of SH3
domains of signaling molecules. Caspr may function as a signaling
component of contactin, enabling recruitment and activation of
intracellular signaling pathways in neurons. The role of the
extracellular domains in neural adhesion and neurite growth
induction was investigated by the use of fusion protein constructs.
The results suggested that binding of glial PTP.zeta. to the
contactin/Nr-CAM complex is important for neurite growth and
neuronal differentiation.
[0047] PTP.zeta. was shown to bind to a heparin-binding growth
factor midkine through the chondroitin sulfate portion of the
receptor. The interactions of pleiotrophin (PTN) with the receptor
in U373-MG cells was also studied. Pleiotrophin was shown to bind
to the spacer domain. Results suggested that PTN signals through
"ligand-dependent receptor inactivation" of PTP.zeta. and disrupts
its normal roles in the regulation of steady-state tyrosine
phosphorylation of downstream signaling molecules. PTN was shown to
bind to and functionally inactivate the catalytic activity of
PTP.zeta.. An active site-containing domain of PTP.zeta. both binds
.beta.-catenin and functionally reduces its levels of tyrosine
phosphorylation when added to lysates of pervanadate-treated cells.
In unstimulated cells, PTP.zeta. was shown to be intrinsically
active, and thought to function as an important regulator in the
reciprocal control of the steady-state tyrosine phosphorylation
levels of .beta.-catenin by tyrosine kinases and phosphatases.
[0048] Using the yeast substrate-trapping system, several substrate
candidates for PTP.zeta. were isolated. The results indicated that
GIT1/Cat-1 is a substrate molecule of PTP.zeta.. In addition,
PTP.zeta. was shown to bind to the PSD-95/SAP90 family through the
second phosphatase domain. Immunohistochemical analysis revealed
that PTP.zeta. and PSD-95/SAP90 are similarly distributed in the
dendrites of pyramidal neurons of the hippocampus and neocortex.
Subcellular fractionation experiments indicated that PTP.zeta. is
concentrated in the postsynaptic density fraction. These results
suggested that PTP.zeta. is involved in the regulation of synaptic
function as postsynaptic macromolecular complexes with
PSD-95/SAP90.
[0049] Voltage-gated sodium channels in brain neurons were also
found to associate with the membrane bound forms of PTP.zeta. and
phosphacan. Both the extracellular domain and the intracellular
catalytic domain of PTP.zeta. interacted with sodium channels.
Sodium channels were tyrosine phosphorylated and were modulated by
the associated catalytic domains of PTP.zeta..
SUMMARY OF THE INVENTION
[0050] The present invention provides novel methods and reagents
for specifically targeting brain tumor neoplastic cells for both
therapeutic and imaging purposes, by targeting brain tumor protein
targets (T.sub.BT). These targets have been identified by the
applicants as being overexpressed in brain tumors, and thus allow
for the selective inhibition of cell function or selective marking
for visualization with therapeutic or visualizing compositions
which have a specific affinity for thes eprotein targets. Each of
angiopoietin related protein 2 (ARP-2,) secreted protein acidic,
rich in cysteine (SPARC,) c-met proto-oncogene (C-MET,) brevican
(BEHAB,) CD-44 antigen (CD-44,) tetraspanin 3 (TSPN3,) pleiotrophin
(PTN,) osteopontin (OPN,) vasoactive intestinal peptide receptor-2
(VIPR-2,) and receptor protein tyrosine phosphatase zeta
(PTP.zeta.) [including the two novel isoforms PTP.zeta. SM1 and
SM2], as the proteins are described below, have been identified as
an independently useful protein target T.sub.BT. In some preferred
embodiments of the invention, either novel isofom PTP.zeta. SM1 or
PTP.zeta. SM2 is the protein target T.sub.BT. Thus, the aspects of
the invention with relation to each of these T.sub.BT are described
generally as follows:
[0051] In a first aspect, the present invention provides T.sub.BT
affinity-based compounds and compositions useful in treating a
brain tumor in a patient. The compositions and compounds of this
aspect of the invention generally fall into two groups:
T.sub.BT-binding conjugate compounds, which comprise a cytotoxic
moiety (C), which inhibits the growth of tumor cells; and
T.sub.BT-binding compound compositions in which the T.sub.BT
binding moiety alters the normal function of the T.sub.BT in or
around the tumor cell, thus inhibiting cell growth and/or
function.
[0052] In a first group of embodiments of this aspect of the
invention, T.sub.BT-binding therapeutic conjugate compounds are
provided. These compounds have the general formula
.alpha.(T.sub.BT)C, wherein .alpha.(T.sub.BT) is one or more
moieties which specifically binds to a T.sub.BT, and C is one or
more cytotoxic moieties. In preferred embodiments,
.alpha.(T.sub.BT) is an antibody or an antibody fragment. In
particularly preferred embodiments, .alpha.(T.sub.BT) is an
antibody or an antibody fragment which elicits a reduced immune
response when administered to a human patient. Preferred cytotoxic
moieties for use in these embodiments of the invention include
radioactive moieties, chemotoxic moieties, and toxin proteins. The
invention also provides compositions comprising these
T.sub.BT-binding therapeutic conjugate compounds in a
pharmaceutically acceptable carrier.
[0053] In a second group of embodiments of this first aspect of the
invention, T.sub.BT-binding therapeutic compounds are provided
which alter the normal function of the T.sub.BT in or around brain
tumor cells and inhibit brain tumor cell growth. These T.sub.BT
-binding therapeutic compounds have the general formula
.alpha.(T.sub.BT), wherein .alpha.(T.sub.BT) is one or more
moieties which specifically binds to a T.sub.BT, and wherein the
binding of .alpha.(T.sub.BT) alters the function of the T.sub.BT.
In preferred embodiments, .alpha.(T.sub.BT) is an antibody or an
antibody fragment. In particularly preferred embodiments,
.alpha.(T.sub.BT) is an antibody or an antibody fragment which
elicits a reduced immune response when administered to a human
patient. It is preferred that the therapeutic compounds of this
second group of embodiments of the first aspect of the invention be
formulated into therapeutic compositions comprising the
T.sub.BT-binding compound in a pharmaceutically acceptable
carrier.
[0054] In a second aspect, the present invention provides methods
for using these compounds and compositions to treat a brain tumor
in a patient. The methods comprise administering an effective
amount of a composition, comprising a T.sub.BT-binding compound
from the first or second group of embodiments of the first aspect
and a pharmaceutically acceptable carrier, to a patient in need
thereof. Brain tumors treated in this fashion may be glioblastomas,
astrocytomas, neuroblastomas, or any type of brain tumor.
Administration of the therapeutic composition may be by any
acceptable means. One preferred method for administration is by
intrathecal administration, although intravascular administration
is also preferred.
[0055] In a third aspect, the present invention provides T.sub.BT
affinity-based compounds and compositions for the visualization of
brain tumors in patients. These compounds have the general formula
.alpha.(T.sub.BT)I, wherein .alpha.(T.sub.BT) is one or more
moieties which specifically binds to a T.sub.BT, and I is one or
more imaging moieties. In preferred embodiments, .alpha.(T.sub.BT)
is an antibody or an antibody fragment. In particularly preferred
embodiments, .alpha.A(T.sub.BT) is an antibody or an antibody
fragment which elicits a reduced immune response when administered
to a human patient. Preferred I moieties include radiographic
moieties (useful in, e.g., x-ray, scintillation, or other radiation
imaging methods,) positron-emitting moieties, magnetic spin
contrast moieties, and optically visible moieties (such as visible
particles, fluorescent dyes, and visible-spectrum dyes.) It is
preferred that the imaging compounds of these embodiments of the
third aspect of the invention be formulated into therapeutic
compositions comprising the T.sub.BT-binding compound in a
pharmaceutically acceptable carrier.
[0056] In a fourth aspect, the present invention provides methods
of using the compounds and compositions of the third aspect of the
invention to visualize a brain tumor in a patient. These methods
generally comprise administering an effective amount of an imaging
compound of the general formula .alpha.(T.sub.BT)I in a
pharmaceutically acceptable carrier to the patient, and then
visualizing the imaging moieties of the compound. Administration of
the imaging composition may be by any acceptable means.
Intravascular administration of the imaging composition is
preferred in these methods, although intrathecal administration is
also preferred. Preferred methods of visualizing the imaging
moieties of the compounds include radiographic imaging techniques
(e.g., x-ray imaging and scintillation imaging techniques),
positron-emission tomography, magnetic resonance imaging
techniques, and direct or indirect (e.g., endoscopic) visual
inspection.
[0057] Various particular embodiments of these aspects of the
invention include:
[0058] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(ARP2)C, wherein .alpha.(ARP2) is one or
more moieties which specifically binds to a human angiopoietin
related protein-2, and C is one or more cytotoxic moieties, and a
pharmaceutically acceptable carrier.
[0059] A compound for the treatment of a brain tumor of the general
formula .alpha.(ARP2)C, wherein .alpha.(ARP2) is one or more
moieties which specifically binds to a human angiopoietin related
protein-2, and C is one or more cytotoxic moieties.
[0060] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(ARP2), wherein .alpha.(ARP2) is one or more
moieties which specifically binds to a human angiopoietin related
protein-2, wherein the binding of .alpha.(ARP2) alters the function
of the angiopoietin related protein-2, and a pharmaceutically
acceptable carrier.
[0061] A method for visualizing a brain tumor in a patient by first
administering to a patient an effective amount of a composition
comprising: a compound of the general formula .alpha.(ARP2)I,
wherein .alpha.(ARP2) is one or more moieties which specifically
binds to a human angiopoietin related protein-2, and I is one or
more imaging moieties and a pharmaceutically acceptable carrier,
and then visualizing the imaging moieties of the compound.
[0062] A composition for the visualization of a brain tumor
comprising a compound of the general formula .alpha.(ARP2)I,
wherein .alpha.(ARP2) is one or more moieties which specifically
binds to a human angiopoietin related protein-2, and I is one or
more imaging moieties, and a pharmaceutically acceptable
carrier.
[0063] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(TSPAN3)C, wherein .alpha.(TSPAN3) is one or
more moieties which specifically binds to a human tetraspanin 3,
and C is one or more cytotoxic moieties, and a pharmaceutically
acceptable carrier.
[0064] A compound for the treatment of a brain tumor of the general
formula .alpha.(TSPAN3)C, wherein .alpha.(TSPAN3) is one or more
moieties which specifically binds to a human tetraspanin 3, and C
is one or more cytotoxic moieties.
[0065] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(TSPAN3), wherein .alpha.(TSPAN3) is one or
more moieties which specifically binds to a human tetraspanin 3,
wherein the binding of .alpha.(TSPAN3) alters the function of the
tetraspanin 3, and a pharmaceutically acceptable carrier.
[0066] A composition for the treatment of a brain tumor comprising:
a compound of the general formula .alpha.(TSPAN3), wherein
.alpha.(TSPAN3) is one or more moieties which specifically binds to
a human tetraspanin 3, wherein the binding of .alpha.(TSPAN3)
alters the function of the tetraspanin 3, and a pharmaceutically
acceptable carrier.
[0067] A method for visualizing a brain tumor in a patient by first
administering to a patient an effective amount of a composition
comprising: a compound of the general formula .alpha.(TSPAN3)I,
wherein .alpha.(TSPAN3) is one or more moieties which specifically
binds to a human tetraspanin 3, and I is one or more imaging
moieties, and a pharmaceutically acceptable carrier, and then
visualizing the imaging moieties of the compound.
[0068] A composition for the visualization of a brain tumor
comprising a compound of the general formula .alpha.(TSPAN3)I,
wherein .alpha.(TSPAN3) is one or more moieties which specifically
binds to a human tetraspanin 3, and I is one or more imaging
moieties, and a pharmaceutically acceptable carrier.
[0069] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(PTP.zeta.)C, wherein .alpha.(PTP.zeta.) is
one or more moieties which specifically binds to a human PTP.zeta.
selected from the group consisting of PTP.zeta. SM1 and PTP.zeta.
SM2, further wherein .alpha.(PTP.zeta.) does not specifically bind
to human PTP.zeta. .alpha., human PTP.zeta. .beta., or phosphacan,
and C is one or more cytotoxic moieties, and a pharmaceutically
acceptable carrier.
[0070] A compound for the treatment of a brain tumor of the general
formula .alpha.(PTP.zeta.)C, wherein .alpha.(PTP.zeta.) is one or
more moieties which specifically binds to a human PTP.zeta.
selected from the group consisting of PTP.zeta. SM1 And PTP.zeta.
SM2, further wherein .alpha.(PTP.zeta.) does not specifically bind
to human PTP.zeta. .alpha., human PTP.zeta. .beta.,or human
phosphacan, and C is one or more cytotoxic moieties.
[0071] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(PTP.zeta.), wherein .alpha.(PTP.zeta.) is
one or more moieties which specifically binds to a human PTP.zeta.
selected from the group consisting of PTP.zeta. SM1 And PTP.zeta.
SM2, further wherein .alpha.(PTP.zeta.) does not specifically bind
to human PTP.zeta. .alpha., human PTP.zeta., or phosphacan, wherein
the binding of .alpha.(PTP.zeta.) alters the function of the human
PTP.zeta.,and a pharmaceutically acceptable carrier.
[0072] A composition for the treatment of a brain tumor comprising
a compound of the general formula .alpha.(PTP.zeta.), wherein
.alpha.(PTP.zeta.) is one or more moieties which specifically binds
to PTP.zeta. selected from the group consisting of PTP.zeta. SM1
And PTP.zeta. SM2, further wherein .alpha.(PTP.zeta.) does not
specifically bind to human PTP.zeta. .alpha., human PTP.zeta.
.alpha., or phosphacan, wherein the binding of .alpha.(PTP.zeta.)
alters the function of the PTP.zeta., and a pharmaceutically
acceptable carrier.
[0073] A method for visualizing a brain tumor in a patient by first
administering to a patient an effective amount of a composition
comprising: a compound of the general formula .alpha.(PTP.zeta.)I,
wherein .alpha.(PTP.zeta.) is one or more moieties which
specifically binds to a human PTP.zeta. selected from the group
consisting of PTP.zeta. SM1 And PTP.zeta. SM2, further wherein
.alpha.(PTP.zeta.) does not specifically bind to human PTP.zeta.
.alpha., human PTP.zeta. .beta., or phosphacan, and I is one or
more imaging moieties and a pharmaceutically acceptable carrier,
and then visualizing the imaging moieties of the compound.
[0074] A composition for the visualization of a brain tumor
comprising a compound of the general formula .alpha.(PTP.zeta.)I,
wherein .alpha.(PTP.zeta.) is one or more moieties which
specifically binds to a human PTP.zeta. selected from the group
consisting of PTP.zeta. SM1 And PTP.zeta. SM2, further wherein
.alpha.(PTP.zeta.) does not specifically bind to human PTP.zeta.
.alpha., human PTP.zeta. .beta., or phosphacan, and I is one or
more imaging moieties, and a pharmaceutically acceptable
carrier.
[0075] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(SPARC)C, wherein .alpha.(SPARC) is one or
more moieties which specifically binds to a human secreted protein,
rich in cysteine, and C is one or more cytotoxic moieties, and a
pharmaceutically acceptable carrier.
[0076] A compound for the treatment of a brain tumor of the general
formula .alpha.(SPARC)C, wherein .alpha.(SPARC) is one or more
moieties which specifically binds to a human secreted protein, rich
in cysteine, and C is one or more cytotoxic moieties.
[0077] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(SPARC), wherein .alpha.(SPARC) is one or
more moieties which specifically binds to a human secreted protein,
rich in cysteine, wherein the binding of .alpha.(SPARC) alters the
function of the secreted protein, rich in cysteine, and a
pharmaceutically acceptable carrier.
[0078] A method for visualizing a brain tumor in a patient by first
administering to a patient an effective amount of a composition
comprising: a compound of the general formula .alpha.(SPARC)I,
wherein .alpha.(SPARC) is one or more moieties which specifically
binds to a human secreted protein, rich in cysteine, and I is one
or more imaging moieties, and a pharmaceutically acceptable
carrier, and then visualizing the imaging moieties of the
compound.
[0079] A composition for the visualization of a brain tumor
comprising a compound of the general formula .alpha.(SPARC)I,
wherein .alpha.(SPARC) is one or more moieties which specifically
binds to a human secreted protein, rich in cysteine, and I is one
or more imaging moieties, and a pharmaceutically acceptable
carrier.
[0080] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(c-MET)C, wherein .alpha.(c-MET) is one or
more moieties which specifically binds to a human c-MET oncogene
product, and C is one or more cytotoxic moieties and a
pharmaceutically acceptable carrier.
[0081] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(c-MET), wherein .alpha.(c-MET) is one or
more moieties which specifically binds to a human c-MET oncogene
product, wherein the binding of .alpha.(TSPAN3) alters the function
of the c-MET oncogene product, and a pharmaceutically acceptable
carrier.
[0082] A method for visualizing a brain tumor in a patient by first
administering to a patient an effective amount of a composition
comprising: a compound of the general formula .alpha.(c-MET)I,
wherein .alpha.(c-MET) is one or more moieties which specifically
binds to a human c-MET oncogene product, and I is one or more
imaging moieties and a pharmaceutically acceptable carrier, and
then visualizing the imaging moieties of the compound.
[0083] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(CD44)C, wherein .alpha.(CD44) is one or
more moieties which specifically binds to a human CD44 antigen, and
C is one or more cytotoxic moieties, and a pharmaceutically
acceptable carrier.
[0084] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(CD44), wherein .alpha.(CD44) is one or more
moieties which specifically binds to a human CD44 antigen, wherein
the binding of .alpha.(CD44) alters the function of the CD44
antigen, and a pharmaceutically acceptable carrier.
[0085] A method for visualizing a brain tumor in a patient by first
administering to a patient an effective amount of a composition
comprising: a compound of the general formula .alpha.(CD44)I,
wherein .alpha.(CD44) is one or more moieties which specifically
binds to a human CD44 antigen, and I is one or more imaging
moieties, and a pharmaceutically acceptable carrier, and then
visualizing the imaging moieties of the compound.
[0086] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(VIPR2)C, wherein .alpha.(VIPR2) is one or
more moieties which specifically binds to a human vasoactive
intestinal peptide receptor-2, and C is one or more cytotoxic
moieties and a pharmaceutically acceptable carrier.
[0087] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(VIPR2), wherein .alpha.(VIPR2) is one or
more moieties which specifically binds to a human vasoactive
intestinal peptide receptor-2, wherein the binding of
.alpha.(VIPR2) alters the function of the vasoactive intestinal
peptide receptor-2, and a pharmaceutically acceptable carrier.
[0088] A method for visualizing a brain tumor in a patient by first
administering to a patient an effective amount of a composition
comprising: a compound of the general formula .alpha.(VIPR2)I,
wherein .alpha.(VIPR2) is one or more moieties which specifically
binds to a human tetraspanin 3, and I is one or more imaging
moieties, and a pharmaceutically acceptable carrier, and then
visualizing the imaging moieties of the compound.
[0089] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(OPN)C, wherein .alpha.(OPN) is one or more
moieties which specifically binds to a human osteopontin, and C is
one or more cytotoxic moieties and a pharmaceutically acceptable
carrier.
[0090] A compound for the treatment of a brain tumor of the general
formula .alpha.(OPN)C, wherein (OPN) is one or more moieties which
specifically binds to a human tetraspanin 3, and C is one or more
cytotoxic moieties.
[0091] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(OPN), wherein .alpha.(OPN) is one or more
moieties which specifically binds to a human osteopontin, wherein
the binding of .alpha.(OPN) alters the function of the osteopontin,
and a pharmaceutically acceptable carrier.
[0092] A method for visualizing a brain tumor in a patient by first
administering to a patient an effective amount of a composition
comprising: a compound of the general formula .alpha.(OPN)I,
wherein .alpha.(OPN) is one or more moieties which specifically
binds to a human osteopontin, and I is one or more imaging
moieties, and a pharmaceutically acceptable carrier, and then
visualizing the imaging moieties of the compound.
[0093] A composition for the visualization of a brain tumor
comprising a compound of the general formula .alpha.(OPN)I, wherein
.alpha.(OPN) is one or more moieties which specifically binds to a
human osteopontin, and I is one or more imaging moieties, and a
pharmaceutically acceptable carrier.
[0094] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(PTN)C, wherein .alpha.(PTN) is one or more
moieties which specifically binds to a human pleiotrophin, and C is
one or more cytotoxic moieties, and a pharmaceutically acceptable
carrier.
[0095] A compound for the treatment of a brain tumor of the general
formula .alpha.(PTN)C, wherein .alpha.(PTN) is one or more moieties
which specifically binds to a human pleiotrophin, and C is one or
more cytotoxic moieties.
[0096] A method to treat a brain tumor by administering a
therapeutic amount of a composition comprising a compound of the
general formula .alpha.(PTN), wherein .alpha.(PTN) is one or more
moieties which specifically binds to a human pleiotrophin, wherein
the binding of .alpha.(PTN) alters the function of the
pleiotrophin, and a pharmaceutically acceptable carrier.
[0097] A method for visualizing a brain tumor in a patient by first
administering to a patient an effective amount of a composition
comprising: a compound of the general formula .alpha.(PTN)I,
wherein .alpha.(PTN) is one or more moieties which specifically
binds to a human pleiotrophin, and I is one or more imaging
moieties, and a pharmaceutically acceptable carrier, and then
visualizing the imaging moieties of the compound.
[0098] A composition for the visualization of a brain tumor
comprising a compound of the general formula .alpha.(PTN)I, wherein
.alpha.(PTN) is one or more moieties which specifically binds to a
human pleiotrophin, and I is one or more imaging moieties, and a
pharmaceutically acceptable carrier.
[0099] Brain tumors are known to be relatively heterogeneous, and
thus all patients may not respond the same to a particular protein
target treatment. Thus, in addition to the independent uses of each
of the T.sub.BT protein targets as described above, the invention
also provides, in yet another aspect, combination therapeutic
and/or visualization agents, compositions, and methods. These
combination embodiments of the invention may utilize as brain tumor
protein targets any two or more of the identified targets
angiopoietin related protein 2 (ARP-2,) secreted protein acidic,
rich in cysteine (SPARC,) c-met proto-oncogene (c-MET,) brevican
(BEHAB,) cd-44 antigen (CD-44,) tetraspanin 3 (TSPN3,) pleiotrophin
(PTN,) osteopontin (OPN,) vasoactive intestinal peptide receptor-2
(VIPR-2,) and receptor protein tyrosine phosphatase zeta
(PTP.zeta.) [including the two novel isoforms PTP.zeta. SM1 and
SM2]. In some preferred embodiments, the brain tumor protein
targets are selected from the group consisting of angiopoietin
related protein 2 (ARP-2,) c-met proto-oncogene (c-MET,) cd-44
antigen (CD-44,) tetraspanin 3 (TSPN3,) osteopontin (OPN,) and
receptor protein tyrosine phosphatase zeta (PTP.zeta.). Embodiments
of the combination aspect of the invention may target a group of
proteins from the identified targets with similar
compartmentalization characteristics. Thus, the combination aspects
may target two or more secreted proteins from the group
angiopoietin related protein 2 (ARP-2,) secreted protein acidic,
rich in cysteine (SPARC,) brevican (BEHAB,) pleiotrophin (PTN,)
secreted forms of receptor protein tyrosine phosphatase zeta
(PTP.zeta.) [including the novel isoform PTP.zeta. SM1]. Or, the
combination aspects may target one or more of the extracellular
matrix binding proteins secreted protein acidic, rich in cysteine
(SPARC,) and/or brevican (BEHAB,) with one or more of the other
identified protein targets. Or, the combination aspects may target
one or more of the membrane-bound proteins from the group c-met
proto-oncogene (c-MET,) cd-44 antigen (CD-44,) tetraspanin 3
(TSPN3,) osteopontin (OPN,) vasoactive intestinal peptide
receptor-2 (VIPR-2,) and membrane bound forms of receptor protein
tyrosine phosphatase zeta (PTP.zeta.) [including the novel isoform
PTP.zeta. SM2].
[0100] In preferred embodiments, at least one of the protein
targets is selected from the proteins angiopoietin related protein
2 (ARP-2,) tetraspanin 3 (TSPN3,), and receptor protein tyrosine
phosphatase zeta (PTP.zeta.) [including the two novel isoforms
PTP.zeta. SM1 and SM2]. In other preferred embodiments, one of
these proteins and another protein selected from the secreted
group, the extracellular matrix group, or the membrane-bound group.
In another group of preferred embodiments are combination aspects
targeting two or more of angiopoietin related protein 2 (ARP-2,)
tetraspanin 3 (TSPN3,), and receptor protein tyrosine phosphatase
zeta (PTP.zeta.) [including the two novel isoforms PTP.zeta. SM1
and SM2]. Another group of preferred embodiments are combination
aspects which target angiopoietin related protein 2 (ARP-2), and
one or more proteins selected from the group secreted protein
acidic, rich in cysteine (SPARC,) c-met proto-oncogene (C-MET,)
brevican (BEHAB,) CD-44 antigen (CD-44,) tetraspanin 3 (TSPN3,)
pleiotrophin (PTN,) osteopontin (OPN,) vasoactive intestinal
peptide receptor-2 (VIPR-2,) and receptor protein tyrosine
phosphatase zeta (PTP.zeta.) [including the two novel isoforms
PTP.zeta. SM1 and SM2]. Another group of preferred embodiments are
combination aspects which target tetraspanin 3 (TSPN3), and one or
more proteins selected from the group secreted protein acidic, rich
in cysteine (SPARC,) c-met proto-oncogene (C-MET,) brevican
(BEHAB,) CD-44 antigen (CD-44,) angiopoietin related protein 2
(ARP-2,) pleiotrophin (PTN,) osteopontin (OPN,) vasoactive
intestinal peptide receptor-2 (VIPR-2,) and receptor protein
tyrosine phosphatase zeta (PTP.zeta.) [including the two novel
isoforms PTP.zeta. SM1 and SM2]. Another group of preferred
embodiments are combination aspects which target receptor protein
tyrosine phosphatase zeta (PTP.zeta.) [including the two novel
isoforms PTP.zeta. SM1 and SM2] and one or more proteins selected
from the group angiopoietin related protein 2 (ARP-2,) secreted
protein acidic, rich in cysteine (SPARC,) c-met proto-oncogene
(C-MET,) brevican (BEHAB,) CD-44 antigen (CD-44,) tetraspanin 3
(TSPN3,) pleiotrophin (PTN,) osteopontin (OPN,) and vasoactive
intestinal peptide receptor-2 (VIPR-2.) As pleiotrophin (PTN) is a
known ligand of PTP.zeta., another preferred embodiment of the
combination aspects of the invention utilizes these proteins as
targets, either alone or in combination with one or more of the
other identified targets.
[0101] In yet another aspect, the present invention provides two
novel splicing isoforms of PTP.zeta., shown to be expressed in
brain tissue. These novel isoforms, PTP.zeta. SM1 and PTP.zeta.
SM2, described in more detail below, differ in structure from the
three known isoforms heretofore disclosed. PTP.zeta. SM1 comprises
the amino acids encoded by the first nine exons of
PTP.zeta.-.alpha., with three unique additional carboxy terminal
amino acids encoded by additional 3' mRNA sequence from the intron
of the gene between exons nine and ten. The mRNA for PTP.zeta. SM2
comprises all exons of PTP.zeta.-.alpha., with a 116 nucleotide
insertion, in the correct reading frame, in the mRNA sequence
between exons 23 and 24, from the from the intron of the gene
between exons 23 and 24. Thus, embodiments of this aspect of the
invention include the mature proteins of PTP.zeta. splice variants
SM1 or SM2, and nucleic acids encoding these novel spice variants,
as well proteins with significant homology to the splice
variants.
[0102] Thus, in one group of embodiments of this aspect, the
invention provides nucleic acid polymers comprising the sequence of
nucleotides 148 to 1272 of SEQ ID NO. 1, the complement of
nucleotides 148 to 1272 of SEQ ID NO. 1, nucleotides 148 to 7209 of
SEQ ID NO. 3, or the complement of nucleotides 148 to 7209 of SEQ
ID NO. 3. In another group of embodiments of this aspect, the
invention provides polypeptides comprising the amino acid sequence
of SEQ ID NO. 2 or the amino acid sequence of SEQ ID NO. 4.
[0103] In an additional related aspect, the invention provides
polypeptides comprising a distinctive portion of the amino acid
sequence of SEQ ID NO. 2 or SEQ ID NO. 4. Such peptides are useful
for the production of antibodies against the PTP.zeta. SM1 or SM2
splicing variants. Preferably, these polypeptides comprise a
portion of the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 4
which is at least 6, more preferably at least 8, more preferably at
least 10, more preferably at least 15, and most preferably at lest
20 amino acids in length. In some preferred embodiments of this
aspect of the invention, the polypeptides comprise the three unique
terminal amino acids of PTP.zeta. SM1 after exon 9. In other
preferred embodiments, the polypeptides comprise a portion of the
unique exon 23a of PTP.zeta. SM2, wherein the portion is preferably
at least 3 amino acids in length, more preferably at least 6 amino
acids in length, more preferably at least 9 amino acids in length,
and most preferably at least 15 amino acids in length.
[0104] In an additional related aspect, the invention also provides
affinity reagents which specifically bind to PTP.zeta. splice
variants SM1 or SM2, but do not bind to the other known splice
variants of PTP.zeta. (e.g., .alpha., .beta., or phosphacan forms).
In preferred embodiments these affinity reagents are antibodies or
antibody fragments.
[0105] In an additional related aspect, the invention also provides
nucleic acid sequences encoding the PTP.zeta. splice variants SM1
or SM2. The invention also encompasses nucleic acid probes which
hybridize to the mRNA encoding PTP.zeta. splice variants SM1 or
SM2, but not mRNA encoding other known splice variants of
PTP.zeta..
BRIEF DESCRIPTION OF THE FIGURES
[0106] FIG. 1: A diagram of the three known splicing variant
isoforms of PTP.zeta.. The approximate position of the domains of
the isoforms is indicated underneath the isoforms, as well as the
approximate exon size (for size reference, exon 12 is 3.6
kilobases.) Isoform PTP.zeta.-.alpha. is the full length isoform,
which contains the primary amino acid sequence aa 25-2314 of SEQ ID
NO. 2 (aa 1-24 are a signal polypeptide). In Isoform
PTP.zeta.-.beta., aa 755-1614 are missing. Isoform PTP.zeta.-S
(phosphacan), is a secreted isoform which comprise the
extracellular domains of PTP.zeta.-.alpha., in which the
transmembrane and cytosol domains are missing.
[0107] FIG. 2: A diagram of the two newly discovered splicing
variant isoforms of PTP.zeta.. The approximate position of the
domains of the isoforms is indicated underneath the isoforms, as
well as the approximate exon size (for size reference, exon 12 is
3.6 kilobases.) SM 1 fails to splice correctly after the 9.sup.th
exon, yielding an mRNA with tow extra codons followed by a stop
codon after the normal terminus of exon 9. SM 2 contains a 116
nucleotide insertion from between exons 23 &24.
[0108] FIG. 3: A diagram comparing the three known PTP.zeta.
isoforms with the two novel isoforms.
DETAILED DESCRIPTION OF THE INVENTION
[0109] Applicants have identified several brain tumor protein
targets and genes which are differentially regulated between brain
cancer tissue (glioblastoma) and normal brain tissue. Applicants
have performed differential cloning between cancerous and normal
brains and have identified the brain tumor protein target genes by
DNA sequence analysis. Based on the observation in other diseases,
particularly other cancers, in which overexpressed genes can
contribute to the pathology of the disease, these overexpressed
genes and their protein products mediate the initiation and
progression of brain tumors. Thus, the overexpressed brain tumor
protein targets, which are presented on the cell surface, provide
excellent targets for immunotherapeutic agents which either deliver
cytotoxic agents to directly promote tumor cell death, or which
alter the function of the brain tumor protein targets to inhibit
the normal physiology of the tumor cell. In addition, immunoimaging
agents targeted to the brain tumor protein targets may be utilized
to visualize the tumor mass either in diagnostic methods (e.g.,
magnetic resonance imaging (MRI) or radiography), or in surgery
(e.g., by the use of optically visual dye moieties in the
immunoimaging agent).
[0110] Applicants have identified the brain tumor protein targets
by a direct examination of the expression level of genes in actual
tumor cells. These samples provide a more accurate and realistic
picture of tumor cell biology, especially on the detailed
transcriptome level, than animal models or established cell tissue
culture cell lines. Several groups have found that cell lines
established from astrocytomas and other cell lines do not exhibit
expression patterns which reflect the actual expression of the
original tumor. For instance, Schreiber, et. al., "Primary brain
tumors differ in their expression of octamer deoxyribonucleic
acid-binding transcription factors from long-term cultured glioma
cell lines." Neurosurgery 34: 129-35 (1994), showed that nervous
system-specific transcription factors known as N-Oct proteins are
differentially expressed in human neuroblastoma and glioblastoma
cell lines in vitro. However, when these results were compared to
freshly isolated human primary and metastatic brain tumors, of the
five astrocytomas and three glioblastomas analyzed, all but two
tumors displayed the complete N-Oct protein profile, irrespective
of histopathological tumor grade. Similarly, Eberle, et al., "The
expression of angiogenin in tissue samples of different brain
tumors and cultured glioma cells."Anticancer Res 20: 1679-84
(2000), could show that angiogenin is detectable in different kinds
of intracranial tumor tissue samples. Although angiogenin could be
detected in primary cultivated glioma cells, it was not detected in
the permanent cell lines. Finally, Hartmann, et al., "The rate of
homozygous CDKN2A/p16 deletions in glioma cell lines and in primary
tumors."Int J Oncol 15: 975-82 (1999), showed that the rate of
homozygous deletions of CDKN2A/p16 is variable between different
tumor entities, but the rate of deletions is higher in established
cell lines in comparison with primary tumors. Hartmann hypothesized
that such incongruity may reflect statistical sampling errors, true
differences depending on tissue derivatization and CDKN2A/p16 loss
under selective pressure in tissue culture. After comparing
established cell lines derived from human glioblastomas and their
corresponding primary tumors by multiplex PCR methodology, they
found that in 2 of 11 cases (18%) the primary tumor had no p16
alteration whereas the corresponding cell lines had a homozygous
p16 deletion, and that CDKN2A/p16 was lost already in the earliest
passages of the cell lines. Thus, Hartmann concluded that the
deletion was the result of selective cell-culture pressures in many
cases.
[0111] These inconsistent results arise because the tumor tissue
samples are obtained from their native milieu, without allowing
them the opportunity to alter their gene expression levels in
response to artificial environmental stimuli. As recently reported
by the Brain Tumor Progress Review group of the National Cancer
Institute in November, 2000, conventionally used glioblastoma cell
lines contain genetic and gene expression alterations that are well
defined and do not necessarily reflect the primary tumors from
which they were derived. In addition, these cell lines are highly
homogenous, unlike a primary brain tumor. Therefore, data derived
soley from a cell line cannot reliably reflect the biology,
heterogeneity, or therapeutic response of a primary brain
tumor.
[0112] Applicants obtained tumor tissue, snap frozen in the
operation hall from unknown patients, which was confirmed as
glioblastoma grade IV by neuropathology. These tissues served as
the experimental sample. Human whole brain tissue (Clontech
Laboratories, Palo Alto, USA) served as control sample.
Poly-A.sup.+ RNA prepared from the cells was converted into
double-stranded cDNA (dscDNA).
[0113] Briefly, the ds-cDNA's from control and disease states were
subjected to kinetic re-annealing hybridization during which
normalization of transcript abundances and enrichment for
differentially expressed transcripts (i.e., subtraction) occurs.
Normalized-subtracted ds-cDNAs were cloned into a plasmid vector, a
large number of recombinant bacterial clones were picked, and their
recombinant inserts were isolated by PCR. High-density cDNA arrays
of those PCR products were screened with cDNA probes derived from
the original control and disease states. Thus, only clones
displaying a significant transcriptional induction and/or
repression were sequenced and carried forward for massive
expression profiling using a variety of temporal, spatial and
disease-related probe sets.
[0114] The selected PCR products (fragments of 200-2000 bp in size)
from clones showing a significant transcriptional induction and/or
repression were sequenced and functionally annotated in AGY's
proprietary database structure (See WO01/13105). Because large
sequence fragments were utilized in the sequencing step, the data
generated has a much higher fidelity and specificity than other
approaches, such as SAGE. The resulting sequence information was
compared to public databases using the BLAST (blastn) and tblastx
algorithm. The results are listed in Table 1, below:
1TABLE 1 RELATIVE EXPRESSION NUMBER OF CLONES PROTEIN LEVEL
ISOLATED (out of 20,000) ARP2 .about.2 times 13 SPARC .about.2-5.6
times 100 CMET .about.1.2-2.5 times 30 CD 44 .about.2.3-3.0 times 6
BEHAB .about.2-6 times 180 TSPAN3 .about.2.0-3.0 times 7 VIPR2
.about.3.0 times 3 OPN .about.2.0-3.0 times 19 PTN
.about..about.1.8-2.6 times 26 PTP.zeta. .about.2.0-4.0 times
20
[0115] As one of skill in the art will appreciate from this data,
each of these proteins is individually useful as a target for the
treatment and/or imaging of brain tumors.
[0116] Characteristics of Protein Targets Utilized in the
Invention
[0117] ARP2
[0118] Given the experiments described above, and the results of
Table 1, ARP-2 was selected as a target for selective
immuno-therapeutic agents in targeting and/or imaging brain tumors.
The mature protein consists of 493 amino acids and contains two
potential consensus glycosylation sites. The complete cDNA sequence
encoding ARP-2 is provided in SEQ ID NO. 7, and the complete amino
acid sequence of ARP-2 is provided in SEQ ID NO. 8. ARP-2 is a 64
kDa, single chain, acidic, angiopoeitin-like protein that includes
multiple functional domains, such as a hydrophobic signal sequence
from amino acids 1-21 (which is typical of secreted proteins), a
coiled-coil domain at the amino terminal end from approximately
amino acid sequences 22-274, and a fibrinogen-like domain, from
approximately about residues 275 through 493. Two major isoforms
have been observed, one 2.4 Kb in size and the other about 4 Kb.
Both forms are abundant in heart, small intestine, spleen and
stomach.
[0119] As used herein, a compound that specifically binds to ARP-2
is any compound (such as an antibody) that has a binding affinity
for any naturally occurring isoform, splice variant, or
polymorphism of ARP-2, explicitly including the isoforms described
herein. As one of ordinary skill in the art will appreciate, such
"specific" binding compounds (e.g., antibodies) may also bind to
other closely related proteins that exhibit significant homology
(such as greater than 90% identity, more preferably greater than
95% identity, and most preferably greater than 99% identity) with
the amino acid sequence of ARP-2. Such proteins include truncated
forms or domains of ARP-2, and recombinantly engineered alterations
of ARP-2. For example, a portion of SEQ ID NO. 8 may be engineered
to include a non-naturally occurring cysteine for cross linking to
an immunoconjugate protein, as described.
[0120] In general, it is preferred that the antibodies utilized in
the compositions and methods of the invention bind to the
fibrinogen domain but need not be restricted to this domain. The
antibody may bind to the extracellular region of ARP-2. It is to be
noted that antibodies which bind to this secreted protein are
useful in the invention as cytotoxic delivery agents, as well as
functional inhibition agents, as one of ordinary skill would expect
that the concentration of ARP-2 would be increased adjacent the
tumor cells which, due to the need for vascularization,
over-express the protein.
[0121] When raising antibodies to ARP-2, the entire protein (either
the unsecreted precursor or the secreted protein), or a portion
thereof, may be utilized. For instance, the carboxyl-terminal
fibrinogen like domain, or any portion of the amino-terminal
coiled-coil domain may be utilized. For instance, amino acids
22-274, which make up the fibrinogen like domain, may be used.
Larger ARP-2 proteins and domains may be produced utilizing any
suitable recombinant vector/protein production system, such as the
baculovirus transfection system outlined below, after being
amplified from a fetal brain cDNA library (as available from, e.g.,
Clontech, Palo Alto, Calif.) or another suitable source.
[0122] When utilizing an entire protein, or a larger section of the
protein, antibodies may be raised by immunizing the production
animal with the protein and a suitable adjuvant (e.g., Fruend's,
Fruend's complete, oil-in-water emulsions, etc.). In these cases,
the ARP-2 protein (or a portion thereof) can serve as the ARP-2
antigen. When a smaller peptide is utilized, it is advantageous to
conjugate the peptide with a larger molecule to make an
immunostimulatory conjugate for use as the ARP-2 antigen. Commonly
utilized conjugate proteins that are commercially available for
such use include bovine serum albumin (BSA) and keyhole limpet
hemocyanin (KLH). In order to raise antibodies to particular
epitopes, peptides derived from the full ARP-2 sequence may be
utilized. Preferably, one or more 8-30 aa peptide portions of the
EC domain of ARP-2 are utilized, with peptides in the range of
10-20 being a more economical choice. Custom-synthesized peptides
in this range are available from a multitude of vendors, and can be
order conjugated to KLH or BSA. Alternatively, peptides in excess
of 30 amino acids may be synthesized by solid-phase methods, or may
be recombinantly produced in a suitable recombinant protein
production system. In order to ensure proper protein glycosylation
and processing, an animal cell system (e.g., Sf9 or other insect
cells, CHO or other mammalian cells) is preferred. Other
information useful in designing an antigen for the production of
antibodies to ARP-2 may be deduced by those of skill in the art by
homology analysis of SEQ ID NO. 8.
[0123] The fibrinogen domain of human ARP-2 is hypothesized to
interact with one or more an unknown receptor for the purposes of
angiogenesis. The interaction of ARP-2 to these molecules may be
through either of the aforementioned structural motifs. Thus, in
alternative embodiments of the compositions and methods of the
invention, antibody moieties are utilized which bind to ARP-2 at a
site on the protein that alters the binding of an extracellular
molecule to ARP-2. Such ARP-2 activity altering antibodies may be
utilized in therapeutic compositions in an unconjugated form (e.g.,
the antibody in an acceptable pharmaceutical carrier), or may be
conjugated to either a therapeutic moiety (creating a double-acting
therapeutic agent) or an imaging moiety (creating a duel
therapeutic/imaging agent).
[0124] Selection of antibodies which alter (enhance or inhibit) the
binding of a ARP-2 to a receptor may be accomplished by a
straightforward binding inhibition/enhancement assay. According to
standard techniques, the binding of a labeled (e.g., fluorescently
or enzyme-labeled) antibody to ARP-2, which has been immobilized in
a microtiter well, is assayed in both the presence and absence of
the appropriate ligand. The change in binding is indicative of
either an enhancer (increased binding) or competitive inhibitor
(decreased binding) relationship between the antibody and the
ligand. Such assays may be carried out in high-throughput formats
(e.g., 384 well plate formats, in robotic systems) for the
automated selection of monoclonal antibody candidates for use as
ARP-2 ligand-binding inhibitors or enhancers.
[0125] In addition, antibodies which are useful for altering the
function of ARP-2 may be assayed in functional formats, such as
endothelial sprouting assays and cell migration assays described in
the examples. Thus, antibodies that exhibit the appropriate
anti-tumor effect may be selected without direct knowledge of a
binding ligand.
[0126] SPARC
[0127] Given the experiments described above, and the results of
Table 1, SPARC was selected as a target for selective
immuno-therapeutic agents in targeting and/or imaging brain tumors.
The mature protein consists of 286 amino acids (after cleavage of
the signal peptide) and contains two potential Asn-X-Thr/Ser
N-glycosylation sites, located at positions 71 and 99 of the mature
protein. The complete cDNA sequence encoding SPARC is provided in
SEQ ID NO. 9, and the complete amino acid sequence of SPARC is
provided in SEQ ID NO. 10. SPARC is an abundant 33 kDa, single
chain, acidic, extracellular calcium binding protein that contains
a flexible N-terminal acidic domain I (.about.50 amino acids), a
follistatin-like (FS) domain (.about.75 residues), and a C-terminal
extracellular calcium-binding (EC) domain with a pair of EF-hand
loops (.about.150 residues). The N-terminal domain shows a low
affinity Ca2+ binding site, a transglutaminase cross linking site,
and inhibits cell spreading in cell culture assays.
Calcium-dependent binding of SPARC to the triple helix of several
fibrillar collagen types and basement membrane collagen type IV has
been mapped to the EC domain. Two isoforms have been described,
bone SPARC with a molecular weight of 31,000 kDa and platelet SPARC
with a molecular weight of 33,000 kDa.
[0128] As used herein, a compound that specifically binds to SPARC
is any compound (such as an antibody) that has a binding affinity
for any naturally occurring isoform, splice variant, or
polymorphism of SPARC, explicitly including the isoforms described
herein. As one of ordinary skill in the art will appreciate, such
"specific" binding compounds (e.g., antibodies) may also bind to
other closely related proteins that exhibit significant homology
(such as greater than 90% identity, more preferably greater than
95% identity, and most preferably greater than 99% identity) with
the amino acid sequence of SPARC. Such proteins include truncated
forms or domains of SPARC, and recombinantly engineered alterations
of SPARC. For example, a portion of SEQ ID NO. 10 may be engineered
to include a non-naturally occurring cysteine for cross linking to
an immunoconjugate protein, as described.
[0129] In general, it is preferred that the antibodies utilized in
the compositions and methods of the invention bind to the
extracellular domain (amino acids 130-280). It is preferable that
this binding inhibit the activity of SPARC. The antibody may bind
to the EF hand which is known to bind Ca2+ with high affinity, but
need not be restricted to this domain. It is to be noted that
antibodies which bind to SPARC are useful in both cytotoxic and
imaging embodiments of the invention, as one of ordinary skill
would expect that the concentration of SPARC in the extracellular
matrix would be increased around tumor cells which over-express the
protein.
[0130] When raising antibodies to SPARC the entire protein (either
the unsecreted precursor or the secreted protein), or a portion
thereof, may be utilized. For instance, the C terminal
extracellular (EC) domain, or any portion of the flexible
N-terminal domain I, or FS domain may be utilized. For instance,
amino acids 125-275, which make up the EC domain, may be used.
Larger SPARC proteins and domains may be produced utilizing any
suitable recombinant vector/protein production system, such as the
baculovirus transfection system outlined below, after being
amplified from a fetal brain cDNA library (as available from, e.g.,
Clontech, Palo Alto, Calif.) or another suitable source.
[0131] When utilizing an entire protein, or a larger section of the
protein, antibodies may be raised by immunizing the production
animal with the protein and a suitable adjuvant (e.g., Fruend's,
Fruend's complete, oil-in-water emulsions, etc.). In these cases,
the SPARC protein (or a portion thereof) can serve as the SPARC
antigen. When a smaller peptide is utilized, it is advantageous to
conjugate the peptide with a larger molecule to make an
immunostimulatory conjugate for use as the SPARC antigen. Commonly
utilized conjugate proteins that are commercially available for
such use include bovine serum albumin (BSA) and keyhole limpet
hemocyanin (KLH). In order to raise antibodies to particular
epitopes, peptides derived from the full SPARC sequence may be
utilized. Preferably, one or more 8-30 aa peptide portions of the
EC domain of SPARC are utilized, with peptides in the range of
10-20 being a more economical choice. Custom-synthesized peptides
in this range are available from a multitude of vendors, and can be
order conjugated to KLH or BSA. Alternatively, peptides in excess
of 30 amino acids may be synthesized by solid-phase methods, or may
be recombinantly produced in a suitable recombinant protein
production system. In order to ensure proper protein glycosylation
and processing, an animal cell system (e.g., Sf9 or other insect
cells, CHO or other mammalian cells) is preferred. Other
information useful in designing an antigen for the production of
antibodies to SPARC, including glycosylation sites, is provided in
SEQ ID NO. 10.
[0132] The EC domain of human SPARC is known to interact with the
collagens I, III, IV and V, and to bind to vitronectin, all of
which are components of the extracellular matrix surrounding
gliomas. The binding of SPARC to these molecules may play a
significant role in the oncogenesis and growth of neoplastic cells
in the brain. Thus, in alternative embodiments of the compositions
and methods of the invention, antibody moieties are utilized which
bind to SPARC at a site on the protein that alters the binding of
an extracellular molecule, such as an ECM molecule, to SPARC. Such
SPARC activity altering antibodies may be utilized in therapeutic
compositions in an unconjugated form (e.g., the antibody in an
acceptable pharmaceutical carrier), or may be conjugated to either
a therapeutic moiety (creating a double-acting therapeutic agent)
or an imaging moiety (creating a duel therapeutic/imaging
agent).
[0133] Selection of antibodies which alter (enhance or inhibit) the
binding of a ligand to SPARC may be accomplished by a
straightforward binding inhibition/enhancement assay. According to
standard techniques, the binding of a labeled (e.g., fluorescently
or enzyme-labeled) antibody to SPARC, which has been immobilized in
a microtiter well, is assayed in both the presence and absence of
the appropriate ligand. The change in binding is indicative of
either an enhancer (increased binding) or competitive inhibitor
(decreased binding) relationship between the antibody and the
ligand. Such assays may be carried out in high-throughput formats
(e.g., 384 well plate formats, in robotic systems) for the
automated selection of monoclonal antibody candidates for use as
SPARC ligand-binding inhibitors or enhancers.
[0134] In addition, antibodies which are useful for altering the
function of SPARC may be assayed in functional formats, such as the
HUVEC tube assay and cell migration assay. Thus, antibodies that
exhibit the appropriate anti-tumor effect may be selected without
direct knowledge of a binding ligand or molecular function.
[0135] c-MET
[0136] Given the experiments described above, and the results of
Table 1, c-MET was selected as a target for selective
immuno-therapeutic agents in targeting and/or imaging brain tumors.
The complete cDNA sequence encoding c-MET is provided in SEQ ID NO.
11, and the complete amino acid sequence of c-MET is provided in
SEQ ID NO. 12. c-MET is a type I membrane protein heterodimer.
Generally, two different receptor variants originate by
post-translational processing of a common singe-chain precursor of
170 kDa. Isoform p190MET is formed of a 50 kDa .alpha.-chain and a
145 kDa .alpha.-chain that are disulfide linked, and isoform
p140Met is formed of a 50 kDa .alpha.-chain and an 85 kDa
.beta.-chain, lacking the cytoplasmic kinase domain. This 85 kDa
.beta. chain is likely a trans-membrane glycoprotein that is bound
to the cell surface. Truncated forms of c-MET containing the 50 kDa
.alpha.-chain and a carboxyl-terminally truncated 75 kDa .beta.
sub-unit have also been described. The 75 kDa form arises by
post-translational proteolytic processing, lacks the trans-membrane
domain, and is secreted from the cell.
[0137] As used herein, a compound that specifically binds to c-MET
is any compound (such as an antibody) that has a binding affinity
for any naturally occurring isoform, splice variant, or
polymorphism of c-MET, explicitly including the three isoforms
described herein. As one of ordinary skill in the art will
appreciate, such "specific" binding compounds (e.g., antibodies)
may also bind to other closely related proteins that exhibit
significant homology (such as greater than 90% identity, more
preferably greater than 95% identity, and most preferably greater
then 99% identity) with the amino acid sequence of c-MET. Such
proteins include truncated forms or domains of c-MET, and
recombinantly engineered alterations of c-MET. For example, a
portion of SEQ ID NO. 12 may be engineered to include a
non-naturally occurring cysteine for cross-linking to an
immunoconjugate protein, as described below.
[0138] In general, it is preferred that the antibodies utilized in
the compositions and methods of the invention bind to the
membrane-bound isoforms of the protein, as this will more
specifically target the cytotoxic therapeutic agent, or the imaging
agent, to the brain tumor cell. However, embodiments that utilize
antibodies that bind to the secreted isoform of the protein are
also useful in the invention, as one of ordinary skill would expect
that the concentration of the secreted isoform would also be
increased adjacent to brain tumor cells which over-express the
protein.
[0139] The amino acid sequence of full length c-MET consists of
1408 amino acids, as the sequence was first deduced by Park et al.,
("Sequence of MET proto-oncogene cDNA has features characteristic
of the tyrosine kinase family of growth-factor receptors" Proc.
nat. Acad. Sci. U.S.A. 84:6379-6383 (1987)) and 1390 amino acids,
as later deduced by Prat et al. ("C-terminal truncated forms of
Met, the Hepatocyte Growth Factor" Mol Cell. Biol. 11:5954-5962
(1991)). According to Prat et al., the first N-terminal amino acids
1-24 of SEQ ID NO. B' [B'] are for the most part hydrophobic, and
could serve as a signal sequence for transporting the protein into
the lumen of the endoplasmic reticulum. The .alpha. chain makes up
the extracellular domain of the mature c-MET protein and spans
amino acids 24-306 of SEQ ID NO. 12. The .beta. chain would consist
of 1,084-5 amino acids with the predicted .beta. chain
extracellular domain being amino acids 306 to 932, the single
transmembrane hydrophobic segment being amino acids 933 to 955, and
the intracellular domain being amino acids 956 to 1390 of SEQ ID NO
12.
[0140] When raising antibodies to c-MET, the entire protein, a
dimeric subunit, or a portion thereof may be utilized. For
instance, the extracellular domain of the .alpha. or .beta.
sub-units or the secreted or extracellular portion of the truncated
forms may be utilized. For instance, amino acids that constitute
the .alpha. sub-unit, amino acids 24-306, may be used. Larger c-MET
proteins and domains may be produced utilizing any suitable
recombinant vector/protein production system, such as the
baculovirus transfection system outlined below, after being
amplified from a fetal brain cDNA library (as available from, e.g.,
Clontech, Palo Alto, Calif.) or another suitable source.
[0141] When utilizing an entire protein, or a larger section of the
protein, antibodies may be raised by immunizing the production
animal with the protein and a suitable adjuvant (e.g., Fruend's,
Fruend's complete, oil-in-water emulsions, etc.). In these cases,
the c-MET protein (or a portion thereof) can serve as the c-MET
antigen. When a smaller peptide is utilized, it is advantageous to
conjugate the peptide with a larger molecule to make an
immunostimulatory conjugate for use as the c-MET antigen. Commonly
utilized conjugate proteins that are commercially available for
such use include bovine serum albumin (BSA) and keyhole limpet
hemocyanin (KLH). In order to raise antibodies to particular
epitopes, peptides derived from the full c-MET sequence may be
utilized. Preferably, one or more 8-30 amino acid peptide portions
of an extracellular domain of c-MET are utilized, with peptides in
the range of 10-20 being a more economical choice.
Custom-synthesized peptides in this range are available from a
multitude of vendors, and can be order conjugated to KLH or BSA.
Alternatively, peptides in excess of 30 amino acids may be
synthesized by solid-phase methods, or may be recombinantly
produced in a suitable recombinant protein production system. In
order to ensure proper protein glycosylation and processing, an
animal cell system (e.g., Sf9 or other insect cells, CHO or other
mammalian cells) is preferred. Other information useful in
designing an antigen for the production of antibodies to c-MET,
including glycosylation sites, is provided in SEQ ID NO. 12.
[0142] The extracellular domain of human c-MET binds hepatocyte
growth factor (HGF).
[0143] Because HGF is largely expressed in mesenchymal and
neuroectodermal tissues and released to the extracellular
compartment, paracrine and/or autocrine signaling implicate tumor
genesis in mesenchymal and neuroectodermal tumors and other tumor
cells that over express the c-MET receptor. Recent studies have
shown that the c-MET proto-oncogene is frequently overexpressed in
many types of epithelial tumors, in spontaneously transformed
NIH/3T3 fibroblasts, and in peripheral nerve sheath tumors. In
alternative embodiments of the compositions and methods of the
invention, antibody moieties are utilized which bind to c-MET at a
site on the protein which alters the binding of an extracellular
ligand molecule, such as HGF, to c-MET. Such c-MET activity
altering antibodies may be utilized in therapeutic compositions in
an unconjugated form (e.g., the antibody in an acceptable
pharmaceutical carrier), or may be conjugated to either a
therapeutic moiety (creating a double-acting therapeutic agent) or
an imaging moiety (creating a duel therapeutic/imaging agent).
[0144] Selection of antibodies that alter (enhance or inhibit) the
binding of a ligand to c-MET may be accomplished by a
straightforward binding inhibition/enhancement assay. According to
standard techniques, the binding of a labeled (e.g., fluorescently
or enzyme-labeled) antibody to c-MET, which has been immobilized in
a microtiter well, is assayed in both the presence and absence of
the ligand. The change in binding is indicative of either an
enhancer (increased binding) or competitive inhibitor (decreased
binding) relationship between the antibody and the ligand. Such
assays may be carried out in high-throughput formats (e.g., 384
well plate formats, in robotic systems) for the automated selection
of monoclonal antibody candidates for use as c-MET ligand-binding
inhibitors or enhancers.
[0145] In addition, antibodies that are useful for altering the
function of c-MET may be assayed in functional formats, such as the
endothelial sprouting assay and cell migration assay. Thus,
antibodies which exhibit the appropriate anti-tumor effect may be
selected without direct knowledge of a molecular function.
[0146] BEHAB
[0147] Given the experiments described above, and the results of
Table 1, BEHAB was selected as a target for selective
immuno-therapeutic agents in targeting and/or imaging brain tumors.
The complete cDNA sequence encoding BEHAB GPI isoform is provided
in SEQ ID NO. 13, and the complete amino acid sequence of this
BEHAB isoform is provided in SEQ ID NO. 14. Two isoforms have been
isolated to date: a full-length isoform that is secreted into the
extracellular matrix and a shorter isoform that has a hydrophobic
carboxy terminus instead of the typical lectican carboxyl terminus,
which predicts a glycophosphatidylinositol (GPI) anchor. BEHAB
contains an N-terminal hyaluronan (HA)-binding domain, which
comprises an immunoglobulin-like loop and two proteoglycan tandem
repeats, a C-terminal epidermal growth factor (EGF)-like repeat, a
C-type lectin-like domain, and a complement regulatory protein
(CRP)-like domain. The central region of the protein contains sites
for glycosylation and proteolytic cleavage (between glu395-Ser396
of the mature protein, after signal peptide cleavage) by
metallo-protease. The complete cDNA of the secreted isoform is 2878
bp encoding 912 amino acids of 99 kDa. The GPI isoform, for which
sequences SEQ ID NO. 13 and SEQ ID NO. 14 are given, is 2558 bp
encoding 672 amino acids of 72 kDa. The GPI-linked form is
generated by a `no splice` event, with the transcript reading
through an exon/intron junction thereby extending the open reading
frame to a stop codon 74 nucleotides further downstream.
[0148] As used herein, a compound that specifically binds to BEHAB
is any compound (such as an antibody) that has a binding affinity
for any naturally occurring isoform, splice variant, or
polymorphism of BEHAB, explicitly including the two splice variants
described herein. As one of ordinary skill in the art will
appreciate, such "specific" binding compounds (e.g., antibodies)
may also bind to other closely related proteins that exhibit
significant homology (such as greater than 90% identity, more
preferably greater than 95% identity, and most preferably greater
then 99% identity) with the amino acid sequence of BEHAB. Such
proteins include truncated forms or domains of BEHAB, and
recombinantly engineered alterations of BEHAB. For example, a
portion of SEQ ID NO. 14 may be engineered to include a
non-naturally occurring cysteine for cross-linking to an
immunoconjugate protein, as described below.
[0149] In general, it is preferred that the antibodies utilized in
the compositions and methods of the invention bind to the
membrane-bound isoform of the protein, as this will more
specifically target the cytotoxic therapeutic agent, or the imaging
agent, to the brain tumor cell. However, embodiments that utilize
antibodies that bind to the secreted isoform of the protein are
also useful in the invention, as one of ordinary skill would expect
that the concentration of the secreted isoform would also be
increased adjacent to brain tumor cells which over-express the
protein.
[0150] When raising antibodies to BEHAB, the entire protein, or a
portion thereof, may be utilized. For instance, any one of the
aforementioned domains of the secreted protein or an extracellular
portion of the truncated, membrane bound GPI form may be utilized.
For instance, amino acids that constitute the hyaluronic acid
binding domain, amino acids 44-247, which includes the Ig like
domain at amino acids 44-140, may be used. Larger BEHAB proteins
and domains may be produced utilizing any suitable recombinant
vector/protein production system, such as the baculovirus
transfection system outlined below, after being amplified from a
fetal brain cDNA library (as available from, e.g., Clontech, Palo
alto, CA) or another suitable source.
[0151] When utilizing an entire protein, or a larger section of the
protein, antibodies may be raised by immunizing the production
animal with the protein and a suitable adjuvant (e.g., Fruend's,
Fruend's complete, oil-in-water emulsions, etc.). In these cases,
the Brevican protein (or a portion thereof) can serve as the BEHAB
antigen. When a smaller peptide is utilized, it is advantageous to
conjugate the peptide with a larger molecule to make an
immunostimulatory conjugate for use as the BEHAB antigen. Commonly
utilized conjugate proteins that are commercially available for
such use include bovine serum albumin (BSA) and keyhole limpet
hemocyanin (KLH). In order to raise antibodies to particular
epitopes, peptides derived from the full Brevican sequence may be
utilized. Preferably, one or more 8-30 amino acid peptide portions
of an extracellular domain of BEHAB are utilized, with peptides in
the range of 10-20 being a more economical choice.
Custom-synthesized peptides in this range are available from a
multitude of vendors, and can be order conjugated to KLH or BSA.
Alternatively, peptides in excess of 30 amino acids may be
synthesized by solid-phase methods, or may be recombinantly
produced in a suitable recombinant protein production system. In
order to ensure proper protein glycosylation and processing, an
animal cell system (e.g., Sf9 or other insect cells, CHO or other
mammalian cells) is preferred.
[0152] The hyaluronic acid binding domain of human BEHAB binds to
hyaluronic acid (HA). Because HA is largely expressed in the ECM
surrounding gliomas and because recent studies have shown that the
BEHAB protein is frequently overexpressed in primary brain tumors,
it is suggested that the up-regulation of BEHAB may be a crucial
step in returning the unmalleable mature extracellular matrix to a
more immature matrix, permissive for cell growth, thereby promoting
the progression of primary brain tumors. Thus, in alternative
embodiments of the compositions and methods of the invention,
antibody moieties are utilized which bind to BEHAB at a site on the
protein which alters the binding of an extracellular ligand
molecule (e.g., HA) to BEHAB. Such BEHAB activity altering
antibodies may be utilized in therapeutic compositions in an
unconjugated form (e.g., the antibody in an acceptable
pharmaceutical carrier), or may be conjugated to either a
therapeutic moiety (creating a double-acting therapeutic agent) or
an imaging moiety (creating a duel therapeutic/imaging agent).
[0153] Selection of antibodies that alter (enhance or inhibit) the
binding of a ligand to BEHAB may be accomplished by a
straightforward binding inhibition/enhancement assay. According to
standard techniques, the binding of a labeled (e.g., fluorescently
or enzyme-labeled) antibody to BEHAB, which has been immobilized in
a microtiter well, is assayed in both the presence and absence of
the ligand. The change in binding is indicative of either an
enhancer (increased binding) or competitive inhibitor (decreased
binding) relationship between the antibody and the ligand. Such
assays may be carried out in high-throughput formats (e.g., 384
well plate formats, in robotic systems) for the automated selection
of monoclonal antibody candidates for use as BEHAB ligand-binding
inhibitors or enhancers.
[0154] In addition, antibodies that are useful for altering the
function of BEHAB may be assayed in functional formats, such as the
HUVEC tube assay and the cell migration assay described below.
Thus, antibodies which exhibit the appropriate anti-tumor effect
may be selected without direct knowledge of molecular function.
[0155] CD-44
[0156] Given the experiments described above, and the results of
Table 1, CD-44 was selected as a target for selective
immuno-therapeutic agents in targeting and/or imaging brain tumors.
The complete cDNA sequence encoding CD-44E is provided in SEQ ID
NO. 15, and the complete amino acid sequence of CD-44, indicating
various splicing variation locations, is provided in SEQ ID NO. 16.
CD-44 is a proteoglycan that is expressed as two major splice
variants. CD-44E is a 150 kDa protein isolated from epithelial
cells. CD-44E has a C-terminal cytoplasmic tail, a hydrophobic
transmembrane domain of 23 amino acids, and an N-terminal
extracellular region of 248 amino acids. The extracellular domain
is O-glycosylated and also binds chondroitin sulfate. In addition,
CD-44E it has two of the three immunodominant epitope clusters of
native gp90Hermes. CD-44E contains an additional 132 amino acids in
the extracellular region. and CD-44H is a 90 kDa protein isolated
from hematopoietic cells. In addition, CD-44R1 and CD-44R2 are 2
isoforms expressed by hematopoietic cells. The complete cDNA
sequence of the 90 kDa CD-44H isoform consist of 1795 bps, encoding
a 341 amino acid protein.
[0157] As used herein, a compound that specifically binds to CD-44
is any compound (such as an antibody) that has a binding affinity
for any naturally occurring isoform, splice variant, or
polymorphism of CD-44, explicitly including the isoforms described
herein. As one of ordinary skill in the art will appreciate, such
"specific" binding compounds (e.g., antibodies) may also bind to
other closely related proteins that exhibit significant homology
(such as greater than 90% identity, more preferably greater than
95% identity, and most preferably greater then 99% identity) with
the amino acid sequence of CD-44. Such proteins include truncated
forms or domains of CD-44, and recombinantly engineered alterations
of CD-44. For example, a portion of SEQ ID NO. 16 may be engineered
to include a non-naturally occurring cysteine for cross-linking to
an immunoconjugate protein, as described below.
[0158] According to the human full length, CD-44H protein has an
overall primary structure of 90 kDa, which consist of 341 amino
acids. The N-terminus is located outside of the cell and the
extracellular domain consist of 248 amino acids. The C-terminus is
located inside of the cell and the intracellular domain consist of
72 amino acids, while the transmembrane region consist of 21 amino
acids. The CD-44 gene contains 20 exons, of which exons 1-5, 15-17
and 19 encode the CD44H isoform. The intervening exons 6, 6a, 7-14
(also designated v1-v10) are alternatively spliced to generate the
variant isoforms with an insertion at the membrane proximal region
of the extracellular domain between amino acids 202 and 203. See
Bajorath (2000). Proteins: structure, function, and genetic,
39:103-111; and Ilangumaram et al. Leukemia and Lymphoma,
35:455-469.
[0159] When raising antibodies to CD-44, the entire protein, or a
portion thereof, may be utilized. For instance, any portion of the
extracellular domain may be utilized. For instance, the amino acids
between the signal sequence and amino acid 202 may be used. Larger
CD-44 proteins and domains may be produced utilizing any suitable
recombinant vector/protein production system, such as the
baculovirus transfection system outlined below, after being
amplified from a fetal brain cDNA library (as available from, e.g.,
Clontech, Palo Alto, Calif.) or another suitable source.
[0160] When utilizing an entire protein, or a larger section of the
protein, antibodies may be raised by immunizing the production
animal with the protein and a suitable adjuvant (e.g., Fruend's,
Fruend's complete, oil-in-water emulsions, etc.). In these cases,
the CD-44 protein (or a portion thereof) can serve as the CD-44
antigen. When a smaller peptide is utilized, it is advantageous to
conjugate the peptide with a larger molecule to make an
immunostimulatory conjugate for use as the CD-44 antigen. Commonly
utilized conjugate proteins that are commercially available for
such use include bovine serum albumin (BSA) and keyhole limpet
hemocyanin (KLH). In order to raise antibodies to particular
epitopes, peptides derived from the full CD-44 sequence may be
utilized. Preferably, one or more 8-30 amino acid peptide portions
of an extracellular domain of CD-44 are utilized, with peptides in
the range of 10-20 being a more economical choice.
Custom-synthesized peptides in this range are available from a
multitude of vendors, and can be order conjugated to KLH or BSA.
Alternatively, peptides in excess of 30 amino acids may be
synthesized by solid-phase methods, or may be recombinantly
produced in a suitable recombinant protein production system. In
order to ensure proper protein glycosylation and processing, an
animal cell system (e.g., Sf9 or other insect cells, CHO or other
mammalian cells) is preferred. Other information useful in
designing an antigen for the production of antibodies to CD-44,
including glycosylation sites, is provided in SEQ ID NO. D'.
[0161] Hyaluronan (HA) is a polymeric glycosaminoglycan and a major
component of the extracellular matrix. CD-44 is one of the
principal receptors for HA. Within the normal CNS, the CD-44
protein has been localized to astrocytes in the white matter.
CD-44H has been shown to be the predominant isoform in normal brain
and neuroectoderm-derived tumors. Hence, the up-regulation of CD-44
may be a crucial step in brain tumor invasiveness and migration.
Thus, in alternative embodiments of the compositions and methods of
the invention, antibody moieties are utilized which bind to CD-44
at a site on the protein which alters the binding of an
extracellular ligand molecule (e.g., HA) to CD-44. Such CD-44
activity altering antibodies may be utilized in therapeutic
compositions in an unconjugated form (e.g., the antibody in an
acceptable pharmaceutical carrier), or may be conjugated to either
a therapeutic moiety (creating a double-acting therapeutic agent)
or an imaging moiety (creating a duel therapeutic/imaging
agent).
[0162] Selection of antibodies that alter (enhance or inhibit) the
binding of a ligand to CD-44 may be accomplished by a
straightforward binding inhibition/enhancement assay. According to
standard techniques, the binding of a labeled (e.g., fluorescently
or enzyme-labeled) antibody to CD-44, which has been immobilized in
a microtiter well, is assayed in both the presence and absence of
the ligand. The change in binding is indicative of either an
enhancer (increased binding) or competitive inhibitor (decreased
binding) relationship between the antibody and the ligand. Such
assays may be carried out in high-throughput formats (e.g., 384
well plate formats, in robotic systems) for the automated selection
of monoclonal antibody candidates for use as CD-44 ligand-binding
inhibitors or enhancers.
[0163] In addition, antibodies that are useful for altering the
function of CD-44 may be assayed in functional formats, such as
endothelial sprouting assay and cell migration assay. Thus,
antibodies which exhibit the appropriate anti-tumor effect may be
selected without direct knowledge of molecular function.
[0164] TSPAN3
[0165] Given the experiments described above, and the results of
Table 1, TSPAN3 was selected as a target for selective
immuno-therapeutic agents in targeting and/or imaging brain tumors.
The complete cDNA sequence encoding TSPAN3 is provided in SEQ ID
NO. 17, and the complete amino acid sequence of TSPAN3 is provided
in SEQ ID NO. 18. Tetraspanin is a 253 amino acid membrane bound
protein. No isoforms have been isolated to date. TSPAN3, as is
characteristic of the tetraspanin family, contains four
transmembrane domains, putatively comprising amino acids 12-32,
51-71, 86-106, and 213-233. The protein has two putative
extracellular domains, amino acids 33-50 and 107-212, and three
putative cytoplasmic domains, amino acids 1-11, 72-85, and 234-235.
Putative N-linked glycosylation sites are listed in SEQ ID NO.
18.
[0166] As used herein, a compound that specifically binds to TSPAN3
is any compound (such as an antibody) that has a binding affinity
for any naturally occurring isoform, splice variant, or
polymorphism of TSPAN3. As one of ordinary skill in the art will
appreciate, such "specific" binding compounds (e.g., antibodies)
may also bind to other closely related proteins that exhibit
significant homology (such as greater than 90% identity, more
preferably greater than 95% identity, and most preferably greater
then 99% identity) with the amino acid sequence of TSPAN3. Such
proteins include truncated forms or domains of TSPAN3, and
recombinantly engineered alterations of TSPAN3. For example, a
portion of SEQ ID NO. 18 may be engineered to include a
non-naturally occurring cysteine for cross-linking to an
immunoconjugate protein, as described below.
[0167] In general, it is preferred that the antibodies utilized in
the compositions and methods of the invention bind to the
membrane-bound isoform of the protein, as this will more
specifically target the cytotoxic therapeutic agent, or the imaging
agent, to the brain tumor cell. The only currently known form of
TSPAN3 is membrane-bound. However, embodiments that utilize
antibodies that bind to any secreted isoform of the protein are
also useful in the invention, as one of ordinary skill would expect
that the concentration of the secreted isoform would also be
increased adjacent to brain tumor cells which over-express the
protein. Likewise, it is preferred that the antibodies utilized in
the invention bind to an extracellular domain of the protein, as
are described in the SEQ ID NO. 18. The cysteine residues at
positions 147, 148, and 197 of SEQ ID NO. 18 in the second
extracellular domain are highly conserved among the tetraspanin
family and are thought to be essential for proper tetraspanin
function. Thus, in some preferred embodiments of the invention, the
antibodies utilized in the invention bind to an epitope comprising,
or alternatively very near to, one of these cysteine residues.
[0168] When raising antibodies to TSPAN3, the entire protein, or a
portion thereof, may be utilized. For instance, any one of the
aforementioned domains of the secreted protein or an extracellular
portion of the truncated, membrane bound GPI form may be utilized.
For instance, amino acids that constitute one of the extracellular
domains, amino acids 33-50 or 107-212, may be used. Larger TSPAN3
proteins and domains may be produced utilizing any suitable
recombinant vector/protein production system, such as the
baculovirus transfection system outlined below, after being
amplified from a fetal brain cDNA library (as available from, e.g.,
Clontech, Palo Alto, Calif.) or another suitable source.
[0169] When utilizing an entire protein, or a larger section of the
protein, antibodies may be raised by immunizing the production
animal with the protein and a suitable adjuvant (e.g., Fruend's,
Fruend's complete, oil-in-water emulsions, etc.). In these cases,
the tetraspanin 3 protein (or a portion thereof) can serve as the
TSPAN3 antigen. When a smaller peptide is utilized, it is
advantageous to conjugate the peptide with a larger molecule to
make an immunostimulatory conjugate for use as the TSPAN3 antigen.
Commonly utilized conjugate proteins that are commercially
available for such use include bovine serum albumin (BSA) and
keyhole limpet hemocyanin (KLH). In order to raise antibodies to
particular epitopes, peptides derived from the full Brevican
sequence may be utilized. Preferably, one or more 8-30 amino acid
peptide portions of an extracellular domain of TSPAN3 are utilized,
with peptides in the range of 10-20 being a more economical choice.
Custom-synthesized peptides in this range are available from a
multitude of vendors, and can be order conjugated to KLH or BSA.
Alternatively, peptides in excess of 30 amino acids may be
synthesized by solid-phase methods, or may be recombinantly
produced in a suitable recombinant protein production system. In
order to ensure proper protein glycosylation and processing, an
animal cell system (e.g., Sf9 or other insect cells, CHO or other
mammalian cells) is preferred. Other information useful in
designing an antigen for the production of antibodies to TSPAN3,
including glycosylation sites, is provided in SEQ ID NO. 18.
[0170] In alternative embodiments of the compositions and methods
of the invention, antibody moieties are utilized which bind to
TSPAN3 at a site on the protein which alters the binding of an
extracellular ligand molecule to TSPAN3. Such TSPAN3 activity
altering antibodies may be utilized in therapeutic compositions in
an unconjugated form (e.g., the antibody in an acceptable
pharmaceutical carrier), or may be conjugated to either a
therapeutic moiety (creating a double-acting therapeutic agent) or
an imaging moiety (creating a duel therapeutic/imaging agent).
[0171] Selection of antibodies that alter (enhance or inhibit) the
binding of a ligand to TSPAN3 may be accomplished by a
straightforward binding inhibition/enhancement assay. According to
standard techniques, the binding of a labeled (e.g., fluorescently
or enzyme-labeled) antibody to TSPAN3, which has been immobilized
in a microtiter well, is assayed in both the presence and absence
of the ligand. The change in binding is indicative of either an
enhancer (increased binding) or competitive inhibitor (decreased
binding) relationship between the antibody and the ligand. Such
assays may be carried out in high-throughput formats (e.g., 384
well plate formats, in robotic systems) for the automated selection
of monoclonal antibody candidates for use as TSPAN3 ligand-binding
inhibitors or enhancers.
[0172] In addition, antibodies that are useful for altering the
function of TSPAN3 may be assayed in functional formats, such as
the HUVEC tube assay and the cell migration assay described below.
Thus, antibodies which exhibit the appropriate anti-tumor effect
may be selected without direct knowledge of molecular function.
[0173] VIPR-2
[0174] Given the experiments described above, and the results of
Table 1, VIPR-2 was selected as a prime target for selective
immuno-therapeutic agents in targeting and/or imaging brain tumors.
The complete cDNA sequence encoding VIPR-2 is provided in SEQ ID
NO. 19, and the complete amino acid sequence of VIPR-2 is provided
in SEQ ID NO. 20. VIPR-2 is a seven transmembrane spanning
G-protein receptor. The complete VIPR-2 protein is encoded by 13
exons. The initiator codon of the approximated 438 amino
acid-encoding open reading frame is located in exon 1 and the
termination signal is located in exon 13. The 5' untranslated
region extends 187 bp upstream of the initiator codon and is
extremely GC-rich (80%). The polyadenylation signal is located 2416
bp downstream of the stop codon. Intron sizes range from 68 bp
(intron 11) to 45 bp (intron 4), the entire human gene spans 117
kb, while the cDNA sequence spans 1317 bp. Recent studies have also
isolated two VIP-2 receptor mRNAs of 4.6 kb and 2.3 kb in size.
[0175] As used herein, a compound that specifically binds to VIPR-2
is any compound (such as an antibody) that has a binding affinity
for any naturally occurring isoform, splice variant, or
polymorphism of VIPR-2, explicitly including any isoforms described
herein. As one of ordinary skill in the art will appreciate, such
"specific" binding compounds (e.g., antibodies) may also bind to
other closely related proteins that exhibit significant homology
(such as greater than 90% identity, more preferably greater than
95% identity, and most preferably greater then 99% identity) with
the amino acid sequence of VIPR-2. Such proteins include truncated
forms or domains of VIPR-2, and recombinantly engineered
alterations of VIPR-2. For example, a portion of SEQ ID NO. 20 may
be engineered to include a non-naturally occurring cysteine for
cross-linking to an immunoconjugate protein, as described
below.
[0176] In general, it is preferred that the antibodies utilized in
the compositions and methods of the invention bind to the
membrane-bound isoforms of the protein, as this will more
specifically target the cytotoxic therapeutic agent, or the imaging
agent, to the brain tumor cell. However, embodiments that utilize
antibodies that bind to the secreted isoform of the protein are
also useful in the invention, as one of ordinary skill would expect
that the concentration of the secreted isoform would also be
increased adjacent to brain tumor cells which over-express the
protein.
[0177] The amino acid sequence of full length VIPR-2 consists of
437 amino acids with a predicted molecular mass is 49 kDa, as the
sequence was first deduced by Lutz et al. FEBS. 334:3-8, 1993. Lutz
et al. predicted that the receptor is a seven membrane spanning
protein where in the first 22 amino acids constitute a typical
hydrophobic signal sequence, and the remaining amino acids
constitute two membrane spanning regions between amino acids 127 to
148 and 158 to 178, two more membrane spanning domains between
amino acids 202 to 227 and 238 to 261, another between 278 to 303,
and two final membrane spanning regions between 327 to 347 and 359
to 380, with three potential N-linked glycosylation sites found in
the amino terminal extracellular domain at residues 57, 87 and 91.
Sreedharan et al. describes the VIPR-2 receptor as being a 457
amino-acid protein encoded by a 2.8 kb cDNA of 52 kDa. Sreedharan
et al. Biochem. Biophys. Res. Commun. 203:141-148, 1994.
[0178] When raising antibodies to VIPR-2, the entire protein or a
portion thereof may be utilized. For instance, the extracellular
domains of any of the seven transmembrane spanning portions of the
protein may be utilized. For instance, amino acids 179 to 201 may
be used. Larger VIPR-2 proteins and domains may be produced
utilizing any suitable recombinant vector/protein production
system, such as the baculovirus transfection system outlined below,
after being amplified from a fetal brain cDNA library (as available
from, e.g., Clontech, Palo Alto, Calif.) or another suitable
source.
[0179] When utilizing an entire protein, or a larger section of the
protein, antibodies may be raised by immunizing the production
animal with the protein and a suitable adjuvant (e.g., Fruend's,
Fruend's complete, oil-in-water emulsions, etc.). In these cases,
the VIPR-2 protein (or a portion thereof) can serve as the VIPR-2
antigen. When a smaller peptide is utilized, it is advantageous to
conjugate the peptide with a larger molecule to make an
immunostimulatory conjugate for use as the VIPR-2 antigen. Commonly
utilized conjugate proteins that are commercially available for
such use include bovine serum albumin (BSA) and keyhole limpet
hemocyanin (KLH). In order to raise antibodies to particular
epitopes, peptides derived from the full VIPR-2 sequence may be
utilized. Preferably, one or more 8-30 amino acid peptide portions
of an extracellular domain of VIPR-2 are utilized, with peptides in
the range of 10-20 being a more economical choice.
Custom-synthesized peptides in this range are available from a
multitude of vendors, and can be order conjugated to KLH or BSA.
Alternatively, peptides in excess of 30 amino acids may be
synthesized by solid-phase methods, or may be recombinantly
produced in a suitable recombinant protein production system. In
order to ensure proper protein glycosylation and processing, an
animal cell system (e.g., Sf9 or other insect cells, CHO or other
mammalian cells) is preferred. Other information useful in
designing an antigen for the production of antibodies to VIPR-2,
including glycosylation sites, is provided in SEQ ID NO. 20.
[0180] The extracellular domain of human VIPR-2 binds PACAP-27,
PACAP-38, VIP and secretin. Because these factors have been found
to affect tumor cell growth, and due to the recent discovery that
the VIPR-2 receptor is overexpressed in glioblastomas (Astrocytoma
grade IV), the binding of these factors to the VIPR-2 receptor may
play a significant role in the oncogenesis and growth of
astrocytoma cells in the brain. Thus, in alternative embodiments of
the of the invention, antibody moieties are utilized which bind to
VIPR-2 at a site on the protein which alters the binding of
extracellular ligand molecules, such as VIP, to VIPR-2. Such VIPR-2
activity altering antibodies may be utilized in therapeutic
compositions in an unconjugated form (e.g., the antibody in an
acceptable pharmaceutical carrier), or may be conjugated to either
a therapeutic moiety (creating a double-acting therapeutic agent)
or an imaging moiety (creating a duel therapeutic/imaging
agent).
[0181] Selection of antibodies that alter (enhance or inhibit) the
binding of a ligand to VIPR-2 may be accomplished by a
straightforward binding inhibition/enhancement assay. According to
standard techniques, the binding of a labeled (e.g., fluorescently
or enzyme-labeled) antibody to VIPR-2, which has been immobilized
in a microtiter well, is assayed in both the presence and absence
of the ligand (e.g., vasoactive intestinal peptide.) The change in
binding is indicative of either an enhancer (increased binding) or
competitive inhibitor (decreased binding) relationship between the
antibody and the ligand. Such assays may be carried out in
high-throughput formats (e.g., 384 well plate formats, in robotic
systems) for the automated selection of monoclonal antibody
candidates for use as VIPR-2 ligand-binding inhibitors or
enhancers.
[0182] In addition, antibodies that are useful for altering the
function of VIPR-2 may be assayed in functional formats, such as
the HUVEC tube assay and cell migration assay. Thus, antibodies
which exhibit the appropriate anti-tumor effect may be selected
without direct knowledge of a binding ligand.
[0183] PTN
[0184] Given the experiments described above, and the results of
Table 1, PTN was selected as a target for selective
immuno-therapeutic agents in targeting and/or imaging brain tumors.
The entire PTN gene spans 65 kb and 7 exons, while the mature
protein is approximately 136 amino acids (after cleavage of a 32
amino acid signal peptide) with distinctive lysine and
arginine-rich clusters within both N- and C-terminal domains. The
complete cDNA sequence encoding PTN is provided in SEQ ID NO. 21,
and the complete amino acid sequence of PTN is provided in SEQ ID
NO. 22. PTN is a 18 kDa, single chain, secreted protein with 10
conserved disulfide linked cysteine residues.
[0185] As used herein, a compound that specifically binds to PTN is
any compound (such as an antibody) that has a binding affinity for
any naturally occurring isoform, splice variant, or polymorphism of
PTN, explicitly including the isoforms described herein. As one of
ordinary skill in the art will appreciate, such "specific" binding
compounds (e.g., antibodies) may also bind to other closely related
proteins that exhibit significant homology (such as greater than
90% identity, more preferably greater than 95% identity, and most
preferably greater than 99% identity) with the amino acid sequence
of PTN. Such proteins include truncated forms or domains of PTN,
and recombinantly engineered alterations of PTN. For example, a
portion of SEQ ID NO. 22 may be engineered to include a
non-naturally occurring cysteine for cross linking to an
immunoconjugate protein, as described.
[0186] According to Milner et al. the gene sequence of PTN isolated
from human genomic DNA consists of five exons and four introns.
While exon 1 does not encode an amino acid sequence, exon 2 encodes
the hydrophobic signal sequence of 32 amino acids, exons 3 and 4
code for the amino terminal and the ten cysteine residues, and exon
5 codes for the highly basic C-terminal domains. Interestingly, the
human cDNA starts toward the end of exon 1, while the coded for
protein begins at exon 2. Thus, the mature protein consist of 136
amino acids encoded by exons 2 to 5. As reported by Kretschmer et
al. the minimum size of the gene is 42 kb, with a mRNA of 1650
nucleotides, spanning five exons, the majority of the protein being
coded for by exon 3 (174 base pairs in length) and exon 4 (162 base
pairs in length). See Kretschmer et al. (1993). Biochem. Biophys.
Res. Commun. 192:420-429.
[0187] When raising antibodies to PTN, the entire protein or a
portion thereof may be utilized. For instance, amino acid domains
encoded for by exons 3 and 4 (i.e. amino acids 7 to 64 or 65 to
118, respectfully). Specifically, residues 41 to 64 may be used to
abolish the transformation potential of PTN. Larger PTN proteins
and domains may be produced utilizing any suitable recombinant
vector/protein production system, such as the baculovirus
transfection system outlined below, after being amplified from a
fetal brain cDNA library (as available from, e.g., Clontech, Palo
Alto, Calif.) or another suitable source. It is to be noted that
antibodies which bind to this secreted protein are useful in
cytotoxic and imaging embodiments of the invention, as one of
ordinary skill would expect that the concentration of the PTN would
be increased adjacent to tumor cells which over-express the
protein.
[0188] When utilizing an entire protein, or a larger section of the
protein, antibodies may be raised by immunizing the production
animal with the protein and a suitable adjuvant (e.g., Fruend's,
Fruend's complete, oil-in-water emulsions, etc.). In these cases,
the PTN protein (or a portion thereof) can serve as the PTN
antigen. When a smaller peptide is utilized, it is advantageous to
conjugate the peptide with a larger molecule to make an
immunostimulatory conjugate for use as the PTN antigen. Commonly
utilized conjugate proteins that are commercially available for
such use include bovine serum albumin (BSA) and keyhole limpet
hemocyanin (KLH). In order to raise antibodies to particular
epitopes, peptides derived from the full PTN sequence may be
utilized. Preferably, one or more 8-30 aa peptide portions of the
protein are utilized, with peptides in the range of 10-20 being a
more economical choice. Custom-synthesized peptides in this range
are available from a multitude of vendors, and can be order
conjugated to KLH or BSA. Alternatively, peptides in excess of 30
amino acids may be synthesized by solid-phase methods, or may be
recombinantly produced in a suitable recombinant protein production
system. In order to ensure proper protein glycosylation and
processing, an animal cell system (e.g., Sf9 or other insect cells,
CHO or other mammalian cells) is preferred. Other information
useful in designing an antigen for the production of antibodies to
PTN, including glycosylation sites, is provided in SEQ ID NO.
22.
[0189] PTN has been shown to bind to extracellular domain of RPTP
beta and zeta. This binding inactivates the catalytic activity of
RPTP, and PTN binds all the three major isoforms pf RPTP beta and
zeta. PTN has also been shown to interact with syndecan-3. Thus, in
alternative embodiments of the compositions and methods of the
invention, antibody moieties are utilized which bind to PTN at a
site on the protein that alters the binding of a cell surface
molecule, such as the ones listed above, to PTN. Such PTN activity
altering antibodies may be utilized in therapeutic compositions in
an unconjugated form (e.g., the antibody in an acceptable
pharmaceutical carrier), or may be conjugated to either a
therapeutic moiety (creating a double-acting therapeutic agent) or
an imaging moiety (creating a duel therapeutic/imaging agent).
[0190] Selection of antibodies which alter (enhance or inhibit) the
binding of a ligand to PTN may be accomplished by a straightforward
binding inhibition/enhancement assay. According to standard
techniques, the binding of a labeled (e.g., fluorescently or
enzyme-labeled) antibody to PTN, which has been immobilized in a
microtiter well, is assayed in both the presence and absence of the
appropriate ligand. The change in binding is indicative of either
an enhancer (increased binding) or competitive inhibitor (decreased
binding) relationship between the antibody and the ligand. Such
assays may be carried out in high-throughput formats (e.g., 384
well plate formats, in robotic systems) for the automated selection
of monoclonal antibody candidates for use as PTN ligand-binding
inhibitors or enhancers.
[0191] In addition, antibodies which are useful for altering the
function of PTN may be assayed in functional formats, such as the
HUVEC tube assay and the cell migration assay described below.
Thus, antibodies that exhibit the appropriate anti-PTN activity may
be selected without direct knowledge of a binding ligand or the
particular biomolecular interactions of PTN.
[0192] OPN
[0193] Given the experiments described above, and the results of
Table 1, OPN was selected as a target for selective
immuno-therapeutic agents in targeting and/or imaging brain tumors.
The mature protein consists of approximately 298 amino acids (after
cleavage of a 16 amino acid signal peptide) and contains two
potential Asn-Xaa-Ser N-glycosylation site, located at positions 65
and 92 of the mature protein. The complete cDNA sequence encoding
OPN is provided in SEQ ID NO. 23, and the complete amino acid
sequence of OPN is provided in SEQ ID NO. 24. OPN is an abundant 34
kDa, single chain, phosphorylated glycoprotein, with a presumed
site for cell attachment at residues 144-148. Three isoforms have
been identified to be generated by post transcriptional
modification, such as alternative splicing, OPN-A, OPN-B, and
OPN-C. OPN-A and OPN-B differ by the addition of 14 amino acids at
residue 58 of the protein. Amino acids 58-71 are absent in OPN-B,
and amino acids 31-57 are absent in OPN-C. OPN is a negatively
charged, highly hydrophilic secreted protein.
[0194] As used herein, a compound that specifically binds to OPN is
any compound (such as an antibody) that has a binding affinity for
any naturally occurring isoform, splice variant, or polymorphism of
OPN, explicitly including the three isoforms described herein. As
one of ordinary skill in the art will appreciate, such "specific"
binding compounds (e.g., antibodies) may also bind to other closely
related proteins that exhibit significant homology (such as greater
than 90% identity, more preferably greater than 95% identity, and
most preferably greater than 99% identity) with the amino acid
sequence of OPN. Such proteins include truncated forms or domains
of OPN, and recombinantly engineered alterations of OPN. For
example, a portion of SEQ ID NO. 24 may be engineered to include a
non-naturally occurring cysteine for cross linking to an
immunoconjugate protein, as described.
[0195] According to Young et al. the cDNA sequence of OPN isolated
from human bone cells (OPN-A) has an overall structure of
approximately 34 kDA that consist of 298 amino acids, which is 14
amino acids less than the cDNA sequence of OPN isolated from human
osteosarcoma by Keifer et al. (OPN-B). The cDNA transcript for
OPN-A is 1.5 kb with an open reading frame of 900 nucleotides, of
which the first 16 amino acids are hydrophobic in nature and
probably constitute a signal sequence for the secreted protein. The
OPN gene contains 7 exons that are alternatively spliced to
generate the variant isoforms, the most common variant being the
addition of a 42 bp (14 amino acid) sequence located at base 280 of
OPN-A. See Young et al. (1990). Genomics, 7:491-502 and Keifer et
al. Nucleic Acids Res. 17:3306.
[0196] When raising antibodies to OPN, the entire protein or a
portion thereof may be utilized. For instance, amino acid domains 4
to 12 (from the N-terminus) or 29 to 37 (from the N-terminus) may
be utilized. Larger OPN proteins and domains may be produced
utilizing any suitable recombinant vector/protein production
system, such as the baculovirus transfection system outlined below,
after being amplified from a fetal brain cDNA library (as available
from, e.g., Clontech, Palo Alto, Calif.) or another suitable
source. It is to be noted that antibodies which bind to this
secreted protein are useful in cytotoxic and imaging embodiments of
the invention, as one of ordinary skill would expect that the
concentration of OPN would be increased adjacent to tumor cells
which over-express the protein.
[0197] When utilizing an entire protein, or a larger section of the
protein, antibodies may be raised by immunizing the production
animal with the protein and a suitable adjuvant (e.g., Fruend's,
Fruend's complete, oil-in-water emulsions, etc.). In these cases,
the OPN protein (or a portion thereof) can serve as the OPN
antigen. When a smaller peptide is utilized, it is advantageous to
conjugate the peptide with a larger molecule to make an
immunostimulatory conjugate for use as the OPN antigen. Commonly
utilized conjugate proteins that are commercially available for
such use include bovine serum albumin (BSA) and keyhole limpet
hemocyanin (KLH). In order to raise antibodies to particular
epitopes, peptides derived from the full OPN sequence may be
utilized. Preferably, one or more 8-30 aa peptide portions of the
protein are utilized, with peptides in the range of 10-20 being a
more economical choice. Custom-synthesized peptides in this range
are available from a multitude of vendors, and can be order
conjugated to KLH or BSA. Alternatively, peptides in excess of 30
amino acids may be synthesized by solid-phase methods, or may be
recombinantly produced in a suitable recombinant protein production
system. In order to ensure proper protein glycosylation and
processing, an animal cell system (e.g., Sf9 or other insect cells,
CHO or other mammalian cells) is preferred. Other information
useful in designing an antigen for the production of antibodies to
OPN, including glycosylation sites, is provided in SEQ ID NO.
22.
[0198] The cell attachment sequence of human OPN (amino acids 144
to 148) is believed to interact with various cell surface proteins
(such as CD-44) to affect cell adhesion, and a highly acidic
stretch composed almost exclusively of aspartic acid residues
(amino acids 72 to 81) is believed to be the mineral binding site
within the protein. Because CD-44 is frequently over expressed on
primary brain tumors and metastases the binding of OPN to these
various cell-surface adhesion protein molecules may play a
significant role in the senescence and growth of tumor cells in the
brain. Thus, in alternative embodiments of the compositions and
methods of the invention, antibody moieties are utilized which bind
to OPN at a site on the protein that alters the binding of a cell
surface molecule, e.g., CD-44, to OPN. Such OPN activity altering
antibodies may be utilized in therapeutic compositions in an
unconjugated form (e.g., the antibody in an acceptable
pharmaceutical carrier), or may be conjugated to either a
therapeutic moiety (creating a double-acting therapeutic agent) or
an imaging moiety (creating a duel therapeutic/imaging agent).
[0199] Selection of antibodies which alter (enhance or inhibit) the
binding of a ligand to OPN may be accomplished by a straightforward
binding inhibition/enhancement assay. According to standard
techniques, the binding of a labeled (e.g., fluorescently or
enzyme-labeled) antibody to OPN, which has been immobilized in a
microtiter well, is assayed in both the presence and absence of the
appropriate ligand. The change in binding is indicative of either
an enhancer (increased binding) or competitive inhibitor (decreased
binding) relationship between the antibody and the ligand. Such
assays may be carried out in high-throughput formats (e.g., 384
well plate formats, in robotic systems) for the automated selection
of monoclonal antibody candidates for use as OPN ligand-binding
inhibitors or enhancers.
[0200] In addition, antibodies which are useful for altering the
function of OPN may be assayed in functional formats, such as the
HUVEC tube assay and the cell migration assay described below.
Thus, antibodies that exhibit the appropriate anti-OPN activity may
be selected without direct knowledge of a the biomolecular role of
OPN.
[0201] PTP.zeta.
[0202] PTP.zeta. was also selected as a prime target for selective
immuno-therapeutic agents in treating or imaging brain tumors. The
complete cDNA sequence encoding PTP.zeta. is provided in SEQ ID NO.
5, and the complete amino acid sequence of PTP.zeta. is provided in
SEQ ID NO. 6. Three different splice variants have been described,
which include two membrane bound variants (full length:
PTP.zeta.-.alpha., and shorter version PTP.zeta.-.beta.) and one
secreted form (Phosphacan). See FIG. 1. Isoform PTP.zeta.-.alpha.
is the full length isoform, which contains the primary amino acid
sequence aa 25-2314 of SEQ ID NO. 6 (aa 1-24 are a signal
polypeptide). This full length long form of PTP.zeta. is a type I
membrane protein. After the signal peptide it contains a carbonic
anhydrase like (CAH) and a fibronectin type III like (FN3) domain,
followed by a long cysteine free spacer (S) domain. This follows a
860 amino acid long insert domain, which can be glycosylated. After
a single transmembrane segment, in the intracellular region it has
2 phosphatase domains, but only the membrane-proximal PTPase domain
is catalytically active (Krueger 1992).
[0203] In Isoform PTP.zeta.-.beta., 755-1614 are missing. Isoform
PTP.zeta.-S (phosphacan), is a secreted isoform, which is comprises
the extracellular domains of PTP.zeta.-.alpha.. Northern Blot
analysis have shown that the PTP zeta is exclusively expressed in
the human central nervous system. In mouse embryos, the PTP.zeta.
transcript was mainly detected in the ventricular and
subventricular zone of the brain and the spinal cord. The same
pattern was detected in adult mice. Detailed studies have shown
that during rat embryogenesis the two transmembrane splice variants
of PTP.zeta. are mainly expressed in glial precursor cells and that
the secretory version (Phosphacan) is more abundant in mature
astrocytes which have already migrated in the ventricle zone.
Applicants have characterized two additional novel slice variants,
PTP.zeta. SM1 and PTP.zeta. SM2, which are described in detail
below.
[0204] As used herein, a compound which specifically binds to human
protein tyrosine phosphatase-zeta (PTP.zeta.) is any compound (such
as an antibody) which has a binding affinity for any naturally
occurring isoform, spice variant, or polymorphism of PTP.zeta.,
explicitly including the three splice variants describe herein. For
example, the compounds which specifically bind to novel isoforms
PTP.zeta. SM1 and PTP.zeta. SM2, described below, are subsets of
compounds which specifically bind to PTP.zeta.. As one of ordinary
skill in the art will appreciate, such "specific" binding compounds
(e.g., antibodies) may also bind to other closely related proteins
which exhibit significant homology (such as greater than 90%
identity, more preferably greater than 95% identity, and most
preferably greater than 99% identity) with the amino acid sequence
of PTP.zeta.. Such proteins include truncated forms or domains of
PTP.zeta., and recombinantly engineered alterations of PTP.zeta..
For example, an portion of SEQ ID NO. 6 may be engineered to
include a non-naturally occurring cysteine for cross-linking to an
immunoconjugate protein, as described below.
[0205] In general, it is preferred that the antibodies utilized in
the compositions and methods of the invention bind to the
membrane-bound isoforms of the protein, as this will more
specifically target the cytotoxic therapeutic agent, or the imaging
agent, to the brain tumor cell. However, embodiments which utilize
antibodies which bind to the secreted isoform of the protein are
also useful in the invention, as one of ordinary skill would expect
that the concentration of the secreted isoform would also be
increased adjacent to brain tumor cells which over-express the
protein.
[0206] The amino acid sequence of full length PTP.zeta. consists of
2307 amino acids, as the sequence was deduced by Levy (in which aa
1722-1728 of SEQ ID NO. 2 were missing) (See also U.S. Pat. Nos.
5,604,094, and 6,160,090, fully incorporated herein by reference),
or 2314 amino acids as the sequence was deduced by Krueger, et al.,
("A human transmembrane protein-tyrosine phosphatase, PTP zeta, is
expressed in brain and has an N-terminal receptor domain homologous
to carbonic anhydrases" Proc. Nat. Acad. Sci. U.S.A. 89:7417-7421
(1992)). Amino acids 1-24 of SEQ ID NO. 6 are a signal sequence
which directs the proper placement of the transmembrane protein.
The extracellular domain of the mature PTP.zeta. protein spans
amino acids 25-1635 of SEQ ID NO. 6 in the long and secreted forms
(this forms the entire secreted form), and amino acids
25-754,1615-1635 in the short isoform. The transmembrane region of
the protein spans amino acids 1636-1661 of SEQ ID NO. 6, and the
balance of the protein forms the cytoplasmic domain, amino acids
1662-2314.
[0207] When raising antibodies to PTP.zeta., the entire protein
(any of the three isoforms) or a portion thereof may be utilized.
For instance, the extracellular domain of the long or short form,
the entire secreted form, or a portion of extracellular domain may
be utilized. For instance, amino acids 25-754, which are common to
both .alpha. and .beta. isoforms, may be used. Such larger
PTP.zeta. proteins and domains may be produced utilizing any
suitable recombinant vector/protein production system, such as the
baculovirus transfection system outlined below, after being
amplified from a fetal brain cDNA library (as available from, e.g.,
Clontech, Palo Alto, Calif.) or another suitable source. When
utilizing an entire protein, or a larger section of the protein,
antibodies may be raised by immunizing the production animal with
the protein and a suitable adjuvant (e.g., Fruend's, Fruend's
complete, oil-in-water emulsions, etc.). In these cases, the
PTP.zeta. protein (or a portion thereof) can serve as the PTP.zeta.
antigen. When a smaller peptide is utilized, it is advantageous to
conjugate the peptide with a larger molecule to make an
immunostimulatory conjugate for use as the PTP.zeta. antigen.
Commonly utilized conjugate proteins which are commercially
available for such use include bovine serum albumin (BSA) and
keyhole limpet hemocyanin (KLH). In order to raise antibodies to
particular epitopes, peptides derived from the full PTP.zeta.
sequence may be utilized. Preferably, one or more 8-30 aa peptide
portions of an extracellular domain of PTP.zeta. are utilized, with
peptides in the range of 10-20 being a more economical choice.
Custom-synthesized peptides in this range are available from a
multitude of vendors, and can be order conjugated to KLH or BSA.
Alternatively, peptides in excess of 30 amino acids may be
synthesized by solid-phase methods, or may be recombinantly
produced in a suitable recombinant protein production system. In
order to ensure proper protein glycosylation and processing, an
animal cell system (e.g., Sf9 or other insect cells, CHO or other
mammalian cells) is preferred. Other information useful in
designing an antigen for the production of antibodies to PTP.zeta.,
including glycosylation sites, is provided in SEQ ID NO. 6.
[0208] The extracellular domain of human PTP.zeta. is known to bind
to tenascin-C, tenascin-R, pleiotrophin (NM.sub.--002825), midkine
(NM.sub.--002391), FGF-2 (XM.sub.--00366), Nr-CAM
1NM.sub.--005010), L1/Ng-CAM, contactin (NM.sub.--001843), N-CAM
(XM.sub.--006332), and axonin-1NM.sub.--005076.) The first 5 of
these molecules are either components of the extracellular matrix
in gliomas or are soluble factors known to be present in gliomas,
and the latter 4 are neuronal surface molecules. The binding of
PTP.zeta. to these molecules may play a significant role in the
oncogenesis and growth of neoplastic cells in the brain. Thus, in
alternative embodiments of the compositions and methods of the
invention, antibody moieties are utilized which bind to PTP.zeta.
at a site on the protein which alters the binding of an
extracellular ligand molecule to PTP.zeta.. Such PTP.zeta. activity
altering antibodies may be utilized in therapeutic compositions in
an unconjugated form (e.g., the antibody in an acceptable
pharmaceutical carrier), or may be conjugated to either a
therapeutic moiety (creating a double-acting therapeutic agent) or
an imaging moiety (creating a duel therapeutic/imaging agent).
[0209] Selection of antibodies which alter (enhance or inhibit) the
binding of a ligand to PTP.zeta. may be accomplished by a
straightforward binding inhibition/enhancement assay. According to
standard techniques, the binding of a labeled (e.g., fluorescently
or enzyme-labeled) antibody to PTP.zeta., which has been
immobilized in a microtiter well, is assayed in both the presence
and absence of the ligand. The change in binding is indicative of
either an enhancer (increased binding) or competitive inhibitor
(decreased binding) relationship between the antibody and the
ligand. Such assays may be carried out in high-throughput formats
(e.g., 384 well plate formats. in robotic systems) for the
automated selection of monoclonal antibody candidates for use as
PTP.zeta. ligand-binding inhibitors or enhancers.
[0210] In addition, antibodies which are useful for altering the
function of PTP.zeta. may be assayed in functional formats, such as
the HUVEC tube assay and the cell migration assay described below.
Thus, antibodies that exhibit the appropriate anti-PTP.zeta.
activity may be selected without direct knowledge of a the
biomolecular role of PTP.zeta..
[0211] Novel PTP.zeta. Splice Variants PTP.zeta. SM1 and PTP.zeta.
SM2
[0212] In addition to the known variants of PTP.zeta. for use in
the invention, applicants have identified two novel splice variant
isoforms of PTP.zeta., SM1 and SM2, from their clone libraries, see
FIG. 2. These novel isoforms, PTP.zeta. SM1 and PTP.zeta. SM2,
differ in structure from the three known isoforms heretofore
disclosed, as is illustrated in FIG. 3. As only cDNA sequences for
the known splice variants had been previously disclosed, rather
than the full gene sequence, applicants verified the location of
the novel sequences by comparison of the known splice variant
sequences and the novel sequences with a publicly available genomic
sequence database.
[0213] The protein PTP.zeta. SM1 (amino acid sequence SEQ ID NO. 2,
cDNA sequence SEQ ID NO. 1) comprises the amino acids encoded by
the first nine exons of PTP.zeta.-.alpha., with three unique
additional carboxy terminal amino acids, see FIG. 2. These are
encoded by additional 3' mRNA sequence (nucleotides 1262-1272 of
SEQ ID NO. 1) from the intron of the gene between exons nine and
ten. The PTP.zeta. SM1 clone was isolated from a human fetal brain
cDNA library, an has been shown to be expressed in several human
glioblastoma cell lines. Expression of the SM1 splice variant has
also been confirmed in primary brain tumor samples. The protein
comprises only extracellular domains of PTP.zeta., and is expected
to be secreted by the cell. Thus, PTP.zeta. SM1 may serve a cell
signaling or messenger function, and may have bind to a receptor on
the surface of cells which are associated with or part of central
nervous system tissues. Thus, antibodies specific for PTP.zeta.
SM1, and not specific for the other splicing isoforms of PTP.zeta.,
may be especially efficacious in the brain tumor therapeutic or
imaging compositions of the invention. The PTP.zeta. SM1 protein
mainly comprises the carbonic anhydrase-like domain which has been
identified in PTP.zeta. .alpha..
[0214] Applicants have explored the relationship between the
putative carbonic anhydrase domain of PTP.zeta. SM1 (SEQ ID NO. 2)
and other human carbonic anhydrase domains from carbonic anhydrase
III (SEQ ID NO. 25), carbonic anhydrase I (SEQ ID NO. 26), and
carbonic anhydrase VIX [e] (SEQ ID NO. 27), shown below:
2 1 50 cah3 human
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out..about..about..about..about..about..about..about..about..about..about.-
.about..about..about..about..about..about..about.AKEW GYASHNGPDH
cah1 human
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ut..about..about..about..about..about..about..about..about..about..about..-
about..about..about..about..about..about.ASPDW GYDDKNGPEQ cahe
human
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..about..about..about..about..about..about..about..about..about.ML
FSALLLEVIW ILAADGGQHW TYEGPHGQDH rptpzetaexon9_frame1 MRILKRFLAC
IQLLCVCRLD WANGYYRQQR KLVEEIG..W SYTGALNQKN 51 100 cah3_human
WHELFPNAKG ENQSPIELHT KDIRHD...P SLQPWSVSYD GGSAKTILNN cah1_human
WSKLYPIANG NNQSPVDIKT SETKHD...T SLKPISVSYN PATAKEIINV cahe_human
WPASYPECGN NAQSPIDIQT DSVTFDPDLP ALQPHGYDQP GTEPLDLHNN
rptpzetaexon9_frame1 WGKKYPTCNS PKQSPINIDE DLTQVNVNLK KLKFQGWDKT
SLENTFIHNT 101 150 cah3_human GKTCRVVFDD TYDRSMLRGG PLPGPYRLRQ
FHLHWGS.S. DDHGSEHTVD cah1_human GHSFHVNFED NDNRSVLKGG PFSDSYRLFQ
FHFHWGS.T. NEHGSEHTVD cahe_human GHTVQLSLP. ....STLYLG GLPRKYVAAQ
LHLHWGQ.KG SPGGSEHQIN rptpzetaexon9_frame1 GKTVEINLTN DYRVS...GG
VSEMVFKASK ITFHWGKCNM SSDGSEHSLE 151 200 cah3_human GVKYAAELHL
VHWN.PKYNT FKEALKQRDG IAVIGIFLKI GH.ENGEFQI cah1_human GVKYSAELHV
AHWNSAKYSS LAEAASKADG LAVIGVLMKV GE.ANPKLQK cahe_human SEATFAELHI
VHYDSDSYDS LSEAAERPQG LAVLGILIEV GETKNIAYEH rptpzetaexon9_frame1
GQKFPLEMQI YCFDADRFSS FEEAVKGKGK LRALSILFEV GTEENLDFKA 201 250
cah3_human FLDALDKIKT KGKEAPFTKF DPSCLFPACR .DYWTYQGSF TTPPCEECIV
cah1_human VLDALQAIKT KGKRAPFTNF DPSTLLPSSL .DFWTYPGSL THPPLYESVT
cahe_human ILSHLHEVRH KDQKTSVPPF NLRELLPKQL GQYFRYNGSL TTPPCYQSVL
rptpzetaexon9_frame1 IIDGVESVSR FGKQAALDPF ILLNLLPNST DKYYIYNGSL
TSPPCTDTVD 251 300 cah3_human WLLLKEPMTV SSDQMAKLRS LLSSAENEPP
VP...LVSNW RPPQPINNRV cah1_human WIICKESISV SSEQLAQFRS LLSNVEGDNA
VP...MQHNN RPTQPLKGRT cahe_human WTVFYRRSQI SMEQLEKLQG TLFSTEEEPS
KL...LVQNY RALQPLNQRM rptpzetaexon9_frame1 WIVFKDTVSI SESQLAVFCE
VLTMQQSGYV MLMDYLQNNF REQQYKFSRQ 301 350 cah3_human
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FAVLYQQLDG EDQTKHEFLT DGYQDLVTI*
[0215] Based on alignment with these catalytically active carbonic
anhydrases, it seems unlikely that the CA domain could function as
a carbonic anhydrase enzyme. Two of the three histidines implicated
in binding of the catalytic zinc are missing from the CA domain of
the receptor. In active enzymes there is a conserved
HxHWG{18,20}ELH motif (the three histidines bind zinc), however, in
the receptor this is modified to TFHWG {18,20}EMQ; i.e. two of the
three critical zinc atoms would be missing. For comparison, it has
been found that a carbonic anhydrase related protein (CAH 8) that
lacks just one of these histidines also lacks catalytic
activity.
[0216] The protein PTP.zeta. SM2 (amino acid sequence SEQ ID NO. 4)
comprises the amino acids encoded by all exons of
PTP.zeta.-.alpha., plus a 116 nucleotide "extra" exon, in the
correct reading frame, between exons 23 and 24 (nucleotides
6229-6345 of SEQ ID NO. 3). This extra exon, designated exon 23a,
contains a portion of the intron sequence between exons 23 and 24
of the PTP.zeta. gene. PTP.zeta. SM2 expression has been verified
in several human glioblastoma cell lines, and has also been
confirmed in primary brain tumor samples. As PTP.zeta. SM2
comprises all the domains of PTP.zeta. .alpha., the protein is
expected to be membrane-bound. The extra exon lies within the
cytoplasmic domain of the protein, and thus may alter the protein
tyrosine phosphatase function of PTP.zeta. SM2.
[0217] A novel splicing variant PTP.zeta. protein having an amino
acid sequence which includes the amino acid sequence of PTP.zeta.
SM1 (SEQ ID NO. 2) or PTP.zeta. SM2 (SEQ. ID NO. 4) may be produced
by recombinant techniques known in the art utilizing any suitable
vector, in any suitable host cell. The term "vector" is intended to
include any physical or biochemical vehicle containing nucleic acid
polymers of interest, by which those nucleic acid polymers are
transferred into a host cell, thereby transfecting that cell with
the introduced nucleic acid polymers. The transfected nucleic acid
sequence preferably contains a control sequence, such as a promoter
sequence, suitable for transcription of the nucleic acid sequence
in the host cell. Examples of vectors include DNA plasmids,
viruses, liposomes, particle gun pellets, and transfection vectors
known to those of skill in the molecular biology arts. The term
"host cell" is intended to mean the target cell for vector
transformation, in which the transferred nucleic acid polymer will
be replicated and/or expressed. Although bacterial cells may be
suitable for production of the proteins for antibody production or
structural study purposes, eukaryotic cell hosts are preferred for
production of the protein for functional assays or therapeutic
purposes. Preferred eukaryotic cell hosts include insect cell lines
(e.g., Sf9, Sf21, or High Five.TM. cell lines), and mammalian cell
lines (e.g., HeLa, CHO-K1, COS-7, COS-1, HEK293, HEPG2, Jurkat,
MDCK, PAE, PC-12, and other acceptable mammalian cell lines). Thus,
the invention also provides vectors incorporating a nucleic acid
sequence encoding PTP.zeta. SM1 or PTP.zeta. SM2, as well as host
cells which express the proteins.
[0218] It is common in the molecular biology arts to utilize
additional functional amino acid domains or proteins fused with a
protein sequence of interest for purification or detection
purposes. Such additional functionalities include, for example,
polyhistidine domains, c-myc domains (specifically comprising amino
acids 410-419 of the human c-myc oncogene product),
.beta.-galactosidase, .beta.-glucuronidase,
glutathione-S-transferase, maltose binding protein, human influenza
virus hemagglutanin, green fluorescent protein, chloramphenicol
acetyltransferase, luciferase, thioredoxin, and others. After
purification (e.g., by antibody-affinity chromatography) or
detection, these extra amino acid sequences may be cleaved (e.g.,
by thrombin, enterokinase, Factor Xa, or other protease) to yield a
functional mature protein. Thus, the PTP.zeta. SM1 and SM2 proteins
of the invention also encompass proteins comprising the amino acid
sequence of SEQ ID NO. 2 or SEQ ID NO. 4 and such additional amino
acid functionalities.
[0219] The invention also provides polypeptides which have a unique
activity of PTP.zeta. SM1 or PTP.zeta. SM2 which is not shared by
the other PTP.zeta. splice variant (e.g., an antigenic epitope) and
which include a portion of the amino acid sequence of PTP.zeta. SM1
or PTP.zeta. SM2 which is at least about 8 to 12 amino acid
residues in length, more preferably at least about 20 amino acids
in length. These polypeptides preferably comprise an amino acid
sequence which is not found in PTP.zeta. .alpha., PTP.zeta. .beta.,
or phosphacan, wherein the included portion of the sequence confers
the unique activity on the polypeptide. Such polypeptides may be
utilized as described above to produce affinity reagents which
specifically bind to PTP.zeta. splice variants SM1 or SM2, but do
not bind to the other known splice variants of PTP.zeta.. The
invention thus provides such specific affinity reagents, which may
be produces from such polypeptides, or from an entire PTP.zeta.
SM1or PTP.zeta. SM2 protein. In preferred embodiments these
affinity reagents are antibodies or antibody fragments.
[0220] In addition, although the understanding of the field of
protein biochemistry is not as complete as that of molecular
genetics, the person or ordinary skill in the art of biochemistry
is capable of predicting, with reasonable certainty, when certain
substitutions to the primary amino acid sequence structure of a
protein will not result in any appreciable modification of a
protein's structure or function. Such conservative substitutions
are made by replacing an amino acid in the sequence with another
containing a side chain with like charge, size, and other
characteristics. Conservative substitutions in a protein sequence
which would be expected to have minimal to no impact on protein
structure or function can be readily devised by a person of
ordinary skill in the biochemical arts. To the extent that such
conservative substitutions can be made while retaining 90%,
preferably 95%, and more preferably 99% or more identity to SEQ. ID
NO. 2 or SEQ ID NO. 4, and maintain the activity of the native
PTP.zeta. SM1 or PTP.zeta. SM2 protein, such altered proteins are
within the scope of the present invention.
[0221] The invention also provides nucleic acid polymers encoding
the PTP.zeta. splice variants SM1 or SM2. These nucleic acid
polymers most preferably comprises a nucleic acid sequence of SEQ.
ID NO. 1 or SEQ ID NO. 3, or the predictable variants thereof which
one of ordinary skill of the art could derive using the degeneracy
of the genetic code. Such nucleic acid polymers are useful for the
production of PTP.zeta. SM1 or PTP.zeta. SM2 by recombinant
methods, as described above.
[0222] The invention also encompasses nucleic acid probes or
primers which hybridize to the mRNA encoding PTP.zeta. splice
variants SM1 or SM2, but not mRNA encoding other known splice
variants of PTP.zeta.. Such probes or primers provided by the
invention are preferably able to hybridize with SEQ. ID NO. 1 or
SEQ. ID NO. 3 (or their complements) under stringent conditions
(e.g., 0.5.times. to 2.times.SSC buffer, 0.1% SDS, and a
temperature of 55-65.degree. C.), but do not hybridize to SEQ ID
NO. 5 (or its complement) under the same conditions. These
PTP.zeta. SM1 or PTP.zeta. SM2 coding sequence specific probes are
preferably from about 16 to about 40 nucleotides in length, more
preferably from about 18 nucleotides to about 30 nucleotides in
length. However, probes may be of a smaller size, preferably from
about 8 to about 15 nucleotides in length, if two ore more probes
are hybridized to adjacent sequences, so that terminal nucleic acid
base-stacking interactions may stabilize their hybridization. In
preferred embodiments of PTP.zeta. SM1 specific nucleic acid
probes, the probes hybridize at or near the novel splice site at
the 3' end of exon 9, or its complement. In preferred embodiments
of PTP.zeta. SM2 specific probes, the probes hybridize at or
adjacent to a location selected from: the novel splice site at the
3' end of exon 23, at least a portion of the novel exon 23a, the
novel splice site at the 5' end of exon 24, or the complement of
any one of these.
[0223] Because PTP.zeta. SM1 and PTP.zeta. SM2 have been shown to
be expressed in glioblastoma cell lines and primary tumors, the
level of the expression of these splice variants may be useful for
staging or characterizing glioblastoma cells. Such cells may be
extracted, for instance, from a primary tumor. Thus, the invention
provides for the monitoring of the relative expression level of
PTP.zeta. SM1 or PTP.zeta. SM2, or both, in relation to each other
or to one or more of the known PTP.zeta. splice variants. In one
preferred embodiment, the level of expression of PTP.zeta. SM1 is
compare to at least one other splice variant selected from
PTP.zeta. SM2, PTP.zeta. .alpha., PTP.zeta. .beta., and phosphacan.
In another preferred embodiment, the level of expression of
PTP.zeta. SM2 is compare to at least one other splice variant
selected from PTP.zeta. SM1, PTP.zeta. .alpha., PTP.zeta. .beta.,
and phosphacan. Such comparison may be made in either a qualitative
or quantitative manner. One means for comparison is by hybridizing
splice-variant specific nucleic acid probes to a sample of nucleic
acids (which may be amplified) obtained from brain tumor cells.
Alternatively, the expression level of the splice variants may be
deduced by the amplification of splice variant nucleic acid
sequences, and the analysis of the size of those amplified products
using methods known in the art. In another alternative embodiment,
protein levels may be studied utilizing splice-variant specific
antibodies in either sandwich immunoassay or in-situ staining
formats. Various expression level assay techniques are known to
those of skill in the molecular biological arts, and thus the
specific techniques mentioned above should be considered merely
exemplary.
[0224] Antibodies for Use in the Antibody-Therapeutics Methods of
the Invention
[0225] Generally, as the term is utilized in the specification,
"antibody" or "antibody moiety" is intended to include any
polypeptide chain-containing molecular structure that has a
specific shape which fits to and recognizes an epitope, where one
or more non-covalent binding interactions stabilize the complex
between the molecular structure and the epitope. Antibodies which
bind specifically to one of the brain tumor protein targets are
referred to as anti-brain tumor protein target antibodies, or
.alpha.(T.sub.BT), or more specifically .alpha.(ARP2),
.alpha.(SPARC), .alpha.(CMET), .alpha.(CD44), .alpha.(BEHAB),
.alpha.(TSPAN3), .alpha.(VIPR2), .alpha.(OPN), .alpha.(PTN), and
.alpha.(PTP.zeta.). The specific or selective fit of a given
structure and its specific epitope is sometimes referred to as a
"lock and key" fit. The archetypal antibody molecule is the
immunoglobulin, and all types of immunoglobulins (IgG, IgM, IgA,
IgE, IgD, etc.), from all sources (e.g., human, rodent, rabbit,
cow, sheep, pig, dog, other mammal, chicken, turkey, emu, other
avians, etc.) are considered to be "antibodies." Antibodies
utilized in the present invention may be polyclonal antibodies,
although monoclonal antibodies are preferred because they may be
reproduced by cell culture or recombinantly, and may be modified to
reduce their antigenicity.
[0226] Polyclonal antibodies may be raised by a standard protocol
by injecting a production animal with an antigenic composition,
formulated as described above. See, e.g., Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988. In one such technique, an T.sub.BT antigen comprising an
antigenic portion of the brain tumor protein targets' polypeptide
is initially injected into any of a wide variety of mammals (e.g.,
mice, rats, rabbits, sheep or goats). Alternatively, in order to
generate antibodies to relatively short peptide portions of the
brain tumor protein target (see discussion above), a superior
immune response may be elicited if the polypeptide is joined to a
carrier protein, such as ovalbumin, BSA or KLH. The
peptide-conjugate is injected into the animal host, preferably
according to a predetermined schedule incorporating one or more
booster immunizations, and the animals are bled periodically.
Polyclonal antibodies specific for the polypeptide may then be
purified from such antisera by, for example, affinity
chromatography using the polypeptide coupled to a suitable solid
support.
[0227] Alternatively, for monoclonal antibodies, hybridomas may be
formed by isolating the stimulated immune cells, such as those from
the spleen of the inoculated animal. These cells are then fused to
immortalized cells, such as myeloma cells or transformed cells,
which are capable of replicating indefinitely in cell culture,
thereby producing an immortal, immunoglobulin-secreting cell line.
The immortal cell line utilized is preferably selected to be
deficient in enzymes necessary for the utilization of certain
nutrients. Many such cell lines (such as myelomas) are known to
those skilled in the art, and include, for example: thymidine
kinase (TK) or hypoxanthine-guanine phosphoriboxyl transferase
(HGPRT). These deficiencies allow selection for fused cells
according to their ability to grow on, for example, hypoxanthine
aminopterinthymidine medium (HAT).
[0228] Preferably, the immortal fusion partners utilized are
derived from a line that does not secrete immunoglobulin. The
resulting fused cells, or hybridomas, are cultured under conditions
that allow for the survival of fused, but not unfused, cells and
the resulting colonies screened for the production of the desired
monoclonal antibodies. Colonies producing such antibodies are
cloned, expanded, and grown so as to produce large quantities of
antibody, see Kohler and Milstein, 1975 Nature 256:495 (the
disclosures of which are hereby incorporated by reference).
[0229] Large quantities of monoclonal antibodies from the secreting
hybridomas may then be produced by injecting the clones into the
peritoneal cavity of mice and harvesting the ascites fluid
therefrom. The mice, preferably primed with pristine, or some other
tumor-promoter, and immunosuppressed chemically or by irradiation,
may be any of various suitable strains known to those in the art.
The ascites fluid is harvested from the mice and the monoclonal
antibody purified therefrom, for example, by CM Sepharose column or
other chromatographic means. Alternatively, the hybridomas may be
cultured in vitro or as suspension cultures. Batch, continuous
culture, or other suitable culture processes may be utilized.
Monoclonal antibodies are then recovered from the culture medium or
supernatant.
[0230] Several monoclonal antibodies against various isoforms of
the brain tumor protein targets are currently available from
commercial sources. For instance, a non-exclusive list of available
commercial antibodies includes: for SPARC/Osteonectin, from Zymed,
mouse anti-bovine MAb (cross-reactivity with human), suitable for
ELISA, WB, IH (paraffin), Cat# 33-5500; for c-MET, from Zymed,
rabbit anti-human polyclonal, suitable for ELISA, WB, IH. Cat#
71-8000, and from RDI, rabbit anti-human MAb, suitable for WB, IP,
IH. Cat# RDI-MET Cabr.; for CD44, from RDI, mouse anti-human MAb,
only for IH and FACS, Cat# RD1-M1676clb., and from Lab vision,
mouse anti-human MAb, known to block binding of hyaluronic acid to
its receptor CD44, "CD44/H-CAM Ab-2"; for Brevican/BEHAB, from BD
Transduction Lab., a mouse anti-human MAb, WB, IF, Cat# B68820; for
VIP 2 receptor, from Exalpha, mouse anti-rat (possible human
cross-specificity, which is easily assayed) MAb, WB, IH. Cat#2140M;
for Laminin receptor 67 kDa, from Lab vision, mouse anti-human MAb,
IH, ELISA, not for WB. "laminin receptor Ab-1"; for Osteopontin,
from Chemicon, rat anti-human MAb, raised against
rh-Osteopontin--recognizes native protein well, WB, IH, ELISA.
"MAB3057"; for Pleiotrophin, from R&D goat anti-human
polyclonal, WB, recognizes rh-Pleiotrophin. "BAF252", and from
Oncogene goat anti-human polyclonal, WB, ELISA, detects
rh-Pleiotrophin. "PC87L".; for PTP.zeta.-.alpha. and
PTP.zeta.-.beta., from BD Transduction Labs, mouse anti-human MAb
(WB, IH, IF), denominated "R20720" and from Chemicon, mouse
anti-human MAb (WB, IH, IP), denominated "MAB5210", which
recognizes both of the transmembrane isoforms, and also recognizes
the soluble isoform (phosphacan, PTP.zeta.-S). These antibodies are
suitable for use in the compositions of the present invention,
especially in Fab fragment form (which eliminates significant
portions of the antigenic mouse constant heavy and light chain
regions). However, it is preferred that such antibodies be
humanized or chimerized according to one of the procedures outlined
below.
[0231] In addition, the antibodies or antigen binding fragments may
be produced by genetic engineering. In this technique, as with the
standard hybridoma procedure, antibody-producing cells are
sensitized to the desired antigen or immunogen. The messenger RNA
isolated from the immune spleen cells or hybridomas is used as a
template to make cDNA using PCR amplification. A library of
vectors, each containing one heavy chain gene and one light chain
gene retaining the initial antigen specificity, is produced by
insertion of appropriate sections of the amplified immunoglobulin
cDNA into the expression vectors. A combinatorial library is
constructed by combining the heavy chain gene library with the
light chain gene library. This results in a library of clones which
co-express a heavy and light chain (resembling the Fab fragment or
antigen binding fragment of an antibody molecule). The vectors that
carry these genes are co-transfected into a host (e.g. bacteria,
insect cells, mammalian cells, or other suitable protein production
host cell.). When antibody gene synthesis is induced in the
transfected host, the heavy and light chain proteins self-assemble
to produce active antibodies that can be detected by screening with
the antigen or immunogen.
[0232] Preferably, recombinant antibodies are produced in a
recombinant protein production system which correctly glycosylates
and processes the immunoglobulin chains, such as insect or
mammalian cells. An advantage to using insect cells which utilize
recombinant baculoviruses for the production of antibodies for use
in the present invention is that the baculovirus system allows
production of mutant antibodies much more rapidly than stably
transfected mammalian cell lines. In addition, insect cells have
been shown to correctly process and glycosylate eukaryotic
proteins, which prokaryotic cells do not. Finally, the baculovirus
expression of foreign protein has been shown to constitute as much
as 50-75% of the total cellular protein late in viral infection,
making this system an excellent means of producing milligram
quantities of the recombinant antibodies.
[0233] The use of the baculovirus Autographia californica nuclear
polyhedrosis virus (AcNPV) and recombinant viral stocks in
Spodoptera frugiperda (Sf9) cells to prepare large quantities of
protein has been described by Smith et al. (1985), Summers and
Smith (1987). A preferred method of preparing recombinant
antibodies is through the expression of DNA encoding recombinant
antibody (produced by screening, as above, or by protein
engineering to include more human-like domains, as discussed below)
via the baculoviral expression system in Sf9 insect cells.
Production of recombinant proteins in Sf9 cells is well known in
the art, and one of ordinary skill would be able to select from a
number of acceptable protocols (e.g., that described in U.S. Pat.
No. 6,603,905).
[0234] It should be noted that antibodies which have a reduced
propensity to induce a violent or detrimental immune response in
humans (such as anaphylactic shock), and which also exhibit a
reduced propensity for priming an immune response which would
prevent repeated dosage with the antibody therapeutic or imaging
agent (e.g., the human-anti-murine-antibo- dy "HAMA" response), are
preferred for use in the invention. These antibodies are preferred
for all administrative routes, including intrathecal
administration. Even through the brain is relatively isolated in
the cranial cavity, behind the blood brain barrier, an immune
response still can occur in the form of increased leukocyte
infiltration, and inflammation. Although some increased immune
response against the tumor is desirable, the concurrent binding and
inactivation of the therapeutic or imaging agent generally
outweighs this benefit. Thus, humanized, chimeric, or xenogenic
human antibodies, which produce less of an immune response when
administered to humans, are preferred for use in the present
invention.
[0235] Chimeric antibodies may be made by recombinant means by
combining the murine variable light and heavy chain regions (VK and
VH), obtained from a murine (or other animal-derived) hybridoma
clone, with the human constant light and heavy chain regions, in
order to produce an antibody with predominantly human domains. The
production of such chimeric antibodies is well known in the art,
and may be achieved by standard means (as described, e.g., in U.S.
Pat. No. 5,624,659, incorporated fully herein by reference).
Humanized antibodies are engineered to contain even more human-like
immunoglobulin domains, and incorporate only the
complementarity-determining regions of the animal-derived antibody.
This is accomplished by carefully examining the sequence of the
hyper-variable loops of the variable regions of the monoclonal
antibody, and fitting them to the structure of the human antibody
chains. Although facially complex, the process is straightforward
in practice. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully
herein by reference.
[0236] Alternatively, polyclonal or monoclonal antibodies may be
produced from animals which have been genetically altered to
produce human immunoglobulins, such as the Abgenix XenoMouse or the
Medarex HuMAb.RTM. technology. The transgenic animal may be
produced by initially producing a "knock-out" animal which does not
produce the animal's natural antibodies, and stably transforming
the animal with a human antibody locus (e.g., by the use of a human
artificial chromosome). Only human antibodies are then made by the
animal. Techniques for generating such animals, and deriving
antibodies therefrom, are described in U.S. Pat. Nos. 6,162,963 and
6,150,584, incorporated fully herein by reference. Such fully human
xenogenic antibodies are a preferred antibody for use in the
methods and compositions of the present invention.
[0237] Alternatively, single chain antibodies (Fv, as described
below) can be produced from phage libraries containing human
variable regions. See U.S. Pat. No. 6,174,708, incorporated fully
herein by reference. Also see Kuan, C. T., Reist, C. J., Foulon, C.
F., Lorimer, I. A., Archer, G., Pegram, C. N., Pastan, I.,
Zalutsky, M. R., and Bigner, D. D. (1999). 125I-labeled
anti-epidermal growth factor receptor-viii single-chain Fv exhibits
specific and high-level targeting of glioma xenografts. Clin Cancer
Res. 5, 1539-49;Lorimer, I. A., Keppler-Hafkemeyer, A., Beers, R.
A., Pegram, C. N., Bigner, D. D., and Pastan, I. (1996).
Recombinant immunotoxins specific for a mutant epidermal growth
factor receptor: targeting with a single chain antibody variable
domain isolated by phage display. Proc. Nat. Acad. Sci. USA 93,
14815-20; Pastan, I. H., Archer, G. E., McLendon, R. E., Friedman,
H. S., Fuchs, H. E., Wang, Q. C., Pai, L. H., Herndon, J., and
Bigner, D. D. (1995). Intrathecal administration of single-chain
immunotoxin, LMB-7 [B3(Fv)-PE38], produces cures of carcinomatous
meningitis in a rat model. Proc Natl. Acad. Sci USA 92, 2765-9, all
of which are incorporated by reference fully herein.
[0238] In addition to entire immunoglobulins (or their recombinant
counterparts), immunoglobulin fragments comprising the epitope
binding site (e.g., Fab', F(ab').sub.2, or other fragments) are
useful as antibody moieties in the present invention. Such antibody
fragments may be generated from whole immunoglobulins by ficin,
pepsin, papain, or other protease cleavage. "Fragment," or minimal
immunoglobulins may be designed utilizing recombinant
immunoglobulin techniques. For instance "Fv" immunoglobulins for
use in the present invention may be produced by linking a variable
light chain region to a variable heavy chain region via a peptide
linker (e.g., poly-glycine or another sequence which does not form
an alpha helix or beta sheet motif).
[0239] Fv fragments are heterodimers of the variable heavy chain
domain (V.sub.H) and the variable light chain domain (V.sub.L). The
heterodimers of heavy and light chain domains that occur in whole
IgG, for example, are connected by a disulfide bond. Recombinant
Fvs in which V.sub.H and V.sub.L are connected by a peptide linker
are typically stable, see, for example, Huston et al., Proc. Natl.
Acad, Sci. USA 85:5879-5883 (1988) and Bird et al., Science
242:423-426 (1988), both fully incorporated herein, by reference.
These are single chain Fvs which have been found to retain
specificity and affinity and have been shown to be useful for
imaging tumors and to make recombinant immunotoxins for tumor
therapy. However, researchers have bound that some of the single
chain Fvs have a reduced affinity for antigen and the peptide
linker can interfere with binding. Improved Fv's have been also
been made which comprise stabilizing disulfide bonds between the
V.sub.H and V.sub.L regions, as described in U.S. Pat. No.
6,147,203, incorporated fully herein by reference. Any of these
minimal antibodies may be utilized in the present invention, and
those which are humanized to avoid HAMA reactions are preferred for
use in embodiments of the invention.
[0240] In addition, derivatized immunoglobulins with added chemical
linkers, detectable moieties [fluorescent dyes, enzymes,
substrates, chemiluminescent moieties], or specific binding
moieties [such as streptavidin, avidin, or biotin] may be utilized
in the methods and compositions of the present invention. For
convenience, the term "antibody" or "antibody moiety" will be used
throughout to generally refer to molecules which specifically bind
to an epitope of the brain tumor protein targets, although the term
will encompass all immunoglobulins, derivatives, fragments,
recombinant or engineered immunoglobulins, and modified
immunoglobulins, as described above.
[0241] Candidate anti-T.sub.BT antibodies can be tested for
anti-T.sub.BT activity by any suitable standard means. As a first
screen, the antibodies may be tested for binding against the brain
tumor protein target antigen utilized to produce them, or against
the entire brain tumor protein target extracellular domain or
protein. As a second screen, anti-T.sub.BT candidates may be tested
for binding to an appropriate glioblastoma cell line (i.e., one
which approximates primary tumor brain tumor protein target
expression), or to primary tumor tissue samples. For these screens,
the anti-T.sub.BT candidate antibody may be labeled for detection
(e.g., with fluorescein or another fluorescent moiety, or with an
enzyme such as horseradish peroxidase). After selective binding to
the brain tumor protein target is established, the candidate
antibody, or an antibody conjugate produced as described below, may
be tested for appropriate activity (i.e., the ability to decrease
tumor cell growth and/or to aid in visualizing tumor cells) in an
in vivo model, such as an appropriate glioblastoma cell line, or in
a mouse or rat human brain tumor model, as described below.
[0242] General Functional Assay Methods for Antibodies for Use in
the Invention
[0243] In addition to the specific binding assays and
protein-specific functional assays described for individual
proteins above, antibodies which are useful for altering the
function of ARP-2, SPARC, c-MET, BEHAB, CD-44, TSPN3, PTN, OPN,
VIPR-2, or PTP.zeta. may be assayed in functional formats, such as
glioblastoma cell culture or mouse/rat CNS tumor model studies. In
glioblastoma cell models of activity, expression of the protein is
first verified in the particular cell strain to be used. If
necessary, the cell line may be stably transfected with a coding
sequence of the protein under the control of an appropriate
constituent promoter, in order to express the protein at a level
comparable to that found in primary tumors. The ability of the
glioblastoma cells to survive in the presence of the candidate
function-altering anti-protein antibody is then determined. In
addition to cell-survival assays, cell migration assays, as
described below in Example 1, may be utilized to determine the
effect of the candidate antibody therapeutic agent on the
tumor-like behavior of the cells. Alternatively, if the brain tumor
protein target is involved in angiogenesis, or endothelial cell
sprouting assays such as described in Example 2 may be utilized to
determine the ability of the candidate antibody therapeutic to
inhibit vascular neogenesis, an important function in tumor
biology.
[0244] Similarly, in vivo models for human brain tumors,
particularly nude mice/SCID mice model or rat models, have been
described [Antunes, L., Angioi-Duprez, K. S., Bracard, S. R.,
Klein-Monhoven, N. A., Le Faou, A. E., Duprez, A. M., and Plenat,
F. M. (2000). Analysis of tissue chimerism in nude mouse brain and
abdominal xenograft models of human glioblastoma multiforme: what
does it tell us about the models and about glioblastoma biology and
therapy? J Histochem Cytochem 48, 847-58; Price, A., Shi, Q.,
Morris, D., Wilcox, M. E., Brasher, P. M., Rewcastle, N. B.,
Shalinsky, D., Zou, H., Appelt, K., Johnston, R. N., Yong, V. W.,
Edwards, D., and Forsyth, P. (1999). Marked inhibition of tumor
growth in a malignant glioma tumor model by a novel synthetic
matrix metalloproteinase inhibitor AG3340. Clin Cancer Res 5,
845-54; and Senner, V., Sturm, A., Hoess, N., Wassmann, H., and
Paulus, W. (2000). In vivo glioma model enabling regulated gene
expression. Acta Neuropathol (Berl) 99, 603-8.] Once correct
expression of the protein in the tumor model is verified, the
effect of the candidate anti-protein antibodies on the tumor masses
in these models can be evaluated, wherein the ability of the
anti-protein antibody candidates to alter protein activity is
indicated by a decrease in tumor growth or a reduction in the tumor
mass. Thus, antibodies that exhibit the appropriate anti-tumor
effect may be selected without direct knowledge of the particular
biomolecular role of the protein in oncogenesis.
[0245] Therapeutic and Imaging Moieties and Methods for Conjugating
Them Qith anti-PTP.zeta. Antibodies to Use in the Compositions and
Methods of the Invention
[0246] As described above, the anti-T.sub.BT antibodies for use in
the present invention may have utility without conjugation when the
native activity of the brain tumor protein target is altered in the
tumor cell. Such antibodies, which may be selected as described
above, may be utilized without further modification to include a
cytotoxic or imaging moiety. These types of compositions have the
advantage of reduced toxicity (in that only the toxicity of the
antibody moieties themselves must be taken into account when
dosing), and are simpler to manufacture. Thus, non-conjugated
activity altering anti-T.sub.BT antibody therapeutics are a
preferred embodiment of the invention. However, the conjugation of
cytotoxic or imaging agents is yet another preferred embodiment
when utilizing these antibodies because the added moieties add
functionality to the therapeutic.
[0247] Thus, in many preferred embodiments of the invention, the
anti-T.sub.BT antibodies may be coupled or conjugated to one or
more therapeutic cytotoxic or imaging moieties. As used herein,
"cytotoxic moiety" (C) simply means a moiety which inhibits cell
growth or promotes cell death when proximate to or absorbed by the
cell. Suitable cytotoxic moieties in this regard include
radioactive isotopes (radionuclides), chemotoxic agents such as
differentiation inducers and small chemotoxic drugs, toxin
proteins, and derivatives thereof. As utilized herein, "imaging
moiety" (I) means a moiety which can be utilized to increase
contrast between a tumor and the surrounding healthy tissue in a
visualization technique (e.g., radiography, positron-emission
tomography, magnetic resonance imaging, direct or indirect visual
inspection). Thus, suitable imaging moieties include radiography
moieties (e.g. heavy metals and radiation emitting moieties),
positron emitting moieties, magnetic resonance contrast moieties,
and optically visible moieties (e.g., fluorescent or
visible-spectrum dyes, visible particles, etc.). It will be
appreciated by one of ordinary skill that some overlap exists
between what is a therapeutic moiety and what is an imaging moiety.
For instance .sup.212Pb and .sup.212Bi are both useful
radioisotopes for therapeutic compositions, but are also
electron-dense, and thus provide contrast for X-ray radiographic
imaging techniques, and can also be utilized in scintillation
imaging techniques.
[0248] In general, therapeutic or imaging agents may be conjugated
to the anti-PTP.zeta. moiety by any suitable technique, with
appropriate consideration of the need for pharmokinetic stability
and reduced overall toxicity to the patient. A therapeutic agent
may be coupled to a suitable antibody moiety either directly or
indirectly (e.g. via a linker group). A direct reaction between an
agent and an antibody is possible when each possesses a functional
group capable of reacting with the other. For example, a
nucleophilic group, such as an amino or sulfhydryl group, may be
capable of reacting with a carbonyl-containing group, such as an
anhydride or an acid halide, or with an alkyl group containing a
good leaving group (e.g., a halide). Alternatively, a suitable
chemical linker group may be used. A linker group can function as a
spacer to distance an antibody from an agent in order to avoid
interference with binding capabilities. A linker group can also
serve to increase the chemical reactivity of a substituent on a
moiety or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
moieties, or functional groups on moieties, which otherwise would
not be possible.
[0249] Suitable linkage chemistries include maleimidyl linkers and
alkyl halide linkers (which react with a sulfhydryl on the antibody
moiety) and succinimidyl linkers (which react with a primary amine
on the antibody moiety). Several primary amine and sulfhydryl
groups are present on immunoglobulins, and additional groups may be
designed into recombinant immunoglobulin molecules. It will be
evident to those skilled in the art that a variety of bifunctional
or polyfunctional reagents, both homo- and hetero-functional (such
as those described in the catalog of the Pierce Chemical Co.,
Rockford, Ill.), may be employed as a linker group. Coupling may be
effected, for example, through amino groups, carboxyl groups,
sulfhydryl groups or oxidized carbohydrate residues. There are
numerous references describing such methodology, e.g., U.S. Pat.
No. 4,671,958. As an alternative coupling method, cytotoxic or
imaging moieties may be coupled to the anti-T.sub.BT antibody
moiety through a an oxidized carbohydrate group at a glycosylation
site, as described in U.S. Pat. Nos. 5,057,313 and 5,156,840. Yet
another alternative method of coupling the antibody moiety to the
cytotoxic or imaging moiety is by the use of a non-covalent binding
pair, such as streptavidin/biotin, or avidin/biotin. In these
embodiments, one member of the pair is covalently coupled to the
antibody moiety and the other member of the binding pair is
covalently coupled to the cytotoxic or imaging moiety.
[0250] Where a cytotoxic moiety is more potent when free from the
antibody portion of the immunoconjugates of the present invention,
it may be desirable to use a linker group which is cleavable during
or upon internalization into a cell, or which is gradually
cleavable over time in the extracellular environment. A number of
different cleavable linker groups have been described. The
mechanisms for the intracellular release of a cytotoxic moiety
agent from these linker groups include cleavage by reduction of a
disulfide bond (e.g., U.S. Pat. No. 4,489,710), by irradiation of a
photolabile bond (e.g., U.S. Pat. No. 4,625,014), by hydrolysis of
derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045),
by serum complement-mediated hydrolysis (e.g., U.S. Pat. No.
4,671,958), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.
4,569,789).
[0251] It may be desirable to couple more than one cytotoxic and/or
imaging moiety to an antibody. By poly-derivatizing the
anti-T.sub.BT antibody, several cytotoxic strategies may be
simultaneously implemented, an antibody may be made useful as a
contrasting agent for several visualization techniques, or a
therapeutic antibody may be labeled for tracking by a visualization
technique. In one embodiment, multiple molecules of an imaging or
cytotoxic moiety are coupled to one antibody molecule. In another
embodiment, more than one type of moiety may be coupled to one
antibody. Regardless of the particular embodiment, immunoconjugates
with more than one moiety may be prepared in a variety of ways. For
example, more than one moiety may be coupled directly to an
antibody molecule, or linkers which provide multiple sites for
attachment (e.g., dendrimers) can be used. Alternatively, a carrier
with the capacity to hold more than one cytotoxic or imaging moiety
can be used.
[0252] A carrier may bear the agents in a variety of ways,
including covalent bonding either directly or via a linker group,
and non-covalent associations. Suitable covalent-bond carriers
include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234),
peptides, and polysaccharides such as aminodextran (e.g., U.S. Pat.
No. 4,699,784), each of which have multiple sites for the
attachment of moieties. A carrier may also bear an agent by
non-covalent associations, such as non-covalent bonding or by
encapsulation, such as within a liposome vesicle (e.g., U.S. Pat.
Nos. 4,429,008 and 4,873,088). Encapsulation carriers are
especially useful for imaging moiety conjugation to anti-T.sub.BT
antibody moieties for use in the invention, as a sufficient amount
of the imaging moiety (dye, magnetic resonance contrast reagent,
etc.) for detection may be more easily associated with the antibody
moiety. In addition, encapsulation carriers are also useful in
chemotoxic therapeutic embodiments, as they can allow the
therapeutic compositions to gradually release a chemotoxic moiety
over time while concentrating it in the vicinity of the tumor
cells.
[0253] Carriers and linkers specific for radionuclide agents (both
for use as cytotoxic moieties or positron-emission imaging
moieties) include radiohalogenated small molecules and chelating
compounds. For example, U.S. Pat. No. 4,735,792 discloses
representative radiohalogenated small molecules and their
synthesis. A radionuclide chelate may be formed from chelating
compounds that include those containing nitrogen and sulfur atoms
as the donor atoms for binding the metal, or metal oxide,
radionuclide. For example, U.S. Pat. No. 4,673,562, to Davison et
al. discloses representative chelating compounds and their
synthesis. Such chelation carriers are also useful for magnetic
spin contrast ions for use in magnetic resonance imaging tumor
visualization methods, and for the chelation of heavy metal ions
for use in radiographic visualization methods.
[0254] Preferred radionuclides for use as cytotoxic moieties are
radionuclides which are suitable for pharmacological
administration. Such radionuclides include .sup.123I, .sup.125I,
.sup.131I, .sup.90Y, .sup.211At, .sup.67Cu, .sup.186Re, .sup.188Re,
.sup.212Pb, and .sup.212Bi. Iodine and astatine isotopes are more
preferred radionuclides for use in the therapeutic compositions of
the present invention, as a large body of literature has been
accumulated regarding their use. .sup.131I is particularly
preferred, as are other .beta.-radiation emitting nuclides, which
have an effective range of several millimeters. .sup.123I,
.sup.125I, .sup.131I, or .sup.211At may be conjugated to antibody
moieties for use in the compositions and methods utilizing any of
several known conjugation reagents, including Iodogen,
N-succinimidyl 3-[.sup.211At]astatobenzoate, N-succinimidyl
3-[.sup.133I]iodobenzoate (SIB), and, N-succinimidyl
5-[.sup.131I]iodob-3-pyridinecarboxylate (SIPC). Any iodine isotope
may be utilized in the recited iodo-reagents. For example, a
suitable antibody for use in the present invention may be easily
made by coupling an Fab fragment of the BD Transduction Labs R20720
anti-PTP.zeta. MAb with .sup.131I Iodogen according to the
manufacturer's instructions. Other radionuclides may be conjugated
to anti-T.sub.BTantibody moieties by suitable chelation agents
known to those of skill in the nuclear medicine arts.
[0255] Preferred chemotoxic agents include small-molecule drugs
such as carboplatin, cisplatin, vincristine, taxanes such as
paclitaxel and doceltaxel, hydroxyurea, gemcitabine, vinorelbine,
irinotecan, tirapazamine, matrilysin, methotrexate, pyrimidine and
purine analogs, and other suitable small toxins known in the art.
Preferred chemotoxin differentiation inducers include phorbol
esters and butyric acid. Chemotoxic moieties may be directly
conjugated to the anti-T.sub.BTantibody moiety via a chemical
linker, or may encapsulated in a carrier, which is in turn coupled
to the anti-T.sub.BT antibody moiety.
[0256] Preferred toxin proteins for use as cytotoxic moieties
include ricins A and B, abrin, diphtheria toxin, bryodin 1 and 2,
momordin, trichokirin, cholera toxin, gelonin, Pseudomonas
exotoxin, Shigella toxin, pokeweed antiviral protein, and other
toxin proteins known in the medicinal biochemistry arts. As these
toxin agents may elicit undesirable immune responses in the
patient, especially if injected intravascularly, it is preferred
that they be encapsulated in a carrier for coupling to the
anti-T.sub.BT antibody moiety.
[0257] Preferred radiographic moieties for use as imaging moieties
in the present invention include compounds and chelates with
relatively large atoms, such as gold, iridium, technetium, barium,
thallium, iodine, and their isotopes. It is preferred that less
toxic radiographic imaging moieties, such as iodine or iodine
isotopes, be utilized in the compositions and methods of the
invention. Examples of such compositions which may be utilized for
x-ray radiography are described in U.S. Pat. No. 5,709,846,
incorporated fully herein by reference. Such moieties may be
conjugated to the anti-T.sub.BT antibody moiety through an
acceptable chemical linker or chelation carrier. In addition,
radionuclides which emit radiation capable of penetrating the scull
may be useful for scintillation imaging techniques. Suitable
radionuclides for conjugation include .sup.99Tc, .sup.111In, and
.sup.67Ga. Positron emitting moieties for use in the present
invention include .sup.18F, which can be easily conjugated by a
fluorination reaction with the anti-T.sub.BT antibody moiety
according to the method described in U.S. Pat. No. 6,187,284.
[0258] Preferred magnetic resonance contrast moieties include
chelates of chromium(III), manganese(II), iron(II), nickel(II),
copper(II), praseodymium(III), neodymium(III), samarium(III) and
ytterbium(III) ion. Because of their very strong magnetic moment,
the gadolinium(III), terbium(III), dysprosium(III), holmium(III),
erbium(III), and iron(III) ions are especially preferred. Examples
of such chelates, suitable for magnetic resonance spin imaging, are
described in U.S. Pat. No. 5,733,522, incorporated fully herein by
reference. Nuclear spin contrast chelates may be conjugated to the
anti-T.sub.BT antibody moieties through a suitable chemical
linker.
[0259] Optically visible moieties for use as imaging moieties
include fluorescent dyes, or visible-spectrum dyes, visible
particles, and other visible labeling moieties. Fluorescent dyes
such as fluorescein, coumarin, rhodamine, bodipy Texas red, and
cyanine dyes, are useful when sufficient excitation energy can be
provided to the site to be inspected visually. Endoscopic
visualization procedures may be more compatible with the use of
such labels. For many procedures where imaging agents are useful,
such as during an operation to resect a brain tumor, visible
spectrum dyes are preferred. Acceptable dyes include FDA-approved
food dyes and colors, which are non-toxic, although
pharmaceutically acceptable dyes which have been approved for
internal administration are preferred. In preferred embodiments,
such dyes are encapsulated in carrier moieties, which are in turn
conjugated to the anti-T.sub.BT antibody. Alternatively, visible
particles, such as colloidal gold particles or latex particles, may
be coupled to the anti-T.sub.BT antibody moiety via a suitable
chemical linker.
[0260] Delivery of Therapeutic and Imaging Agents to the
Patient:
[0261] The Blood Brain Barrier (BBB) and Administration
Strategies:
[0262] At one time, the BBB was not considered to present a problem
in the diagnosis and treatment of brain tumors, because early scans
of human brain tumors suggested that the BTB (blood tumor barrier)
was "leaky." However, as the size of the molecule increases, the
rate of movement across the barrier decreases. The BBB has been
demonstrated to be heterogeneous in experimental human tumor
xenograft animal models and in human patients. This lack of
uniformity is because of the reduced integrity of tight junctions
in the capillary endothelial cells of the tumor neovasculature,
intratumoral variation in permeability, and altered intratumoral
blood flow (Fuchs et al, 1990, Cancer research 50, 1954-59,
Groothuis et al., 1984, Prog. Exp. Tumor Res.) Thus, although the
BBB may not pose a delivery problem for some tumors in some
patients, this cannot be said for all brain tumors. In addition, a
preferred mode of administration of the therapeutics of the
invention is after removal of the main tumor mass (resection of the
tumor), which destroys much of the "leaky" neovasculature.
Moreover, as brain carcinomas are usually pervasive throughout the
organ, therapies which are directed towards eradicating all
tumor-producing cells cannot rely exclusively on the localized
tumor neovasculature.
[0263] A first strategy for drug delivery through the BBB entails
disruption of the BBB, either by osmotic means such as mannitol or
leukotrienes, or biochemically by the use of vasoactive substances
such as bradykinin. The potential for using BBB opening to target
specific agents to brain tumors is also an option. In preferred
embodiments, a BBB disrupting agent is co-administered with the
therapeutic or imaging compositions of the invention when the
compositions are administered by intravascular injection. Other
strategies to go through the BBB may entail the use of endogenous
transport systems, including carrier-mediated transporters such as
glucose and amino acid carriers, receptor-mediated transcytosis for
insulin or transferrin, and active efflux transporters such as
p-glycoprotein. Active transport moieties may also be conjugated to
the therapeutic or imaging compounds for use in the invention to
facilitate transport across the epithelial wall of the blood
vessel. However, the best current strategy for drug delivery behind
the BBB is by intrathecal delivery of therapeutics or imaging
agents directly to the cranium, as through an Ommaya reservoir.
[0264] Delivery/Administration of Therapeutic Antibodies:
[0265] For administration, the antibody-therapeutic or
antibody-imaging agent will generally be mixed, prior to
administration, with a non-toxic, pharmaceutically acceptable
carrier substance. Usually, this will be an aqueous solution, such
as normal saline or phosphate-buffered saline (PBS), Ringer's
solution, lactate-Ringer's solution, or any isotonic
physiologically acceptable solution for administration by the
chosen means. Preferably, the solution is sterile and pyrogen-free,
and is manufactured and packaged under current Good Manufacturing
Processes (GMPs), as approved by the FDA. The clinician of ordinary
skill is familiar with appropriate ranges for pH, tonicity, and
additives or preservatives when formulating pharmaceutical
compositions for administration by intravascular injection,
intrathecal injection, injection into the cerebro-spinal fluid,
direct injection into the tumor, or by other routes. In addition to
additives for adjusting pH or tonicity, the antibody-therapeutics
and antibody-imaging agents may be stabilized against aggregation
and polymerization with amino acids and non-ionic detergents,
polysorbate, and polyethylene glycol. Optionally, additional
stabilizers may include various physiologically-acceptable
carbohydrates and salts. Also, polyvinylpyrrolidone may be added in
addition to the amino acid. Suitable therapeutic immunoglobulin
solutions which are stabilized for storage and administration to
humans are described in U.S. Pat. No. 5,945,098, incorporated fully
herein by reference. Other agents, such as human serum albumin
(HSA), may be added to the therapeutic or imaging composition to
stabilize the antibody conjugates.
[0266] The compositions of the invention may be administered using
any medically appropriate procedure, e.g., intravascular
(intravenous, intraarterial, intracapillary) administration,
injection into the cerebrospinal fluid, intracavity or direct
injection in the tumor. Intrathecal administration maybe carried
out through the use of an Ommaya reservoir, in accordance with
known techniques. (F. Balis et al., Am J. Pediatr. Hematol. Oncol.
11, 74, 76 (1989). For the imaging compositions of the invention,
administration via intravascular injection is preferred for
pre-operative visualization of the tumor. Post-operative
visualization or visualization concurrent with an operation may be
through intrathecal or intracavity administration, as through an
Ommaya reservoir, or also by intravascular administration.
[0267] Intravascular injection may be by intravenous or
intraarterial injection: carotid artery injection is thought to
assist in administration to the brain, and is thus preferred.
Antibody-agents injected into the blood stream have been shown to
cross the blood-brain barrier and to infiltrate the cranial cavity
to some extent, usually in the range of 10.sup.-4 to 10.sup.-3%
[?UNITS?] injected dose per gram. This rate of uptake may be
sufficient for imaging reagents, and also may be useful for tumor
cell specific cytotoxic agents (e.g, those specifically directed to
the inhibition of the function of tumor-cell overexpressed
proteins). However, in order to achieve therapeutic concentrations
of the antibody-therapeutic agents without unacceptable toxicity to
the patient, it is preferred that the therapeutics compositions be
administered by intrathecal injection, direct injection, or
injection into the cerebro-spinal fluid.
[0268] Thus, a preferred method for administration of the
therapeutic compositions of the invention is by depositing it into
the inner cavity of a cystic tumor by any suitable technique, such
as by direct injection (aided by stereotaxic positioning of an
injection syringe, if necessary) or by placing the tip of an Ommaya
reservoir into a cavity, or cyst, for administration. Where the
tumor is a solid tumor, the antibody may be administered by first
creating a resection cavity in the location of the tumor. This
procedure differs from an ordinary craniotomy and tumor resection
only in a few minor respects. As tumor resection is a common
treatment procedure, and is often indicated to relieve pressure,
administration of the therapeutic compositions of the invention
following tumor resection is a preferred embodiment of the
treatment methods of the invention. Following gross total resection
in a standard neurosurgical fashion, the cavity is preferable
rinsed with saline until all bleeding is stopped by cauterization.
Next the pia-arachnoid membrane, surrounding the tumor cavity at
the surface, is cauterized to enhance the formation of fibroblastic
reaction and scarring in the pia-arachnoid area. The result is the
formation of an enclosed, fluid-filled cavity within the brain
tissue at the location from where the tumor was removed. After the
cyst has been formed, either the tip of an Ommaya reservoir or a
micro catheter, which is connected to a pump device and allows the
continues infusion of an antibody solution into the cavity, can be
placed into the cavity. See, e.g., U.S. Pat. No. 5,558,852,
incorporated fully herein by reference.
[0269] Alternatively, a convection-enhanced delivery catheter may
be implanted directly into the tumor mass, into a natural or
surgically created cyst, or into the normal brain mass. Such
convection-enhanced pharmaceutical composition delivery devices
greatly improve the diffusion of the composition throughout the
brain mass. The implanted catheters of these delivery devices
utilize high-flow microinfusion (with flow rates in the range of
about 0.5 to 15.0 .mu.l/minute), rather than diffusive flow, to
deliver the therapeutic or imaging composition to the brain and/or
tumor mass. Such devices are described in U.S. Pat. No. 5,720,720,
incorporated fully herein by reference.
[0270] The effective amount of the therapeutic antibody-conjugate
composition or of the imaging antibody-conjugate compositions to be
given to a particular patient will depend on a variety of factors,
several of which will be different from patient to patient. A
competent clinician will be able to determine an effective amount
of a therapeutic antibody-conjugate composition to administer to a
patient to retard the growth and promote the death of tumor cells,
or an effective amount of an imaging composition to administer to a
patient to facilitate the visualization of a tumor. Dosage of the
antibody-conjugate will depend on the treatment of the tumor, route
of administration, the nature of the therapeutics, sensitivity of
the tumor to the therapeutics, etc. Utilizing LD.sub.50 animal
data, and other information available for the conjugated cytotoxic
or imaging moiety, a clinician can determine the maximum safe dose
for an individual, depending on the route of administration. For
instance, an intravenously administered dose may be more than an
intrathecally administered dose, given the greater body of fluid
into which the therapeutic composition is being administered.
Similarly, compositions which are rapidly cleared from the body may
be administered at higher doses, or in repeated doses, in order to
maintain a therapeutic concentration. Imaging moieties are
typically less toxic than cytotoxic moieties and may be
administered in higher doses in some embodiments. Utilizing
ordinary skill, the competent clinician will be able to optimize
the dosage of a particular therapeutic or imaging composition in
the course of routine clinical trials.
[0271] Typically the dosage will be 0.001 to 100 milligrams of
conjugate per kilogram subject body weight. Doses in the range of
0.01 to 1 mg per kilogram of patient body weight may be utilized
for a radionuclide therapeutic composition which is administered
intrathecally. Relatively large doses, in the range of 0.1 to 10 mg
per kilogram of patient body weight, may used for imaging
conjugates with a relatively non-toxic imaging moiety. The amount
utilized will depend on the sensitivity of the imaging method, and
the relative toxicity of the imaging moiety. In a therapeutic
example, where the therapeutic composition comprises a .sup.131I
cytotoxic moiety, the dosage to the patient will typically start at
a lower range of 10 mCi, and go up to 100, 300 or even 500 mCi.
Stated otherwise, where the therapeutic agent is .sup.131I, the
dosage to the patient will typically be from 5,000 Rads to 100,000
Rads (preferably at least 13,000 Rads, or even at least 50,000
Rads). Doses for other radionuclides are typically selected so that
the tumoricidal dose will be equivalent to the foregoing range for
.sup.131I. Similarly, chemotoxic or toxin protein doses may be
scaled accordingly.
[0272] The antibody conjugate can be administered to the subject in
a series of more than one administration. For therapeutic
compositions, regular periodic administration (e.g., every 2-3
days) will sometimes be required, or may be desirable to reduce
toxicity. For therapeutic compositions which will be utilized in
repeated-dose regimens, antibody moieties which do not provoke HAMA
or other immune responses are preferred. The imaging antibody
conjugate compositions may be administered at an appropriate time
before the visualization technique. For example, administration
within an hour before direct visual inspection may be appropriate,
or administration within twelve hours before an MRI scan may be
appropriate. Care should be taken, however, to not allow too much
time to pass between administration and visualization, as the
imaging compound may eventually be cleared from the patient's
system.
[0273] In addition to the use of imaging antibody conjugates for
simple visualization, these compositions may be utilized as a "dry
run" for more toxic cytotoxic antibody conjugates. If the same
antibody moiety is utilized for the imaging conjugate as for the
therapeutic conjugate, the physician may first use a visualization
technique to determine precisely where in the brain the cytotoxic
conjugate will concentrate. If a sufficient degree of tissue
selectivity is not achieved (e.g, if the tumor cells are too
disperse in the normal tissue, or if the particular brain tumor
protein target chosen is not sufficiently overexpressed in the
particular patient's tumor cells), then the physician may choose
another brain tumor protein target. The provision of numerous brain
tumor protein targets by the present invention, along with both
imaging and therapeutic agents, allows a high degree of flexibility
in designing an effective treatment regimen for the individual
patient.
[0274] Combination Therapies of the Invention
[0275] As mentioned previously, brain tumors tend to be
heterogeneous in character, and pervasive throughout the brain
tissue. This combination often makes them difficult to treat, as
individual portions of the tumor cells in any particular patient
may have differing biological characteristic. Thus, in some cases,
it may be preferred to use various combinations of therapeutic or
imaging agents, as described above in the Summary of Invention, in
order to more fully target all of the cells exhibiting tumorigenic
characteristics. Such combination treatments may be by
administering blended antibody therapeutic or imaging compositions,
individually prepared as described above, and administering the
blended therapeutic to the patient as described. The skilled
administering physician will be able to take such factors as
combined toxicity, and individual antibody agent efficacy, into
account when administering such combined agents. Additionally,
those of skill in the art will be able to screen the antibodies to
avoid potential cross-reaction with each other, in order to assure
full efficacy of each antibody therapeutic or imaging agent.
[0276] Alternatively, several individual brain tumor protein target
compositions may be administered simultaneously or in succession
for a combined therapy. This may be desirable to avoid accumulated
toxicity from several antibody conjugate reagents, or to more
closely monitor potential adverse reactions to the individual
antibody reagents. Thus, cycles such as where a first antibody
therapeutic agent is administered on day one, followed by a second
on day two, then a period with out administration, followed by
re-administration of the antibody therapeutics on different
successive days, is comprehended within the present invention.
EXAMPLES
Example 1
Identification of Two New Splicing Variant Isoforms of PTP.zeta.:
PTP.zeta. SM1 and SM2
[0277] The mRNA nucleotide sequence for PTP.zeta. SM1 was
identified in a human fetal brain phage cDNA library by
sequencing.
[0278] The mRNA nucleotide sequence for PTP.zeta. SM2 was
identified by PCR amplification of adult human brain cDNA, and
sequencing of the resulting nucleic acids.
[0279] For the RT-PCR analyses performed below, total RNA was
isolated from either cells (glioblastoma cultured lines) or tissue
using Trizole (Gibco Life Technologies, Inc.), following the
manufacture's protocol. cDNA was generated from total RNA using the
1.sup.st Strand synthesis kit from Gibco Life Technologies, Inc.,
and an oligo dT.sub.30 anchored primer. For each RT-PCR reaction, 1
.mu.l of cDNA was utilized. The PCR reaction was carried out using
an Advantage 2 kit (Clontech) under standard conditions. The
products of the PCR reactions were confirmed via sequencing.
[0280] Both clones were verified by RT-PCR analysis of glioblastoma
cell lines and primary tumors. For PTP.zeta. SM1, primers
CAGCAGTTGGATGGAAGAGGAC [SEQ ID NO. 28] and CACTGAGATTCTGGCACTATTC
[SEQ ID NO. 29] were used, producing an identifiable 1116 bp
product. RT-PCR analysis was performed, confirming expression of
the SM1 splice variant in 11 of 17 different glioblastoma cell
lines tested, fetal brain, and adult brain, using the unique 3 end
and portion of the 3' untranslated region as the hybridization
target for the probe. In addition, RT-PCR analysis was performed on
28 primary brain tumor samples, confirming expression of the
PTP.zeta. SM1 variant in 16 of the 28 tumors.
[0281] For PTP.zeta. SM2, primers AACAATTCCAGGGTCTCACTC [SEQ ID NO.
30] and TTGACTGGCTCAGGAGTATAG [SEQ ID NO. 31] were used, which
produce a 130 bp product when the extra exon 23a is present, and a
no product when the exon 23a is absent. RT-PCR analysis was
performed, confirming expression in 6 of 17 different glioblastoma
cell lines tested. In addition, RT-PCR analysis was performed on 28
primary brain tumor samples, confirming expression of the PTP.zeta.
SM1 variant in 19 of the 28 tumors.
[0282] For comparison, RT-PCR analysis was also done for the
expression of PTP.zeta.-.alpha. (primers CTGATAATGAGGGCTCCCAAC [SEQ
ID NO. 32] and CTCTGCACTTCCTGGTAAAACTCT [SEQ ID NO. 33]) and
PTP.zeta.-.beta. (primers CAGCAGTTGGATGGAAGAGGAC [SEQ ID NO. 34]
and CTCTGCACTTCCTGGTAAAACTCT [SEQ ID NO. 35]) in the 28 brain tumor
tissue samples. PTP.zeta.-.alpha. was shown to be expressed in 16
of the 28 samples, and the short form PTP.zeta.-.beta. was shown to
be expressed in 19 of the 28 samples.
[0283] The nucleotide sequence alignment of the two new splice
variants with the reference sequence for PTP.zeta.-.alpha. is shown
in the following table:
3TABLE 2 PTP 5' PTP 3' PAC 1 5' PAC 1 3' Corresponding Exon Key: 1
48 87274 87321 5' UTR PAC 1: RPS-1062J16 BAC: RP11-384A20 70 205
87343 87487 1 PAC 2: RPS-1049N15 205 272 142076 142143 2 BAC 5' BAC
3' 291 451 24001 24161 * 3 *88 nt deletion seen in 5' PCR clone
from PTP 363-451 450 603 28570 28723 4 602 701 32814 32888 5 698
772 32814 32888 6 766 924 39695 39853 7 922 1075 39995 40148 8 1074
1261 52411 52598 * 9 *not spliced at 1261 in phage library clones
1260 1387 53910 54037 10 1387 1435 60644 60692 11 1432 2346 66362
67276 5' 12(end of BAC) PAC 2 5' PAC 2 3' 2147 4409 1 2263 mid 12
4437 4987 2294 2844 3' 12 4925 5133 8027 8224 13 5131 5224 17505
17598 14 5223 5310 20427 20514 15 5309 5332 23048 23071 16 5329
5428 23234 23333 17 5429 5512 25555 25638 18 5512 5646 27710 27844
19 5572 5602 42925 42955 * Duplicate of mid 19 *duplicated regions
of exons 19 5646 5768 28408 28530 most of 20 (-12 bp 3') and 26
vary by one aa/two nt 5791 5945 29770 29934 21 (-10 bp 5') 5943
6082 31560 31699 22 6080 6228 33375 33523 .about. 23 .about.116 nt
insert seen b/w exons 23 & 24 in 3' PCR clone: maps to PAC b/w
23 & 24 6225 6322 40379 40476 .about. 24 PTP location PAC 2 5'
PAC 2 3' 6322 6397 40820 40895 25 6228 36744 36629 6396 6526 42864
42994 26 6457 6487 27770 27800 * Duplicate of mid 26 6525 6673
43895 44043 27 6671 6816 47753 47898 28 6816 6952 48708 48844 29
**BOUNDARIES DETERMINED FROM HOMO SAPIENS CHROMOSOME 7 WORKING
DRAFT (NT_007845.3)** Nucleotide location refers to position in
full length RPTPZ (accession M93426)
Example 2
Cell Migration Assay For Determining Antibody Activity on Protein
Targets
[0284] Tumor cells are known to migrate more rapidly towards
chemoattractants. The cell migration assay measures the ability of
a cell to migrate. The ability to migrate is taken as a measure of
tumorigenicity. Chemoattractants generally used are fetal bovine
serum, pleiotrophin, bFGF, and VEGF. Thus, this assay can be used
to determine migration capability of a cell in which the gene has
been knocked down or the gene of interest is being
overexpressed.
[0285] The ChemoTx.RTM. disposable chemotaxis system (Neuroprobe,
Inc., Gaithersburg, Md.) is used according to the manufacturer's
instructions, with a few modifications. Briefly, glioblastoma
cultured cells from cell line G55T2 are prepared by splitting the
cells the day before the assay is performed. A ChemoTx.RTM. chamber
with the following specifications is used: Pore size 8 .mu.m,
exposed filter area 8 mm, exposed filter area diameter 3.2
mm.sup.2. The plate configuration is: 30 .mu.l per well, 96 well
plate. The membrane type is: Track-etched polycarbonate.
[0286] In preparation for the assays, the filter membrane is coated
in 100 ml PBS containing 0.1% acetic acid and 3.5 ml Vitrogen 100
(from Cohesion) at 37.degree. C. overnight. About 30 minutes before
starting the assay the coated membrane is washed and rinsed with
PBS containing 0.1% BSA. Cells are harvested by using the standard
technique (trypsin-EDTA). The cells are washed once with DMEM 10%
FBS, and then spun at 1000 RPM, for 5 minutes at room temperature.
The pellet is resuspended in DMEM without serum, containing 0.1%
BSA (serum free medium). The cells are spun and resuspended again
in serum free medium, and then spun and resuspended in the amount
of serum free medium needed to provide a concentration of 1 mio.
cells/ml, or 25,000 cells per 25 ul. Just prior to the assay, a
suitable amount of the antibody to be tested for anti-target
function activity is added to the cell suspension.
[0287] For the assay, a standard chemoattractant is used to measure
the mobility of the cells. The chemoattractants are diluted in
serum free medium. A suitable unspecific chemoattractant is DMEM
with 5% FBS. The chemoattractant solutions and control solutions
without chemoattractant are pipetted (29 .mu.l) into the lower
plate wells. After placing and securing the filter plate over the
lower wells, ensuring contact with the solution in the bottom
wells, serial dilutions of the cell suspension are pipetted onto
each site on the filter top. The plates are them covered and
incubated at 37.degree. C., 5% CO.sub.2, for 3-4 hours.
[0288] After incubation, the upper filter side is rinsed with PBS
and exposed upper filter areas are cleaned with wet cotton swabs.
The filter is stained using Diff-Quik.TM. (VWR) dye kit, according
to the manufacturer's instructions. The migrated cells are counted
on the lower filter side using a microscope (Magnification
200.times.), by counting of 5 high power field sections per
well.
Example 2
HUVEC(Human Umbilical Vein Endothelial Cells) Endothelial Sprouting
Assay For Determining Antibody Activity on Protein Targets
[0289] Cell-sprouting morphology can be utilized as an easily
visualized assay to determine the inhibitory effect of a candidate
antibody on the protein target function for protein targets which
stimulate endothelial cell sprouting, such as ARP2. Such assays
have been described extensively in the literature (Nehls, V., et
al., Histochem. Cell Biol. 104: 459-466 (1995); Koblizek, T. I., et
al., Curr. Biol. 8: 529-532 (1988); and Kwak, H. J., et al., FEBS
Lett. 448: 249-253). Briefly, a endothelial cells from a suitable
source, such as HUVECs or PPAECs (porcine pulmonary artery
endothelial cells) are grown to confluence on microcarrier (MC)
beads (diameter 175 .mu.m, available from Sigma) and placed into a
2.5 mg/ml fibrinogen gel containing the protein target at an
appropriate effective concentration (200 ng/ml is an suitable
starting concentration, which the skilled practitioner may
optimize) and the antibody in an appropriate range of
concentrations (this will depend on antibody titer and affinity for
the target), and 200 units/ml Trasylol (available from Bayer).
Fibrin gels are incubated in M-199 with a daily supplement of the
same amount of recombinant protein and antibody, 2.0%
heat-inactivated fetal bovine serum, and 200 units/ml Trasylol.
After three days, the extent of sprouting is determined using a
phase-contrast microscope (such as those available from Zeiss). A
decrease in cell sprouting as compared to controls without antibody
indicates a reduction in protein target activity by the
antibody.
[0290] The foregoing is intended to be illustrative of the
embodiments of the present invention, and are not intended to limit
the invention in any way. Although the invention has been described
with respect to specific modifications, the details thereof are not
to be construed as limitations, for it will be apparent that
various equivalents, changes and modifications may be resorted to
without departing from the spirit and scope thereof and it is
understood that such equivalent embodiments are to be included
herein. All publications and patent applications are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
Sequence CWU 1
1
35 1 3091 DNA Homo sapiens gene (1)..(3091) PTP-zeta SM1 exon 9
variant 1 cacacatacg cacgcacgat ctcacttcga tctatacact ggaggattaa
aacaaacaaa 60 caaaaaaaac atttccttcg ctccccctcc ctctccactc
tgagaagcag aggagccgca 120 cggcgagggg ccgcagaccg tctggaa atg cga atc
cta aag cgt ttc ctc gct 174 Met Arg Ile Leu Lys Arg Phe Leu Ala 1 5
tgc att cag ctc ctc tgt gtt tgc cgc ctg gat tgg gct aat gga tac 222
Cys Ile Gln Leu Leu Cys Val Cys Arg Leu Asp Trp Ala Asn Gly Tyr 10
15 20 25 tac aga caa cag aga aaa ctt gtt gaa gag att ggc tgg tcc
tat aca 270 Tyr Arg Gln Gln Arg Lys Leu Val Glu Glu Ile Gly Trp Ser
Tyr Thr 30 35 40 gga gca ctg aat caa aaa aat tgg gga aag aaa tat
cca aca tgt aat 318 Gly Ala Leu Asn Gln Lys Asn Trp Gly Lys Lys Tyr
Pro Thr Cys Asn 45 50 55 agc cca aaa caa tct cct atc aat att gat
gaa gat ctt aca caa gta 366 Ser Pro Lys Gln Ser Pro Ile Asn Ile Asp
Glu Asp Leu Thr Gln Val 60 65 70 aat gtg aat ctt aag aaa ctt aaa
ttt cag ggt tgg gat aaa aca tca 414 Asn Val Asn Leu Lys Lys Leu Lys
Phe Gln Gly Trp Asp Lys Thr Ser 75 80 85 ttg gaa aac aca ttc att
cat aac act ggg aaa aca gtg gaa att aat 462 Leu Glu Asn Thr Phe Ile
His Asn Thr Gly Lys Thr Val Glu Ile Asn 90 95 100 105 ctc act aat
gac tac cgt gtc agc gga gga gtt tca gaa atg gtg ttt 510 Leu Thr Asn
Asp Tyr Arg Val Ser Gly Gly Val Ser Glu Met Val Phe 110 115 120 aaa
gca agc aag ata act ttt cac tgg gga aaa tgc aat atg tca tct 558 Lys
Ala Ser Lys Ile Thr Phe His Trp Gly Lys Cys Asn Met Ser Ser 125 130
135 gat gga tca gag cat agt tta gaa gga caa aaa ttt cca ctt gag atg
606 Asp Gly Ser Glu His Ser Leu Glu Gly Gln Lys Phe Pro Leu Glu Met
140 145 150 caa atc tac tgc ttt gat gcg gac cga ttt tca agt ttt gag
gaa gca 654 Gln Ile Tyr Cys Phe Asp Ala Asp Arg Phe Ser Ser Phe Glu
Glu Ala 155 160 165 gtc aaa gga aaa ggg aag tta aga gct tta tcc att
ttg ttt gag gtt 702 Val Lys Gly Lys Gly Lys Leu Arg Ala Leu Ser Ile
Leu Phe Glu Val 170 175 180 185 ggg aca gaa gaa aat ttg gat ttc aaa
gcg att att gat gga gtc gaa 750 Gly Thr Glu Glu Asn Leu Asp Phe Lys
Ala Ile Ile Asp Gly Val Glu 190 195 200 agt gtt agt cgt ttt ggg aag
cag gct gct tta gat cca ttc ata ctg 798 Ser Val Ser Arg Phe Gly Lys
Gln Ala Ala Leu Asp Pro Phe Ile Leu 205 210 215 ttg aac ctt ctg cca
aac tca act gac aag tat tac att tac aat ggc 846 Leu Asn Leu Leu Pro
Asn Ser Thr Asp Lys Tyr Tyr Ile Tyr Asn Gly 220 225 230 tca ttg aca
tct cct ccc tgc aca gac aca gtt gac tgg att gtt ttt 894 Ser Leu Thr
Ser Pro Pro Cys Thr Asp Thr Val Asp Trp Ile Val Phe 235 240 245 aaa
gat aca gtt agc atc tct gaa agc cag ttg gct gtt ttt tgt gaa 942 Lys
Asp Thr Val Ser Ile Ser Glu Ser Gln Leu Ala Val Phe Cys Glu 250 255
260 265 gtt ctt aca atg caa caa tct ggt tat gtc atg ctg atg gac tac
tta 990 Val Leu Thr Met Gln Gln Ser Gly Tyr Val Met Leu Met Asp Tyr
Leu 270 275 280 caa aac aat ttt cga gag caa cag tac aag ttc tct aga
cag gtg ttt 1038 Gln Asn Asn Phe Arg Glu Gln Gln Tyr Lys Phe Ser
Arg Gln Val Phe 285 290 295 tcc tca tac act gga aag gaa gag att cat
gaa gca gtt tgt agt tca 1086 Ser Ser Tyr Thr Gly Lys Glu Glu Ile
His Glu Ala Val Cys Ser Ser 300 305 310 gaa cca gaa aat gtt cag gct
gac cca gag aat tat acc agc ctt ctt 1134 Glu Pro Glu Asn Val Gln
Ala Asp Pro Glu Asn Tyr Thr Ser Leu Leu 315 320 325 gtt aca tgg gaa
aga cct cga gtc gtt tat gat acc atg att gag aag 1182 Val Thr Trp
Glu Arg Pro Arg Val Val Tyr Asp Thr Met Ile Glu Lys 330 335 340 345
ttt gca gtt ttg tac cag cag ttg gat gga gag gac caa acc aag cat
1230 Phe Ala Val Leu Tyr Gln Gln Leu Asp Gly Glu Asp Gln Thr Lys
His 350 355 360 gaa ttt ttg aca gat ggc tat caa gac ttg gta act ata
tga 1272 Glu Phe Leu Thr Asp Gly Tyr Gln Asp Leu Val Thr Ile 365
370 tcagttgttt tacatagggt aacattataa tttaatttcc aaggtaagaa
cttacaaatg 1332 gttgtatatt attttcctcc attactttta gactttatgt
gaaggtgggg taggctgagt 1392 atttttaaat ttaaaaaaaa attttaaatt
agaagctata ctaaattatg tttaaagtta 1452 catttaatta aatggatatc
ataactttgc caacaataac actatagagt agatacatat 1512 gacttatgaa
ctggagatca tttagtgtgg cctttcttaa gatttcagtt gtagaatagt 1572
gccagaatct cagtgccctg atacatttta tattgtgtct tccattacgc tatatcagca
1632 caggaaaagt agagtagggg acatacaagt cctctttgtt gcaccaaaaa
attttcagat 1692 aacagctggg aagtcatgat tgggtcagaa ctttggggat
gtaagaaaac atttcttaca 1752 aaaagatcca cccctgcctc cctccaccag
cgcatgcgaa taaagtacag attccctttg 1812 tggcctgagc atgtcagtat
taaactttgc tctggtaggg aagtgttggc catagattag 1872 ggtgtagttg
acaaaccttc atctggatgt aggtccagaa agtccccact gcaggttaaa 1932
ggacactgga ctctgcactc aggcacctag agtcctgcaa gtcctgggaa cctgcattta
1992 aataaaaatg cactattaat tatgtttcat atcatgtgga caaaatggat
aaaattttag 2052 taacctttta attcagttgc ctggaatatg gagacacaat
gacctgggaa aatcgtgaaa 2112 taaatagtaa taaaaatgtt tatttcataa
ttacgtgaag aagataattc tattactgtt 2172 cttgcatata tattgtcaag
aaaaagagat aacttagttg ttcacttttt cacattgctc 2232 cttgtttgca
aatgcccccc atttatttgt ctaaaatatt aatttttagt ttgtagtact 2292
aatttatgaa tttgatgagt tctggctaaa aatgaaactt cctgaaacta aatctgattt
2352 ttaaaaagca aaaaaaaaaa aaaagcctag ctttccagtt cttcataatt
cacaaatacc 2412 acaagtttaa ctaagcaaca ttgcataaac ttttccttag
gttaataaaa tagaagtatt 2472 ttccacggac cagggagaaa aagttttcta
ggaaagatac ctagtgtgtt ggtagtccta 2532 tgagaataac atttgtataa
ttactaacat ctttctttta gggtgctatt ctcaataatt 2592 tgctacccaa
tatgagttat gttcttcaga tagtagccat atgcactaat ggcttatatg 2652
gaaaatacag cgaccaactg attgtcgaca tgcctactga taatcctggt aagtgccacc
2712 agatacatct atatattaac tcaataaatg aggttagttt aattactgta
tgcattgatg 2772 ctttctctct atattctttt ggccaaaagg caaagtgatt
ttctcttaag tctggattgc 2832 cgggtaattt tttggggcat gggacccatt
tctcattcag caggtctggt gccagacaat 2892 aagtaaactt atccttaata
ttggagttta ccatttgtaa aataagagtg actaaacata 2952 tttataacat
tgtaataatc attaaatgaa aattgctatg taaatgttga gactgttatt 3012
ttggataatt aagagttggt ttaatttgta tttatttcct cttttcagcc cccaaagcat
3072 tatgtagtaa gtgtataca 3091 2 374 PRT Homo sapiens gene
(1)..(374) PTP-zeta SM1 exon 9 variant 2 Met Arg Ile Leu Lys Arg
Phe Leu Ala Cys Ile Gln Leu Leu Cys Val 1 5 10 15 Cys Arg Leu Asp
Trp Ala Asn Gly Tyr Tyr Arg Gln Gln Arg Lys Leu 20 25 30 Val Glu
Glu Ile Gly Trp Ser Tyr Thr Gly Ala Leu Asn Gln Lys Asn 35 40 45
Trp Gly Lys Lys Tyr Pro Thr Cys Asn Ser Pro Lys Gln Ser Pro Ile 50
55 60 Asn Ile Asp Glu Asp Leu Thr Gln Val Asn Val Asn Leu Lys Lys
Leu 65 70 75 80 Lys Phe Gln Gly Trp Asp Lys Thr Ser Leu Glu Asn Thr
Phe Ile His 85 90 95 Asn Thr Gly Lys Thr Val Glu Ile Asn Leu Thr
Asn Asp Tyr Arg Val 100 105 110 Ser Gly Gly Val Ser Glu Met Val Phe
Lys Ala Ser Lys Ile Thr Phe 115 120 125 His Trp Gly Lys Cys Asn Met
Ser Ser Asp Gly Ser Glu His Ser Leu 130 135 140 Glu Gly Gln Lys Phe
Pro Leu Glu Met Gln Ile Tyr Cys Phe Asp Ala 145 150 155 160 Asp Arg
Phe Ser Ser Phe Glu Glu Ala Val Lys Gly Lys Gly Lys Leu 165 170 175
Arg Ala Leu Ser Ile Leu Phe Glu Val Gly Thr Glu Glu Asn Leu Asp 180
185 190 Phe Lys Ala Ile Ile Asp Gly Val Glu Ser Val Ser Arg Phe Gly
Lys 195 200 205 Gln Ala Ala Leu Asp Pro Phe Ile Leu Leu Asn Leu Leu
Pro Asn Ser 210 215 220 Thr Asp Lys Tyr Tyr Ile Tyr Asn Gly Ser Leu
Thr Ser Pro Pro Cys 225 230 235 240 Thr Asp Thr Val Asp Trp Ile Val
Phe Lys Asp Thr Val Ser Ile Ser 245 250 255 Glu Ser Gln Leu Ala Val
Phe Cys Glu Val Leu Thr Met Gln Gln Ser 260 265 270 Gly Tyr Val Met
Leu Met Asp Tyr Leu Gln Asn Asn Phe Arg Glu Gln 275 280 285 Gln Tyr
Lys Phe Ser Arg Gln Val Phe Ser Ser Tyr Thr Gly Lys Glu 290 295 300
Glu Ile His Glu Ala Val Cys Ser Ser Glu Pro Glu Asn Val Gln Ala 305
310 315 320 Asp Pro Glu Asn Tyr Thr Ser Leu Leu Val Thr Trp Glu Arg
Pro Arg 325 330 335 Val Val Tyr Asp Thr Met Ile Glu Lys Phe Ala Val
Leu Tyr Gln Gln 340 345 350 Leu Asp Gly Glu Asp Gln Thr Lys His Glu
Phe Leu Thr Asp Gly Tyr 355 360 365 Gln Asp Leu Val Thr Ile 370 3
8058 DNA Homo sapiens gene (1)..(8058) PTP-zeta SM2 exon 23a
variant 3 cacacatacg cacgcacgat ctcacttcga tctatacact ggaggattaa
aacaaacaaa 60 caaaaaaaac atttccttcg ctccccctcc ctctccactc
tgagaagcag aggagccgca 120 cggcgagggg ccgcagaccg tctggaaatg
cgaatcctaa agcgtttcct cgcttgcatt 180 cagctcctct gtgtttgccg
cctggattgg gctaatggat actacagaca acagagaaaa 240 cttgttgaag
agattggctg gtcctataca ggagcactga atcaaaaaaa ttggggaaag 300
aaatatccaa catgtaatag cccaaaacaa tctcctatca atattgatga agatcttaca
360 caagtaaatg tgaatcttaa gaaacttaaa tttcagggtt gggataaaac
atcattggaa 420 aacacattca ttcataacac tgggaaaaca gtggaaatta
atctcactaa tgactaccgt 480 gtcagcggag gagtttcaga aatggtgttt
aaagcaagca agataacttt tcactgggga 540 aaatgcaata tgtcatctga
tggatcagag catagtttag aaggacaaaa atttccactt 600 gagatgcaaa
tctactgctt tgatgcggac cgattttcaa gttttgagga agcagtcaaa 660
ggaaaaggga agttaagagc tttatccatt ttgtttgagg ttgggacaga agaaaatttg
720 gatttcaaag cgattattga tggagtcgaa agtgttagtc gttttgggaa
gcaggctgct 780 ttagatccat tcatactgtt gaaccttctg ccaaactcaa
ctgacaagta ttacatttac 840 aatggctcat tgacatctcc tccctgcaca
gacacagttg actggattgt ttttaaagat 900 acagttagca tctctgaaag
ccagttggct gttttttgtg aagttcttac aatgcaacaa 960 tctggttatg
tcatgctgat ggactactta caaaacaatt ttcgagagca acagtacaag 1020
ttctctagac aggtgttttc ctcatacact ggaaaggaag agattcatga agcagtttgt
1080 agttcagaac cagaaaatgt tcaggctgac ccagagaatt ataccagcct
tcttgttaca 1140 tgggaaagac ctcgagtcgt ttatgatacc atgattgaga
agtttgcagt tttgtaccag 1200 cagttggatg gagaggacca aaccaagcat
gaatttttga cagatggcta tcaagacttg 1260 ggtgctattc tcaataattt
gctacccaat atgagttatg ttcttcagat agtagccata 1320 tgcactaatg
gcttatatgg aaaatacagc gaccaactga ttgtcgacat gcctactgat 1380
aatcctgaac ttgatctttt ccctgaatta attggaactg aagaaataat caaggaggag
1440 gaagagggaa aagacattga agaaggcgct attgtgaatc ctggtagaga
cagtgctaca 1500 aaccaaatca ggaaaaagga accccagatt tctaccacaa
cacactacaa tcgcataggg 1560 acgaaataca atgaagccaa gactaaccga
tccccaacaa gaggaagtga attctctgga 1620 aagggtgatg ttcccaatac
atctttaaat tccacttccc aaccagtcac taaattagcc 1680 acagaaaaag
atatttcctt gacttctcag actgtgactg aactgccacc tcacactgtg 1740
gaaggtactt cagcctcttt aaatgatggc tctaaaactg ttcttagatc tccacatatg
1800 aacttgtcgg ggactgcaga atccttaaat acagtttcta taacagaata
tgaggaggag 1860 agtttattga ccagtttcaa gcttgatact ggagctgaag
attcttcagg ctccagtccc 1920 gcaacttctg ctatcccatt catctctgag
aacatatccc aagggtatat attttcctcc 1980 gaaaacccag agacaataac
atatgatgtc cttataccag aatctgctag aaatgcttcc 2040 gaagattcaa
cttcatcagg ttcagaagaa tcactaaagg atccttctat ggagggaaat 2100
gtgtggtttc ctagctctac agacataaca gcacagcccg atgttggatc aggcagagag
2160 agctttctcc agactaatta cactgagata cgtgttgatg aatctgagaa
gacaaccaag 2220 tccttttctg caggcccagt gatgtcacag ggtccctcag
ttacagatct ggaaatgcca 2280 cattattcta cctttgccta cttcccaact
gaggtaacac ctcatgcttt taccccatcc 2340 tccagacaac aggatttggt
ctccacggtc aacgtggtat actcgcagac aacccaaccg 2400 gtatacaatg
gtgagacacc tcttcaacct tcctacagta gtgaagtctt tcctctagtc 2460
acccctttgt tgcttgacaa tcagatcctc aacactaccc ctgctgcttc aagtagtgat
2520 tcggccttgc atgctacgcc tgtatttccc agtgtcgatg tgtcatttga
atccatcctg 2580 tcttcctatg atggtgcacc tttgcttcca ttttcctctg
cttccttcag tagtgaattg 2640 tttcgccatc tgcatacagt ttctcaaatc
cttccacaag ttacttcagc taccgagagt 2700 gataaggtgc ccttgcatgc
ttctctgcca gtggctgggg gtgatttgct attagagccc 2760 agccttgctc
agtattctga tgtgctgtcc actactcatg ctgcttcaga gacgctggaa 2820
tttggtagtg aatctggtgt tctttataaa acgcttatgt tttctcaagt tgaaccaccc
2880 agcagtgatg ccatgatgca tgcacgttct tcagggcctg aaccttctta
tgccttgtct 2940 gataatgagg gctcccaaca catcttcact gtttcttaca
gttctgcaat acctgtgcat 3000 gattctgtgg gtgtaactta tcagggttcc
ttatttagcg gccctagcca tataccaata 3060 cctaagtctt cgttaataac
cccaactgca tcattactgc agcctactca tgccctctct 3120 ggtgatgggg
aatggtctgg agcctcttct gatagtgaat ttcttttacc tgacacagat 3180
gggctgacag cccttaacat ttcttcacct gtttctgtag ctgaatttac atatacaaca
3240 tctgtgtttg gtgatgataa taaggcgctt tctaaaagtg aaataatata
tggaaatgag 3300 actgaactgc aaattccttc tttcaatgag atggtttacc
cttctgaaag cacagtcatg 3360 cccaacatgt atgataatgt aaataagttg
aatgcgtctt tacaagaaac ctctgtttcc 3420 atttctagca ccaagggcat
gtttccaggg tcccttgctc ataccaccac taaggttttt 3480 gatcatgaga
ttagtcaagt tccagaaaat aacttttcag ttcaacctac acatactgtc 3540
tctcaagcat ctggtgacac ttcgcttaaa cctgtgctta gtgcaaactc agagccagca
3600 tcctctgacc ctgcttctag tgaaatgtta tctccttcaa ctcagctctt
attttatgag 3660 acctcagctt cttttagtac tgaagtattg ctacaacctt
cctttcaggc ttctgatgtt 3720 gacaccttgc ttaaaactgt tcttccagct
gtgcccagtg atccaatatt ggttgaaacc 3780 cccaaagttg ataaaattag
ttctacaatg ttgcatctca ttgtatcaaa ttctgcttca 3840 agtgaaaaca
tgctgcactc tacatctgta ccagtttttg atgtgtcgcc tacttctcat 3900
atgcactctg cttcacttca aggtttgacc atttcctatg caagtgagaa atatgaacca
3960 gttttgttaa aaagtgaaag ttcccaccaa gtggtacctt ctttgtacag
taatgatgag 4020 ttgttccaaa cggccaattt ggagattaac caggcccatc
ccccaaaagg aaggcatgta 4080 tttgctacac ctgttttatc aattgatgaa
ccattaaata cactaataaa taagcttata 4140 cattccgatg aaattttaac
ctccaccaaa agttctgtta ctggtaaggt atttgctggt 4200 attccaacag
ttgcttctga tacatttgta tctactgatc attctgttcc tataggaaat 4260
gggcatgttg ccattacagc tgtttctccc cacagagatg gttctgtaac ctcaacaaag
4320 ttgctgtttc cttctaaggc aacttctgag ctgagtcata gtgccaaatc
tgatgccggt 4380 ttagtgggtg gtggtgaaga tggtgacact gatgatgatg
gtgatgatga tgatgacaga 4440 gatagtgatg gcttatccat tcataagtgt
atgtcatgct catcctatag agaatcacag 4500 gaaaaggtaa tgaatgattc
agacacccac gaaaacagtc ttatggatca gaataatcca 4560 atctcatact
cactatctga gaattctgaa gaagataata gagtcacaag tgtatcctca 4620
gacagtcaaa ctggtatgga cagaagtcct ggtaaatcac catcagcaaa tgggctatcc
4680 caaaagcaca atgatggaaa agaggaaaat gacattcaga ctggtagtgc
tctgcttcct 4740 ctcagccctg aatctaaagc atgggcagtt ctgacaagtg
atgaagaaag tggatcaggg 4800 caaggtacct cagatagcct taatgagaat
gagacttcca cagatttcag ttttgcagac 4860 actaatgaaa aagatgctga
tgggatcctg gcagcaggtg actcagaaat aactcctgga 4920 ttcccacagt
ccccaacatc atctgttact agcgagaact cagaagtgtt ccacgtttca 4980
gaggcagagg ccagtaatag tagccatgag tctcgtattg gtctagctga ggggttggaa
5040 tccgagaaga aggcagttat accccttgtg atcgtgtcag ccctgacttt
tatctgtcta 5100 gtggttcttg tgggtattct catctactgg aggaaatgct
tccagactgc acacttttac 5160 ttagaggaca gtacatcccc tagagttata
tccacacctc caacacctat ctttccaatt 5220 tcagatgatg tcggagcaat
tccaataaag cactttccaa agcatgttgc agatttacat 5280 gcaagtagtg
ggtttactga agaatttgag acactgaaag agttttacca ggaagtgcag 5340
agctgtactg ttgacttagg tattacagca gacagctcca accacccaga caacaagcac
5400 aagaatcgat acataaatat cgttgcctat gatcatagca gggttaagct
agcacagctt 5460 gctgaaaagg atggcaaact gactgattat atcaatgcca
attatgttga tggctacaac 5520 agaccaaaag cttatattgc tgcccaaggc
ccactgaaat ccacagctga agatttctgg 5580 agaatgatat gggaacataa
tgtggaagtt attgtcatga taacaaacct cgtggagaaa 5640 ggaaggagaa
aatgtgatca gtactggcct gccgatggga gtgaggagta cgggaacttt 5700
ctggtcactc agaagagtgt gcaagtgctt gcctattata ctgtgaggaa ttttactcta
5760 agaaacacaa aaataaaaaa gggctcccag aaaggaagac ccagtggacg
tgtggtcaca 5820 cagtatcact acacgcagtg gcctgacatg ggagtaccag
agtactccct gccagtgctg 5880 acctttgtga gaaaggcagc ctatgccaag
cgccatgcag tggggcctgt tgtcgtccac 5940 tgcagtgctg gagttggaag
aacaggcaca tatattgtgc tagacagtat gttgcagcag 6000 attcaacacg
aaggaactgt caacatattt ggcttcttaa aacacatccg ttcacaaaga 6060
aattatttgg tacaaactga ggagcaatat gtcttcattc atgatacact ggttgaggcc
6120 atacttagta aagaaactga ggtgctggac agtcatattc atgcctatgt
taatgcactc 6180 ctcattcctg gaccagcagg caaaacaaag ctagagaaac
aattccaggg tctcactctg 6240 tcacccaggc tggagtgcag aggcacaatc
tcggctcact gcaaccttcc tctccctggc 6300 ttaactgatc ctcctacctc
agcctcccga gtggctggga ctatactcct gagccagtca 6360 aatatacagc
agagtgacta ttctgcagcc ctaaagcaat gcaacaggga aaagaatcga 6420
acttcttcta tcatccctgt ggaaagatca agggttggca tttcatccct gagtggagaa
6480 ggcacagact acatcaatgc ctcctatatc atgggctatt accagagcaa
tgaattcatc 6540 attacccagc accctctcct tcataccatc aaggatttct
ggaggatgat atgggaccat 6600 aatgcccaac tggtggttat gattcctgat
ggccaaaaca tggcagaaga tgaatttgtt 6660 tactggccaa ataaagatga
gcctataaat tgtgagagct ttaaggtcac tcttatggct 6720 gaagaacaca
aatgtctatc taatgaggaa aaacttataa ttcaggactt tatcttagaa 6780
gctacacagg atgattatgt acttgaagtg aggcactttc agtgtcctaa atggccaaat
6840 ccagatagcc
ccattagtaa aacttttgaa cttataagtg ttataaaaga agaagctgcc 6900
aatagggatg ggcctatgat tgttcatgat gagcatggag gagtgacggc aggaactttc
6960 tgtgctctga caacccttat gcaccaacta gaaaaagaaa attccgtgga
tgtttaccag 7020 gtagccaaga tgatcaatct gatgaggcca ggagtctttg
ctgacattga gcagtatcag 7080 tttctctaca aagtgatcct cagccttgtg
agcacaaggc aggaagagaa tccatccacc 7140 tctctggaca gtaatggtgc
agcattgcct gatggaaata tagctgagag cttagagtct 7200 ttagtttaac
acagaaaggg gtggggggac tcacatctga gcattgtttt cctcttccta 7260
aaattaggca ggaaaatcag tctagttctg ttatctgttg atttcccatc acctgacagt
7320 aactttcatg acataggatt ctgccgccaa atttatatca ttaacaatgt
gtgccttttt 7380 gcaagacttg taatttactt attatgtttg aactaaaatg
attgaatttt acagtatttc 7440 taagaatgga attgtggtat ttttttctgt
attgatttta acagaaaatt tcaatttata 7500 gaggttagga attccaaact
acagaaaatg tttgttttta gtgtcaaatt tttagctgta 7560 tttgtagcaa
ttatcaggtt tgctagaaat ataactttta atacagtagc ctgtaaataa 7620
aacactcttc catatgatat tcaacatttt acaactgcag tattcaccta aagtagaaat
7680 aatctgttac ttattgtaaa tactgcccta gtgtctccat ggaccaaatt
tatatttata 7740 attgtagatt tttatatttt actactgagt caagttttct
agttctgtgt aattgtttag 7800 tttaatgacg tagttcatta gctggtctta
ctctaccagt tttctgacat tgtattgtgt 7860 tacctaagtc attaactttg
tttcagcatg taattttaac ttttgtggaa aatagaaata 7920 ccttcatttt
gaaagaagtt tttatgagaa taacacctta ccaaacattg ttcaaatggt 7980
ttttatccaa ggaattgcaa aaataaatat aaatattgcc attaaaaaaa aaaaaaaaaa
8040 aaaaaaaaaa aaaaaaaa 8058 4 2353 PRT Homo sapiens gene
(1)..(2353) PTP-zeta SM2 23a exon variant 4 Met Arg Ile Leu Lys Arg
Phe Leu Ala Cys Ile Gln Leu Leu Cys Val 1 5 10 15 Cys Arg Leu Asp
Trp Ala Asn Gly Tyr Tyr Arg Gln Gln Arg Lys Leu 20 25 30 Val Glu
Glu Ile Gly Trp Ser Tyr Thr Gly Ala Leu Asn Gln Lys Asn 35 40 45
Trp Gly Lys Lys Tyr Pro Thr Cys Asn Ser Pro Lys Gln Ser Pro Ile 50
55 60 Asn Ile Asp Glu Asp Leu Thr Gln Val Asn Val Asn Leu Lys Lys
Leu 65 70 75 80 Lys Phe Gln Gly Trp Asp Lys Thr Ser Leu Glu Asn Thr
Phe Ile His 85 90 95 Asn Thr Gly Lys Thr Val Glu Ile Asn Leu Thr
Asn Asp Tyr Arg Val 100 105 110 Ser Gly Gly Val Ser Glu Met Val Phe
Lys Ala Ser Lys Ile Thr Phe 115 120 125 His Trp Gly Lys Cys Asn Met
Ser Ser Asp Gly Ser Glu His Ser Leu 130 135 140 Glu Gly Gln Lys Phe
Pro Leu Glu Met Gln Ile Tyr Cys Phe Asp Ala 145 150 155 160 Asp Arg
Phe Ser Ser Phe Glu Glu Ala Val Lys Gly Lys Gly Lys Leu 165 170 175
Arg Ala Leu Ser Ile Leu Phe Glu Val Gly Thr Glu Glu Asn Leu Asp 180
185 190 Phe Lys Ala Ile Ile Asp Gly Val Glu Ser Val Ser Arg Phe Gly
Lys 195 200 205 Gln Ala Ala Leu Asp Pro Phe Ile Leu Leu Asn Leu Leu
Pro Asn Ser 210 215 220 Thr Asp Lys Tyr Tyr Ile Tyr Asn Gly Ser Leu
Thr Ser Pro Pro Cys 225 230 235 240 Thr Asp Thr Val Asp Trp Ile Val
Phe Lys Asp Thr Val Ser Ile Ser 245 250 255 Glu Ser Gln Leu Ala Val
Phe Cys Glu Val Leu Thr Met Gln Gln Ser 260 265 270 Gly Tyr Val Met
Leu Met Asp Tyr Leu Gln Asn Asn Phe Arg Glu Gln 275 280 285 Gln Tyr
Lys Phe Ser Arg Gln Val Phe Ser Ser Tyr Thr Gly Lys Glu 290 295 300
Glu Ile His Glu Ala Val Cys Ser Ser Glu Pro Glu Asn Val Gln Ala 305
310 315 320 Asp Pro Glu Asn Tyr Thr Ser Leu Leu Val Thr Trp Glu Arg
Pro Arg 325 330 335 Val Val Tyr Asp Thr Met Ile Glu Lys Phe Ala Val
Leu Tyr Gln Gln 340 345 350 Leu Asp Gly Glu Asp Gln Thr Lys His Glu
Phe Leu Thr Asp Gly Tyr 355 360 365 Gln Asp Leu Gly Ala Ile Leu Asn
Asn Leu Leu Pro Asn Met Ser Tyr 370 375 380 Val Leu Gln Ile Val Ala
Ile Cys Thr Asn Gly Leu Tyr Gly Lys Tyr 385 390 395 400 Ser Asp Gln
Leu Ile Val Asp Met Pro Thr Asp Asn Pro Glu Leu Asp 405 410 415 Leu
Phe Pro Glu Leu Ile Gly Thr Glu Glu Ile Ile Lys Glu Glu Glu 420 425
430 Glu Gly Lys Asp Ile Glu Glu Gly Ala Ile Val Asn Pro Gly Arg Asp
435 440 445 Ser Ala Thr Asn Gln Ile Arg Lys Lys Glu Pro Gln Ile Ser
Thr Thr 450 455 460 Thr His Tyr Asn Arg Ile Gly Thr Lys Tyr Asn Glu
Ala Lys Thr Asn 465 470 475 480 Arg Ser Pro Thr Arg Gly Ser Glu Phe
Ser Gly Lys Gly Asp Val Pro 485 490 495 Asn Thr Ser Leu Asn Ser Thr
Ser Gln Pro Val Thr Lys Leu Ala Thr 500 505 510 Glu Lys Asp Ile Ser
Leu Thr Ser Gln Thr Val Thr Glu Leu Pro Pro 515 520 525 His Thr Val
Glu Gly Thr Ser Ala Ser Leu Asn Asp Gly Ser Lys Thr 530 535 540 Val
Leu Arg Ser Pro His Met Asn Leu Ser Gly Thr Ala Glu Ser Leu 545 550
555 560 Asn Thr Val Ser Ile Thr Glu Tyr Glu Glu Glu Ser Leu Leu Thr
Ser 565 570 575 Phe Lys Leu Asp Thr Gly Ala Glu Asp Ser Ser Gly Ser
Ser Pro Ala 580 585 590 Thr Ser Ala Ile Pro Phe Ile Ser Glu Asn Ile
Ser Gln Gly Tyr Ile 595 600 605 Phe Ser Ser Glu Asn Pro Glu Thr Ile
Thr Tyr Asp Val Leu Ile Pro 610 615 620 Glu Ser Ala Arg Asn Ala Ser
Glu Asp Ser Thr Ser Ser Gly Ser Glu 625 630 635 640 Glu Ser Leu Lys
Asp Pro Ser Met Glu Gly Asn Val Trp Phe Pro Ser 645 650 655 Ser Thr
Asp Ile Thr Ala Gln Pro Asp Val Gly Ser Gly Arg Glu Ser 660 665 670
Phe Leu Gln Thr Asn Tyr Thr Glu Ile Arg Val Asp Glu Ser Glu Lys 675
680 685 Thr Thr Lys Ser Phe Ser Ala Gly Pro Val Met Ser Gln Gly Pro
Ser 690 695 700 Val Thr Asp Leu Glu Met Pro His Tyr Ser Thr Phe Ala
Tyr Phe Pro 705 710 715 720 Thr Glu Val Thr Pro His Ala Phe Thr Pro
Ser Ser Arg Gln Gln Asp 725 730 735 Leu Val Ser Thr Val Asn Val Val
Tyr Ser Gln Thr Thr Gln Pro Val 740 745 750 Tyr Asn Gly Glu Thr Pro
Leu Gln Pro Ser Tyr Ser Ser Glu Val Phe 755 760 765 Pro Leu Val Thr
Pro Leu Leu Leu Asp Asn Gln Ile Leu Asn Thr Thr 770 775 780 Pro Ala
Ala Ser Ser Ser Asp Ser Ala Leu His Ala Thr Pro Val Phe 785 790 795
800 Pro Ser Val Asp Val Ser Phe Glu Ser Ile Leu Ser Ser Tyr Asp Gly
805 810 815 Ala Pro Leu Leu Pro Phe Ser Ser Ala Ser Phe Ser Ser Glu
Leu Phe 820 825 830 Arg His Leu His Thr Val Ser Gln Ile Leu Pro Gln
Val Thr Ser Ala 835 840 845 Thr Glu Ser Asp Lys Val Pro Leu His Ala
Ser Leu Pro Val Ala Gly 850 855 860 Gly Asp Leu Leu Leu Glu Pro Ser
Leu Ala Gln Tyr Ser Asp Val Leu 865 870 875 880 Ser Thr Thr His Ala
Ala Ser Glu Thr Leu Glu Phe Gly Ser Glu Ser 885 890 895 Gly Val Leu
Tyr Lys Thr Leu Met Phe Ser Gln Val Glu Pro Pro Ser 900 905 910 Ser
Asp Ala Met Met His Ala Arg Ser Ser Gly Pro Glu Pro Ser Tyr 915 920
925 Ala Leu Ser Asp Asn Glu Gly Ser Gln His Ile Phe Thr Val Ser Tyr
930 935 940 Ser Ser Ala Ile Pro Val His Asp Ser Val Gly Val Thr Tyr
Gln Gly 945 950 955 960 Ser Leu Phe Ser Gly Pro Ser His Ile Pro Ile
Pro Lys Ser Ser Leu 965 970 975 Ile Thr Pro Thr Ala Ser Leu Leu Gln
Pro Thr His Ala Leu Ser Gly 980 985 990 Asp Gly Glu Trp Ser Gly Ala
Ser Ser Asp Ser Glu Phe Leu Leu Pro 995 1000 1005 Asp Thr Asp Gly
Leu Thr Ala Leu Asn Ile Ser Ser Pro Val Ser 1010 1015 1020 Val Ala
Glu Phe Thr Tyr Thr Thr Ser Val Phe Gly Asp Asp Asn 1025 1030 1035
Lys Ala Leu Ser Lys Ser Glu Ile Ile Tyr Gly Asn Glu Thr Glu 1040
1045 1050 Leu Gln Ile Pro Ser Phe Asn Glu Met Val Tyr Pro Ser Glu
Ser 1055 1060 1065 Thr Val Met Pro Asn Met Tyr Asp Asn Val Asn Lys
Leu Asn Ala 1070 1075 1080 Ser Leu Gln Glu Thr Ser Val Ser Ile Ser
Ser Thr Lys Gly Met 1085 1090 1095 Phe Pro Gly Ser Leu Ala His Thr
Thr Thr Lys Val Phe Asp His 1100 1105 1110 Glu Ile Ser Gln Val Pro
Glu Asn Asn Phe Ser Val Gln Pro Thr 1115 1120 1125 His Thr Val Ser
Gln Ala Ser Gly Asp Thr Ser Leu Lys Pro Val 1130 1135 1140 Leu Ser
Ala Asn Ser Glu Pro Ala Ser Ser Asp Pro Ala Ser Ser 1145 1150 1155
Glu Met Leu Ser Pro Ser Thr Gln Leu Leu Phe Tyr Glu Thr Ser 1160
1165 1170 Ala Ser Phe Ser Thr Glu Val Leu Leu Gln Pro Ser Phe Gln
Ala 1175 1180 1185 Ser Asp Val Asp Thr Leu Leu Lys Thr Val Leu Pro
Ala Val Pro 1190 1195 1200 Ser Asp Pro Ile Leu Val Glu Thr Pro Lys
Val Asp Lys Ile Ser 1205 1210 1215 Ser Thr Met Leu His Leu Ile Val
Ser Asn Ser Ala Ser Ser Glu 1220 1225 1230 Asn Met Leu His Ser Thr
Ser Val Pro Val Phe Asp Val Ser Pro 1235 1240 1245 Thr Ser His Met
His Ser Ala Ser Leu Gln Gly Leu Thr Ile Ser 1250 1255 1260 Tyr Ala
Ser Glu Lys Tyr Glu Pro Val Leu Leu Lys Ser Glu Ser 1265 1270 1275
Ser His Gln Val Val Pro Ser Leu Tyr Ser Asn Asp Glu Leu Phe 1280
1285 1290 Gln Thr Ala Asn Leu Glu Ile Asn Gln Ala His Pro Pro Lys
Gly 1295 1300 1305 Arg His Val Phe Ala Thr Pro Val Leu Ser Ile Asp
Glu Pro Leu 1310 1315 1320 Asn Thr Leu Ile Asn Lys Leu Ile His Ser
Asp Glu Ile Leu Thr 1325 1330 1335 Ser Thr Lys Ser Ser Val Thr Gly
Lys Val Phe Ala Gly Ile Pro 1340 1345 1350 Thr Val Ala Ser Asp Thr
Phe Val Ser Thr Asp His Ser Val Pro 1355 1360 1365 Ile Gly Asn Gly
His Val Ala Ile Thr Ala Val Ser Pro His Arg 1370 1375 1380 Asp Gly
Ser Val Thr Ser Thr Lys Leu Leu Phe Pro Ser Lys Ala 1385 1390 1395
Thr Ser Glu Leu Ser His Ser Ala Lys Ser Asp Ala Gly Leu Val 1400
1405 1410 Gly Gly Gly Glu Asp Gly Asp Thr Asp Asp Asp Gly Asp Asp
Asp 1415 1420 1425 Asp Asp Arg Asp Ser Asp Gly Leu Ser Ile His Lys
Cys Met Ser 1430 1435 1440 Cys Ser Ser Tyr Arg Glu Ser Gln Glu Lys
Val Met Asn Asp Ser 1445 1450 1455 Asp Thr His Glu Asn Ser Leu Met
Asp Gln Asn Asn Pro Ile Ser 1460 1465 1470 Tyr Ser Leu Ser Glu Asn
Ser Glu Glu Asp Asn Arg Val Thr Ser 1475 1480 1485 Val Ser Ser Asp
Ser Gln Thr Gly Met Asp Arg Ser Pro Gly Lys 1490 1495 1500 Ser Pro
Ser Ala Asn Gly Leu Ser Gln Lys His Asn Asp Gly Lys 1505 1510 1515
Glu Glu Asn Asp Ile Gln Thr Gly Ser Ala Leu Leu Pro Leu Ser 1520
1525 1530 Pro Glu Ser Lys Ala Trp Ala Val Leu Thr Ser Asp Glu Glu
Ser 1535 1540 1545 Gly Ser Gly Gln Gly Thr Ser Asp Ser Leu Asn Glu
Asn Glu Thr 1550 1555 1560 Ser Thr Asp Phe Ser Phe Ala Asp Thr Asn
Glu Lys Asp Ala Asp 1565 1570 1575 Gly Ile Leu Ala Ala Gly Asp Ser
Glu Ile Thr Pro Gly Phe Pro 1580 1585 1590 Gln Ser Pro Thr Ser Ser
Val Thr Ser Glu Asn Ser Glu Val Phe 1595 1600 1605 His Val Ser Glu
Ala Glu Ala Ser Asn Ser Ser His Glu Ser Arg 1610 1615 1620 Ile Gly
Leu Ala Glu Gly Leu Glu Ser Glu Lys Lys Ala Val Ile 1625 1630 1635
Pro Leu Val Ile Val Ser Ala Leu Thr Phe Ile Cys Leu Val Val 1640
1645 1650 Leu Val Gly Ile Leu Ile Tyr Trp Arg Lys Cys Phe Gln Thr
Ala 1655 1660 1665 His Phe Tyr Leu Glu Asp Ser Thr Ser Pro Arg Val
Ile Ser Thr 1670 1675 1680 Pro Pro Thr Pro Ile Phe Pro Ile Ser Asp
Asp Val Gly Ala Ile 1685 1690 1695 Pro Ile Lys His Phe Pro Lys His
Val Ala Asp Leu His Ala Ser 1700 1705 1710 Ser Gly Phe Thr Glu Glu
Phe Glu Thr Leu Lys Glu Phe Tyr Gln 1715 1720 1725 Glu Val Gln Ser
Cys Thr Val Asp Leu Gly Ile Thr Ala Asp Ser 1730 1735 1740 Ser Asn
His Pro Asp Asn Lys His Lys Asn Arg Tyr Ile Asn Ile 1745 1750 1755
Val Ala Tyr Asp His Ser Arg Val Lys Leu Ala Gln Leu Ala Glu 1760
1765 1770 Lys Asp Gly Lys Leu Thr Asp Tyr Ile Asn Ala Asn Tyr Val
Asp 1775 1780 1785 Gly Tyr Asn Arg Pro Lys Ala Tyr Ile Ala Ala Gln
Gly Pro Leu 1790 1795 1800 Lys Ser Thr Ala Glu Asp Phe Trp Arg Met
Ile Trp Glu His Asn 1805 1810 1815 Val Glu Val Ile Val Met Ile Thr
Asn Leu Val Glu Lys Gly Arg 1820 1825 1830 Arg Lys Cys Asp Gln Tyr
Trp Pro Ala Asp Gly Ser Glu Glu Tyr 1835 1840 1845 Gly Asn Phe Leu
Val Thr Gln Lys Ser Val Gln Val Leu Ala Tyr 1850 1855 1860 Tyr Thr
Val Arg Asn Phe Thr Leu Arg Asn Thr Lys Ile Lys Lys 1865 1870 1875
Gly Ser Gln Lys Gly Arg Pro Ser Gly Arg Val Val Thr Gln Tyr 1880
1885 1890 His Tyr Thr Gln Trp Pro Asp Met Gly Val Pro Glu Tyr Ser
Leu 1895 1900 1905 Pro Val Leu Thr Phe Val Arg Lys Ala Ala Tyr Ala
Lys Arg His 1910 1915 1920 Ala Val Gly Pro Val Val Val His Cys Ser
Ala Gly Val Gly Arg 1925 1930 1935 Thr Gly Thr Tyr Ile Val Leu Asp
Ser Met Leu Gln Gln Ile Gln 1940 1945 1950 His Glu Gly Thr Val Asn
Ile Phe Gly Phe Leu Lys His Ile Arg 1955 1960 1965 Ser Gln Arg Asn
Tyr Leu Val Gln Thr Glu Glu Gln Tyr Val Phe 1970 1975 1980 Ile His
Asp Thr Leu Val Glu Ala Ile Leu Ser Lys Glu Thr Glu 1985 1990 1995
Val Leu Asp Ser His Ile His Ala Tyr Val Asn Ala Leu Leu Ile 2000
2005 2010 Pro Gly Pro Ala Gly Lys Thr Lys Leu Glu Lys Gln Phe Gln
Gly 2015 2020 2025 Leu Thr Leu Ser Pro Arg Leu Glu Cys Arg Gly Thr
Ile Ser Ala 2030 2035 2040 His Cys Asn Leu Pro Leu Pro Gly Leu Thr
Asp Pro Pro Thr Ser 2045 2050 2055 Ala Ser Arg Val Ala Gly Thr Ile
Leu Leu Ser Gln Ser Asn Ile 2060 2065 2070 Gln Gln Ser Asp Tyr Ser
Ala Ala Leu Lys Gln Cys Asn Arg Glu 2075 2080 2085 Lys Asn Arg Thr
Ser Ser Ile Ile Pro Val Glu Arg Ser Arg Val 2090 2095 2100 Gly Ile
Ser Ser Leu Ser Gly Glu Gly Thr Asp Tyr Ile Asn Ala 2105 2110 2115
Ser Tyr Ile Met Gly Tyr Tyr Gln Ser Asn Glu Phe Ile Ile Thr 2120
2125 2130 Gln His Pro Leu Leu His Thr Ile Lys Asp Phe Trp Arg Met
Ile 2135 2140 2145 Trp Asp His Asn Ala Gln Leu Val Val Met Ile Pro
Asp Gly Gln 2150 2155 2160 Asn Met Ala Glu Asp Glu Phe Val Tyr Trp
Pro Asn Lys Asp Glu 2165 2170 2175 Pro Ile Asn Cys Glu Ser Phe Lys
Val Thr Leu Met Ala Glu Glu 2180 2185 2190 His Lys Cys Leu Ser Asn
Glu Glu Lys Leu Ile Ile Gln Asp Phe 2195 2200 2205 Ile Leu Glu
Ala
Thr Gln Asp Asp Tyr Val Leu Glu Val Arg His 2210 2215 2220 Phe Gln
Cys Pro Lys Trp Pro Asn Pro Asp Ser Pro Ile Ser Lys 2225 2230 2235
Thr Phe Glu Leu Ile Ser Val Ile Lys Glu Glu Ala Ala Asn Arg 2240
2245 2250 Asp Gly Pro Met Ile Val His Asp Glu His Gly Gly Val Thr
Ala 2255 2260 2265 Gly Thr Phe Cys Ala Leu Thr Thr Leu Met His Gln
Leu Glu Lys 2270 2275 2280 Glu Asn Ser Val Asp Val Tyr Gln Val Ala
Lys Met Ile Asn Leu 2285 2290 2295 Met Arg Pro Gly Val Phe Ala Asp
Ile Glu Gln Tyr Gln Phe Leu 2300 2305 2310 Tyr Lys Val Ile Leu Ser
Leu Val Ser Thr Arg Gln Glu Glu Asn 2315 2320 2325 Pro Ser Thr Ser
Leu Asp Ser Asn Gly Ala Ala Leu Pro Asp Gly 2330 2335 2340 Asn Ile
Ala Glu Ser Leu Glu Ser Leu Val 2345 2350 5 7941 DNA Homo sapiens
CDS (148)..(7092) 5 cacacatacg cacgcacgat ctcacttcga tctatacact
ggaggattaa aacaaacaaa 60 caaaaaaaac atttccttcg ctccccctcc
ctctccactc tgagaagcag aggagccgca 120 cggcgagggg ccgcagaccg tctggaa
atg cga atc cta aag cgt ttc ctc gct 174 Met Arg Ile Leu Lys Arg Phe
Leu Ala 1 5 tgc att cag ctc ctc tgt gtt tgc cgc ctg gat tgg gct aat
gga tac 222 Cys Ile Gln Leu Leu Cys Val Cys Arg Leu Asp Trp Ala Asn
Gly Tyr 10 15 20 25 tac aga caa cag aga aaa ctt gtt gaa gag att ggc
tgg tcc tat aca 270 Tyr Arg Gln Gln Arg Lys Leu Val Glu Glu Ile Gly
Trp Ser Tyr Thr 30 35 40 gga gca ctg aat caa aaa aat tgg gga aag
aaa tat cca aca tgt aat 318 Gly Ala Leu Asn Gln Lys Asn Trp Gly Lys
Lys Tyr Pro Thr Cys Asn 45 50 55 agc cca aaa caa tct cct atc aat
att gat gaa gat ctt aca caa gta 366 Ser Pro Lys Gln Ser Pro Ile Asn
Ile Asp Glu Asp Leu Thr Gln Val 60 65 70 aat gtg aat ctt aag aaa
ctt aaa ttt cag ggt tgg gat aaa aca tca 414 Asn Val Asn Leu Lys Lys
Leu Lys Phe Gln Gly Trp Asp Lys Thr Ser 75 80 85 ttg gaa aac aca
ttc att cat aac act ggg aaa aca gtg gaa att aat 462 Leu Glu Asn Thr
Phe Ile His Asn Thr Gly Lys Thr Val Glu Ile Asn 90 95 100 105 ctc
act aat gac tac cgt gtc agc gga gga gtt tca gaa atg gtg ttt 510 Leu
Thr Asn Asp Tyr Arg Val Ser Gly Gly Val Ser Glu Met Val Phe 110 115
120 aaa gca agc aag ata act ttt cac tgg gga aaa tgc aat atg tca tct
558 Lys Ala Ser Lys Ile Thr Phe His Trp Gly Lys Cys Asn Met Ser Ser
125 130 135 gat gga tca gag cat agt tta gaa gga caa aaa ttt cca ctt
gag atg 606 Asp Gly Ser Glu His Ser Leu Glu Gly Gln Lys Phe Pro Leu
Glu Met 140 145 150 caa atc tac tgc ttt gat gcg gac cga ttt tca agt
ttt gag gaa gca 654 Gln Ile Tyr Cys Phe Asp Ala Asp Arg Phe Ser Ser
Phe Glu Glu Ala 155 160 165 gtc aaa gga aaa ggg aag tta aga gct tta
tcc att ttg ttt gag gtt 702 Val Lys Gly Lys Gly Lys Leu Arg Ala Leu
Ser Ile Leu Phe Glu Val 170 175 180 185 ggg aca gaa gaa aat ttg gat
ttc aaa gcg att att gat gga gtc gaa 750 Gly Thr Glu Glu Asn Leu Asp
Phe Lys Ala Ile Ile Asp Gly Val Glu 190 195 200 agt gtt agt cgt ttt
ggg aag cag gct gct tta gat cca ttc ata ctg 798 Ser Val Ser Arg Phe
Gly Lys Gln Ala Ala Leu Asp Pro Phe Ile Leu 205 210 215 ttg aac ctt
ctg cca aac tca act gac aag tat tac att tac aat ggc 846 Leu Asn Leu
Leu Pro Asn Ser Thr Asp Lys Tyr Tyr Ile Tyr Asn Gly 220 225 230 tca
ttg aca tct cct ccc tgc aca gac aca gtt gac tgg att gtt ttt 894 Ser
Leu Thr Ser Pro Pro Cys Thr Asp Thr Val Asp Trp Ile Val Phe 235 240
245 aaa gat aca gtt agc atc tct gaa agc cag ttg gct gtt ttt tgt gaa
942 Lys Asp Thr Val Ser Ile Ser Glu Ser Gln Leu Ala Val Phe Cys Glu
250 255 260 265 gtt ctt aca atg caa caa tct ggt tat gtc atg ctg atg
gac tac tta 990 Val Leu Thr Met Gln Gln Ser Gly Tyr Val Met Leu Met
Asp Tyr Leu 270 275 280 caa aac aat ttt cga gag caa cag tac aag ttc
tct aga cag gtg ttt 1038 Gln Asn Asn Phe Arg Glu Gln Gln Tyr Lys
Phe Ser Arg Gln Val Phe 285 290 295 tcc tca tac act gga aag gaa gag
att cat gaa gca gtt tgt agt tca 1086 Ser Ser Tyr Thr Gly Lys Glu
Glu Ile His Glu Ala Val Cys Ser Ser 300 305 310 gaa cca gaa aat gtt
cag gct gac cca gag aat tat acc agc ctt ctt 1134 Glu Pro Glu Asn
Val Gln Ala Asp Pro Glu Asn Tyr Thr Ser Leu Leu 315 320 325 gtt aca
tgg gaa aga cct cga gtc gtt tat gat acc atg att gag aag 1182 Val
Thr Trp Glu Arg Pro Arg Val Val Tyr Asp Thr Met Ile Glu Lys 330 335
340 345 ttt gca gtt ttg tac cag cag ttg gat gga gag gac caa acc aag
cat 1230 Phe Ala Val Leu Tyr Gln Gln Leu Asp Gly Glu Asp Gln Thr
Lys His 350 355 360 gaa ttt ttg aca gat ggc tat caa gac ttg ggt gct
att ctc aat aat 1278 Glu Phe Leu Thr Asp Gly Tyr Gln Asp Leu Gly
Ala Ile Leu Asn Asn 365 370 375 ttg cta ccc aat atg agt tat gtt ctt
cag ata gta gcc ata tgc act 1326 Leu Leu Pro Asn Met Ser Tyr Val
Leu Gln Ile Val Ala Ile Cys Thr 380 385 390 aat ggc tta tat gga aaa
tac agc gac caa ctg att gtc gac atg cct 1374 Asn Gly Leu Tyr Gly
Lys Tyr Ser Asp Gln Leu Ile Val Asp Met Pro 395 400 405 act gat aat
cct gaa ctt gat ctt ttc cct gaa tta att gga act gaa 1422 Thr Asp
Asn Pro Glu Leu Asp Leu Phe Pro Glu Leu Ile Gly Thr Glu 410 415 420
425 gaa ata atc aag gag gag gaa gag gga aaa gac att gaa gaa ggc gct
1470 Glu Ile Ile Lys Glu Glu Glu Glu Gly Lys Asp Ile Glu Glu Gly
Ala 430 435 440 att gtg aat cct ggt aga gac agt gct aca aac caa atc
agg aaa aag 1518 Ile Val Asn Pro Gly Arg Asp Ser Ala Thr Asn Gln
Ile Arg Lys Lys 445 450 455 gaa ccc cag att tct acc aca aca cac tac
aat cgc ata ggg acg aaa 1566 Glu Pro Gln Ile Ser Thr Thr Thr His
Tyr Asn Arg Ile Gly Thr Lys 460 465 470 tac aat gaa gcc aag act aac
cga tcc cca aca aga gga agt gaa ttc 1614 Tyr Asn Glu Ala Lys Thr
Asn Arg Ser Pro Thr Arg Gly Ser Glu Phe 475 480 485 tct gga aag ggt
gat gtt ccc aat aca tct tta aat tcc act tcc caa 1662 Ser Gly Lys
Gly Asp Val Pro Asn Thr Ser Leu Asn Ser Thr Ser Gln 490 495 500 505
cca gtc act aaa tta gcc aca gaa aaa gat att tcc ttg act tct cag
1710 Pro Val Thr Lys Leu Ala Thr Glu Lys Asp Ile Ser Leu Thr Ser
Gln 510 515 520 act gtg act gaa ctg cca cct cac act gtg gaa ggt act
tca gcc tct 1758 Thr Val Thr Glu Leu Pro Pro His Thr Val Glu Gly
Thr Ser Ala Ser 525 530 535 tta aat gat ggc tct aaa act gtt ctt aga
tct cca cat atg aac ttg 1806 Leu Asn Asp Gly Ser Lys Thr Val Leu
Arg Ser Pro His Met Asn Leu 540 545 550 tcg ggg act gca gaa tcc tta
aat aca gtt tct ata aca gaa tat gag 1854 Ser Gly Thr Ala Glu Ser
Leu Asn Thr Val Ser Ile Thr Glu Tyr Glu 555 560 565 gag gag agt tta
ttg acc agt ttc aag ctt gat act gga gct gaa gat 1902 Glu Glu Ser
Leu Leu Thr Ser Phe Lys Leu Asp Thr Gly Ala Glu Asp 570 575 580 585
tct tca ggc tcc agt ccc gca act tct gct atc cca ttc atc tct gag
1950 Ser Ser Gly Ser Ser Pro Ala Thr Ser Ala Ile Pro Phe Ile Ser
Glu 590 595 600 aac ata tcc caa ggg tat ata ttt tcc tcc gaa aac cca
gag aca ata 1998 Asn Ile Ser Gln Gly Tyr Ile Phe Ser Ser Glu Asn
Pro Glu Thr Ile 605 610 615 aca tat gat gtc ctt ata cca gaa tct gct
aga aat gct tcc gaa gat 2046 Thr Tyr Asp Val Leu Ile Pro Glu Ser
Ala Arg Asn Ala Ser Glu Asp 620 625 630 tca act tca tca ggt tca gaa
gaa tca cta aag gat cct tct atg gag 2094 Ser Thr Ser Ser Gly Ser
Glu Glu Ser Leu Lys Asp Pro Ser Met Glu 635 640 645 gga aat gtg tgg
ttt cct agc tct aca gac ata aca gca cag ccc gat 2142 Gly Asn Val
Trp Phe Pro Ser Ser Thr Asp Ile Thr Ala Gln Pro Asp 650 655 660 665
gtt gga tca ggc aga gag agc ttt ctc cag act aat tac act gag ata
2190 Val Gly Ser Gly Arg Glu Ser Phe Leu Gln Thr Asn Tyr Thr Glu
Ile 670 675 680 cgt gtt gat gaa tct gag aag aca acc aag tcc ttt tct
gca ggc cca 2238 Arg Val Asp Glu Ser Glu Lys Thr Thr Lys Ser Phe
Ser Ala Gly Pro 685 690 695 gtg atg tca cag ggt ccc tca gtt aca gat
ctg gaa atg cca cat tat 2286 Val Met Ser Gln Gly Pro Ser Val Thr
Asp Leu Glu Met Pro His Tyr 700 705 710 tct acc ttt gcc tac ttc cca
act gag gta aca cct cat gct ttt acc 2334 Ser Thr Phe Ala Tyr Phe
Pro Thr Glu Val Thr Pro His Ala Phe Thr 715 720 725 cca tcc tcc aga
caa cag gat ttg gtc tcc acg gtc aac gtg gta tac 2382 Pro Ser Ser
Arg Gln Gln Asp Leu Val Ser Thr Val Asn Val Val Tyr 730 735 740 745
tcg cag aca acc caa ccg gta tac aat ggt gag aca cct ctt caa cct
2430 Ser Gln Thr Thr Gln Pro Val Tyr Asn Gly Glu Thr Pro Leu Gln
Pro 750 755 760 tcc tac agt agt gaa gtc ttt cct cta gtc acc cct ttg
ttg ctt gac 2478 Ser Tyr Ser Ser Glu Val Phe Pro Leu Val Thr Pro
Leu Leu Leu Asp 765 770 775 aat cag atc ctc aac act acc cct gct gct
tca agt agt gat tcg gcc 2526 Asn Gln Ile Leu Asn Thr Thr Pro Ala
Ala Ser Ser Ser Asp Ser Ala 780 785 790 ttg cat gct acg cct gta ttt
ccc agt gtc gat gtg tca ttt gaa tcc 2574 Leu His Ala Thr Pro Val
Phe Pro Ser Val Asp Val Ser Phe Glu Ser 795 800 805 atc ctg tct tcc
tat gat ggt gca cct ttg ctt cca ttt tcc tct gct 2622 Ile Leu Ser
Ser Tyr Asp Gly Ala Pro Leu Leu Pro Phe Ser Ser Ala 810 815 820 825
tcc ttc agt agt gaa ttg ttt cgc cat ctg cat aca gtt tct caa atc
2670 Ser Phe Ser Ser Glu Leu Phe Arg His Leu His Thr Val Ser Gln
Ile 830 835 840 ctt cca caa gtt act tca gct acc gag agt gat aag gtg
ccc ttg cat 2718 Leu Pro Gln Val Thr Ser Ala Thr Glu Ser Asp Lys
Val Pro Leu His 845 850 855 gct tct ctg cca gtg gct ggg ggt gat ttg
cta tta gag ccc agc ctt 2766 Ala Ser Leu Pro Val Ala Gly Gly Asp
Leu Leu Leu Glu Pro Ser Leu 860 865 870 gct cag tat tct gat gtg ctg
tcc act act cat gct gct tca gag acg 2814 Ala Gln Tyr Ser Asp Val
Leu Ser Thr Thr His Ala Ala Ser Glu Thr 875 880 885 ctg gaa ttt ggt
agt gaa tct ggt gtt ctt tat aaa acg ctt atg ttt 2862 Leu Glu Phe
Gly Ser Glu Ser Gly Val Leu Tyr Lys Thr Leu Met Phe 890 895 900 905
tct caa gtt gaa cca ccc agc agt gat gcc atg atg cat gca cgt tct
2910 Ser Gln Val Glu Pro Pro Ser Ser Asp Ala Met Met His Ala Arg
Ser 910 915 920 tca ggg cct gaa cct tct tat gcc ttg tct gat aat gag
ggc tcc caa 2958 Ser Gly Pro Glu Pro Ser Tyr Ala Leu Ser Asp Asn
Glu Gly Ser Gln 925 930 935 cac atc ttc act gtt tct tac agt tct gca
ata cct gtg cat gat tct 3006 His Ile Phe Thr Val Ser Tyr Ser Ser
Ala Ile Pro Val His Asp Ser 940 945 950 gtg ggt gta act tat cag ggt
tcc tta ttt agc ggc cct agc cat ata 3054 Val Gly Val Thr Tyr Gln
Gly Ser Leu Phe Ser Gly Pro Ser His Ile 955 960 965 cca ata cct aag
tct tcg tta ata acc cca act gca tca tta ctg cag 3102 Pro Ile Pro
Lys Ser Ser Leu Ile Thr Pro Thr Ala Ser Leu Leu Gln 970 975 980 985
cct act cat gcc ctc tct ggt gat ggg gaa tgg tct gga gcc tct tct
3150 Pro Thr His Ala Leu Ser Gly Asp Gly Glu Trp Ser Gly Ala Ser
Ser 990 995 1000 gat agt gaa ttt ctt tta cct gac aca gat ggg ctg
aca gcc ctt 3195 Asp Ser Glu Phe Leu Leu Pro Asp Thr Asp Gly Leu
Thr Ala Leu 1005 1010 1015 aac att tct tca cct gtt tct gta gct gaa
ttt aca tat aca aca 3240 Asn Ile Ser Ser Pro Val Ser Val Ala Glu
Phe Thr Tyr Thr Thr 1020 1025 1030 tct gtg ttt ggt gat gat aat aag
gcg ctt tct aaa agt gaa ata 3285 Ser Val Phe Gly Asp Asp Asn Lys
Ala Leu Ser Lys Ser Glu Ile 1035 1040 1045 ata tat gga aat gag act
gaa ctg caa att cct tct ttc aat gag 3330 Ile Tyr Gly Asn Glu Thr
Glu Leu Gln Ile Pro Ser Phe Asn Glu 1050 1055 1060 atg gtt tac cct
tct gaa agc aca gtc atg ccc aac atg tat gat 3375 Met Val Tyr Pro
Ser Glu Ser Thr Val Met Pro Asn Met Tyr Asp 1065 1070 1075 aat gta
aat aag ttg aat gcg tct tta caa gaa acc tct gtt tcc 3420 Asn Val
Asn Lys Leu Asn Ala Ser Leu Gln Glu Thr Ser Val Ser 1080 1085 1090
att tct agc acc aag ggc atg ttt cca ggg tcc ctt gct cat acc 3465
Ile Ser Ser Thr Lys Gly Met Phe Pro Gly Ser Leu Ala His Thr 1095
1100 1105 acc act aag gtt ttt gat cat gag att agt caa gtt cca gaa
aat 3510 Thr Thr Lys Val Phe Asp His Glu Ile Ser Gln Val Pro Glu
Asn 1110 1115 1120 aac ttt tca gtt caa cct aca cat act gtc tct caa
gca tct ggt 3555 Asn Phe Ser Val Gln Pro Thr His Thr Val Ser Gln
Ala Ser Gly 1125 1130 1135 gac act tcg ctt aaa cct gtg ctt agt gca
aac tca gag cca gca 3600 Asp Thr Ser Leu Lys Pro Val Leu Ser Ala
Asn Ser Glu Pro Ala 1140 1145 1150 tcc tct gac cct gct tct agt gaa
atg tta tct cct tca act cag 3645 Ser Ser Asp Pro Ala Ser Ser Glu
Met Leu Ser Pro Ser Thr Gln 1155 1160 1165 ctc tta ttt tat gag acc
tca gct tct ttt agt act gaa gta ttg 3690 Leu Leu Phe Tyr Glu Thr
Ser Ala Ser Phe Ser Thr Glu Val Leu 1170 1175 1180 cta caa cct tcc
ttt cag gct tct gat gtt gac acc ttg ctt aaa 3735 Leu Gln Pro Ser
Phe Gln Ala Ser Asp Val Asp Thr Leu Leu Lys 1185 1190 1195 act gtt
ctt cca gct gtg ccc agt gat cca ata ttg gtt gaa acc 3780 Thr Val
Leu Pro Ala Val Pro Ser Asp Pro Ile Leu Val Glu Thr 1200 1205 1210
ccc aaa gtt gat aaa att agt tct aca atg ttg cat ctc att gta 3825
Pro Lys Val Asp Lys Ile Ser Ser Thr Met Leu His Leu Ile Val 1215
1220 1225 tca aat tct gct tca agt gaa aac atg ctg cac tct aca tct
gta 3870 Ser Asn Ser Ala Ser Ser Glu Asn Met Leu His Ser Thr Ser
Val 1230 1235 1240 cca gtt ttt gat gtg tcg cct act tct cat atg cac
tct gct tca 3915 Pro Val Phe Asp Val Ser Pro Thr Ser His Met His
Ser Ala Ser 1245 1250 1255 ctt caa ggt ttg acc att tcc tat gca agt
gag aaa tat gaa cca 3960 Leu Gln Gly Leu Thr Ile Ser Tyr Ala Ser
Glu Lys Tyr Glu Pro 1260 1265 1270 gtt ttg tta aaa agt gaa agt tcc
cac caa gtg gta cct tct ttg 4005 Val Leu Leu Lys Ser Glu Ser Ser
His Gln Val Val Pro Ser Leu 1275 1280 1285 tac agt aat gat gag ttg
ttc caa acg gcc aat ttg gag att aac 4050 Tyr Ser Asn Asp Glu Leu
Phe Gln Thr Ala Asn Leu Glu Ile Asn 1290 1295 1300 cag gcc cat ccc
cca aaa gga agg cat gta ttt gct aca cct gtt 4095 Gln Ala His Pro
Pro Lys Gly Arg His Val Phe Ala Thr Pro Val 1305 1310 1315 tta tca
att gat gaa cca tta aat aca cta ata aat aag ctt ata 4140 Leu Ser
Ile Asp Glu Pro Leu Asn Thr Leu Ile Asn Lys Leu Ile 1320 1325 1330
cat tcc gat gaa att tta acc tcc acc aaa agt tct gtt act ggt 4185
His Ser Asp Glu Ile Leu Thr Ser Thr Lys Ser Ser Val Thr Gly 1335
1340 1345 aag gta ttt gct ggt att cca aca gtt gct tct gat aca ttt
gta 4230 Lys Val Phe Ala Gly Ile Pro Thr Val Ala Ser Asp Thr Phe
Val 1350 1355 1360 tct act gat cat tct gtt cct ata gga aat ggg cat
gtt gcc att 4275 Ser Thr Asp His Ser Val Pro Ile Gly Asn Gly His
Val Ala Ile 1365 1370 1375 aca gct gtt tct ccc
cac aga gat ggt tct gta acc tca aca aag 4320 Thr Ala Val Ser Pro
His Arg Asp Gly Ser Val Thr Ser Thr Lys 1380 1385 1390 ttg ctg ttt
cct tct aag gca act tct gag ctg agt cat agt gcc 4365 Leu Leu Phe
Pro Ser Lys Ala Thr Ser Glu Leu Ser His Ser Ala 1395 1400 1405 aaa
tct gat gcc ggt tta gtg ggt ggt ggt gaa gat ggt gac act 4410 Lys
Ser Asp Ala Gly Leu Val Gly Gly Gly Glu Asp Gly Asp Thr 1410 1415
1420 gat gat gat ggt gat gat gat gat gac aga gat agt gat ggc tta
4455 Asp Asp Asp Gly Asp Asp Asp Asp Asp Arg Asp Ser Asp Gly Leu
1425 1430 1435 tcc att cat aag tgt atg tca tgc tca tcc tat aga gaa
tca cag 4500 Ser Ile His Lys Cys Met Ser Cys Ser Ser Tyr Arg Glu
Ser Gln 1440 1445 1450 gaa aag gta atg aat gat tca gac acc cac gaa
aac agt ctt atg 4545 Glu Lys Val Met Asn Asp Ser Asp Thr His Glu
Asn Ser Leu Met 1455 1460 1465 gat cag aat aat cca atc tca tac tca
cta tct gag aat tct gaa 4590 Asp Gln Asn Asn Pro Ile Ser Tyr Ser
Leu Ser Glu Asn Ser Glu 1470 1475 1480 gaa gat aat aga gtc aca agt
gta tcc tca gac agt caa act ggt 4635 Glu Asp Asn Arg Val Thr Ser
Val Ser Ser Asp Ser Gln Thr Gly 1485 1490 1495 atg gac aga agt cct
ggt aaa tca cca tca gca aat ggg cta tcc 4680 Met Asp Arg Ser Pro
Gly Lys Ser Pro Ser Ala Asn Gly Leu Ser 1500 1505 1510 caa aag cac
aat gat gga aaa gag gaa aat gac att cag act ggt 4725 Gln Lys His
Asn Asp Gly Lys Glu Glu Asn Asp Ile Gln Thr Gly 1515 1520 1525 agt
gct ctg ctt cct ctc agc cct gaa tct aaa gca tgg gca gtt 4770 Ser
Ala Leu Leu Pro Leu Ser Pro Glu Ser Lys Ala Trp Ala Val 1530 1535
1540 ctg aca agt gat gaa gaa agt gga tca ggg caa ggt acc tca gat
4815 Leu Thr Ser Asp Glu Glu Ser Gly Ser Gly Gln Gly Thr Ser Asp
1545 1550 1555 agc ctt aat gag aat gag act tcc aca gat ttc agt ttt
gca gac 4860 Ser Leu Asn Glu Asn Glu Thr Ser Thr Asp Phe Ser Phe
Ala Asp 1560 1565 1570 act aat gaa aaa gat gct gat ggg atc ctg gca
gca ggt gac tca 4905 Thr Asn Glu Lys Asp Ala Asp Gly Ile Leu Ala
Ala Gly Asp Ser 1575 1580 1585 gaa ata act cct gga ttc cca cag tcc
cca aca tca tct gtt act 4950 Glu Ile Thr Pro Gly Phe Pro Gln Ser
Pro Thr Ser Ser Val Thr 1590 1595 1600 agc gag aac tca gaa gtg ttc
cac gtt tca gag gca gag gcc agt 4995 Ser Glu Asn Ser Glu Val Phe
His Val Ser Glu Ala Glu Ala Ser 1605 1610 1615 aat agt agc cat gag
tct cgt att ggt cta gct gag ggg ttg gaa 5040 Asn Ser Ser His Glu
Ser Arg Ile Gly Leu Ala Glu Gly Leu Glu 1620 1625 1630 tcc gag aag
aag gca gtt ata ccc ctt gtg atc gtg tca gcc ctg 5085 Ser Glu Lys
Lys Ala Val Ile Pro Leu Val Ile Val Ser Ala Leu 1635 1640 1645 act
ttt atc tgt cta gtg gtt ctt gtg ggt att ctc atc tac tgg 5130 Thr
Phe Ile Cys Leu Val Val Leu Val Gly Ile Leu Ile Tyr Trp 1650 1655
1660 agg aaa tgc ttc cag act gca cac ttt tac tta gag gac agt aca
5175 Arg Lys Cys Phe Gln Thr Ala His Phe Tyr Leu Glu Asp Ser Thr
1665 1670 1675 tcc cct aga gtt ata tcc aca cct cca aca cct atc ttt
cca att 5220 Ser Pro Arg Val Ile Ser Thr Pro Pro Thr Pro Ile Phe
Pro Ile 1680 1685 1690 tca gat gat gtc gga gca att cca ata aag cac
ttt cca aag cat 5265 Ser Asp Asp Val Gly Ala Ile Pro Ile Lys His
Phe Pro Lys His 1695 1700 1705 gtt gca gat tta cat gca agt agt ggg
ttt act gaa gaa ttt gag 5310 Val Ala Asp Leu His Ala Ser Ser Gly
Phe Thr Glu Glu Phe Glu 1710 1715 1720 aca ctg aaa gag ttt tac cag
gaa gtg cag agc tgt act gtt gac 5355 Thr Leu Lys Glu Phe Tyr Gln
Glu Val Gln Ser Cys Thr Val Asp 1725 1730 1735 tta ggt att aca gca
gac agc tcc aac cac cca gac aac aag cac 5400 Leu Gly Ile Thr Ala
Asp Ser Ser Asn His Pro Asp Asn Lys His 1740 1745 1750 aag aat cga
tac ata aat atc gtt gcc tat gat cat agc agg gtt 5445 Lys Asn Arg
Tyr Ile Asn Ile Val Ala Tyr Asp His Ser Arg Val 1755 1760 1765 aag
cta gca cag ctt gct gaa aag gat ggc aaa ctg act gat tat 5490 Lys
Leu Ala Gln Leu Ala Glu Lys Asp Gly Lys Leu Thr Asp Tyr 1770 1775
1780 atc aat gcc aat tat gtt gat ggc tac aac aga cca aaa gct tat
5535 Ile Asn Ala Asn Tyr Val Asp Gly Tyr Asn Arg Pro Lys Ala Tyr
1785 1790 1795 att gct gcc caa ggc cca ctg aaa tcc aca gct gaa gat
ttc tgg 5580 Ile Ala Ala Gln Gly Pro Leu Lys Ser Thr Ala Glu Asp
Phe Trp 1800 1805 1810 aga atg ata tgg gaa cat aat gtg gaa gtt att
gtc atg ata aca 5625 Arg Met Ile Trp Glu His Asn Val Glu Val Ile
Val Met Ile Thr 1815 1820 1825 aac ctc gtg gag aaa gga agg aga aaa
tgt gat cag tac tgg cct 5670 Asn Leu Val Glu Lys Gly Arg Arg Lys
Cys Asp Gln Tyr Trp Pro 1830 1835 1840 gcc gat ggg agt gag gag tac
ggg aac ttt ctg gtc act cag aag 5715 Ala Asp Gly Ser Glu Glu Tyr
Gly Asn Phe Leu Val Thr Gln Lys 1845 1850 1855 agt gtg caa gtg ctt
gcc tat tat act gtg agg aat ttt act cta 5760 Ser Val Gln Val Leu
Ala Tyr Tyr Thr Val Arg Asn Phe Thr Leu 1860 1865 1870 aga aac aca
aaa ata aaa aag ggc tcc cag aaa gga aga ccc agt 5805 Arg Asn Thr
Lys Ile Lys Lys Gly Ser Gln Lys Gly Arg Pro Ser 1875 1880 1885 gga
cgt gtg gtc aca cag tat cac tac acg cag tgg cct gac atg 5850 Gly
Arg Val Val Thr Gln Tyr His Tyr Thr Gln Trp Pro Asp Met 1890 1895
1900 gga gta cca gag tac tcc ctg cca gtg ctg acc ttt gtg aga aag
5895 Gly Val Pro Glu Tyr Ser Leu Pro Val Leu Thr Phe Val Arg Lys
1905 1910 1915 gca gcc tat gcc aag cgc cat gca gtg ggg cct gtt gtc
gtc cac 5940 Ala Ala Tyr Ala Lys Arg His Ala Val Gly Pro Val Val
Val His 1920 1925 1930 tgc agt gct gga gtt gga aga aca ggc aca tat
att gtg cta gac 5985 Cys Ser Ala Gly Val Gly Arg Thr Gly Thr Tyr
Ile Val Leu Asp 1935 1940 1945 agt atg ttg cag cag att caa cac gaa
gga act gtc aac ata ttt 6030 Ser Met Leu Gln Gln Ile Gln His Glu
Gly Thr Val Asn Ile Phe 1950 1955 1960 ggc ttc tta aaa cac atc cgt
tca caa aga aat tat ttg gta caa 6075 Gly Phe Leu Lys His Ile Arg
Ser Gln Arg Asn Tyr Leu Val Gln 1965 1970 1975 act gag gag caa tat
gtc ttc att cat gat aca ctg gtt gag gcc 6120 Thr Glu Glu Gln Tyr
Val Phe Ile His Asp Thr Leu Val Glu Ala 1980 1985 1990 ata ctt agt
aaa gaa act gag gtg ctg gac agt cat att cat gcc 6165 Ile Leu Ser
Lys Glu Thr Glu Val Leu Asp Ser His Ile His Ala 1995 2000 2005 tat
gtt aat gca ctc ctc att cct gga cca gca ggc aaa aca aag 6210 Tyr
Val Asn Ala Leu Leu Ile Pro Gly Pro Ala Gly Lys Thr Lys 2010 2015
2020 cta gag aaa caa ttc cag ctc ctg agc cag tca aat ata cag cag
6255 Leu Glu Lys Gln Phe Gln Leu Leu Ser Gln Ser Asn Ile Gln Gln
2025 2030 2035 agt gac tat tct gca gcc cta aag caa tgc aac agg gaa
aag aat 6300 Ser Asp Tyr Ser Ala Ala Leu Lys Gln Cys Asn Arg Glu
Lys Asn 2040 2045 2050 cga act tct tct atc atc cct gtg gaa aga tca
agg gtt ggc att 6345 Arg Thr Ser Ser Ile Ile Pro Val Glu Arg Ser
Arg Val Gly Ile 2055 2060 2065 tca tcc ctg agt gga gaa ggc aca gac
tac atc aat gcc tcc tat 6390 Ser Ser Leu Ser Gly Glu Gly Thr Asp
Tyr Ile Asn Ala Ser Tyr 2070 2075 2080 atc atg ggc tat tac cag agc
aat gaa ttc atc att acc cag cac 6435 Ile Met Gly Tyr Tyr Gln Ser
Asn Glu Phe Ile Ile Thr Gln His 2085 2090 2095 cct ctc ctt cat acc
atc aag gat ttc tgg agg atg ata tgg gac 6480 Pro Leu Leu His Thr
Ile Lys Asp Phe Trp Arg Met Ile Trp Asp 2100 2105 2110 cat aat gcc
caa ctg gtg gtt atg att cct gat ggc caa aac atg 6525 His Asn Ala
Gln Leu Val Val Met Ile Pro Asp Gly Gln Asn Met 2115 2120 2125 gca
gaa gat gaa ttt gtt tac tgg cca aat aaa gat gag cct ata 6570 Ala
Glu Asp Glu Phe Val Tyr Trp Pro Asn Lys Asp Glu Pro Ile 2130 2135
2140 aat tgt gag agc ttt aag gtc act ctt atg gct gaa gaa cac aaa
6615 Asn Cys Glu Ser Phe Lys Val Thr Leu Met Ala Glu Glu His Lys
2145 2150 2155 tgt cta tct aat gag gaa aaa ctt ata att cag gac ttt
atc tta 6660 Cys Leu Ser Asn Glu Glu Lys Leu Ile Ile Gln Asp Phe
Ile Leu 2160 2165 2170 gaa gct aca cag gat gat tat gta ctt gaa gtg
agg cac ttt cag 6705 Glu Ala Thr Gln Asp Asp Tyr Val Leu Glu Val
Arg His Phe Gln 2175 2180 2185 tgt cct aaa tgg cca aat cca gat agc
ccc att agt aaa act ttt 6750 Cys Pro Lys Trp Pro Asn Pro Asp Ser
Pro Ile Ser Lys Thr Phe 2190 2195 2200 gaa ctt ata agt gtt ata aaa
gaa gaa gct gcc aat agg gat ggg 6795 Glu Leu Ile Ser Val Ile Lys
Glu Glu Ala Ala Asn Arg Asp Gly 2205 2210 2215 cct atg att gtt cat
gat gag cat gga gga gtg acg gca gga act 6840 Pro Met Ile Val His
Asp Glu His Gly Gly Val Thr Ala Gly Thr 2220 2225 2230 ttc tgt gct
ctg aca acc ctt atg cac caa cta gaa aaa gaa aat 6885 Phe Cys Ala
Leu Thr Thr Leu Met His Gln Leu Glu Lys Glu Asn 2235 2240 2245 tcc
gtg gat gtt tac cag gta gcc aag atg atc aat ctg atg agg 6930 Ser
Val Asp Val Tyr Gln Val Ala Lys Met Ile Asn Leu Met Arg 2250 2255
2260 cca gga gtc ttt gct gac att gag cag tat cag ttt ctc tac aaa
6975 Pro Gly Val Phe Ala Asp Ile Glu Gln Tyr Gln Phe Leu Tyr Lys
2265 2270 2275 gtg atc ctc agc ctt gtg agc aca agg cag gaa gag aat
cca tcc 7020 Val Ile Leu Ser Leu Val Ser Thr Arg Gln Glu Glu Asn
Pro Ser 2280 2285 2290 acc tct ctg gac agt aat ggt gca gca ttg cct
gat gga aat ata 7065 Thr Ser Leu Asp Ser Asn Gly Ala Ala Leu Pro
Asp Gly Asn Ile 2295 2300 2305 gct gag agc tta gag tct tta gtt taa
cacagaaagg ggtgggggga 7112 Ala Glu Ser Leu Glu Ser Leu Val 2310
ctcacatctg agcattgttt tcctcttcct aaaattaggc aggaaaatca gtctagttct
7172 gttatctgtt gatttcccat cacctgacag taactttcat gacataggat
tctgccgcca 7232 aatttatatc attaacaatg tgtgcctttt tgcaagactt
gtaatttact tattatgttt 7292 gaactaaaat gattgaattt tacagtattt
ctaagaatgg aattgtggta tttttttctg 7352 tattgatttt aacagaaaat
ttcaatttat agaggttagg aattccaaac tacagaaaat 7412 gtttgttttt
agtgtcaaat ttttagctgt atttgtagca attatcaggt ttgctagaaa 7472
tataactttt aatacagtag cctgtaaata aaacactctt ccatatgata ttcaacattt
7532 tacaactgca gtattcacct aaagtagaaa taatctgtta cttattgtaa
atactgccct 7592 agtgtctcca tggaccaaat ttatatttat aattgtagat
ttttatattt tactactgag 7652 tcaagttttc tagttctgtg taattgttta
gtttaatgac gtagttcatt agctggtctt 7712 actctaccag ttttctgaca
ttgtattgtg ttacctaagt cattaacttt gtttcagcat 7772 gtaattttaa
cttttgtgga aaatagaaat accttcattt tgaaagaagt ttttatgaga 7832
ataacacctt accaaacatt gttcaaatgg tttttatcca aggaattgca aaaataaata
7892 taaatattgc cattaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 7941 6
2314 PRT Homo sapiens gene (1)..(2314) PTP-zeta 6 Met Arg Ile Leu
Lys Arg Phe Leu Ala Cys Ile Gln Leu Leu Cys Val 1 5 10 15 Cys Arg
Leu Asp Trp Ala Asn Gly Tyr Tyr Arg Gln Gln Arg Lys Leu 20 25 30
Val Glu Glu Ile Gly Trp Ser Tyr Thr Gly Ala Leu Asn Gln Lys Asn 35
40 45 Trp Gly Lys Lys Tyr Pro Thr Cys Asn Ser Pro Lys Gln Ser Pro
Ile 50 55 60 Asn Ile Asp Glu Asp Leu Thr Gln Val Asn Val Asn Leu
Lys Lys Leu 65 70 75 80 Lys Phe Gln Gly Trp Asp Lys Thr Ser Leu Glu
Asn Thr Phe Ile His 85 90 95 Asn Thr Gly Lys Thr Val Glu Ile Asn
Leu Thr Asn Asp Tyr Arg Val 100 105 110 Ser Gly Gly Val Ser Glu Met
Val Phe Lys Ala Ser Lys Ile Thr Phe 115 120 125 His Trp Gly Lys Cys
Asn Met Ser Ser Asp Gly Ser Glu His Ser Leu 130 135 140 Glu Gly Gln
Lys Phe Pro Leu Glu Met Gln Ile Tyr Cys Phe Asp Ala 145 150 155 160
Asp Arg Phe Ser Ser Phe Glu Glu Ala Val Lys Gly Lys Gly Lys Leu 165
170 175 Arg Ala Leu Ser Ile Leu Phe Glu Val Gly Thr Glu Glu Asn Leu
Asp 180 185 190 Phe Lys Ala Ile Ile Asp Gly Val Glu Ser Val Ser Arg
Phe Gly Lys 195 200 205 Gln Ala Ala Leu Asp Pro Phe Ile Leu Leu Asn
Leu Leu Pro Asn Ser 210 215 220 Thr Asp Lys Tyr Tyr Ile Tyr Asn Gly
Ser Leu Thr Ser Pro Pro Cys 225 230 235 240 Thr Asp Thr Val Asp Trp
Ile Val Phe Lys Asp Thr Val Ser Ile Ser 245 250 255 Glu Ser Gln Leu
Ala Val Phe Cys Glu Val Leu Thr Met Gln Gln Ser 260 265 270 Gly Tyr
Val Met Leu Met Asp Tyr Leu Gln Asn Asn Phe Arg Glu Gln 275 280 285
Gln Tyr Lys Phe Ser Arg Gln Val Phe Ser Ser Tyr Thr Gly Lys Glu 290
295 300 Glu Ile His Glu Ala Val Cys Ser Ser Glu Pro Glu Asn Val Gln
Ala 305 310 315 320 Asp Pro Glu Asn Tyr Thr Ser Leu Leu Val Thr Trp
Glu Arg Pro Arg 325 330 335 Val Val Tyr Asp Thr Met Ile Glu Lys Phe
Ala Val Leu Tyr Gln Gln 340 345 350 Leu Asp Gly Glu Asp Gln Thr Lys
His Glu Phe Leu Thr Asp Gly Tyr 355 360 365 Gln Asp Leu Gly Ala Ile
Leu Asn Asn Leu Leu Pro Asn Met Ser Tyr 370 375 380 Val Leu Gln Ile
Val Ala Ile Cys Thr Asn Gly Leu Tyr Gly Lys Tyr 385 390 395 400 Ser
Asp Gln Leu Ile Val Asp Met Pro Thr Asp Asn Pro Glu Leu Asp 405 410
415 Leu Phe Pro Glu Leu Ile Gly Thr Glu Glu Ile Ile Lys Glu Glu Glu
420 425 430 Glu Gly Lys Asp Ile Glu Glu Gly Ala Ile Val Asn Pro Gly
Arg Asp 435 440 445 Ser Ala Thr Asn Gln Ile Arg Lys Lys Glu Pro Gln
Ile Ser Thr Thr 450 455 460 Thr His Tyr Asn Arg Ile Gly Thr Lys Tyr
Asn Glu Ala Lys Thr Asn 465 470 475 480 Arg Ser Pro Thr Arg Gly Ser
Glu Phe Ser Gly Lys Gly Asp Val Pro 485 490 495 Asn Thr Ser Leu Asn
Ser Thr Ser Gln Pro Val Thr Lys Leu Ala Thr 500 505 510 Glu Lys Asp
Ile Ser Leu Thr Ser Gln Thr Val Thr Glu Leu Pro Pro 515 520 525 His
Thr Val Glu Gly Thr Ser Ala Ser Leu Asn Asp Gly Ser Lys Thr 530 535
540 Val Leu Arg Ser Pro His Met Asn Leu Ser Gly Thr Ala Glu Ser Leu
545 550 555 560 Asn Thr Val Ser Ile Thr Glu Tyr Glu Glu Glu Ser Leu
Leu Thr Ser 565 570 575 Phe Lys Leu Asp Thr Gly Ala Glu Asp Ser Ser
Gly Ser Ser Pro Ala 580 585 590 Thr Ser Ala Ile Pro Phe Ile Ser Glu
Asn Ile Ser Gln Gly Tyr Ile 595 600 605 Phe Ser Ser Glu Asn Pro Glu
Thr Ile Thr Tyr Asp Val Leu Ile Pro 610 615 620 Glu Ser Ala Arg Asn
Ala Ser Glu Asp Ser Thr Ser Ser Gly Ser Glu 625 630 635 640 Glu Ser
Leu Lys Asp Pro Ser Met Glu Gly Asn Val Trp Phe Pro Ser 645 650 655
Ser Thr Asp Ile Thr Ala Gln Pro Asp Val Gly Ser Gly Arg Glu Ser 660
665 670 Phe Leu Gln Thr Asn Tyr Thr Glu Ile Arg Val
Asp Glu Ser Glu Lys 675 680 685 Thr Thr Lys Ser Phe Ser Ala Gly Pro
Val Met Ser Gln Gly Pro Ser 690 695 700 Val Thr Asp Leu Glu Met Pro
His Tyr Ser Thr Phe Ala Tyr Phe Pro 705 710 715 720 Thr Glu Val Thr
Pro His Ala Phe Thr Pro Ser Ser Arg Gln Gln Asp 725 730 735 Leu Val
Ser Thr Val Asn Val Val Tyr Ser Gln Thr Thr Gln Pro Val 740 745 750
Tyr Asn Gly Glu Thr Pro Leu Gln Pro Ser Tyr Ser Ser Glu Val Phe 755
760 765 Pro Leu Val Thr Pro Leu Leu Leu Asp Asn Gln Ile Leu Asn Thr
Thr 770 775 780 Pro Ala Ala Ser Ser Ser Asp Ser Ala Leu His Ala Thr
Pro Val Phe 785 790 795 800 Pro Ser Val Asp Val Ser Phe Glu Ser Ile
Leu Ser Ser Tyr Asp Gly 805 810 815 Ala Pro Leu Leu Pro Phe Ser Ser
Ala Ser Phe Ser Ser Glu Leu Phe 820 825 830 Arg His Leu His Thr Val
Ser Gln Ile Leu Pro Gln Val Thr Ser Ala 835 840 845 Thr Glu Ser Asp
Lys Val Pro Leu His Ala Ser Leu Pro Val Ala Gly 850 855 860 Gly Asp
Leu Leu Leu Glu Pro Ser Leu Ala Gln Tyr Ser Asp Val Leu 865 870 875
880 Ser Thr Thr His Ala Ala Ser Glu Thr Leu Glu Phe Gly Ser Glu Ser
885 890 895 Gly Val Leu Tyr Lys Thr Leu Met Phe Ser Gln Val Glu Pro
Pro Ser 900 905 910 Ser Asp Ala Met Met His Ala Arg Ser Ser Gly Pro
Glu Pro Ser Tyr 915 920 925 Ala Leu Ser Asp Asn Glu Gly Ser Gln His
Ile Phe Thr Val Ser Tyr 930 935 940 Ser Ser Ala Ile Pro Val His Asp
Ser Val Gly Val Thr Tyr Gln Gly 945 950 955 960 Ser Leu Phe Ser Gly
Pro Ser His Ile Pro Ile Pro Lys Ser Ser Leu 965 970 975 Ile Thr Pro
Thr Ala Ser Leu Leu Gln Pro Thr His Ala Leu Ser Gly 980 985 990 Asp
Gly Glu Trp Ser Gly Ala Ser Ser Asp Ser Glu Phe Leu Leu Pro 995
1000 1005 Asp Thr Asp Gly Leu Thr Ala Leu Asn Ile Ser Ser Pro Val
Ser 1010 1015 1020 Val Ala Glu Phe Thr Tyr Thr Thr Ser Val Phe Gly
Asp Asp Asn 1025 1030 1035 Lys Ala Leu Ser Lys Ser Glu Ile Ile Tyr
Gly Asn Glu Thr Glu 1040 1045 1050 Leu Gln Ile Pro Ser Phe Asn Glu
Met Val Tyr Pro Ser Glu Ser 1055 1060 1065 Thr Val Met Pro Asn Met
Tyr Asp Asn Val Asn Lys Leu Asn Ala 1070 1075 1080 Ser Leu Gln Glu
Thr Ser Val Ser Ile Ser Ser Thr Lys Gly Met 1085 1090 1095 Phe Pro
Gly Ser Leu Ala His Thr Thr Thr Lys Val Phe Asp His 1100 1105 1110
Glu Ile Ser Gln Val Pro Glu Asn Asn Phe Ser Val Gln Pro Thr 1115
1120 1125 His Thr Val Ser Gln Ala Ser Gly Asp Thr Ser Leu Lys Pro
Val 1130 1135 1140 Leu Ser Ala Asn Ser Glu Pro Ala Ser Ser Asp Pro
Ala Ser Ser 1145 1150 1155 Glu Met Leu Ser Pro Ser Thr Gln Leu Leu
Phe Tyr Glu Thr Ser 1160 1165 1170 Ala Ser Phe Ser Thr Glu Val Leu
Leu Gln Pro Ser Phe Gln Ala 1175 1180 1185 Ser Asp Val Asp Thr Leu
Leu Lys Thr Val Leu Pro Ala Val Pro 1190 1195 1200 Ser Asp Pro Ile
Leu Val Glu Thr Pro Lys Val Asp Lys Ile Ser 1205 1210 1215 Ser Thr
Met Leu His Leu Ile Val Ser Asn Ser Ala Ser Ser Glu 1220 1225 1230
Asn Met Leu His Ser Thr Ser Val Pro Val Phe Asp Val Ser Pro 1235
1240 1245 Thr Ser His Met His Ser Ala Ser Leu Gln Gly Leu Thr Ile
Ser 1250 1255 1260 Tyr Ala Ser Glu Lys Tyr Glu Pro Val Leu Leu Lys
Ser Glu Ser 1265 1270 1275 Ser His Gln Val Val Pro Ser Leu Tyr Ser
Asn Asp Glu Leu Phe 1280 1285 1290 Gln Thr Ala Asn Leu Glu Ile Asn
Gln Ala His Pro Pro Lys Gly 1295 1300 1305 Arg His Val Phe Ala Thr
Pro Val Leu Ser Ile Asp Glu Pro Leu 1310 1315 1320 Asn Thr Leu Ile
Asn Lys Leu Ile His Ser Asp Glu Ile Leu Thr 1325 1330 1335 Ser Thr
Lys Ser Ser Val Thr Gly Lys Val Phe Ala Gly Ile Pro 1340 1345 1350
Thr Val Ala Ser Asp Thr Phe Val Ser Thr Asp His Ser Val Pro 1355
1360 1365 Ile Gly Asn Gly His Val Ala Ile Thr Ala Val Ser Pro His
Arg 1370 1375 1380 Asp Gly Ser Val Thr Ser Thr Lys Leu Leu Phe Pro
Ser Lys Ala 1385 1390 1395 Thr Ser Glu Leu Ser His Ser Ala Lys Ser
Asp Ala Gly Leu Val 1400 1405 1410 Gly Gly Gly Glu Asp Gly Asp Thr
Asp Asp Asp Gly Asp Asp Asp 1415 1420 1425 Asp Asp Arg Asp Ser Asp
Gly Leu Ser Ile His Lys Cys Met Ser 1430 1435 1440 Cys Ser Ser Tyr
Arg Glu Ser Gln Glu Lys Val Met Asn Asp Ser 1445 1450 1455 Asp Thr
His Glu Asn Ser Leu Met Asp Gln Asn Asn Pro Ile Ser 1460 1465 1470
Tyr Ser Leu Ser Glu Asn Ser Glu Glu Asp Asn Arg Val Thr Ser 1475
1480 1485 Val Ser Ser Asp Ser Gln Thr Gly Met Asp Arg Ser Pro Gly
Lys 1490 1495 1500 Ser Pro Ser Ala Asn Gly Leu Ser Gln Lys His Asn
Asp Gly Lys 1505 1510 1515 Glu Glu Asn Asp Ile Gln Thr Gly Ser Ala
Leu Leu Pro Leu Ser 1520 1525 1530 Pro Glu Ser Lys Ala Trp Ala Val
Leu Thr Ser Asp Glu Glu Ser 1535 1540 1545 Gly Ser Gly Gln Gly Thr
Ser Asp Ser Leu Asn Glu Asn Glu Thr 1550 1555 1560 Ser Thr Asp Phe
Ser Phe Ala Asp Thr Asn Glu Lys Asp Ala Asp 1565 1570 1575 Gly Ile
Leu Ala Ala Gly Asp Ser Glu Ile Thr Pro Gly Phe Pro 1580 1585 1590
Gln Ser Pro Thr Ser Ser Val Thr Ser Glu Asn Ser Glu Val Phe 1595
1600 1605 His Val Ser Glu Ala Glu Ala Ser Asn Ser Ser His Glu Ser
Arg 1610 1615 1620 Ile Gly Leu Ala Glu Gly Leu Glu Ser Glu Lys Lys
Ala Val Ile 1625 1630 1635 Pro Leu Val Ile Val Ser Ala Leu Thr Phe
Ile Cys Leu Val Val 1640 1645 1650 Leu Val Gly Ile Leu Ile Tyr Trp
Arg Lys Cys Phe Gln Thr Ala 1655 1660 1665 His Phe Tyr Leu Glu Asp
Ser Thr Ser Pro Arg Val Ile Ser Thr 1670 1675 1680 Pro Pro Thr Pro
Ile Phe Pro Ile Ser Asp Asp Val Gly Ala Ile 1685 1690 1695 Pro Ile
Lys His Phe Pro Lys His Val Ala Asp Leu His Ala Ser 1700 1705 1710
Ser Gly Phe Thr Glu Glu Phe Glu Thr Leu Lys Glu Phe Tyr Gln 1715
1720 1725 Glu Val Gln Ser Cys Thr Val Asp Leu Gly Ile Thr Ala Asp
Ser 1730 1735 1740 Ser Asn His Pro Asp Asn Lys His Lys Asn Arg Tyr
Ile Asn Ile 1745 1750 1755 Val Ala Tyr Asp His Ser Arg Val Lys Leu
Ala Gln Leu Ala Glu 1760 1765 1770 Lys Asp Gly Lys Leu Thr Asp Tyr
Ile Asn Ala Asn Tyr Val Asp 1775 1780 1785 Gly Tyr Asn Arg Pro Lys
Ala Tyr Ile Ala Ala Gln Gly Pro Leu 1790 1795 1800 Lys Ser Thr Ala
Glu Asp Phe Trp Arg Met Ile Trp Glu His Asn 1805 1810 1815 Val Glu
Val Ile Val Met Ile Thr Asn Leu Val Glu Lys Gly Arg 1820 1825 1830
Arg Lys Cys Asp Gln Tyr Trp Pro Ala Asp Gly Ser Glu Glu Tyr 1835
1840 1845 Gly Asn Phe Leu Val Thr Gln Lys Ser Val Gln Val Leu Ala
Tyr 1850 1855 1860 Tyr Thr Val Arg Asn Phe Thr Leu Arg Asn Thr Lys
Ile Lys Lys 1865 1870 1875 Gly Ser Gln Lys Gly Arg Pro Ser Gly Arg
Val Val Thr Gln Tyr 1880 1885 1890 His Tyr Thr Gln Trp Pro Asp Met
Gly Val Pro Glu Tyr Ser Leu 1895 1900 1905 Pro Val Leu Thr Phe Val
Arg Lys Ala Ala Tyr Ala Lys Arg His 1910 1915 1920 Ala Val Gly Pro
Val Val Val His Cys Ser Ala Gly Val Gly Arg 1925 1930 1935 Thr Gly
Thr Tyr Ile Val Leu Asp Ser Met Leu Gln Gln Ile Gln 1940 1945 1950
His Glu Gly Thr Val Asn Ile Phe Gly Phe Leu Lys His Ile Arg 1955
1960 1965 Ser Gln Arg Asn Tyr Leu Val Gln Thr Glu Glu Gln Tyr Val
Phe 1970 1975 1980 Ile His Asp Thr Leu Val Glu Ala Ile Leu Ser Lys
Glu Thr Glu 1985 1990 1995 Val Leu Asp Ser His Ile His Ala Tyr Val
Asn Ala Leu Leu Ile 2000 2005 2010 Pro Gly Pro Ala Gly Lys Thr Lys
Leu Glu Lys Gln Phe Gln Leu 2015 2020 2025 Leu Ser Gln Ser Asn Ile
Gln Gln Ser Asp Tyr Ser Ala Ala Leu 2030 2035 2040 Lys Gln Cys Asn
Arg Glu Lys Asn Arg Thr Ser Ser Ile Ile Pro 2045 2050 2055 Val Glu
Arg Ser Arg Val Gly Ile Ser Ser Leu Ser Gly Glu Gly 2060 2065 2070
Thr Asp Tyr Ile Asn Ala Ser Tyr Ile Met Gly Tyr Tyr Gln Ser 2075
2080 2085 Asn Glu Phe Ile Ile Thr Gln His Pro Leu Leu His Thr Ile
Lys 2090 2095 2100 Asp Phe Trp Arg Met Ile Trp Asp His Asn Ala Gln
Leu Val Val 2105 2110 2115 Met Ile Pro Asp Gly Gln Asn Met Ala Glu
Asp Glu Phe Val Tyr 2120 2125 2130 Trp Pro Asn Lys Asp Glu Pro Ile
Asn Cys Glu Ser Phe Lys Val 2135 2140 2145 Thr Leu Met Ala Glu Glu
His Lys Cys Leu Ser Asn Glu Glu Lys 2150 2155 2160 Leu Ile Ile Gln
Asp Phe Ile Leu Glu Ala Thr Gln Asp Asp Tyr 2165 2170 2175 Val Leu
Glu Val Arg His Phe Gln Cys Pro Lys Trp Pro Asn Pro 2180 2185 2190
Asp Ser Pro Ile Ser Lys Thr Phe Glu Leu Ile Ser Val Ile Lys 2195
2200 2205 Glu Glu Ala Ala Asn Arg Asp Gly Pro Met Ile Val His Asp
Glu 2210 2215 2220 His Gly Gly Val Thr Ala Gly Thr Phe Cys Ala Leu
Thr Thr Leu 2225 2230 2235 Met His Gln Leu Glu Lys Glu Asn Ser Val
Asp Val Tyr Gln Val 2240 2245 2250 Ala Lys Met Ile Asn Leu Met Arg
Pro Gly Val Phe Ala Asp Ile 2255 2260 2265 Glu Gln Tyr Gln Phe Leu
Tyr Lys Val Ile Leu Ser Leu Val Ser 2270 2275 2280 Thr Arg Gln Glu
Glu Asn Pro Ser Thr Ser Leu Asp Ser Asn Gly 2285 2290 2295 Ala Ala
Leu Pro Asp Gly Asn Ile Ala Glu Ser Leu Glu Ser Leu 2300 2305 2310
Val 7 1518 DNA Homo sapiens gene (1)..(1518) Angiopoietin-like 2
(ANGPTL2), mRNA 7 aaccaccatt ttgcaaggac c atg agg cca ctg tgc gtg
aca tgc tgg tgg 51 Met Arg Pro Leu Cys Val Thr Cys Trp Trp 1 5 10
ctc gga ctg ctg gct gcc atg gga gct gtt gca ggc cag gag gac ggt 99
Leu Gly Leu Leu Ala Ala Met Gly Ala Val Ala Gly Gln Glu Asp Gly 15
20 25 ttt gag ggc act gag gag ggc tcg cca aga gag ttc att tac cta
aac 147 Phe Glu Gly Thr Glu Glu Gly Ser Pro Arg Glu Phe Ile Tyr Leu
Asn 30 35 40 agg tac aag cgg gcg ggc gag tcc cag gac aag tgc acc
tac acc ttc 195 Arg Tyr Lys Arg Ala Gly Glu Ser Gln Asp Lys Cys Thr
Tyr Thr Phe 45 50 55 att gtg ccc cag cag cgg gtc acg ggt gcc atc
tgc gtc aac tcc aag 243 Ile Val Pro Gln Gln Arg Val Thr Gly Ala Ile
Cys Val Asn Ser Lys 60 65 70 gag cct gag gtg ctt ctg gag aac cga
gtg cat aag cag gag cta gag 291 Glu Pro Glu Val Leu Leu Glu Asn Arg
Val His Lys Gln Glu Leu Glu 75 80 85 90 ctg ctc aac aat gag ctg ctc
aag cag aag cgg cag atc gag aca ctg 339 Leu Leu Asn Asn Glu Leu Leu
Lys Gln Lys Arg Gln Ile Glu Thr Leu 95 100 105 cag cag ctg gtg gag
gtg gac ggc ggc att gtg agc gag gtg aag ctg 387 Gln Gln Leu Val Glu
Val Asp Gly Gly Ile Val Ser Glu Val Lys Leu 110 115 120 ctg cgc aag
gag agc cgc aac atg aac tcg cgg gtc acg cag ctc tac 435 Leu Arg Lys
Glu Ser Arg Asn Met Asn Ser Arg Val Thr Gln Leu Tyr 125 130 135 atg
cag ctc ctg cac gag atc atc cgc aag cgg gac aac gcg ttg gag 483 Met
Gln Leu Leu His Glu Ile Ile Arg Lys Arg Asp Asn Ala Leu Glu 140 145
150 ctc tcc cag ctg gag aac agg atc ctg aac cag aca gcc gac atg ctg
531 Leu Ser Gln Leu Glu Asn Arg Ile Leu Asn Gln Thr Ala Asp Met Leu
155 160 165 170 cag ctg gcc agc aag tac aag gac ctg gag cac aag tac
cag cac ctg 579 Gln Leu Ala Ser Lys Tyr Lys Asp Leu Glu His Lys Tyr
Gln His Leu 175 180 185 gcc aca ctg gcc cac aac caa tca gag atc atc
gcg cag ctt gag gag 627 Ala Thr Leu Ala His Asn Gln Ser Glu Ile Ile
Ala Gln Leu Glu Glu 190 195 200 cac tgc cag agg gtg ccc tcg gcc agg
ccc gtc ccc cag cca ccc ccc 675 His Cys Gln Arg Val Pro Ser Ala Arg
Pro Val Pro Gln Pro Pro Pro 205 210 215 gct gcc ccg ccc cgg gtc tac
caa cca ccc acc tac aac cgc atc atc 723 Ala Ala Pro Pro Arg Val Tyr
Gln Pro Pro Thr Tyr Asn Arg Ile Ile 220 225 230 aac cag atc tct acc
aac gag atc cag agt gac cag aac ctg aag gtg 771 Asn Gln Ile Ser Thr
Asn Glu Ile Gln Ser Asp Gln Asn Leu Lys Val 235 240 245 250 ctg cca
ccc cct ctg ccc act atg ccc act ctc acc agc ctc cca tct 819 Leu Pro
Pro Pro Leu Pro Thr Met Pro Thr Leu Thr Ser Leu Pro Ser 255 260 265
tcc acc gac aag ccg tcg ggc cca tgg aga gac tgc ctg cag gcc ctg 867
Ser Thr Asp Lys Pro Ser Gly Pro Trp Arg Asp Cys Leu Gln Ala Leu 270
275 280 gag gat ggc cac gac acc agc tcc atc tac ctg gtg aag ccg gag
aac 915 Glu Asp Gly His Asp Thr Ser Ser Ile Tyr Leu Val Lys Pro Glu
Asn 285 290 295 acc aac cgc ctc atg cag gtg tgg tgc gac cag aga cac
gac ccc ggg 963 Thr Asn Arg Leu Met Gln Val Trp Cys Asp Gln Arg His
Asp Pro Gly 300 305 310 ggc tgg acc gtc atc cag aga cgc ctg gat ggc
tct gtt aac ttc ttc 1011 Gly Trp Thr Val Ile Gln Arg Arg Leu Asp
Gly Ser Val Asn Phe Phe 315 320 325 330 agg aac tgg gag acg tac aag
caa ggg ttt ggg aac att gat ggc gaa 1059 Arg Asn Trp Glu Thr Tyr
Lys Gln Gly Phe Gly Asn Ile Asp Gly Glu 335 340 345 tac tgg ctg ggc
ctg gag aac att tac tgg ctg acg aac caa ggc aac 1107 Tyr Trp Leu
Gly Leu Glu Asn Ile Tyr Trp Leu Thr Asn Gln Gly Asn 350 355 360 tac
aaa ctc ctg gtg acc atg gag gac tgg tcc ggc cgc aaa gtc ttt 1155
Tyr Lys Leu Leu Val Thr Met Glu Asp Trp Ser Gly Arg Lys Val Phe 365
370 375 gca gaa tac gcc agt ttc cgc ctg gaa cct gag agc gag tat tat
aag 1203 Ala Glu Tyr Ala Ser Phe Arg Leu Glu Pro Glu Ser Glu Tyr
Tyr Lys 380 385 390 ctg cgg ctg ggg cgc tac cat ggc aat gcg ggt gac
tcc ttt aca tgg 1251 Leu Arg Leu Gly Arg Tyr His Gly Asn Ala Gly
Asp Ser Phe Thr Trp 395 400 405 410 cac aac ggc aag cag ttc acc acc
ctg gac aga gat cat gat gtc tac 1299 His Asn Gly Lys Gln Phe Thr
Thr Leu Asp Arg Asp His Asp Val Tyr 415 420 425 aca gga aac tgt gcc
cac tac cag aag gga ggc tgg tgg tat aac gcc 1347 Thr Gly Asn Cys
Ala His Tyr Gln Lys Gly Gly Trp Trp Tyr Asn Ala 430 435 440 tgt gcc
cac tcc aac ctc aac ggg gtc tgg tac cgc ggg ggc cat tac 1395 Cys
Ala His Ser Asn Leu Asn Gly Val Trp Tyr Arg Gly Gly His Tyr 445 450
455 cgg agc cgc tac cag gac gga gtc tac tgg gct gag ttc cga gga ggc
1443 Arg Ser Arg Tyr Gln Asp Gly Val Tyr Trp Ala Glu Phe Arg Gly
Gly 460 465 470 tct tac tca ctc aag
aaa gtg gtg atg atg atc cga ccg aac ccc aac 1491 Ser Tyr Ser Leu
Lys Lys Val Val Met Met Ile Arg Pro Asn Pro Asn 475 480 485 490 acc
ttc cac taa gccagctccc cctcc 1518 Thr Phe His 8 493 PRT Homo
sapiens gene (1)..(493) Angiopoietin-like 2 (ANGPTL2), protein 8
Met Arg Pro Leu Cys Val Thr Cys Trp Trp Leu Gly Leu Leu Ala Ala 1 5
10 15 Met Gly Ala Val Ala Gly Gln Glu Asp Gly Phe Glu Gly Thr Glu
Glu 20 25 30 Gly Ser Pro Arg Glu Phe Ile Tyr Leu Asn Arg Tyr Lys
Arg Ala Gly 35 40 45 Glu Ser Gln Asp Lys Cys Thr Tyr Thr Phe Ile
Val Pro Gln Gln Arg 50 55 60 Val Thr Gly Ala Ile Cys Val Asn Ser
Lys Glu Pro Glu Val Leu Leu 65 70 75 80 Glu Asn Arg Val His Lys Gln
Glu Leu Glu Leu Leu Asn Asn Glu Leu 85 90 95 Leu Lys Gln Lys Arg
Gln Ile Glu Thr Leu Gln Gln Leu Val Glu Val 100 105 110 Asp Gly Gly
Ile Val Ser Glu Val Lys Leu Leu Arg Lys Glu Ser Arg 115 120 125 Asn
Met Asn Ser Arg Val Thr Gln Leu Tyr Met Gln Leu Leu His Glu 130 135
140 Ile Ile Arg Lys Arg Asp Asn Ala Leu Glu Leu Ser Gln Leu Glu Asn
145 150 155 160 Arg Ile Leu Asn Gln Thr Ala Asp Met Leu Gln Leu Ala
Ser Lys Tyr 165 170 175 Lys Asp Leu Glu His Lys Tyr Gln His Leu Ala
Thr Leu Ala His Asn 180 185 190 Gln Ser Glu Ile Ile Ala Gln Leu Glu
Glu His Cys Gln Arg Val Pro 195 200 205 Ser Ala Arg Pro Val Pro Gln
Pro Pro Pro Ala Ala Pro Pro Arg Val 210 215 220 Tyr Gln Pro Pro Thr
Tyr Asn Arg Ile Ile Asn Gln Ile Ser Thr Asn 225 230 235 240 Glu Ile
Gln Ser Asp Gln Asn Leu Lys Val Leu Pro Pro Pro Leu Pro 245 250 255
Thr Met Pro Thr Leu Thr Ser Leu Pro Ser Ser Thr Asp Lys Pro Ser 260
265 270 Gly Pro Trp Arg Asp Cys Leu Gln Ala Leu Glu Asp Gly His Asp
Thr 275 280 285 Ser Ser Ile Tyr Leu Val Lys Pro Glu Asn Thr Asn Arg
Leu Met Gln 290 295 300 Val Trp Cys Asp Gln Arg His Asp Pro Gly Gly
Trp Thr Val Ile Gln 305 310 315 320 Arg Arg Leu Asp Gly Ser Val Asn
Phe Phe Arg Asn Trp Glu Thr Tyr 325 330 335 Lys Gln Gly Phe Gly Asn
Ile Asp Gly Glu Tyr Trp Leu Gly Leu Glu 340 345 350 Asn Ile Tyr Trp
Leu Thr Asn Gln Gly Asn Tyr Lys Leu Leu Val Thr 355 360 365 Met Glu
Asp Trp Ser Gly Arg Lys Val Phe Ala Glu Tyr Ala Ser Phe 370 375 380
Arg Leu Glu Pro Glu Ser Glu Tyr Tyr Lys Leu Arg Leu Gly Arg Tyr 385
390 395 400 His Gly Asn Ala Gly Asp Ser Phe Thr Trp His Asn Gly Lys
Gln Phe 405 410 415 Thr Thr Leu Asp Arg Asp His Asp Val Tyr Thr Gly
Asn Cys Ala His 420 425 430 Tyr Gln Lys Gly Gly Trp Trp Tyr Asn Ala
Cys Ala His Ser Asn Leu 435 440 445 Asn Gly Val Trp Tyr Arg Gly Gly
His Tyr Arg Ser Arg Tyr Gln Asp 450 455 460 Gly Val Tyr Trp Ala Glu
Phe Arg Gly Gly Ser Tyr Ser Leu Lys Lys 465 470 475 480 Val Val Met
Met Ile Arg Pro Asn Pro Asn Thr Phe His 485 490 9 2133 DNA Homo
sapiens gene (1)..(2133) Human SPARC/osteonectin mRNA 9 cgggagagcg
cgctctgcct gccgcctgcc tgcctgccac tgagggttcc cagcacc 57 atg agg gcc
tgg atc ttc ttt ctc ctt tgc ctg gcc ggg agg gcc ttg 105 Met Arg Ala
Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu 1 5 10 15 gca
gcc cct cag caa gaa gcc ctg cct gat gag aca gag gtg gtg gaa 153 Ala
Ala Pro Gln Gln Glu Ala Leu Pro Asp Glu Thr Glu Val Val Glu 20 25
30 gaa act gtg gca gag gtg act gag gta tct gtg gga gct aat cct gtc
201 Glu Thr Val Ala Glu Val Thr Glu Val Ser Val Gly Ala Asn Pro Val
35 40 45 cag gtg gaa gta gga gaa ttt gat gat ggt gca gag gaa acc
gaa gag 249 Gln Val Glu Val Gly Glu Phe Asp Asp Gly Ala Glu Glu Thr
Glu Glu 50 55 60 gag gtg gtg gcg gaa aat ccc tgc cag aac cac cac
tgc aaa cac ggc 297 Glu Val Val Ala Glu Asn Pro Cys Gln Asn His His
Cys Lys His Gly 65 70 75 80 aag gtg tgc gag ctg gat gag aac aac acc
ccc atg tgc gtg tgc cag 345 Lys Val Cys Glu Leu Asp Glu Asn Asn Thr
Pro Met Cys Val Cys Gln 85 90 95 gac ccc acc agc tgc cca gcc ccc
att ggc gag ttt gag aag gtg tgc 393 Asp Pro Thr Ser Cys Pro Ala Pro
Ile Gly Glu Phe Glu Lys Val Cys 100 105 110 agc aat gac aac aag acc
ttc gac tct tcc tgc cac ttc ttt gcc aca 441 Ser Asn Asp Asn Lys Thr
Phe Asp Ser Ser Cys His Phe Phe Ala Thr 115 120 125 aag tgc acc ctg
gag ggc acc aag aag ggc cac aag ctc cac ctg gac 489 Lys Cys Thr Leu
Glu Gly Thr Lys Lys Gly His Lys Leu His Leu Asp 130 135 140 tac atc
ggg cct tgc aaa tac atc ccc cct tgc ctg gac tct gag ctg 537 Tyr Ile
Gly Pro Cys Lys Tyr Ile Pro Pro Cys Leu Asp Ser Glu Leu 145 150 155
160 acc gaa ttc ccc ctg cgc atg cgg gac tgg ctc aag aac gtc ctg gtc
585 Thr Glu Phe Pro Leu Arg Met Arg Asp Trp Leu Lys Asn Val Leu Val
165 170 175 acc ctg tat gag agg gat gag gac aac aac ctt ctg act gag
aag cag 633 Thr Leu Tyr Glu Arg Asp Glu Asp Asn Asn Leu Leu Thr Glu
Lys Gln 180 185 190 aag ctg cgg gtg aag aag atc cat gag aat gag aag
cgc ctg gag gca 681 Lys Leu Arg Val Lys Lys Ile His Glu Asn Glu Lys
Arg Leu Glu Ala 195 200 205 gga gac cac ccc gtg gag ctg ctg gcc cgg
gac ttc gag aag aac tat 729 Gly Asp His Pro Val Glu Leu Leu Ala Arg
Asp Phe Glu Lys Asn Tyr 210 215 220 aac atg tac atc ttc cct gta cac
tgg cag ttc ggc cag ctg gac cag 777 Asn Met Tyr Ile Phe Pro Val His
Trp Gln Phe Gly Gln Leu Asp Gln 225 230 235 240 cac ccc att gac ggg
tac ctc tcc cac acc gag ctg gct cca ctg cgt 825 His Pro Ile Asp Gly
Tyr Leu Ser His Thr Glu Leu Ala Pro Leu Arg 245 250 255 gct ccc ctc
atc ccc atg gag cat tgc acc acc cgc ttt ttc gag acc 873 Ala Pro Leu
Ile Pro Met Glu His Cys Thr Thr Arg Phe Phe Glu Thr 260 265 270 tgt
gac ctg gac aat gac aag tac atc gcc ctg gat gag tgg gcc ggc 921 Cys
Asp Leu Asp Asn Asp Lys Tyr Ile Ala Leu Asp Glu Trp Ala Gly 275 280
285 tgc ttc ggc atc aag cag aag gat atc gac aag gat ctt gtg atc taa
969 Cys Phe Gly Ile Lys Gln Lys Asp Ile Asp Lys Asp Leu Val Ile 290
295 300 atccactcct tccacagtac cggattctct ctttaaccct ccccttcgtg
tttcccccaa 1029 tgtttaaaat gtttggatgg tttgttgttc tgcctggaga
caaggtgcta acatagattt 1089 aagtgaatac attaacggtg ctaaaaatga
aaattctaac ccaagacatg acattcttag 1149 ctgtaactta actattaagg
ccttttccac acgcattaat agtcccattt ttctcttgcc 1209 atttgtagct
ttgcccattg tcttattggc acatgggtgg acacggatct gctgggctct 1269
gccttaaaca cacattgcag cttcaacttt tctctttagt gttctgtttg aaactaatac
1329 ttaccgagtc agactttgtg ttcatttcat ttcagggtct tggctgcctg
tgggcttccc 1389 caggtggcct ggaggtgggc aaagggaagt aacagacaca
cgatgttgtc aaggatggtt 1449 ttgggactag aggctcagtg gtgggagaga
tccctgcaga atccaccaac cagaacgtgg 1509 tttgcctgag gctgtaactg
agagaaagat tctggggctg tcttatgaaa atatagacat 1569 tctcacataa
gcccagttca tcaccatttc ctcctttacc tttcagtgca gtttcttttc 1629
acattaggct gttggttcaa acttttggga gcacggactg tcagttctct gggaagtggt
1689 cagcgcatcc tgcagggctt ctcctcctct gtcttttgga gaaccagggc
tcttctcagg 1749 ggctctaggg actgccaggc tgtttcagcc aggaaggcca
aaatcaagag tgagatgtag 1809 aaagttgtaa aatagaaaaa gtggagttgg
tgaatcggtt gttctttcct cacatttgga 1869 tgattgtcat aaggttttta
gcatgttcct ccttttcttc accctcccct ttgttcttct 1929 attaatcaag
agaaacttca aagttaatgg gatggtcgga tctcacaggc tgagaactcg 1989
ttcacctcca agcatttcat gaaaaagctg cttcttatta atcatacaaa ctctcaccat
2049 gatgtgaaga gtttcacaaa tctttcaaaa taaaaagtaa tgacttagaa
actgaaaaaa 2109 aaaaaaaaaa aaaaaaaaaa aaaa 2133 10 303 PRT Homo
sapiens SIGNAL (1)..(17) 10 Met Arg Ala Trp Ile Phe Phe Leu Leu Cys
Leu Ala Gly Arg Ala Leu 1 5 10 15 Ala Ala Pro Gln Gln Glu Ala Leu
Pro Asp Glu Thr Glu Val Val Glu 20 25 30 Glu Thr Val Ala Glu Val
Thr Glu Val Ser Val Gly Ala Asn Pro Val 35 40 45 Gln Val Glu Val
Gly Glu Phe Asp Asp Gly Ala Glu Glu Thr Glu Glu 50 55 60 Glu Val
Val Ala Glu Asn Pro Cys Gln Asn His His Cys Lys His Gly 65 70 75 80
Lys Val Cys Glu Leu Asp Glu Asn Asn Thr Pro Met Cys Val Cys Gln 85
90 95 Asp Pro Thr Ser Cys Pro Ala Pro Ile Gly Glu Phe Glu Lys Val
Cys 100 105 110 Ser Asn Asp Asn Lys Thr Phe Asp Ser Ser Cys His Phe
Phe Ala Thr 115 120 125 Lys Cys Thr Leu Glu Gly Thr Lys Lys Gly His
Lys Leu His Leu Asp 130 135 140 Tyr Ile Gly Pro Cys Lys Tyr Ile Pro
Pro Cys Leu Asp Ser Glu Leu 145 150 155 160 Thr Glu Phe Pro Leu Arg
Met Arg Asp Trp Leu Lys Asn Val Leu Val 165 170 175 Thr Leu Tyr Glu
Arg Asp Glu Asp Asn Asn Leu Leu Thr Glu Lys Gln 180 185 190 Lys Leu
Arg Val Lys Lys Ile His Glu Asn Glu Lys Arg Leu Glu Ala 195 200 205
Gly Asp His Pro Val Glu Leu Leu Ala Arg Asp Phe Glu Lys Asn Tyr 210
215 220 Asn Met Tyr Ile Phe Pro Val His Trp Gln Phe Gly Gln Leu Asp
Gln 225 230 235 240 His Pro Ile Asp Gly Tyr Leu Ser His Thr Glu Leu
Ala Pro Leu Arg 245 250 255 Ala Pro Leu Ile Pro Met Glu His Cys Thr
Thr Arg Phe Phe Glu Thr 260 265 270 Cys Asp Leu Asp Asn Asp Lys Tyr
Ile Ala Leu Asp Glu Trp Ala Gly 275 280 285 Cys Phe Gly Ile Lys Gln
Lys Asp Ile Asp Lys Asp Leu Val Ile 290 295 300 11 4586 DNA Homo
sapiens gene (1)..(4586) mRNA for met proto-oncogene 11 gaattccgcc
ctcgccgccc gcggcgcccc gagcgctttg tgagcagatg cggagccgag 60
tggagggcgc gagccagatg cggggcgaca gctgacttgc tgagaggagg cggggaggcg
120 cggagcgcgc gtgtggtcct tgcgccgctg acttctccac tggttcctgg
gcaccgaaag 180 ataaacctct cata atg aag gcc ccc gct gtg ctt gca cct
ggc atc ctc 230 Met Lys Ala Pro Ala Val Leu Ala Pro Gly Ile Leu 1 5
10 gtg ctc ctg ttt acc ttg gtg cag agg agc aat ggg gag tgt aaa gag
278 Val Leu Leu Phe Thr Leu Val Gln Arg Ser Asn Gly Glu Cys Lys Glu
15 20 25 gca cta gca aag tcc gag atg aat gtg aat atg aag tat cag
ctt ccc 326 Ala Leu Ala Lys Ser Glu Met Asn Val Asn Met Lys Tyr Gln
Leu Pro 30 35 40 aac ttc acc gcg gaa aca ccc atc cag aat gtc att
cta cat gag cat 374 Asn Phe Thr Ala Glu Thr Pro Ile Gln Asn Val Ile
Leu His Glu His 45 50 55 60 cac att ttc ctt ggt gcc act aac tac att
tat gtt tta aat gag gaa 422 His Ile Phe Leu Gly Ala Thr Asn Tyr Ile
Tyr Val Leu Asn Glu Glu 65 70 75 gac ctt cag aag gtt gct gag tac
aag act ggg cct gtg ctg gaa cac 470 Asp Leu Gln Lys Val Ala Glu Tyr
Lys Thr Gly Pro Val Leu Glu His 80 85 90 cca gat tgt ttc cca tgt
cag gac tgc agc agc aaa gcc aat tta tca 518 Pro Asp Cys Phe Pro Cys
Gln Asp Cys Ser Ser Lys Ala Asn Leu Ser 95 100 105 gga ggt gtt tgg
aaa gat aac atc aac atg gct cta gtt gtc gac acc 566 Gly Gly Val Trp
Lys Asp Asn Ile Asn Met Ala Leu Val Val Asp Thr 110 115 120 tac tat
gat gat caa ctc att agc tgt ggc agc gtc aac aga ggg acc 614 Tyr Tyr
Asp Asp Gln Leu Ile Ser Cys Gly Ser Val Asn Arg Gly Thr 125 130 135
140 tgc cag cga cat gtc ttt ccc cac aat cat act gct gac ata cag tcg
662 Cys Gln Arg His Val Phe Pro His Asn His Thr Ala Asp Ile Gln Ser
145 150 155 gag gtt cac tgc ata ttc tcc cca cag ata gaa gag ccc agc
cag tgt 710 Glu Val His Cys Ile Phe Ser Pro Gln Ile Glu Glu Pro Ser
Gln Cys 160 165 170 cct gac tgt gtg gtg agc gcc ctg gga gcc aaa gtc
ctt tca tct gta 758 Pro Asp Cys Val Val Ser Ala Leu Gly Ala Lys Val
Leu Ser Ser Val 175 180 185 aag gac cgg ttc atc aac ttc ttt gta ggc
aat acc ata aat tct tct 806 Lys Asp Arg Phe Ile Asn Phe Phe Val Gly
Asn Thr Ile Asn Ser Ser 190 195 200 tat ttc cca gat cat cca ttg cat
tcg ata tca gtg aga agg cta aag 854 Tyr Phe Pro Asp His Pro Leu His
Ser Ile Ser Val Arg Arg Leu Lys 205 210 215 220 gaa acg aaa gat ggt
ttt atg ttt ttg acg gac cag tcc tac att gat 902 Glu Thr Lys Asp Gly
Phe Met Phe Leu Thr Asp Gln Ser Tyr Ile Asp 225 230 235 gtt tta cct
gag ttc aga gat tct tac ccc att aag tat gtc cat gcc 950 Val Leu Pro
Glu Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Val His Ala 240 245 250 ttt
gaa agc aac aat ttt att tac ttc ttg acg gtc caa agg gaa act 998 Phe
Glu Ser Asn Asn Phe Ile Tyr Phe Leu Thr Val Gln Arg Glu Thr 255 260
265 cta gat gct cag act ttt cac aca aga ata atc agg ttc tgt tcc ata
1046 Leu Asp Ala Gln Thr Phe His Thr Arg Ile Ile Arg Phe Cys Ser
Ile 270 275 280 aac tct gga ttg cat tcc tac atg gaa atg cct ctg gag
tgt att ctc 1094 Asn Ser Gly Leu His Ser Tyr Met Glu Met Pro Leu
Glu Cys Ile Leu 285 290 295 300 aca gaa aag aga aaa aag aga tcc aca
aag aag gaa gtg ttt aat ata 1142 Thr Glu Lys Arg Lys Lys Arg Ser
Thr Lys Lys Glu Val Phe Asn Ile 305 310 315 ctt cag gct gcg tat gtc
agc aag cct ggg gcc cag ctt gct aga caa 1190 Leu Gln Ala Ala Tyr
Val Ser Lys Pro Gly Ala Gln Leu Ala Arg Gln 320 325 330 ata gga gcc
agc ctg aat gat gac att ctt ttc ggg gtg ttc gca caa 1238 Ile Gly
Ala Ser Leu Asn Asp Asp Ile Leu Phe Gly Val Phe Ala Gln 335 340 345
agc aag cca gat tct gcc gaa cca atg gat cga tct gcc atg tgt gca
1286 Ser Lys Pro Asp Ser Ala Glu Pro Met Asp Arg Ser Ala Met Cys
Ala 350 355 360 ttc cct atc aaa tat gtc aac gac ttc ttc aac aag atc
gtc aac aaa 1334 Phe Pro Ile Lys Tyr Val Asn Asp Phe Phe Asn Lys
Ile Val Asn Lys 365 370 375 380 aac aat gtg aga tgt ctc cag cat ttt
tac gga ccc aat cat gag cac 1382 Asn Asn Val Arg Cys Leu Gln His
Phe Tyr Gly Pro Asn His Glu His 385 390 395 tgc ttt aat agg aca ctt
ctg aga aat tca tca ggc tgt gaa gcg cgc 1430 Cys Phe Asn Arg Thr
Leu Leu Arg Asn Ser Ser Gly Cys Glu Ala Arg 400 405 410 cgt gat gaa
tat cga aca gag ttt acc aca gct ttg cag cgc gtt gac 1478 Arg Asp
Glu Tyr Arg Thr Glu Phe Thr Thr Ala Leu Gln Arg Val Asp 415 420 425
tta ttc atg ggt caa ttc agc gaa gtc ctc tta aca tct ata tcc acc
1526 Leu Phe Met Gly Gln Phe Ser Glu Val Leu Leu Thr Ser Ile Ser
Thr 430 435 440 ttc att aaa gga gac ctc acc ata gct aat ctt ggg aca
tca gag ggt 1574 Phe Ile Lys Gly Asp Leu Thr Ile Ala Asn Leu Gly
Thr Ser Glu Gly 445 450 455 460 cgc ttc atg cag gtt gtg gtt tct cga
tca gga cca tca acc cct cat 1622 Arg Phe Met Gln Val Val Val Ser
Arg Ser Gly Pro Ser Thr Pro His 465 470 475 gtg aat ttt ctc ctg gac
tcc cat cca gtg tct cca gaa gtg att gtg 1670 Val Asn Phe Leu Leu
Asp Ser His Pro Val Ser Pro Glu Val Ile Val 480 485 490 gag cat aca
tta aac caa aat ggc tac aca ctg gtt atc act ggg aag 1718 Glu His
Thr Leu Asn Gln Asn Gly Tyr Thr Leu Val Ile Thr Gly Lys 495 500 505
aag atc acg aag atc cca ttg aat ggc ttg ggc tgc aga cat ttc cag
1766 Lys Ile Thr Lys Ile Pro Leu Asn Gly Leu Gly Cys Arg His Phe
Gln 510 515 520 tcc tgc agt caa tgc ctc tct gcc cca ccc ttt gtt cag
tgt ggc tgg 1814 Ser Cys
Ser Gln Cys Leu Ser Ala Pro Pro Phe Val Gln Cys Gly Trp 525 530 535
540 tgc cac gac aaa tgt gtg cga tcg gag gaa tgc ctg agc ggg aca tgg
1862 Cys His Asp Lys Cys Val Arg Ser Glu Glu Cys Leu Ser Gly Thr
Trp 545 550 555 act caa cag atc tgt ctg cct gca atc tac aag gtt ttc
cca aat agt 1910 Thr Gln Gln Ile Cys Leu Pro Ala Ile Tyr Lys Val
Phe Pro Asn Ser 560 565 570 gca ccc ctt gaa gga ggg aca agg ctg acc
ata tgt ggc tgg gac ttt 1958 Ala Pro Leu Glu Gly Gly Thr Arg Leu
Thr Ile Cys Gly Trp Asp Phe 575 580 585 gga ttt cgg agg aat aat aaa
ttt gat tta aag aaa act aga gtt ctc 2006 Gly Phe Arg Arg Asn Asn
Lys Phe Asp Leu Lys Lys Thr Arg Val Leu 590 595 600 ctt gga aat gag
agc tgc acc ttg act tta agt gag agc acg atg aat 2054 Leu Gly Asn
Glu Ser Cys Thr Leu Thr Leu Ser Glu Ser Thr Met Asn 605 610 615 620
aca ttg aaa tgc aca gtt ggt cct gcc atg aat aag cat ttc aat atg
2102 Thr Leu Lys Cys Thr Val Gly Pro Ala Met Asn Lys His Phe Asn
Met 625 630 635 tcc ata att att tca aat ggc cac ggg aca aca caa tac
agt aca ttc 2150 Ser Ile Ile Ile Ser Asn Gly His Gly Thr Thr Gln
Tyr Ser Thr Phe 640 645 650 tcc tat gtg gat cct gta ata aca agt att
tcg ccg aaa tac ggt cct 2198 Ser Tyr Val Asp Pro Val Ile Thr Ser
Ile Ser Pro Lys Tyr Gly Pro 655 660 665 atg gct ggt ggc act tta ctt
act tta act gga aat tac cta aac agt 2246 Met Ala Gly Gly Thr Leu
Leu Thr Leu Thr Gly Asn Tyr Leu Asn Ser 670 675 680 ggg aat tct aga
cac att tca att ggt gga aaa aca tgt act tta aaa 2294 Gly Asn Ser
Arg His Ile Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys 685 690 695 700
agt gtg tca aac agt att ctt gaa tgt tat acc cca gcc caa acc att
2342 Ser Val Ser Asn Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Thr
Ile 705 710 715 tca act gag ttt gct gtt aaa ttg aaa att gac tta gcc
aac cga gag 2390 Ser Thr Glu Phe Ala Val Lys Leu Lys Ile Asp Leu
Ala Asn Arg Glu 720 725 730 aca agc atc ttc agt tac cgt gaa gat ccc
att gtc tat gaa att cat 2438 Thr Ser Ile Phe Ser Tyr Arg Glu Asp
Pro Ile Val Tyr Glu Ile His 735 740 745 cca acc aaa tct ttt att agt
ggt ggg agc aca ata aca ggt gtt ggg 2486 Pro Thr Lys Ser Phe Ile
Ser Gly Gly Ser Thr Ile Thr Gly Val Gly 750 755 760 aaa aac ctg aat
tca gtt agt gtc ccg aga atg gtc ata aat gtg cat 2534 Lys Asn Leu
Asn Ser Val Ser Val Pro Arg Met Val Ile Asn Val His 765 770 775 780
gaa gca gga agg aac ttt aca gtg gca tgt caa cat cgc tct aat tca
2582 Glu Ala Gly Arg Asn Phe Thr Val Ala Cys Gln His Arg Ser Asn
Ser 785 790 795 gag ata atc tgt tgt acc act cct tcc ctg caa cag ctg
aat ctg caa 2630 Glu Ile Ile Cys Cys Thr Thr Pro Ser Leu Gln Gln
Leu Asn Leu Gln 800 805 810 ctc ccc ctg aaa acc aaa gcc ttt ttc atg
tta gat ggg atc ctt tcc 2678 Leu Pro Leu Lys Thr Lys Ala Phe Phe
Met Leu Asp Gly Ile Leu Ser 815 820 825 aaa tac ttt gat ctc att tat
gta cat aat cct gtg ttt aag cct ttt 2726 Lys Tyr Phe Asp Leu Ile
Tyr Val His Asn Pro Val Phe Lys Pro Phe 830 835 840 gaa aag cca gtg
atg atc tca atg ggc aat gaa aat gta ctg gaa att 2774 Glu Lys Pro
Val Met Ile Ser Met Gly Asn Glu Asn Val Leu Glu Ile 845 850 855 860
aag gga aat gat att gac cct gaa gca gtt aaa ggt gaa gtg tta aaa
2822 Lys Gly Asn Asp Ile Asp Pro Glu Ala Val Lys Gly Glu Val Leu
Lys 865 870 875 gtt gga aat aag agc tgt gag aat ata cac tta cat tct
gaa gcc gtt 2870 Val Gly Asn Lys Ser Cys Glu Asn Ile His Leu His
Ser Glu Ala Val 880 885 890 tta tgc acg gtc ccc aat gac ctg ctg aaa
ttg aac agc gag cta aat 2918 Leu Cys Thr Val Pro Asn Asp Leu Leu
Lys Leu Asn Ser Glu Leu Asn 895 900 905 ata gag tgg aag caa gca att
tct tca acc gtc ctt gga aaa gta ata 2966 Ile Glu Trp Lys Gln Ala
Ile Ser Ser Thr Val Leu Gly Lys Val Ile 910 915 920 gtt caa cca gat
cag aat ttc aca gga ttg att gct ggt gtt gtc tca 3014 Val Gln Pro
Asp Gln Asn Phe Thr Gly Leu Ile Ala Gly Val Val Ser 925 930 935 940
ata tca aca gca ctg tta tta cta ctt ggg ttt ttc ctg tgg ctg aaa
3062 Ile Ser Thr Ala Leu Leu Leu Leu Leu Gly Phe Phe Leu Trp Leu
Lys 945 950 955 aag aga aag caa att aaa gat ctg ggc agt gaa tta gtt
cgc tac gat 3110 Lys Arg Lys Gln Ile Lys Asp Leu Gly Ser Glu Leu
Val Arg Tyr Asp 960 965 970 gca aga gta cac act cct cat ttg gat agg
ctt gta agt gcc cga agt 3158 Ala Arg Val His Thr Pro His Leu Asp
Arg Leu Val Ser Ala Arg Ser 975 980 985 gta agc cca act aca gaa atg
gtt tca aat gaa tct gta gac tac cga 3206 Val Ser Pro Thr Thr Glu
Met Val Ser Asn Glu Ser Val Asp Tyr Arg 990 995 1000 gct act ttt
cca gaa gat cag ttt cct aat tca tct cag aac ggt 3251 Ala Thr Phe
Pro Glu Asp Gln Phe Pro Asn Ser Ser Gln Asn Gly 1005 1010 1015 tca
tgc cga caa gtg cag tat cct ctg aca gac atg tcc ccc atc 3296 Ser
Cys Arg Gln Val Gln Tyr Pro Leu Thr Asp Met Ser Pro Ile 1020 1025
1030 cta act agt ggg gac tct gat ata tcc agt cca tta ctg caa aat
3341 Leu Thr Ser Gly Asp Ser Asp Ile Ser Ser Pro Leu Leu Gln Asn
1035 1040 1045 act gtc cac att gac ctc agt gct cta aat cca gag ctg
gtc cag 3386 Thr Val His Ile Asp Leu Ser Ala Leu Asn Pro Glu Leu
Val Gln 1050 1055 1060 gca gtg cag cat gta gtg att ggg ccc agt agc
ctg att gtg cat 3431 Ala Val Gln His Val Val Ile Gly Pro Ser Ser
Leu Ile Val His 1065 1070 1075 ttc aat gaa gtc ata gga aga ggg cat
ttt ggt tgt gta tat cat 3476 Phe Asn Glu Val Ile Gly Arg Gly His
Phe Gly Cys Val Tyr His 1080 1085 1090 ggg act ttg ttg gac aat gat
ggc aag aaa att cac tgt gct gtg 3521 Gly Thr Leu Leu Asp Asn Asp
Gly Lys Lys Ile His Cys Ala Val 1095 1100 1105 aaa tcc ttg aac aga
atc act gac ata gga gaa gtt tcc caa ttt 3566 Lys Ser Leu Asn Arg
Ile Thr Asp Ile Gly Glu Val Ser Gln Phe 1110 1115 1120 ctg acc gag
gga atc atc atg aaa gat ttt agt cat ccc aat gtc 3611 Leu Thr Glu
Gly Ile Ile Met Lys Asp Phe Ser His Pro Asn Val 1125 1130 1135 ctc
tcg ctc ctg gga atc tgc ctg cga agt gaa ggg tct ccg ctg 3656 Leu
Ser Leu Leu Gly Ile Cys Leu Arg Ser Glu Gly Ser Pro Leu 1140 1145
1150 gtg gtc cta cca tac atg aaa cat gga gat ctt cga aat ttc att
3701 Val Val Leu Pro Tyr Met Lys His Gly Asp Leu Arg Asn Phe Ile
1155 1160 1165 cga aat gag act cat aat cca act gta aaa gat ctt att
ggc ttt 3746 Arg Asn Glu Thr His Asn Pro Thr Val Lys Asp Leu Ile
Gly Phe 1170 1175 1180 ggt ctt caa gta gcc aaa ggc atg aaa tat ctt
gca agc aaa aag 3791 Gly Leu Gln Val Ala Lys Gly Met Lys Tyr Leu
Ala Ser Lys Lys 1185 1190 1195 ttt gtc cac aga gac ttg gct gca aga
aac tgt atg ctg gat gaa 3836 Phe Val His Arg Asp Leu Ala Ala Arg
Asn Cys Met Leu Asp Glu 1200 1205 1210 aaa ttc aca gtc aag gtt gct
gat ttt ggt ctt gcc aga gac atg 3881 Lys Phe Thr Val Lys Val Ala
Asp Phe Gly Leu Ala Arg Asp Met 1215 1220 1225 tat gat aaa gaa tac
tat agt gta cac aac aaa aca ggt gca aag 3926 Tyr Asp Lys Glu Tyr
Tyr Ser Val His Asn Lys Thr Gly Ala Lys 1230 1235 1240 ctg cca gtg
aag tgg atg gct ttg gaa agt ctg caa act caa aag 3971 Leu Pro Val
Lys Trp Met Ala Leu Glu Ser Leu Gln Thr Gln Lys 1245 1250 1255 ttt
acc acc aag tca gat gtg tgg tcc ttt ggc gtc gtc ctc tgg 4016 Phe
Thr Thr Lys Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp 1260 1265
1270 gag ctg atg aca aga gga gcc cca cct tat cct gac gta aac acc
4061 Glu Leu Met Thr Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr
1275 1280 1285 ttt gat ata act gtt tac ttg ttg caa ggg aga aga ctc
cta caa 4106 Phe Asp Ile Thr Val Tyr Leu Leu Gln Gly Arg Arg Leu
Leu Gln 1290 1295 1300 ccc gaa tac tgc cca gac ccc tta tat gaa gta
atg cta aaa tgc 4151 Pro Glu Tyr Cys Pro Asp Pro Leu Tyr Glu Val
Met Leu Lys Cys 1305 1310 1315 tgg cac cct aaa gcc gaa atg cgc cca
tcc ttt tct gaa ctg gtg 4196 Trp His Pro Lys Ala Glu Met Arg Pro
Ser Phe Ser Glu Leu Val 1320 1325 1330 tcc cgg ata tca gcg atc ttc
tct act ttc att ggg gag cac tat 4241 Ser Arg Ile Ser Ala Ile Phe
Ser Thr Phe Ile Gly Glu His Tyr 1335 1340 1345 gtc cat gtg aac gct
act tat gtg aac gta aaa tgt gtc gct ccg 4286 Val His Val Asn Ala
Thr Tyr Val Asn Val Lys Cys Val Ala Pro 1350 1355 1360 tat cct tct
ctg ttg tca tca gaa gat aac gct gat gat gag gtg 4331 Tyr Pro Ser
Leu Leu Ser Ser Glu Asp Asn Ala Asp Asp Glu Val 1365 1370 1375 gac
aca cga cca gcc tcc ttc tgg gag aca tca tag tgctagtact 4377 Asp Thr
Arg Pro Ala Ser Phe Trp Glu Thr Ser 1380 1385 1390 atgtcaaagc
aacagtccac actttgtcca atggtttttt cactgcctga cctttaaaag 4437
gccatcgata ttctttgctc cttgccaaat tgcactatta ataggacttg tattgttatt
4497 taaattactg gattctaagg aatttcttat ctgacagagc atcagaacca
gaggcttggt 4557 cccacaggcc agggaccaat gcgctgcag 4586 12 1390 PRT
Homo sapiens SIGNAL (1)..(24) 12 Met Lys Ala Pro Ala Val Leu Ala
Pro Gly Ile Leu Val Leu Leu Phe 1 5 10 15 Thr Leu Val Gln Arg Ser
Asn Gly Glu Cys Lys Glu Ala Leu Ala Lys 20 25 30 Ser Glu Met Asn
Val Asn Met Lys Tyr Gln Leu Pro Asn Phe Thr Ala 35 40 45 Glu Thr
Pro Ile Gln Asn Val Ile Leu His Glu His His Ile Phe Leu 50 55 60
Gly Ala Thr Asn Tyr Ile Tyr Val Leu Asn Glu Glu Asp Leu Gln Lys 65
70 75 80 Val Ala Glu Tyr Lys Thr Gly Pro Val Leu Glu His Pro Asp
Cys Phe 85 90 95 Pro Cys Gln Asp Cys Ser Ser Lys Ala Asn Leu Ser
Gly Gly Val Trp 100 105 110 Lys Asp Asn Ile Asn Met Ala Leu Val Val
Asp Thr Tyr Tyr Asp Asp 115 120 125 Gln Leu Ile Ser Cys Gly Ser Val
Asn Arg Gly Thr Cys Gln Arg His 130 135 140 Val Phe Pro His Asn His
Thr Ala Asp Ile Gln Ser Glu Val His Cys 145 150 155 160 Ile Phe Ser
Pro Gln Ile Glu Glu Pro Ser Gln Cys Pro Asp Cys Val 165 170 175 Val
Ser Ala Leu Gly Ala Lys Val Leu Ser Ser Val Lys Asp Arg Phe 180 185
190 Ile Asn Phe Phe Val Gly Asn Thr Ile Asn Ser Ser Tyr Phe Pro Asp
195 200 205 His Pro Leu His Ser Ile Ser Val Arg Arg Leu Lys Glu Thr
Lys Asp 210 215 220 Gly Phe Met Phe Leu Thr Asp Gln Ser Tyr Ile Asp
Val Leu Pro Glu 225 230 235 240 Phe Arg Asp Ser Tyr Pro Ile Lys Tyr
Val His Ala Phe Glu Ser Asn 245 250 255 Asn Phe Ile Tyr Phe Leu Thr
Val Gln Arg Glu Thr Leu Asp Ala Gln 260 265 270 Thr Phe His Thr Arg
Ile Ile Arg Phe Cys Ser Ile Asn Ser Gly Leu 275 280 285 His Ser Tyr
Met Glu Met Pro Leu Glu Cys Ile Leu Thr Glu Lys Arg 290 295 300 Lys
Lys Arg Ser Thr Lys Lys Glu Val Phe Asn Ile Leu Gln Ala Ala 305 310
315 320 Tyr Val Ser Lys Pro Gly Ala Gln Leu Ala Arg Gln Ile Gly Ala
Ser 325 330 335 Leu Asn Asp Asp Ile Leu Phe Gly Val Phe Ala Gln Ser
Lys Pro Asp 340 345 350 Ser Ala Glu Pro Met Asp Arg Ser Ala Met Cys
Ala Phe Pro Ile Lys 355 360 365 Tyr Val Asn Asp Phe Phe Asn Lys Ile
Val Asn Lys Asn Asn Val Arg 370 375 380 Cys Leu Gln His Phe Tyr Gly
Pro Asn His Glu His Cys Phe Asn Arg 385 390 395 400 Thr Leu Leu Arg
Asn Ser Ser Gly Cys Glu Ala Arg Arg Asp Glu Tyr 405 410 415 Arg Thr
Glu Phe Thr Thr Ala Leu Gln Arg Val Asp Leu Phe Met Gly 420 425 430
Gln Phe Ser Glu Val Leu Leu Thr Ser Ile Ser Thr Phe Ile Lys Gly 435
440 445 Asp Leu Thr Ile Ala Asn Leu Gly Thr Ser Glu Gly Arg Phe Met
Gln 450 455 460 Val Val Val Ser Arg Ser Gly Pro Ser Thr Pro His Val
Asn Phe Leu 465 470 475 480 Leu Asp Ser His Pro Val Ser Pro Glu Val
Ile Val Glu His Thr Leu 485 490 495 Asn Gln Asn Gly Tyr Thr Leu Val
Ile Thr Gly Lys Lys Ile Thr Lys 500 505 510 Ile Pro Leu Asn Gly Leu
Gly Cys Arg His Phe Gln Ser Cys Ser Gln 515 520 525 Cys Leu Ser Ala
Pro Pro Phe Val Gln Cys Gly Trp Cys His Asp Lys 530 535 540 Cys Val
Arg Ser Glu Glu Cys Leu Ser Gly Thr Trp Thr Gln Gln Ile 545 550 555
560 Cys Leu Pro Ala Ile Tyr Lys Val Phe Pro Asn Ser Ala Pro Leu Glu
565 570 575 Gly Gly Thr Arg Leu Thr Ile Cys Gly Trp Asp Phe Gly Phe
Arg Arg 580 585 590 Asn Asn Lys Phe Asp Leu Lys Lys Thr Arg Val Leu
Leu Gly Asn Glu 595 600 605 Ser Cys Thr Leu Thr Leu Ser Glu Ser Thr
Met Asn Thr Leu Lys Cys 610 615 620 Thr Val Gly Pro Ala Met Asn Lys
His Phe Asn Met Ser Ile Ile Ile 625 630 635 640 Ser Asn Gly His Gly
Thr Thr Gln Tyr Ser Thr Phe Ser Tyr Val Asp 645 650 655 Pro Val Ile
Thr Ser Ile Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly 660 665 670 Thr
Leu Leu Thr Leu Thr Gly Asn Tyr Leu Asn Ser Gly Asn Ser Arg 675 680
685 His Ile Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser Asn
690 695 700 Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Thr Ile Ser Thr
Glu Phe 705 710 715 720 Ala Val Lys Leu Lys Ile Asp Leu Ala Asn Arg
Glu Thr Ser Ile Phe 725 730 735 Ser Tyr Arg Glu Asp Pro Ile Val Tyr
Glu Ile His Pro Thr Lys Ser 740 745 750 Phe Ile Ser Gly Gly Ser Thr
Ile Thr Gly Val Gly Lys Asn Leu Asn 755 760 765 Ser Val Ser Val Pro
Arg Met Val Ile Asn Val His Glu Ala Gly Arg 770 775 780 Asn Phe Thr
Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile Ile Cys 785 790 795 800
Cys Thr Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro Leu Lys 805
810 815 Thr Lys Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys Tyr Phe
Asp 820 825 830 Leu Ile Tyr Val His Asn Pro Val Phe Lys Pro Phe Glu
Lys Pro Val 835 840 845 Met Ile Ser Met Gly Asn Glu Asn Val Leu Glu
Ile Lys Gly Asn Asp 850 855 860 Ile Asp Pro Glu Ala Val Lys Gly Glu
Val Leu Lys Val Gly Asn Lys 865 870 875 880 Ser Cys Glu Asn Ile His
Leu His Ser Glu Ala Val Leu Cys Thr Val 885 890 895 Pro Asn Asp Leu
Leu Lys Leu Asn Ser Glu Leu Asn Ile Glu Trp Lys 900 905 910 Gln Ala
Ile Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln Pro Asp 915 920 925
Gln Asn Phe Thr Gly Leu Ile Ala Gly Val Val Ser Ile Ser Thr Ala 930
935 940 Leu Leu Leu Leu Leu Gly Phe Phe Leu Trp Leu Lys Lys Arg Lys
Gln 945 950 955 960 Ile Lys Asp Leu Gly Ser Glu Leu Val Arg Tyr Asp
Ala Arg Val His 965 970 975 Thr Pro His Leu Asp Arg Leu Val Ser Ala
Arg Ser Val Ser Pro Thr 980 985 990 Thr Glu Met Val Ser Asn Glu Ser
Val Asp Tyr Arg Ala Thr Phe Pro 995 1000
1005 Glu Asp Gln Phe Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg Gln
1010 1015 1020 Val Gln Tyr Pro Leu Thr Asp Met Ser Pro Ile Leu Thr
Ser Gly 1025 1030 1035 Asp Ser Asp Ile Ser Ser Pro Leu Leu Gln Asn
Thr Val His Ile 1040 1045 1050 Asp Leu Ser Ala Leu Asn Pro Glu Leu
Val Gln Ala Val Gln His 1055 1060 1065 Val Val Ile Gly Pro Ser Ser
Leu Ile Val His Phe Asn Glu Val 1070 1075 1080 Ile Gly Arg Gly His
Phe Gly Cys Val Tyr His Gly Thr Leu Leu 1085 1090 1095 Asp Asn Asp
Gly Lys Lys Ile His Cys Ala Val Lys Ser Leu Asn 1100 1105 1110 Arg
Ile Thr Asp Ile Gly Glu Val Ser Gln Phe Leu Thr Glu Gly 1115 1120
1125 Ile Ile Met Lys Asp Phe Ser His Pro Asn Val Leu Ser Leu Leu
1130 1135 1140 Gly Ile Cys Leu Arg Ser Glu Gly Ser Pro Leu Val Val
Leu Pro 1145 1150 1155 Tyr Met Lys His Gly Asp Leu Arg Asn Phe Ile
Arg Asn Glu Thr 1160 1165 1170 His Asn Pro Thr Val Lys Asp Leu Ile
Gly Phe Gly Leu Gln Val 1175 1180 1185 Ala Lys Gly Met Lys Tyr Leu
Ala Ser Lys Lys Phe Val His Arg 1190 1195 1200 Asp Leu Ala Ala Arg
Asn Cys Met Leu Asp Glu Lys Phe Thr Val 1205 1210 1215 Lys Val Ala
Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu 1220 1225 1230 Tyr
Tyr Ser Val His Asn Lys Thr Gly Ala Lys Leu Pro Val Lys 1235 1240
1245 Trp Met Ala Leu Glu Ser Leu Gln Thr Gln Lys Phe Thr Thr Lys
1250 1255 1260 Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu Leu
Met Thr 1265 1270 1275 Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr
Phe Asp Ile Thr 1280 1285 1290 Val Tyr Leu Leu Gln Gly Arg Arg Leu
Leu Gln Pro Glu Tyr Cys 1295 1300 1305 Pro Asp Pro Leu Tyr Glu Val
Met Leu Lys Cys Trp His Pro Lys 1310 1315 1320 Ala Glu Met Arg Pro
Ser Phe Ser Glu Leu Val Ser Arg Ile Ser 1325 1330 1335 Ala Ile Phe
Ser Thr Phe Ile Gly Glu His Tyr Val His Val Asn 1340 1345 1350 Ala
Thr Tyr Val Asn Val Lys Cys Val Ala Pro Tyr Pro Ser Leu 1355 1360
1365 Leu Ser Ser Glu Asp Asn Ala Asp Asp Glu Val Asp Thr Arg Pro
1370 1375 1380 Ala Ser Phe Trp Glu Thr Ser 1385 1390 13 2558 DNA
Homo sapiens gene (1)..(2558) Chondroitin sulfate proteoglycan
BEHAB/brevican mRNA, GPI isoform, complete cds 13 tgtggcactg
cctgcgtacc caaccccagc cctgggtagc ctgcagc atg gcc cag 56 Met Ala Gln
1 ctg ttc ctg ccc ctg ctg gca gcc ctg gtc ctg gcc cag gct cct gca
104 Leu Phe Leu Pro Leu Leu Ala Ala Leu Val Leu Ala Gln Ala Pro Ala
5 10 15 gct tta gca gat gtt ctg gaa gga gac agc tca gag gac cgc gct
ttt 152 Ala Leu Ala Asp Val Leu Glu Gly Asp Ser Ser Glu Asp Arg Ala
Phe 20 25 30 35 cgc gtg cgc atc gcg ggc gac gcg cca ctg cag ggc gtg
ctc ggc ggc 200 Arg Val Arg Ile Ala Gly Asp Ala Pro Leu Gln Gly Val
Leu Gly Gly 40 45 50 gcc ctc acc atc cct tgc cac gtc cac tac ctg
cgg cca ccg ccg agc 248 Ala Leu Thr Ile Pro Cys His Val His Tyr Leu
Arg Pro Pro Pro Ser 55 60 65 cgc cgg gct gtg ctg ggc tct ccg cgg
gtc aag tgg act ttc ctg tcc 296 Arg Arg Ala Val Leu Gly Ser Pro Arg
Val Lys Trp Thr Phe Leu Ser 70 75 80 cgg ggc cgg gag gca gag gtg
ctg gtg gcg cgg gga gtg cgc gtc aag 344 Arg Gly Arg Glu Ala Glu Val
Leu Val Ala Arg Gly Val Arg Val Lys 85 90 95 gtg aac gag gcc tac
cgg ttc cgc gtg gca ctg cct gcg tac cca gcg 392 Val Asn Glu Ala Tyr
Arg Phe Arg Val Ala Leu Pro Ala Tyr Pro Ala 100 105 110 115 tcg ctc
acc gac gtc tcc ctg gcg ctg agc gag ctg cgc ccc aac gac 440 Ser Leu
Thr Asp Val Ser Leu Ala Leu Ser Glu Leu Arg Pro Asn Asp 120 125 130
tca ggt atc tat cgc tgt gag gtc cag cac ggc atc gat gac agc agc 488
Ser Gly Ile Tyr Arg Cys Glu Val Gln His Gly Ile Asp Asp Ser Ser 135
140 145 gac gct gtg gag gtc aag gtc aaa ggg gtc gtc ttt ctc tac cga
gag 536 Asp Ala Val Glu Val Lys Val Lys Gly Val Val Phe Leu Tyr Arg
Glu 150 155 160 ggc tct gcc cgc tat gct ttc tcc ttt tct ggg gcc cag
gag gcc tgt 584 Gly Ser Ala Arg Tyr Ala Phe Ser Phe Ser Gly Ala Gln
Glu Ala Cys 165 170 175 gcc cgc att gga gcc cac atc gcc acc ccg gag
cag ctc tat gcc gcc 632 Ala Arg Ile Gly Ala His Ile Ala Thr Pro Glu
Gln Leu Tyr Ala Ala 180 185 190 195 tac ctt ggg ggc tat gag caa tgt
gat gct ggc tgg ctg tcg gat cag 680 Tyr Leu Gly Gly Tyr Glu Gln Cys
Asp Ala Gly Trp Leu Ser Asp Gln 200 205 210 acc gtg agg tat ccc atc
cag acc cca cga gag gcc tgt tac gga gac 728 Thr Val Arg Tyr Pro Ile
Gln Thr Pro Arg Glu Ala Cys Tyr Gly Asp 215 220 225 atg gat ggc ttc
ccc ggg gtc cgg aac tat ggt gtg gtg gac ccg gat 776 Met Asp Gly Phe
Pro Gly Val Arg Asn Tyr Gly Val Val Asp Pro Asp 230 235 240 gac ctc
tat gat gtg tac tgt tat gct gaa gac cta aat gga gaa ttg 824 Asp Leu
Tyr Asp Val Tyr Cys Tyr Ala Glu Asp Leu Asn Gly Glu Leu 245 250 255
ttc ctg ggt gac cct cca gag aag ctg aca ttg gag gaa gca cgg gcg 872
Phe Leu Gly Asp Pro Pro Glu Lys Leu Thr Leu Glu Glu Ala Arg Ala 260
265 270 275 tac tgc cag gag cgg ggt gca gag att gcc acc acg ggc caa
ctg tat 920 Tyr Cys Gln Glu Arg Gly Ala Glu Ile Ala Thr Thr Gly Gln
Leu Tyr 280 285 290 gca gcc tgg gat ggt ggc ctg gac cac tgc agc cca
ggg tgg cta gct 968 Ala Ala Trp Asp Gly Gly Leu Asp His Cys Ser Pro
Gly Trp Leu Ala 295 300 305 gat ggc agt gtg cgc tac ccc atc gtc aca
ccc agc cag cgc tgt ggt 1016 Asp Gly Ser Val Arg Tyr Pro Ile Val
Thr Pro Ser Gln Arg Cys Gly 310 315 320 ggg ggc ttg cct ggt gtc aag
act ctc ttc ctc ttc ccc aac cag act 1064 Gly Gly Leu Pro Gly Val
Lys Thr Leu Phe Leu Phe Pro Asn Gln Thr 325 330 335 ggc ttc ccc aat
aag cac agc cgc ttc aac gtc tac tgc ttc cga gac 1112 Gly Phe Pro
Asn Lys His Ser Arg Phe Asn Val Tyr Cys Phe Arg Asp 340 345 350 355
tcg gcc cag cct tct gcc atc cct gag gcc tcc aac cca gcc tcc aac
1160 Ser Ala Gln Pro Ser Ala Ile Pro Glu Ala Ser Asn Pro Ala Ser
Asn 360 365 370 cca gcc tct gat gga cta gag gct atc gtc aca gtg aca
gag acc ctg 1208 Pro Ala Ser Asp Gly Leu Glu Ala Ile Val Thr Val
Thr Glu Thr Leu 375 380 385 gag gaa ctg cag ctg cct cag gaa gcc aca
gag agt gaa tcc cgt ggg 1256 Glu Glu Leu Gln Leu Pro Gln Glu Ala
Thr Glu Ser Glu Ser Arg Gly 390 395 400 gcc atc tac tcc atc ccc atc
atg gag gac gga gga ggt gga agc tcc 1304 Ala Ile Tyr Ser Ile Pro
Ile Met Glu Asp Gly Gly Gly Gly Ser Ser 405 410 415 act cca gaa gac
cca gca gag gcc cct agg acg ctc cta gaa ttt gaa 1352 Thr Pro Glu
Asp Pro Ala Glu Ala Pro Arg Thr Leu Leu Glu Phe Glu 420 425 430 435
aca caa tcc atg gta ccg ccc acg ggg ttc tca gaa gag gaa ggt aag
1400 Thr Gln Ser Met Val Pro Pro Thr Gly Phe Ser Glu Glu Glu Gly
Lys 440 445 450 gca ttg gag gaa gaa gag aaa tat gaa gat gaa gaa gag
aaa gag gag 1448 Ala Leu Glu Glu Glu Glu Lys Tyr Glu Asp Glu Glu
Glu Lys Glu Glu 455 460 465 gaa gaa gaa gag gag gag gtg gag gat gag
gct ctg tgg gca tgg ccc 1496 Glu Glu Glu Glu Glu Glu Val Glu Asp
Glu Ala Leu Trp Ala Trp Pro 470 475 480 agc gag ctc agc agc ccg ggc
cct gag gcc tct ctc ccc act gag cca 1544 Ser Glu Leu Ser Ser Pro
Gly Pro Glu Ala Ser Leu Pro Thr Glu Pro 485 490 495 gca gcc cag gag
gag tca ctc tcc cag gcg cca gca agg gca gtc ctg 1592 Ala Ala Gln
Glu Glu Ser Leu Ser Gln Ala Pro Ala Arg Ala Val Leu 500 505 510 515
cag cct ggt gca tca cca ctt cct gat gga gag tca gaa gct tcc agg
1640 Gln Pro Gly Ala Ser Pro Leu Pro Asp Gly Glu Ser Glu Ala Ser
Arg 520 525 530 cct cca agg gtc cat gga cca cct act gag act ctg ccc
act ccc agg 1688 Pro Pro Arg Val His Gly Pro Pro Thr Glu Thr Leu
Pro Thr Pro Arg 535 540 545 gag agg aac cta gca tcc cca tca cct tcc
act ctg gtt gag gca aga 1736 Glu Arg Asn Leu Ala Ser Pro Ser Pro
Ser Thr Leu Val Glu Ala Arg 550 555 560 gag gtg ggg gag gca act ggt
ggt cct gag cta tct ggg gtc cct cga 1784 Glu Val Gly Glu Ala Thr
Gly Gly Pro Glu Leu Ser Gly Val Pro Arg 565 570 575 gga gag agc gag
gag aca gga agc tcc gag ggt gcc cct tcc ctg ctt 1832 Gly Glu Ser
Glu Glu Thr Gly Ser Ser Glu Gly Ala Pro Ser Leu Leu 580 585 590 595
cca gcc aca cgg gcc cct gag ggt acc agg gag ctg gag gcc ccc tct
1880 Pro Ala Thr Arg Ala Pro Glu Gly Thr Arg Glu Leu Glu Ala Pro
Ser 600 605 610 gaa gat aat tct gga aga act gcc cca gca ggg acc tca
gtg cag gcc 1928 Glu Asp Asn Ser Gly Arg Thr Ala Pro Ala Gly Thr
Ser Val Gln Ala 615 620 625 cag cca gtg ctg ccc act gac agc gcc agc
cga ggt gga gtg gcc gtg 1976 Gln Pro Val Leu Pro Thr Asp Ser Ala
Ser Arg Gly Gly Val Ala Val 630 635 640 gtc ccc gca tca ggt aat tct
gcc caa ggc tca act gcc ctc tct atc 2024 Val Pro Ala Ser Gly Asn
Ser Ala Gln Gly Ser Thr Ala Leu Ser Ile 645 650 655 cta ctc ctt ttc
ttc ccc ctg cag ctc tgg gtc acc tga cctgtagtcc 2073 Leu Leu Leu Phe
Phe Pro Leu Gln Leu Trp Val Thr 660 665 670 tttaacccac catcatccca
aactctcctg tcctttgcct tcattctctt acccacctct 2133 acctatgggt
ctccaatctc ggatatccac cttgtgggta tctcagctct ccgcgtcttt 2193
accctgtgat cccagccccg ccactgacca tctgtgaccc ttccctgcca ttgggccctc
2253 cacctgtggc tcacatctcg ccagccccac agagcatcct caggcctctc
caagggtcct 2313 catcacctat tgcagccttc agggctcggc ctattttcca
ctactccctt catccgcctg 2373 tgtgccgtcc cctttagctg cctcctattg
atctcaggga agcctgggag tcccttctca 2433 cccctcaacc tccggagtcc
aggagaaccc gtacccccac agagccttaa gcaactactt 2493 ctgtgaagta
ttttttgact gtttcatgga aaacaagcct tggaaataaa tctctattaa 2553 accgc
2558 14 671 PRT Homo sapiens gene (1)..(671) Chondroitin sulfate
proteoglycan BEHAB/brevican 14 Met Ala Gln Leu Phe Leu Pro Leu Leu
Ala Ala Leu Val Leu Ala Gln 1 5 10 15 Ala Pro Ala Ala Leu Ala Asp
Val Leu Glu Gly Asp Ser Ser Glu Asp 20 25 30 Arg Ala Phe Arg Val
Arg Ile Ala Gly Asp Ala Pro Leu Gln Gly Val 35 40 45 Leu Gly Gly
Ala Leu Thr Ile Pro Cys His Val His Tyr Leu Arg Pro 50 55 60 Pro
Pro Ser Arg Arg Ala Val Leu Gly Ser Pro Arg Val Lys Trp Thr 65 70
75 80 Phe Leu Ser Arg Gly Arg Glu Ala Glu Val Leu Val Ala Arg Gly
Val 85 90 95 Arg Val Lys Val Asn Glu Ala Tyr Arg Phe Arg Val Ala
Leu Pro Ala 100 105 110 Tyr Pro Ala Ser Leu Thr Asp Val Ser Leu Ala
Leu Ser Glu Leu Arg 115 120 125 Pro Asn Asp Ser Gly Ile Tyr Arg Cys
Glu Val Gln His Gly Ile Asp 130 135 140 Asp Ser Ser Asp Ala Val Glu
Val Lys Val Lys Gly Val Val Phe Leu 145 150 155 160 Tyr Arg Glu Gly
Ser Ala Arg Tyr Ala Phe Ser Phe Ser Gly Ala Gln 165 170 175 Glu Ala
Cys Ala Arg Ile Gly Ala His Ile Ala Thr Pro Glu Gln Leu 180 185 190
Tyr Ala Ala Tyr Leu Gly Gly Tyr Glu Gln Cys Asp Ala Gly Trp Leu 195
200 205 Ser Asp Gln Thr Val Arg Tyr Pro Ile Gln Thr Pro Arg Glu Ala
Cys 210 215 220 Tyr Gly Asp Met Asp Gly Phe Pro Gly Val Arg Asn Tyr
Gly Val Val 225 230 235 240 Asp Pro Asp Asp Leu Tyr Asp Val Tyr Cys
Tyr Ala Glu Asp Leu Asn 245 250 255 Gly Glu Leu Phe Leu Gly Asp Pro
Pro Glu Lys Leu Thr Leu Glu Glu 260 265 270 Ala Arg Ala Tyr Cys Gln
Glu Arg Gly Ala Glu Ile Ala Thr Thr Gly 275 280 285 Gln Leu Tyr Ala
Ala Trp Asp Gly Gly Leu Asp His Cys Ser Pro Gly 290 295 300 Trp Leu
Ala Asp Gly Ser Val Arg Tyr Pro Ile Val Thr Pro Ser Gln 305 310 315
320 Arg Cys Gly Gly Gly Leu Pro Gly Val Lys Thr Leu Phe Leu Phe Pro
325 330 335 Asn Gln Thr Gly Phe Pro Asn Lys His Ser Arg Phe Asn Val
Tyr Cys 340 345 350 Phe Arg Asp Ser Ala Gln Pro Ser Ala Ile Pro Glu
Ala Ser Asn Pro 355 360 365 Ala Ser Asn Pro Ala Ser Asp Gly Leu Glu
Ala Ile Val Thr Val Thr 370 375 380 Glu Thr Leu Glu Glu Leu Gln Leu
Pro Gln Glu Ala Thr Glu Ser Glu 385 390 395 400 Ser Arg Gly Ala Ile
Tyr Ser Ile Pro Ile Met Glu Asp Gly Gly Gly 405 410 415 Gly Ser Ser
Thr Pro Glu Asp Pro Ala Glu Ala Pro Arg Thr Leu Leu 420 425 430 Glu
Phe Glu Thr Gln Ser Met Val Pro Pro Thr Gly Phe Ser Glu Glu 435 440
445 Glu Gly Lys Ala Leu Glu Glu Glu Glu Lys Tyr Glu Asp Glu Glu Glu
450 455 460 Lys Glu Glu Glu Glu Glu Glu Glu Glu Val Glu Asp Glu Ala
Leu Trp 465 470 475 480 Ala Trp Pro Ser Glu Leu Ser Ser Pro Gly Pro
Glu Ala Ser Leu Pro 485 490 495 Thr Glu Pro Ala Ala Gln Glu Glu Ser
Leu Ser Gln Ala Pro Ala Arg 500 505 510 Ala Val Leu Gln Pro Gly Ala
Ser Pro Leu Pro Asp Gly Glu Ser Glu 515 520 525 Ala Ser Arg Pro Pro
Arg Val His Gly Pro Pro Thr Glu Thr Leu Pro 530 535 540 Thr Pro Arg
Glu Arg Asn Leu Ala Ser Pro Ser Pro Ser Thr Leu Val 545 550 555 560
Glu Ala Arg Glu Val Gly Glu Ala Thr Gly Gly Pro Glu Leu Ser Gly 565
570 575 Val Pro Arg Gly Glu Ser Glu Glu Thr Gly Ser Ser Glu Gly Ala
Pro 580 585 590 Ser Leu Leu Pro Ala Thr Arg Ala Pro Glu Gly Thr Arg
Glu Leu Glu 595 600 605 Ala Pro Ser Glu Asp Asn Ser Gly Arg Thr Ala
Pro Ala Gly Thr Ser 610 615 620 Val Gln Ala Gln Pro Val Leu Pro Thr
Asp Ser Ala Ser Arg Gly Gly 625 630 635 640 Val Ala Val Val Pro Ala
Ser Gly Asn Ser Ala Gln Gly Ser Thr Ala 645 650 655 Leu Ser Ile Leu
Leu Leu Phe Phe Pro Leu Gln Leu Trp Val Thr 660 665 670 15 2316 DNA
Homo sapiens gene (1)..(2316) Human mRNA for CD44E (epithelial
form) 15 agcggacccc agcctctgcc aggttcggtc cgccatcctc gtcccgtcct
ccgccggccc 60 ctgccccgcg cccagggatc ctccagctcc tttcgcccgc
gccctccgtt cgctccggac 120 acc atg gac aag ttt tgg tgg cac gca gcc
tgg gga ctc tgc ctc gtg 168 Met Asp Lys Phe Trp Trp His Ala Ala Trp
Gly Leu Cys Leu Val 1 5 10 15 ccg ctg agc ctg gcg cag atc gat ttg
aat ata acc tgc cgc ttt gca 216 Pro Leu Ser Leu Ala Gln Ile Asp Leu
Asn Ile Thr Cys Arg Phe Ala 20 25 30 ggt gta ttc cac gtg gag aaa
aat ggt cgc tac agc atc tct cgg acg 264 Gly Val Phe His Val Glu Lys
Asn Gly Arg Tyr Ser Ile Ser Arg Thr 35 40 45 gag gcc gct gac ctc
tgc aag gct ttc aat agc acc ttg ccc aca atg 312 Glu Ala Ala Asp Leu
Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr Met 50 55 60 gcc cag atg
gag aaa gct ctg agc atc gga ttt gag acc tgc agg tat 360 Ala Gln Met
Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr Cys Arg Tyr 65 70 75 ggg
ttc ata gaa ggg cat gtg gtg att
ccc cgg atc cac ccc aac tcc 408 Gly Phe Ile Glu Gly His Val Val Ile
Pro Arg Ile His Pro Asn Ser 80 85 90 95 atc tgt gca gca aac aac aca
ggg gtg tac atc ctc aca tac aac acc 456 Ile Cys Ala Ala Asn Asn Thr
Gly Val Tyr Ile Leu Thr Tyr Asn Thr 100 105 110 tcc cag tat gac aca
tat tgc ttc aat gct tca gct cca cct gaa gaa 504 Ser Gln Tyr Asp Thr
Tyr Cys Phe Asn Ala Ser Ala Pro Pro Glu Glu 115 120 125 gat tgt aca
tca gtc aca gac ctg ccc aat gcc ttt gat gga cca att 552 Asp Cys Thr
Ser Val Thr Asp Leu Pro Asn Ala Phe Asp Gly Pro Ile 130 135 140 acc
ata act att gtt aac cgt gat ggc acc cgc tat gtc cag aaa gga 600 Thr
Ile Thr Ile Val Asn Arg Asp Gly Thr Arg Tyr Val Gln Lys Gly 145 150
155 gaa tac aga acg aat cct gaa gac atc tac ccc agc aac cct act gat
648 Glu Tyr Arg Thr Asn Pro Glu Asp Ile Tyr Pro Ser Asn Pro Thr Asp
160 165 170 175 gat gac gtg agc agc ggc tcc tcc agt gaa agg agc agc
act tca gga 696 Asp Asp Val Ser Ser Gly Ser Ser Ser Glu Arg Ser Ser
Thr Ser Gly 180 185 190 ggt tac atc ttt tac acc ttt tct act gta cac
ccc atc cca gac gaa 744 Gly Tyr Ile Phe Tyr Thr Phe Ser Thr Val His
Pro Ile Pro Asp Glu 195 200 205 gac agt ccc tgg atc acc gac agc aca
gac aga atc cct cgt acc aat 792 Asp Ser Pro Trp Ile Thr Asp Ser Thr
Asp Arg Ile Pro Arg Thr Asn 210 215 220 atg gac tcc agt cat agt aca
acg ctt cag cct act gca aat cca aac 840 Met Asp Ser Ser His Ser Thr
Thr Leu Gln Pro Thr Ala Asn Pro Asn 225 230 235 aca ggt ttg gtg gaa
gat ttg gac agg aca gga cct ctt tca atg aca 888 Thr Gly Leu Val Glu
Asp Leu Asp Arg Thr Gly Pro Leu Ser Met Thr 240 245 250 255 acg cag
cag agt aat tct cag agc ttc tct aca tca cat gaa ggc ttg 936 Thr Gln
Gln Ser Asn Ser Gln Ser Phe Ser Thr Ser His Glu Gly Leu 260 265 270
gaa gaa gat aaa gac cat cca aca act tct act ctg aca tca agc aat 984
Glu Glu Asp Lys Asp His Pro Thr Thr Ser Thr Leu Thr Ser Ser Asn 275
280 285 agg aat gat gtc aca ggt gga aga aga gac cca aat cat tct gaa
ggc 1032 Arg Asn Asp Val Thr Gly Gly Arg Arg Asp Pro Asn His Ser
Glu Gly 290 295 300 tca act cat tta ctg gaa ggt tat acc tct cat tac
cca cac acg aag 1080 Ser Thr His Leu Leu Glu Gly Tyr Thr Ser His
Tyr Pro His Thr Lys 305 310 315 gaa agc agg acc ttc atc cca gtg acc
tca gct aag act ggg tcc ttt 1128 Glu Ser Arg Thr Phe Ile Pro Val
Thr Ser Ala Lys Thr Gly Ser Phe 320 325 330 335 gga gtt act gca gtt
act gtt gga gat tcc aac tct aat gtc aat cgt 1176 Gly Val Thr Ala
Val Thr Val Gly Asp Ser Asn Ser Asn Val Asn Arg 340 345 350 tcc tta
tca gga gac caa gac aca ttc cac ccc agt ggg ggg tcc cat 1224 Ser
Leu Ser Gly Asp Gln Asp Thr Phe His Pro Ser Gly Gly Ser His 355 360
365 acc act cat gga tct gaa tca gat gga cac tca cat ggg agt caa gaa
1272 Thr Thr His Gly Ser Glu Ser Asp Gly His Ser His Gly Ser Gln
Glu 370 375 380 ggt gga gca aac aca acc tct ggt cct ata agg aca ccc
caa att cca 1320 Gly Gly Ala Asn Thr Thr Ser Gly Pro Ile Arg Thr
Pro Gln Ile Pro 385 390 395 gaa tgg ctg atc atc ttg gca tcc ctc ttg
gcc ttg gct ttg att ctt 1368 Glu Trp Leu Ile Ile Leu Ala Ser Leu
Leu Ala Leu Ala Leu Ile Leu 400 405 410 415 gca gtt tgc att gca gtc
aac agt cga aga agg tgt ggg cag aag aaa 1416 Ala Val Cys Ile Ala
Val Asn Ser Arg Arg Arg Cys Gly Gln Lys Lys 420 425 430 aag cta gtg
atc aac agt ggc aat gga gct gtg gag gac aga aag cca 1464 Lys Leu
Val Ile Asn Ser Gly Asn Gly Ala Val Glu Asp Arg Lys Pro 435 440 445
agt gga ctc aac gga gag gcc agc aag tct cag gaa atg gtg cat ttg
1512 Ser Gly Leu Asn Gly Glu Ala Ser Lys Ser Gln Glu Met Val His
Leu 450 455 460 gtg aac aag gag tcg tca gaa act cca gac cag ttt atg
aca gct gat 1560 Val Asn Lys Glu Ser Ser Glu Thr Pro Asp Gln Phe
Met Thr Ala Asp 465 470 475 gag aca agg aac ctg cag aat gtg gac atg
aag att ggg gtg taa 1605 Glu Thr Arg Asn Leu Gln Asn Val Asp Met
Lys Ile Gly Val 480 485 490 cacctacacc attatcttgg aaagaaacaa
cgttggaaac ataaccatta caggggagct 1665 gggacactta acagatgcaa
tgtgctactg attgtttcat ttcgaatcta taatagcata 1725 aaattttcta
ctctttttgt tttttgtgtt ttgttcttta aagtcaggtc caatttgtaa 1785
aaacagcatt gctttctgaa attagggccc aattaataat cagcaagaat tttgatcgtt
1845 tcagttcccc acttggaggc ctttcatccc tcgggtgtgc tatggatggc
ttctaacaaa 1905 aacctaccac atagttattc ctgatcgcca accttgcccc
ccaccagcta aggacatttc 1965 cagggttaat agggcctggt cctgggagga
aatttgaatg ggtcattttg cccttccatt 2025 agcctaatcc ctgggcattg
ctttccactg aggttggggg ttggggtgta ctagttacac 2085 atcttcaaca
gaccccctct agaaattttt cagatgcttc tgggagacac ccaaagggta 2145
agtctattta tctgtagtaa actatttatc tgtgtttttg aaatattaaa ccctggatca
2205 gtccttttat tcagtataat tttttaaagt tactttgtca gaggcacaaa
aagggtttaa 2265 actgattcat aataaatatc tgtaccttct tcgaaaaaaa
aaaaaaaaaa a 2316 16 742 PRT Homo sapiens SIGNAL (1)..(20) BY
SIMILARITY 16 Met Asp Lys Phe Trp Trp His Ala Ala Trp Gly Leu Cys
Leu Val Pro 1 5 10 15 Leu Ser Leu Ala Gln Ile Asp Leu Asn Ile Thr
Cys Arg Phe Ala Gly 20 25 30 Val Phe His Val Glu Lys Asn Gly Arg
Tyr Ser Ile Ser Arg Thr Glu 35 40 45 Ala Ala Asp Leu Cys Lys Ala
Phe Asn Ser Thr Leu Pro Thr Met Ala 50 55 60 Gln Met Glu Lys Ala
Leu Ser Ile Gly Phe Glu Thr Cys Arg Tyr Gly 65 70 75 80 Phe Ile Glu
Gly His Val Val Ile Pro Arg Ile His Pro Asn Ser Ile 85 90 95 Cys
Ala Ala Asn Asn Thr Gly Val Tyr Ile Leu Thr Ser Asn Thr Ser 100 105
110 Gln Tyr Asp Thr Tyr Cys Phe Asn Ala Ser Ala Pro Pro Glu Glu Asp
115 120 125 Cys Thr Ser Val Thr Asp Leu Pro Asn Ala Phe Asp Gly Pro
Ile Thr 130 135 140 Ile Thr Ile Val Asn Arg Asp Gly Thr Arg Tyr Val
Gln Lys Gly Glu 145 150 155 160 Tyr Arg Thr Asn Pro Glu Asp Ile Tyr
Pro Ser Asn Pro Thr Asp Asp 165 170 175 Asp Val Ser Ser Gly Ser Ser
Ser Glu Arg Ser Ser Thr Ser Gly Gly 180 185 190 Tyr Ile Phe Tyr Thr
Phe Ser Thr Val His Pro Ile Pro Asp Glu Asp 195 200 205 Ser Pro Trp
Ile Thr Asp Ser Thr Asp Arg Ile Pro Ala Thr Thr Leu 210 215 220 Met
Ser Thr Ser Ala Thr Ala Thr Glu Thr Ala Thr Lys Arg Gln Glu 225 230
235 240 Thr Trp Asp Trp Phe Ser Trp Leu Phe Leu Pro Ser Glu Ser Lys
Asn 245 250 255 His Leu His Thr Thr Thr Gln Met Ala Gly Thr Ser Ser
Asn Thr Ile 260 265 270 Ser Ala Gly Trp Glu Pro Asn Glu Glu Asn Glu
Asp Glu Arg Asp Arg 275 280 285 His Leu Ser Phe Ser Gly Ser Gly Ile
Asp Asp Asp Glu Asp Phe Ile 290 295 300 Ser Ser Thr Ile Ser Thr Thr
Pro Arg Ala Phe Asp His Thr Lys Gln 305 310 315 320 Asn Gln Asp Trp
Thr Gln Trp Asn Pro Ser His Ser Asn Pro Glu Val 325 330 335 Leu Leu
Gln Thr Thr Thr Arg Met Thr Asp Val Asp Arg Asn Gly Thr 340 345 350
Thr Ala Tyr Glu Gly Asn Trp Asn Pro Glu Ala His Pro Pro Leu Ile 355
360 365 His His Glu His His Glu Glu Glu Glu Thr Pro His Ser Thr Ser
Thr 370 375 380 Ile Gln Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala
Thr Gln Lys 385 390 395 400 Glu Gln Trp Phe Gly Asn Arg Trp His Glu
Gly Tyr Arg Gln Thr Pro 405 410 415 Arg Glu Asp Ser His Ser Thr Thr
Gly Thr Ala Ala Ala Ser Ala His 420 425 430 Thr Ser His Pro Met Gln
Gly Arg Thr Thr Pro Ser Pro Glu Asp Ser 435 440 445 Ser Trp Thr Asp
Phe Phe Asn Pro Ile Ser His Pro Met Gly Arg Gly 450 455 460 His Gln
Ala Gly Arg Arg Met Asp Met Asp Ser Ser His Ser Thr Thr 465 470 475
480 Leu Gln Pro Thr Ala Asn Pro Asn Thr Gly Leu Val Glu Asp Leu Asp
485 490 495 Arg Thr Gly Pro Leu Ser Met Thr Thr Gln Gln Ser Asn Ser
Gln Ser 500 505 510 Phe Ser Thr Ser His Glu Gly Leu Glu Glu Asp Lys
Asp His Pro Thr 515 520 525 Thr Ser Thr Leu Thr Ser Ser Asn Arg Asn
Asp Val Thr Gly Gly Arg 530 535 540 Arg Asp Pro Asn His Ser Glu Gly
Ser Thr Thr Leu Leu Glu Gly Tyr 545 550 555 560 Thr Ser His Tyr Pro
His Thr Lys Glu Ser Arg Thr Phe Ile Pro Val 565 570 575 Thr Ser Ala
Lys Thr Gly Ser Phe Gly Val Thr Ala Val Thr Val Gly 580 585 590 Asp
Ser Asn Ser Asn Val Asn Arg Ser Leu Ser Gly Asp Gln Asp Thr 595 600
605 Phe His Pro Ser Gly Gly Ser His Thr Thr His Gly Ser Glu Ser Asp
610 615 620 Gly His Ser His Gly Ser Gln Glu Gly Gly Ala Asn Thr Thr
Ser Gly 625 630 635 640 Pro Ile Arg Thr Pro Gln Ile Pro Glu Trp Leu
Ile Ile Leu Ala Ser 645 650 655 Leu Leu Ala Leu Ala Leu Ile Leu Ala
Val Cys Ile Ala Val Asn Ser 660 665 670 Arg Arg Arg Cys Gly Gln Lys
Lys Lys Leu Val Ile Asn Ser Gly Asn 675 680 685 Gly Ala Val Glu Asp
Arg Lys Pro Ser Gly Leu Asn Gly Glu Ala Ser 690 695 700 Lys Ser Gln
Glu Met Val His Leu Val Asn Lys Glu Ser Ser Glu Thr 705 710 715 720
Pro Asp Gln Phe Met Thr Ala Asp Glu Thr Arg Asn Leu Gln Asn Val 725
730 735 Asp Met Lys Ile Gly Val 740 17 930 DNA Homo sapiens Gene
(1)..(930) Tetraspan TM4SF (TSPAN-3), mRNA 17 atg ggc cag tgc ggc
atc acc tcc tcc aag acc gtg ctg gtc ttt ctc 48 Met Gly Gln Cys Gly
Ile Thr Ser Ser Lys Thr Val Leu Val Phe Leu 1 5 10 15 aac ctc atc
ttc tgg ggg gca gct ggc att tta tgc tat gtg gga gcc 96 Asn Leu Ile
Phe Trp Gly Ala Ala Gly Ile Leu Cys Tyr Val Gly Ala 20 25 30 tat
gtc ttc atc act tat gat gac tat gac cac ttc ttt gaa gat gtg 144 Tyr
Val Phe Ile Thr Tyr Asp Asp Tyr Asp His Phe Phe Glu Asp Val 35 40
45 tac acg ctc atc cct gct gta gtg atc ata gct gta gga gcc ctg ctt
192 Tyr Thr Leu Ile Pro Ala Val Val Ile Ile Ala Val Gly Ala Leu Leu
50 55 60 ttc atc att ggg cta att ggc tgc tgt gcc aca atc cgg gaa
agt cgc 240 Phe Ile Ile Gly Leu Ile Gly Cys Cys Ala Thr Ile Arg Glu
Ser Arg 65 70 75 80 tgt gga ctt gcc acg ttt gtc atc atc ctg ctc ttg
gtt ttt gtc aca 288 Cys Gly Leu Ala Thr Phe Val Ile Ile Leu Leu Leu
Val Phe Val Thr 85 90 95 gaa gtt gtt gta gtg gtt ttg gga tat gtt
tac aga gca aag gtg gaa 336 Glu Val Val Val Val Val Leu Gly Tyr Val
Tyr Arg Ala Lys Val Glu 100 105 110 aat gag gtt gat cgc agc att cag
aaa gtg tat aag acc tac aat gga 384 Asn Glu Val Asp Arg Ser Ile Gln
Lys Val Tyr Lys Thr Tyr Asn Gly 115 120 125 acc aac cct gat gct gct
agc cgg gct att gat tat gta cag aga cag 432 Thr Asn Pro Asp Ala Ala
Ser Arg Ala Ile Asp Tyr Val Gln Arg Gln 130 135 140 ctg cat tgt tgt
gga att cac aac tac tca gac tgg gaa aat aca gat 480 Leu His Cys Cys
Gly Ile His Asn Tyr Ser Asp Trp Glu Asn Thr Asp 145 150 155 160 tgg
ttc aaa gaa acc aaa aac cag agt gtc cct ctt agc tgc tgc aga 528 Trp
Phe Lys Glu Thr Lys Asn Gln Ser Val Pro Leu Ser Cys Cys Arg 165 170
175 gag act gcc agc aat tgt aat ggc agc ctg gcc cac cct tcc gac ctc
576 Glu Thr Ala Ser Asn Cys Asn Gly Ser Leu Ala His Pro Ser Asp Leu
180 185 190 tat gct gag ggg tgt gag gct cta gtt gtg aag aag cta caa
gaa atc 624 Tyr Ala Glu Gly Cys Glu Ala Leu Val Val Lys Lys Leu Gln
Glu Ile 195 200 205 atg atg cat gtg atc tgg gcc gca ctg gca ttt gca
gct att cag ctg 672 Met Met His Val Ile Trp Ala Ala Leu Ala Phe Ala
Ala Ile Gln Leu 210 215 220 ctg ggc atg ctg tgt gct tgc atc gtg ttg
tgc aga agg agt aga gat 720 Leu Gly Met Leu Cys Ala Cys Ile Val Leu
Cys Arg Arg Ser Arg Asp 225 230 235 240 cct gct tac gag ctc ctc atc
act ggc gga acc tat gca tag 762 Pro Ala Tyr Glu Leu Leu Ile Thr Gly
Gly Thr Tyr Ala 245 250 ttgacaactc ttgcctgagc tttttggtct tgttctgatt
tggaaggtga attgagcagg 822 tctgctgctg ttggcctctg gagttcattt
agttaaagca catgtacact ggtgttggac 882 agagcagctt ggcttttcat
gtgcccaact acttactact actgcgat 930 18 253 PRT Homo sapiens DOMAIN
(1)..(11) Cytoplasmic (Potential) 18 Met Gly Gln Cys Gly Ile Thr
Ser Ser Lys Thr Val Leu Val Phe Leu 1 5 10 15 Asn Leu Ile Phe Trp
Gly Ala Ala Gly Ile Leu Cys Tyr Val Gly Ala 20 25 30 Tyr Val Phe
Ile Thr Tyr Asp Asp Tyr Asp His Phe Phe Glu Asp Val 35 40 45 Tyr
Thr Leu Ile Pro Ala Val Val Ile Ile Ala Val Gly Ala Leu Leu 50 55
60 Phe Ile Ile Gly Leu Ile Gly Cys Cys Ala Thr Ile Arg Glu Ser Arg
65 70 75 80 Cys Gly Leu Ala Thr Phe Val Ile Ile Leu Leu Leu Val Phe
Val Thr 85 90 95 Glu Val Val Val Val Val Leu Gly Tyr Val Tyr Arg
Ala Lys Val Glu 100 105 110 Asn Glu Val Asp Arg Ser Ile Gln Lys Val
Tyr Lys Thr Tyr Asn Gly 115 120 125 Thr Asn Pro Asp Ala Ala Ser Arg
Ala Ile Asp Tyr Val Gln Arg Gln 130 135 140 Leu His Cys Cys Gly Ile
His Asn Tyr Ser Asp Trp Glu Asn Thr Asp 145 150 155 160 Trp Phe Lys
Glu Thr Lys Asn Gln Ser Val Pro Leu Ser Cys Cys Arg 165 170 175 Glu
Thr Ala Ser Asn Cys Asn Gly Ser Leu Ala His Pro Ser Asp Leu 180 185
190 Tyr Ala Glu Gly Cys Glu Ala Leu Val Val Lys Lys Leu Gln Glu Ile
195 200 205 Met Met His Val Ile Trp Ala Ala Leu Ala Phe Ala Ala Ile
Gln Leu 210 215 220 Leu Gly Met Leu Cys Ala Cys Ile Val Leu Cys Arg
Arg Ser Arg Asp 225 230 235 240 Pro Ala Tyr Glu Leu Leu Ile Thr Gly
Gly Thr Tyr Ala 245 250 19 1317 DNA Homo sapiens Gene (1)..(1317)
Vasoactive Intestinal Peptide Receptor-2 19 atg cgg acg ctg ctg cct
ccc gcg ctg ctg acc tgc tgg ctg ctc gcc 48 Met Arg Thr Leu Leu Pro
Pro Ala Leu Leu Thr Cys Trp Leu Leu Ala 1 5 10 15 ccc gtg aac agc
att cac cca gaa tgc cga ttt cat ctg gaa ata cag 96 Pro Val Asn Ser
Ile His Pro Glu Cys Arg Phe His Leu Glu Ile Gln 20 25 30 gag gaa
gaa aca aaa tgt aca gag ctt ctg agg tct caa aca gaa aaa 144 Glu Glu
Glu Thr Lys Cys Thr Glu Leu Leu Arg Ser Gln Thr Glu Lys 35 40 45
cac aaa gcc tgc agt ggc gtc tgg gac aac atc acg tgc tgg cgg cct 192
His Lys Ala Cys Ser Gly Val Trp Asp Asn Ile Thr Cys Trp Arg Pro 50
55 60 gcc aat gtg gga gag acc gtc acg gtg ccc tgc cca aaa gtc ttc
agc 240 Ala Asn Val Gly Glu Thr Val Thr Val Pro Cys Pro Lys Val Phe
Ser 65 70 75 80 aat ttt tac agc aaa gca gga aac ata agc aaa aac tgt
acg agt gac 288 Asn Phe Tyr Ser Lys Ala Gly Asn Ile Ser Lys Asn Cys
Thr Ser Asp 85 90 95 gga tgg tca gag acg ttc cca gat ttc gtc gat
gcc tgt ggc tac agc 336 Gly Trp Ser Glu Thr Phe Pro Asp Phe Val Asp
Ala Cys Gly Tyr Ser 100 105 110 gac ccg gag gat gag agc aag atc acg
ttt tat att ctg gtg aag gcc 384 Asp Pro Glu Asp Glu Ser Lys Ile Thr
Phe Tyr Ile Leu Val
Lys Ala 115 120 125 att tat acc ctg ggc tac agt gtc tct ctg atg tct
ctt gca aca gga 432 Ile Tyr Thr Leu Gly Tyr Ser Val Ser Leu Met Ser
Leu Ala Thr Gly 130 135 140 agc ata att ctg tgc ctc ttc agg aag ctg
cac tgc acc agg aat tac 480 Ser Ile Ile Leu Cys Leu Phe Arg Lys Leu
His Cys Thr Arg Asn Tyr 145 150 155 160 atc cac ctg aac ctg ttc ctg
tcc ttc atc ctg aga gcc atc tca gtg 528 Ile His Leu Asn Leu Phe Leu
Ser Phe Ile Leu Arg Ala Ile Ser Val 165 170 175 ctg gtc aag gac gac
gtt ctc tac tcc agc tct ggc acg ttg cac tgc 576 Leu Val Lys Asp Asp
Val Leu Tyr Ser Ser Ser Gly Thr Leu His Cys 180 185 190 cct gac cag
cca tcc tcc tgg gtg ggc tgc aag ctg agc ctg gtc ttc 624 Pro Asp Gln
Pro Ser Ser Trp Val Gly Cys Lys Leu Ser Leu Val Phe 195 200 205 ctg
cag tac tgc atc atg gcc aac ttc ttc tgg ctg ctg gtg gag ggg 672 Leu
Gln Tyr Cys Ile Met Ala Asn Phe Phe Trp Leu Leu Val Glu Gly 210 215
220 ctc tac ctc cac acc ctc ctg gtg gcc atg ctc ccc cct aga agg tgc
720 Leu Tyr Leu His Thr Leu Leu Val Ala Met Leu Pro Pro Arg Arg Cys
225 230 235 240 ttc ctg gcc tac ctc ctg atc gga tgg ggc ctc ccc acc
gtc tgc atc 768 Phe Leu Ala Tyr Leu Leu Ile Gly Trp Gly Leu Pro Thr
Val Cys Ile 245 250 255 ggt gca tgg act gcg gcc agg ctc tac tta gaa
gac acc ggt tgc tgg 816 Gly Ala Trp Thr Ala Ala Arg Leu Tyr Leu Glu
Asp Thr Gly Cys Trp 260 265 270 gat aca aac gac cac agt gtg ccc tgg
tgg gtc ata cga ata ccg att 864 Asp Thr Asn Asp His Ser Val Pro Trp
Trp Val Ile Arg Ile Pro Ile 275 280 285 tta att tcc atc atc gtc aat
ttt gtc ctt ttc att agt att ata cga 912 Leu Ile Ser Ile Ile Val Asn
Phe Val Leu Phe Ile Ser Ile Ile Arg 290 295 300 att ttg ctg cag aag
tta aca tcc cca gat gtc ggc ggc aac gac cag 960 Ile Leu Leu Gln Lys
Leu Thr Ser Pro Asp Val Gly Gly Asn Asp Gln 305 310 315 320 tct cag
tac aag agg ctg gcc aag tcc acg ctc ctg ctt atc ccg ctg 1008 Ser
Gln Tyr Lys Arg Leu Ala Lys Ser Thr Leu Leu Leu Ile Pro Leu 325 330
335 ttc ggc gtc cac tac atg gtg ttt gcc gtg ttt ccc atc agc atc tcc
1056 Phe Gly Val His Tyr Met Val Phe Ala Val Phe Pro Ile Ser Ile
Ser 340 345 350 tcc aaa tac cag ata ctg ttt gag ctg tgc ctc ggg tcg
ttc cag ggc 1104 Ser Lys Tyr Gln Ile Leu Phe Glu Leu Cys Leu Gly
Ser Phe Gln Gly 355 360 365 ctg gtg gtg gcc gtc ctc tac tgt ttc ctg
aac agt gag gtg cag tgc 1152 Leu Val Val Ala Val Leu Tyr Cys Phe
Leu Asn Ser Glu Val Gln Cys 370 375 380 gag ctg aag cga aaa tgg cga
agc cgg tgc ccg acc ccg tcc gcg agc 1200 Glu Leu Lys Arg Lys Trp
Arg Ser Arg Cys Pro Thr Pro Ser Ala Ser 385 390 395 400 cgg gat tac
agg gtc tgc ggt tcc tcc ttc tcc cac aac ggc tcg gag 1248 Arg Asp
Tyr Arg Val Cys Gly Ser Ser Phe Ser His Asn Gly Ser Glu 405 410 415
ggc gcc ctg cag ttc cac cgc gcg tcc cga gcc cag tcc ttc ctg caa
1296 Gly Ala Leu Gln Phe His Arg Ala Ser Arg Ala Gln Ser Phe Leu
Gln 420 425 430 acg gag acc tcg gtc atc tag 1317 Thr Glu Thr Ser
Val Ile 435 20 438 PRT Homo sapiens SIGNAL (1)..(23) Potential 20
Met Arg Thr Leu Leu Pro Pro Ala Leu Leu Thr Cys Trp Leu Leu Ala 1 5
10 15 Pro Val Asn Ser Ile His Pro Glu Cys Arg Phe His Leu Glu Ile
Gln 20 25 30 Glu Glu Glu Thr Lys Cys Ala Glu Leu Leu Arg Ser Gln
Thr Glu Lys 35 40 45 His Lys Ala Cys Ser Gly Val Trp Asp Asn Ile
Thr Cys Trp Arg Pro 50 55 60 Ala Asn Val Gly Glu Thr Val Thr Val
Pro Cys Pro Lys Val Phe Ser 65 70 75 80 Asn Phe Tyr Ser Lys Ala Gly
Asn Ile Ser Lys Asn Cys Thr Ser Asp 85 90 95 Gly Trp Ser Glu Thr
Phe Pro Asp Phe Val Asp Ala Cys Gly Tyr Ser 100 105 110 Asp Pro Glu
Asp Glu Ser Lys Ile Thr Phe Tyr Ile Leu Val Lys Ala 115 120 125 Ile
Tyr Thr Leu Gly Tyr Ser Val Ser Leu Met Ser Leu Ala Thr Gly 130 135
140 Ser Ile Ile Leu Cys Leu Phe Arg Lys Leu His Cys Thr Arg Asn Tyr
145 150 155 160 Ile His Leu Asn Leu Phe Leu Ser Phe Ile Leu Arg Ala
Ile Ser Val 165 170 175 Leu Val Lys Asp Asp Val Leu Tyr Ser Ser Ser
Gly Thr Leu His Cys 180 185 190 Pro Asp Gln Pro Ser Ser Trp Val Gly
Cys Lys Leu Ser Leu Val Phe 195 200 205 Leu Gln Tyr Cys Ile Met Ala
Asn Phe Phe Trp Leu Leu Val Glu Gly 210 215 220 Leu Tyr Leu His Thr
Leu Leu Val Ala Met Leu Pro Pro Arg Arg Cys 225 230 235 240 Phe Leu
Ala Tyr Leu Leu Ile Gly Trp Gly Leu Pro Thr Val Cys Ile 245 250 255
Gly Ala Trp Thr Ala Ala Arg Leu Tyr Leu Glu Asp Thr Gly Cys Trp 260
265 270 Asp Thr Asn Asp His Ser Val Pro Trp Trp Val Ile Arg Ile Pro
Ile 275 280 285 Leu Ile Ser Ile Ile Val Asn Phe Val Leu Phe Ile Ser
Ile Ile Arg 290 295 300 Ile Leu Leu Gln Lys Leu Thr Ser Pro Asp Val
Gly Gly Asn Asp Gln 305 310 315 320 Ser Gln Tyr Lys Arg Leu Ala Lys
Ser Thr Leu Leu Leu Ile Pro Leu 325 330 335 Phe Gly Val His Tyr Met
Val Phe Ala Val Phe Pro Ile Ser Ile Ser 340 345 350 Ser Lys Tyr Gln
Ile Leu Phe Glu Leu Cys Leu Gly Ser Phe Gln Gly 355 360 365 Leu Val
Val Ala Val Leu Tyr Cys Phe Leu Asn Ser Glu Val Gln Cys 370 375 380
Glu Leu Lys Arg Lys Trp Arg Ser Arg Cys Pro Thr Pro Ser Ala Ser 385
390 395 400 Arg Asp Tyr Arg Val Cys Gly Ser Ser Phe Ser Arg Asn Gly
Ser Glu 405 410 415 Gly Ala Leu Gln Phe His Arg Gly Ser Arg Ala Gln
Ser Phe Leu Gln 420 425 430 Thr Glu Thr Ser Val Ile 435 21 889 DNA
Homo sapiens Gene (1)..(889) Pleiotrophin 21 gtcaaaggca ggatcaggtt
ccccgccttc cagtccaaaa atcccgccaa gagagcccca 60 gagcagagga
aaatccaaag tggagagagg ggaagaaaga gaccagtgag tcatccgtcc 120
agaaggcggg gagagcagca gcggcccaag caggagctgc agcgagccgg gtacctggac
180 tcagcggtag caacctcgcc ccttgcaaca aaggcagact gagcgccaga
gaggacgttt 240 ccaactcaaa a atg cag gct caa cag tac cag cag cag cgt
cga aaa ttt 290 Met Gln Ala Gln Gln Tyr Gln Gln Gln Arg Arg Lys Phe
1 5 10 gca gct gcc ttc ttg gca ttc att ttc ata ctg gca gct gtg gat
act 338 Ala Ala Ala Phe Leu Ala Phe Ile Phe Ile Leu Ala Ala Val Asp
Thr 15 20 25 gct gaa gca ggg aag aaa gag aaa cca gaa aaa aaa gtg
aag aag tct 386 Ala Glu Ala Gly Lys Lys Glu Lys Pro Glu Lys Lys Val
Lys Lys Ser 30 35 40 45 gac tgt gga gaa tgg cag tgg agt gtg tgt gtg
ccc acc agt gga gac 434 Asp Cys Gly Glu Trp Gln Trp Ser Val Cys Val
Pro Thr Ser Gly Asp 50 55 60 tgt ggg ctg ggc aca cgg gag ggc act
cgg act gga gct gag tgc aag 482 Cys Gly Leu Gly Thr Arg Glu Gly Thr
Arg Thr Gly Ala Glu Cys Lys 65 70 75 caa acc atg aag acc cag aga
tgt aag atc ccc tgc aac tgg aag aag 530 Gln Thr Met Lys Thr Gln Arg
Cys Lys Ile Pro Cys Asn Trp Lys Lys 80 85 90 caa ttt ggc gcg gag
tgc aaa tac cag ttc cag gcc tgg gga gaa tgt 578 Gln Phe Gly Ala Glu
Cys Lys Tyr Gln Phe Gln Ala Trp Gly Glu Cys 95 100 105 gac ctg aac
aca gcc ctg aag acc aga act gga agt ctg aag cga gcc 626 Asp Leu Asn
Thr Ala Leu Lys Thr Arg Thr Gly Ser Leu Lys Arg Ala 110 115 120 125
ctg cac aat gcc gaa tgc cag aag act gtc acc atc tcc aag ccc tgt 674
Leu His Asn Ala Glu Cys Gln Lys Thr Val Thr Ile Ser Lys Pro Cys 130
135 140 ggc aaa ctg acc aag ccc aaa cct caa gca gaa tct aag aag aag
aaa 722 Gly Lys Leu Thr Lys Pro Lys Pro Gln Ala Glu Ser Lys Lys Lys
Lys 145 150 155 aag gaa ggc aag aaa cag gag aag atg ctg gat taa
aagatgtcac 768 Lys Glu Gly Lys Lys Gln Glu Lys Met Leu Asp 160 165
ctgtggaaca taaaaaggac atcagcaaac aggatcagtt aactattgca tttatatgta
828 ccgtaggctt tgtattcaaa aattatctat agctaagtac acaataagca
aaaacaaaaa 888 g 889 22 168 PRT Homo sapiens SIGNAL (1)..(32) 22
Met Gln Ala Gln Gln Tyr Gln Gln Gln Arg Arg Lys Phe Ala Ala Ala 1 5
10 15 Phe Leu Ala Phe Ile Phe Ile Leu Ala Ala Val Asp Thr Ala Glu
Ala 20 25 30 Gly Lys Lys Glu Lys Pro Glu Lys Lys Val Lys Lys Ser
Asp Cys Gly 35 40 45 Glu Trp Gln Trp Ser Val Cys Val Pro Thr Ser
Gly Asp Cys Gly Leu 50 55 60 Gly Thr Arg Glu Gly Thr Arg Thr Gly
Ala Glu Cys Lys Gln Thr Met 65 70 75 80 Lys Thr Gln Arg Cys Lys Ile
Pro Cys Asn Trp Lys Lys Gln Phe Gly 85 90 95 Ala Glu Cys Lys Tyr
Gln Phe Gln Ala Trp Gly Glu Cys Asp Leu Asn 100 105 110 Thr Ala Leu
Lys Thr Arg Thr Gly Ser Leu Lys Arg Ala Leu His Asn 115 120 125 Ala
Glu Cys Gln Lys Thr Val Thr Ile Ser Lys Pro Cys Gly Lys Leu 130 135
140 Thr Lys Pro Lys Pro Gln Ala Glu Ser Lys Lys Lys Lys Lys Glu Gly
145 150 155 160 Lys Lys Gln Glu Lys Met Leu Asp 165 23 3143 DNA
Homo sapiens Gene (1)..(3143) Osteopontin 23 ggggaagtgt gggagcaggt
gggctgggca gtggcagaaa cctgatgaca caatctcgcc 60 gcctccctgt
gttggtggag gatgtctgca gcagcattta aattctggga gggcttggtt 120
gtcagcagca gcaggaggag gcagagacag catcgtcggg accagactcg tctcaggcca
180 gttgcagcct tctcagccaa acgccgacca aggtacagct tcagtttgct
actgggttgt 240 gcattcagct gaatttcatg gggaagtcca aattctaagg
aaaaaaatgt ggtagtataa 300 aaaggtatca ctgttgtaac ctatgaagat
gtcagctatt cctttgaaat attttgcagg 360 aaaactcact acc atg aga att gca
gtg att tgc ttt tgc ctc cta ggc 409 Met Arg Ile Ala Val Ile Cys Phe
Cys Leu Leu Gly 1 5 10 atc acc tgt gcc ata cca gtgagtacag
ttgcatctta aagaaaattc 457 Ile Thr Cys Ala Ile Pro 15 ctgaaaataa
ctgaattgtg tgcttccatg tgctaggagg acattcttgt aatctttctt 517
catcttttct gtttctaag gtt aaa cag gct gat tct gga agt tct gag gaa
569 Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu 20 25 aag cag
gtaagcatct tttatgtttt tatatagtta aatcatttac tcaattatgg 625 Lys Gln
30 cgagaggtgc aagaaacgta tttgctgcga tcaaatgagt tcatatttgt
aaagcaattt 685 gaaagagtgc ctagcccaca gtaagtgcta cataagagtt
tgttaaatga atctgcaaaa 745 aaaaaaaaaa ttacaaaaag gtacctaagg
gtccgggtga ctatatgctt ccatcaagac 805 tagtgaagaa tggttgtttt
ttccattcat ccctacattt ctttttttaa taatgataaa 865 catgcaactt ttttgtag
ctt tac aac aaa tac cca gat gct gtg gcc aca 916 Leu Tyr Asn Lys Tyr
Pro Asp Ala Val Ala Thr 35 40 tgg cta aac cct gac cca tct cag aag
cag aat ctc cta gcc cca cag 964 Trp Leu Asn Pro Asp Pro Ser Gln Lys
Gln Asn Leu Leu Ala Pro Gln 45 50 55 gtatttttaa acttctcata
attaaactac agtgatgaaa gatagccaca ctcaggccat 1024 ttgggctgct
cagatgaatc ctgccctgcc tgctggcaaa catgtgctta ggacattgac 1084
tgatctgcca tgttggcttc tctctgtgtt aagccatcca cagatgaggc tgaaaaataa
1144 aaactgcttt ggattaaaaa ggttaacttt tgaataaaaa agctaggcat
gtgtgatgcg 1204 cactaacacg tgccattcct tcttcag aat gct gtg tcc tct
gaa gaa acc aat 1258 Asn Ala Val Ser Ser Glu Glu Thr Asn 60 65 gac
ttt aaa caa gag gtaagttctc attttcaatc agaggcccat catgccttga 1313
Asp Phe Lys Gln Glu 70 agagatgaaa gaaggcattg cctggattct cttctgatga
aatttcatta gcaagttttc 1373 cagctaattg gcagtctaaa acttgctcat
aaataaaaca tgtatttact aaatatcaga 1433 aatactaggt ttcctcggat
aacctaaaag ccatggtatg tactgtgaat gcaaagattc 1493 tgaaactaaa
taaaaagaaa gatagtaaaa gactaatgtg ctataaaggc taagggaaaa 1553
taaaaaccca tatattaatt ttcccggcca tcttaatttt cag acc ctt cca agt
1608 Thr Leu Pro Ser 75 aag tcc aac gaa agc cat gac cac atg gat gat
atg gat gat gaa gat 1656 Lys Ser Asn Glu Ser His Asp His Met Asp
Asp Met Asp Asp Glu Asp 80 85 90 gat gat gac cat gtg gac agc cag
gac tcc att gac tcg aac gac tct 1704 Asp Asp Asp His Val Asp Ser
Gln Asp Ser Ile Asp Ser Asn Asp Ser 95 100 105 gat gat gta gat gac
act gat gat tct cac cag tct gat gag tct cac 1752 Asp Asp Val Asp
Asp Thr Asp Asp Ser His Gln Ser Asp Glu Ser His 110 115 120 cat tct
gat gaa tct gat gaa ctg gtc act gat ttt ccc acg gac ctg 1800 His
Ser Asp Glu Ser Asp Glu Leu Val Thr Asp Phe Pro Thr Asp Leu 125 130
135 140 cca gca acc gaa gtt ttc act cca gtt gtc ccc aca gta gac aca
tat 1848 Pro Ala Thr Glu Val Phe Thr Pro Val Val Pro Thr Val Asp
Thr Tyr 145 150 155 gat ggc cga ggt gat agt gtg gtt tat gga ctg agg
tca aaa tct aag 1896 Asp Gly Arg Gly Asp Ser Val Val Tyr Gly Leu
Arg Ser Lys Ser Lys 160 165 170 aag ttt cgc aga cct gac atc cag
gtaaatcctt taacagacac acctgatggt 1950 Lys Phe Arg Arg Pro Asp Ile
Gln 175 180 tctgactagc gctcaagtct aggaaaccac agtttgcata ttcattcatt
cattcatcca 2010 ttcattcatc cattcagcaa gaattcattc atattctact
ttatgaccat tgaatacaaa 2070 tctttttctg cttggcggtt tttgtaagtc
tacataattt ctctctagat ttgattctca 2130 aacacaattc tactttttga
aatcctggat caaagtaaca tgctagtatt atttcagcca 2190 gatttagaca
atttttagta taagatgacc taaaagctag agagtggaaa aggattacca 2250
tattcccatc cctagccgtt catataatta ttcttcattt gtgccgtgat tcag tac
2307 Tyr cct gat gct aca gac gag gac atc acc tca cac atg gaa agc
gag gag 2355 Pro Asp Ala Thr Asp Glu Asp Ile Thr Ser His Met Glu
Ser Glu Glu 185 190 195 ttg aat ggt gca tac aag gcc atc ccc gtt gcc
cag gac ctg aac gcg 2403 Leu Asn Gly Ala Tyr Lys Ala Ile Pro Val
Ala Gln Asp Leu Asn Ala 200 205 210 cct tct gat tgg gac agc cgt ggg
aag gac agt tat gaa acg agt cag 2451 Pro Ser Asp Trp Asp Ser Arg
Gly Lys Asp Ser Tyr Glu Thr Ser Gln 215 220 225 ctg gat gac cag agt
gct gaa acc cac agc cac aag cag tcc aga tta 2499 Leu Asp Asp Gln
Ser Ala Glu Thr His Ser His Lys Gln Ser Arg Leu 230 235 240 245 tat
aag cgg aaa gcc aat gat gag agc aat gag cat tcc gat gtg att 2547
Tyr Lys Arg Lys Ala Asn Asp Glu Ser Asn Glu His Ser Asp Val Ile 250
255 260 gat agt cag gaa ctt tcc aaa gtc agc cgt gaa ttc cac agc cat
gaa 2595 Asp Ser Gln Glu Leu Ser Lys Val Ser Arg Glu Phe His Ser
His Glu 265 270 275 ttt cac agc cat gaa gat atg ctg gtt gta gac ccc
aaa agt aag gaa 2643 Phe His Ser His Glu Asp Met Leu Val Val Asp
Pro Lys Ser Lys Glu 280 285 290 gaa gat aaa cac ctg aaa ttt cgt att
tct cat gaa tta gat agt gca 2691 Glu Asp Lys His Leu Lys Phe Arg
Ile Ser His Glu Leu Asp Ser Ala 295 300 305 tct tct gag gtc aat taa
aaggagaaaa aatacaattt ctcactttgc 2739 Ser Ser Glu Val Asn 310
atttagtcaa aagaaaaaat gctttatagc aaaatgaaag agaacatgaa atgcttcttt
2799 ctcagtttat tggttgaatg tgtatctatt tgagtctgga aataactaat
gtgtttgata 2859 attagtttag tttgtggctt catggaaact ccctgtaaac
aaaagcttca gggttatgtc 2919 tatgttcatt ctatagaaga aatgcaaact
atcactgtat tttaatattt gttattctct 2979 catgaataga aatttatgta
gaagcaaaca aaatactttt acccacttaa aaagagaata 3039 taacatttta
tgtcactata atcttttgtt ttttaagtta gtgtatattt tgttgtgatt 3099
atcttttgtg gtgtgaataa atcttttatc ttgaatgtaa taag 3143 24 314 PRT
Homo sapiens SIGNAL (1)..(16) Potential 24 Met Arg Ile Ala Val Ile
Cys Phe Cys Leu Leu Gly Ile Thr Cys Ala 1 5 10 15 Ile Pro Val Lys
Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln Leu 20 25 30 Tyr Asn
Lys Tyr Pro Asp Ala Val Ala Thr Trp Leu Asn Pro Asp Pro
35 40 45 Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln Asn Ala Val Ser
Ser Glu 50 55 60 Glu Thr Asn Asp Phe Lys Gln Glu Thr Leu Pro Ser
Lys Ser Asn Glu 65 70 75 80 Ser His Asp His Met Asp Asp Met Asp Asp
Glu Asp Asp Asp Asp His 85 90 95 Val Asp Ser Gln Asp Ser Ile Asp
Ser Asn Asp Ser Asp Asp Val Asp 100 105 110 Asp Thr Asp Asp Ser His
Gln Ser Asp Glu Ser His His Ser Asp Glu 115 120 125 Ser Asp Glu Leu
Val Thr Asp Phe Pro Thr Asp Leu Pro Ala Thr Glu 130 135 140 Val Phe
Thr Pro Val Val Pro Thr Val Asp Thr Tyr Asp Gly Arg Gly 145 150 155
160 Asp Ser Val Val Tyr Gly Leu Arg Ser Lys Ser Lys Lys Phe Arg Arg
165 170 175 Pro Asp Ile Gln Tyr Pro Asp Ala Thr Asp Glu Asp Ile Thr
Ser His 180 185 190 Met Glu Ser Glu Glu Leu Asn Gly Ala Tyr Lys Ala
Ile Pro Val Ala 195 200 205 Gln Asp Leu Asn Ala Pro Ser Asp Trp Asp
Ser Arg Gly Lys Asp Ser 210 215 220 Tyr Glu Thr Ser Gln Leu Asp Asp
Gln Ser Ala Glu Thr His Ser His 225 230 235 240 Lys Gln Ser Arg Leu
Tyr Lys Arg Lys Ala Asn Asp Glu Ser Asn Glu 245 250 255 His Ser Asp
Val Ile Asp Ser Gln Glu Leu Ser Lys Val Ser Arg Glu 260 265 270 Phe
His Ser His Glu Phe His Ser His Glu Asp Met Leu Val Val Asp 275 280
285 Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys Phe Arg Ile Ser His
290 295 300 Glu Leu Asp Ser Ala Ser Ser Glu Val Asn 305 310 25 259
PRT Homo sapiens Gene (1)..(259) Carbonic Anhydrase domain of human
carbonic anhydrase III 25 Ala Lys Glu Trp Gly Tyr Ala Ser His Asn
Gly Pro Asp His Trp His 1 5 10 15 Glu Leu Phe Pro Asn Ala Lys Gly
Glu Asn Gln Ser Pro Ile Glu Leu 20 25 30 His Thr Lys Asp Ile Arg
His Asp Pro Ser Leu Gln Pro Trp Ser Val 35 40 45 Ser Tyr Asp Gly
Gly Ser Ala Lys Thr Ile Leu Asn Asn Gly Lys Thr 50 55 60 Cys Arg
Val Val Phe Asp Asp Thr Tyr Asp Arg Ser Met Leu Arg Gly 65 70 75 80
Gly Pro Leu Pro Gly Pro Tyr Arg Leu Arg Gln Phe His Leu His Trp 85
90 95 Gly Ser Ser Asp Asp His Gly Ser Glu His Thr Val Asp Gly Val
Lys 100 105 110 Tyr Ala Ala Glu Leu His Leu Val His Trp Asn Pro Lys
Tyr Asn Thr 115 120 125 Phe Lys Glu Ala Leu Lys Gln Arg Asp Gly Ile
Ala Val Ile Gly Ile 130 135 140 Phe Leu Lys Ile Gly His Glu Asn Gly
Glu Phe Gln Ile Phe Leu Asp 145 150 155 160 Ala Leu Asp Lys Ile Lys
Thr Lys Gly Lys Glu Ala Pro Phe Thr Lys 165 170 175 Phe Asp Pro Ser
Cys Leu Phe Pro Ala Cys Arg Asp Tyr Trp Thr Tyr 180 185 190 Gln Gly
Ser Phe Thr Thr Pro Pro Cys Glu Glu Cys Ile Val Trp Leu 195 200 205
Leu Leu Lys Glu Pro Met Thr Val Ser Ser Asp Gln Met Ala Lys Leu 210
215 220 Arg Ser Leu Leu Ser Ser Ala Glu Asn Glu Pro Pro Val Pro Leu
Val 225 230 235 240 Ser Asn Trp Arg Pro Pro Gln Pro Ile Asn Asn Arg
Val Val Arg Ala 245 250 255 Ser Phe Lys 26 260 PRT Homo sapiens
Gene (1)..(260) Carbonic anhydrase domain of human carbonic
anhydrase I 26 Ala Ser Pro Asp Trp Gly Tyr Asp Asp Lys Asn Gly Pro
Glu Gln Trp 1 5 10 15 Ser Lys Leu Tyr Pro Ile Ala Asn Gly Asn Asn
Gln Ser Pro Val Asp 20 25 30 Ile Lys Thr Ser Glu Thr Lys His Asp
Thr Ser Leu Lys Pro Ile Ser 35 40 45 Val Ser Tyr Asn Pro Ala Thr
Ala Lys Glu Ile Ile Asn Val Gly His 50 55 60 Ser Phe His Val Asn
Phe Glu Asp Asn Asp Asn Arg Ser Val Leu Lys 65 70 75 80 Gly Gly Pro
Phe Ser Asp Ser Tyr Arg Leu Phe Gln Phe His Phe His 85 90 95 Trp
Gly Ser Thr Asn Glu His Gly Ser Glu His Thr Val Asp Gly Val 100 105
110 Lys Tyr Ser Ala Glu Leu His Val Ala His Trp Asn Ser Ala Lys Tyr
115 120 125 Ser Ser Leu Ala Glu Ala Ala Ser Lys Ala Asp Gly Leu Ala
Val Ile 130 135 140 Gly Val Leu Met Lys Val Gly Glu Ala Asn Pro Lys
Leu Gln Lys Val 145 150 155 160 Leu Asp Ala Leu Gln Ala Ile Lys Thr
Lys Gly Lys Arg Ala Pro Phe 165 170 175 Thr Asn Phe Asp Pro Ser Thr
Leu Leu Pro Ser Ser Leu Asp Phe Trp 180 185 190 Thr Tyr Pro Gly Ser
Leu Thr His Pro Pro Leu Tyr Glu Ser Val Thr 195 200 205 Trp Ile Ile
Cys Lys Glu Ser Ile Ser Val Ser Ser Glu Gln Leu Ala 210 215 220 Gln
Phe Arg Ser Leu Leu Ser Asn Val Glu Gly Asp Asn Ala Val Pro 225 230
235 240 Met Gln His Asn Asn Arg Pro Thr Gln Pro Leu Lys Gly Arg Thr
Val 245 250 255 Arg Ala Ser Phe 260 27 337 PRT Homo sapiens Gene
(1)..(337) Carbonic anhydrase domain of human carbonic anhydrase
VIX 27 Met Leu Phe Ser Ala Leu Leu Leu Glu Val Ile Trp Ile Leu Ala
Ala 1 5 10 15 Asp Gly Gly Gln His Trp Thr Tyr Glu Gly Pro His Gly
Gln Asp His 20 25 30 Trp Pro Ala Ser Tyr Pro Glu Cys Gly Asn Asn
Ala Gln Ser Pro Ile 35 40 45 Asp Ile Gln Thr Asp Ser Val Thr Phe
Asp Pro Asp Leu Pro Ala Leu 50 55 60 Gln Pro His Gly Tyr Asp Gln
Pro Gly Thr Glu Pro Leu Asp Leu His 65 70 75 80 Asn Asn Gly His Thr
Val Gln Leu Ser Leu Pro Ser Thr Leu Tyr Leu 85 90 95 Gly Gly Leu
Pro Arg Lys Tyr Val Ala Ala Gln Leu His Leu His Trp 100 105 110 Gly
Gln Lys Gly Ser Pro Gly Gly Ser Glu His Gln Ile Asn Ser Glu 115 120
125 Ala Thr Phe Ala Glu Leu His Ile Val His Tyr Asp Ser Asp Ser Tyr
130 135 140 Asp Ser Leu Ser Glu Ala Ala Glu Arg Pro Gln Gly Leu Ala
Val Leu 145 150 155 160 Gly Ile Leu Ile Glu Val Gly Glu Thr Lys Asn
Ile Ala Tyr Glu His 165 170 175 Ile Leu Ser His Leu His Glu Val Arg
His Lys Asp Gln Lys Thr Ser 180 185 190 Val Pro Pro Phe Asn Leu Arg
Glu Leu Leu Pro Lys Gln Leu Gly Gln 195 200 205 Tyr Phe Arg Tyr Asn
Gly Ser Leu Thr Thr Pro Pro Cys Tyr Gln Ser 210 215 220 Val Leu Trp
Thr Val Phe Tyr Arg Arg Ser Gln Ile Ser Met Glu Gln 225 230 235 240
Leu Glu Lys Leu Gln Gly Thr Leu Phe Ser Thr Glu Glu Glu Pro Ser 245
250 255 Lys Leu Leu Val Gln Asn Tyr Arg Ala Leu Gln Pro Leu Asn Gln
Arg 260 265 270 Met Val Phe Ala Ser Phe Ile Gln Ala Gly Ser Ser Tyr
Thr Thr Gly 275 280 285 Glu Met Leu Ser Leu Gly Val Gly Ile Leu Val
Gly Cys Leu Cys Leu 290 295 300 Leu Leu Ala Val Tyr Phe Ile Ala Arg
Lys Ile Arg Lys Lys Arg Leu 305 310 315 320 Glu Asn Arg Lys Ser Val
Val Phe Thr Ser Ala Gln Ala Thr Thr Glu 325 330 335 Ala 28 22 DNA
Artificial sequence Primer 28 cagcagttgg atggaagagg ac 22 29 22 DNA
Artificial sequence Primer 29 cactgagatt ctggcactat tc 22 30 21 DNA
Artificial sequence Primer 30 aacaattcca gggtctcact c 21 31 21 DNA
Artificial sequence Primer 31 ttgactggct caggagtata g 21 32 21 DNA
Artificial sequence Primer 32 ctgataatga gggctcccaa c 21 33 24 DNA
Artificial sequence Primer 33 ctctgcactt cctggtaaaa ctct 24 34 22
DNA Artificial sequence Primer 34 cagcagttgg atggaagagg ac 22 35 24
DNA Artificial sequence Primer 35 ctctgcactt cctggtaaaa ctct 24
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