U.S. patent application number 11/135663 was filed with the patent office on 2006-07-06 for dna encoding mck-10, a novel receptor tyrosine kinase.
This patent application is currently assigned to Max-Planck-Gesellschaft zur Foederung der Wissenschaften. Invention is credited to Frauke Hildegard Elisabeth Alves, Axel Ullrich.
Application Number | 20060147372 11/135663 |
Document ID | / |
Family ID | 22547060 |
Filed Date | 2006-07-06 |
United States Patent
Application |
20060147372 |
Kind Code |
A1 |
Ullrich; Axel ; et
al. |
July 6, 2006 |
DNA encoding MCK-10, a novel receptor tyrosine kinase
Abstract
The present invention relates to the novel family of receptor
tyrosine kinases, herein referred to as MCK-10, to nucleotide
sequences and expression vectors encoding MCK-10, and to methods of
inhibiting MCK-10 activity. The invention relates to differentially
spliced isoforms of MCK-10 and to other members of the MCK-10
receptor tyrosine kinase family. Genetically engineered host cells
that express MCK-10 may be used to evaluate and screen drugs
involved in MCK-10 activation and regulation. The invention relates
to the use of such drugs, in the treatment of disorders, including
cancer, by modulating the activity of MCK-10.
Inventors: |
Ullrich; Axel; (Muenchen,
DE) ; Alves; Frauke Hildegard Elisabeth; (Goettingen,
DE) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Max-Planck-Gesellschaft zur
Foederung der Wissenschaften
|
Family ID: |
22547060 |
Appl. No.: |
11/135663 |
Filed: |
May 24, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09551188 |
Apr 17, 2000 |
6897029 |
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11135663 |
May 24, 2005 |
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08153397 |
Nov 16, 1993 |
6051397 |
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09551188 |
Apr 17, 2000 |
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Current U.S.
Class: |
424/1.49 ;
530/388.26; 530/391.1 |
Current CPC
Class: |
A61K 38/00 20130101;
A61K 51/103 20130101; C12N 2799/027 20130101; C07K 2317/34
20130101; C07K 16/2863 20130101; C07K 2319/00 20130101; C07K 14/705
20130101 |
Class at
Publication: |
424/001.49 ;
530/388.26; 530/391.1 |
International
Class: |
C07K 16/46 20060101
C07K016/46; C07K 16/40 20060101 C07K016/40; A61K 51/00 20060101
A61K051/00 |
Claims
1-20. (canceled)
21. A monoclonal antibody which immunospecifically binds to an
epitope of the MCK-10.
22. The monoclonal antibody of claim 21 which competitively
inhibits the binding of ligand to the MCK-10.
23. The monoclonal antibody of claim 21 which is linked to a
cytotoxic agent.
24. The monoclonal antibody of claim 21 which is liked to a
radioisotope.
25-74. (canceled)
Description
1. INTRODUCTION
[0001] The present invention relates to the novel family of
receptor tyrosine kinases, herein referred to as MCK-10, to
nucleotide sequences and expression vectors encoding MCK-10, and to
methods of inhibiting MCK-10 activity. The invention relates to
differentially spliced isoforms of MCK-10 and to other members of
the MCK-10 receptor tyrosine kinase family. Genetically engineered
host cells that express MCK-10 may be used to evaluate and screen
drugs involved in MCK-10 activation and regulation. The invention
relates to the use of such drugs, in the treatment of disorders,
including cancer, by modulating the activity of MCK-10.
2. BACKGROUND
[0002] Receptor tyrosine kinases comprise a large family of
transmembrane receptors which are comprised of an extracellular
ligand-binding domain and an intracellular tyrosine-kinase domain
responsible for mediating receptor activity. The receptor tyrosine
kinases are involved in a variety of normal cellular responses
which include proliferation, alterations in gene expression, and
changes in cell shape.
[0003] The binding of ligand to its cognate receptor induces the
formation of receptor dimers leading to activation of receptor
kinase activity. The activation of kinase activity results in
phosphorylation of multiple cellular substrates involved in the
cascade of events leading to cellular responses such as cell
proliferation.
[0004] Genetic alterations in growth factor mediated signalling
pathways have been linked to a number of different diseases,
including human cancer. For example, the normal homologs of many
oncogenes have been found to encode growth factors or growth factor
receptors. This is illustrated by the discovery that the B chain of
human PDGF is homologous to the transforming protein of simian
sarcoma virus (SSV), the EGF (epidermal growth factor) receptor to
erb B; the CSF (colony stimulating factor) receptor to fms; and the
NGF (nerve growth factor) receptor to trk. In addition, growth
factor receptors are often found amplified and/or overexpressed in
cancer cells as exemplified by the observation that the EGF
receptor is often found amplified or overexpressed in squamous cell
carcinomas and glioblastomas. Similarly, amplification and
overexpression of the met gene, encoding the HGF receptor, has been
detected in stomach carcinomas.
[0005] Recently, a number of cDNAs have been identified that encode
receptor tyrosine kinases. One such clone, referred to as DDR
(discoidin domain receptor), was isolated from a breast carcinoma
cDNA library (Johnson et al., 1993, Proc. Natl. Acad. Sci. USA, 90,
5677-57681) and is homologous to MCK-10. In addition, a mouse
homologue of MCK-10 has recently been cloned and characterized
(Yerlin, M. et al., 1993, Oncongene, 8:2731-2739).
[0006] The discovery of novel receptor tyrosine kinase receptors,
whose expression is associated with proliferative diseases such as
cancer, will provide opportunities for development of novel
diagnostic reagents. In addition, the identification of aberrantly
expressed receptor tyrosine kinases will lead to the development of
therapeutic applications designed to inhibit the activity of that
receptor, which may be useful for treatment of proliferative
diseases such as cancer.
3. SUMMARY OF THE INVENTION
[0007] The present invention relates to a novel family of receptor
tyrosine kinases, herein referred to as MCK-10 (mammary carcinoma
kinase 10), to nucleotide sequences and expression vectors encoding
MCK-10, and to methods of inhibiting MCK-10 activity. The invention
is based on the isolation of cDNA clones from a human mammary
carcinoma cDNA library encoding the MCK-10 receptor tyrosine
kinase.
[0008] The invention also relates to differentially spliced
isoforms of MCK-10 and to other members of the MCK-10 family of
receptor tyrosine kinases. More specifically, the invention relates
to members of the MCK-10 family of receptors tyrosine kinases that
are defined, herein, as those receptors demonstrating 80% homology
at the amino acid level in substantial stretches of DNA sequences
with MCK-10. In addition, members of the MCK-10 family of tyrosine
kinase receptors are defined as those receptors containing an
intracellular tyrosine kinase domain and consensus sequences near
the extracellular N-terminus of the protein for the discoidin I
like family of proteins. The invention as it relates to the members
of the MCK-10 family of receptor tyrosine kinases, is based on the
isolation and characterization of a cDNA, herein referred to as
CCK-2, encoding a member of the MCK-10 family of receptor tyrosine
kinases.
[0009] Northern blot analysis and in situ hybridization indicates
that MCK-10 is expressed in a wide variety of cancer cell lines and
tumor tissue. The MCK-10 or CCK-2 coding sequence may be used for
diagnostic purposes for detection of aberrant expression of these
genes. For example the MCK-10 or CCK-2 DNA sequence may be used in
hybridization assays of biopsied tissue to diagnose abnormalities
in gene expression.
[0010] The present invention also relates to inhibitors of MCK-10
or CCK-2 receptor activity which may have therapeutic value in the
treatment of proliferative diseases such as cancer. Such inhibitors
include antibodies to epitopes of recombinantly expressed MCK-10 or
CCK-2 receptor that neutralize the activity of the receptor. In
another embodiment of the invention, MCK-10 or CCK-2 anti-sense
oligonucleotides may be designed to inhibit synthesis of the
encoded proteins through inhibition of translation. In addition,
random peptide libraries may be screened using recombinantly
produced MCK-10 or CCK-2 protein to identify peptides that inhibit
the biological activity of the receptor through binding to the
ligand binding sites or other functional domains of the MCK-10 or
CCK-2 receptor. In a further embodiment of the invention, mutated
forms of MCK-10 and CCK-2, having a dominant negative effect, may
be expressed in targeted cell populations to inhibit the activity
of the endogenously expressed receptors.
4. BRIEF DESCRIPTION OF THE FIGURES
[0011] FIGS. 1A, 1B and 1C. Human MCK-10 nucleotide sequence and
deduced amino acid sequence. Regions of interest include the signal
sequence (amino acids (aa) 1-18); the Discoidin I-like domain (aa
31-185); the putative precursor cleavage site (aa 304-307); the
transmembrane region (aa 417-439); the alternatively spliced
sequence I (aa 505-541); the alternatively spliced sequence II (aa
666-671); and the peptide antibody recognition sequences:
NT.alpha.:aa 25-42, NT.beta.:aa 309-321, CT.beta.:aa 902-919.
[0012] FIG. 2. MCK-10 splice variants.
[0013] FIGS. 3A, 3B, 3C and 3D. Human CCK-2 nucleotide sequence and
deduced amino acid sequence.
[0014] FIG. 4A. Shared sequence homology between MCK-10 and
CCK-2.
[0015] FIG. 4B. Shared regions of homology between MCK-10 and
CCK-2.
[0016] FIG. 5A. Northern blot analysis of MCK-10 mRNA in different
human tissues. Three micrograms of poly (A).sup.+ RNA are loaded
per lane. The blot is hybridized with a cDNA restriction fragment
corresponding to nucleotide 278 to 1983 of MCK-10 (FIGS. 1A, 1B and
1C) (excluding the 111 bp insertion). As a control, the blot was
rehybridized with a glyceraldehyde phosphate dehydrogenase (GAPDH)
cDNA probe (lower panel).
[0017] FIG. 5B. Northern blot analysis of MCK-10 gene in various
human breast cancer cell lines. Samples containing three micrograms
of poly (A).sup.+ RNA isolated from different human breast cancer
cell lines were analyzed. The position of 28S and 18S ribosomal
RNAs is indicated, the lower panel shows the rehybridization with a
GAPDH cDNA probe.
[0018] FIG. 5C. Northern blot analysis of MCK-10 mRNA in different
human tissues and cell lines of tumor origin. Size markers are
indicating 28S and 18S ribosomal RNAs (upper panel).
Rehybridization is performed with a GAPDH cDNA probe (lower
panel).
[0019] FIG. 6A. Tyrosine phosphorylation of overexpressed MCK-10.
The coding cDNAs of MCK-10-1 and MCK-10-2 were cloned into an
expression vector and transiently overexpressed in the 293 cell
line (human embryonic kidney fibroblasts, ATCC CRL 1573). Portions
of cell lysate from either MCK-10-1 or -2 transfected cells or
control plasmid transfected cells (mock) were separated on a 7-12%
gradient polyacrylamide gel and transferred to nitrocellulose and
probed with anti-phosphotyrosine antibodies (.alpha.PY). The
incubation of cells with 1 mM sodium ortho-vanadate 90 min. prior
to lysis is indicated by .+-.; (left panel). After removal of the
.alpha.PY antibody the blot was reprobed with an affinity purified
polyclonal antiserum raised against the C-terminal octapeptide of
MCK-10 (.alpha. MCK-10-C); (right panel). Molecular size markers
are indicated in kD.
[0020] FIG. 6B. Distinct glycosylation of overexpressed MCK-10
splice variants. 293 cells were transfected with MCK-10-1 and -2 as
before, metabolically labeled with [.sup.35S]-L-methionine and
treated with 10 .mu.g/ml tunicamycin overnight as indicated (+),
lysed and immunoprecipitated with antisera generated against the
N-terminal and C-terminal peptides of MCK-10 (.alpha. MCK-10-N and
.alpha. MCK-10-C). The autoradiograph of the SDS-PAGE analysis is
shown. Molecular size markers are indicated in kD.
[0021] FIG. 7. In situ hybridization showing specific expression of
MCK-10 in epithelial cells of the distal tubuli of the kidney.
[0022] FIG. 8. In situ hybridization showing expression of MCK-10
only in epithelial cells of the distal tubular cells of the
kidney.
[0023] FIG. 9. In situ hybridization showing specific expression of
MCK-10 in tumor cells of a renal cell carcinoma.
[0024] FIG. 10. In situ hybridization of MCK-10 in the ductal
epithelial cells of normal breast tissue.
[0025] FIG. 11. In situ hybridization showing MCK-10 expression in
infiltrating tumor cells of a breast carcinoma. The tumor
infiltrates the surrounding fat tissue, which is negative for
MCK-10 expression.
[0026] FIG. 12. In situ hybridization showing MCK-10 expression in
infiltrating tumor cells of a breast carcinoma. The tumor
infiltrates the surrounding fat tissue, which is negative for
MCK-10 expression.
[0027] FIG. 13. In situ hybridization showing expression of MCK-10
expression in the islet cells of the pancreas.
[0028] FIG. 14. In situ hybridization showing expression of MCK-10
expression in the islet cells of the pancreas.
[0029] FIG. 15. In situ hybridization showing selective expression
of MCK-10 in the surface epithelium of the colon in contrast to
connective tissue.
[0030] FIG. 16. In situ hybridization showing expression of MCK-10
in the tumor cells of an adenocarcinoma of the colon.
[0031] FIG. 17. In situ hybridization showing expression of MCK-10
in the tumor cells of an adenocarcinoma of the colon.
[0032] FIG. 18. In situ hybridization showing expression of MCK-10
in meningiothelial tumor cells.
[0033] FIG. 19. In situ hybridization showing expression of MCK-10
in cells of a glioblastoma (glioma), a tumor of the neuroepithelial
tissue.
[0034] FIG. 20. In situ hybridization showing expression of MCK-10
in cells of a medulloblastoma with hyperchromatic atypical nuclei.
Expression of MCK-10 is predominantly in cells with well developed
cytoplasm.
[0035] FIG. 21. In situ hybridization showing the expression of
MCK-10 in cells of a medulloblastoma with hyperchromatic atypical
nuclei. Expression of MCK-10 is predominantly in cells with well
developed cytoplasm.
5. DETAILED DESCRIPTION
[0036] The present invention relates to a novel family of receptor
tyrosine kinases referred to herein as MCK-10. The invention
relates to differentially spliced isoforms of MCK-10 and to
additional members of the MCK-10 family of receptor tyrosine
kinases such as the CCK-gene described herein. The invention is
based, in part, on the isolation of a cDNA clone encoding the
MCK-10 receptor tyrosine kinase and the discovery of differentially
spliced isoforms of MCK-10. The invention also relates to the
isolation of a cDNA encoding on additional member of MCK-10
receptor tyrosine kinase family, herein referred to as CCK-2.
[0037] Results from Northern Blot analysis and in situ
hybridization indicates that MCK-10 is expressed in epithelial
cells. In addition, MCK-10 expression can be detected in a wide
variety of cancer cells lines and in all tested tumors. The
invention relates to, expression and production of MCK-10 protein,
as well as to inhibitors of MCK-10 receptor activity which may have
therapeutic value in the treatment of diseases such as cancer.
[0038] For clarity of discussion, the invention is described in the
subsections below by way of example for the MCK-10 gene depicted in
FIGS. 1A, 1B and 1C and the CCK-2 gene depicted in FIGS. 3A, 3B, 3C
and 3D. However, the principles may be analogously applied to
differentially spliced isoforms of MCK-10 and to other members of
the MCK-10 family of receptors.
5.1. The MCK-10 Coding Sequence
[0039] The nucleotide coding sequence and deduced amino acid
sequence of the human MCK-10 gene is depicted in FIGS. 1A, 1B and
1C (SEQ. ID NO. 1). In accordance with the invention, any
nucleotide sequence which encodes the amino acid sequence of the
MCK-10 gene product can be used to generate recombinant molecules
which direct the expression of MCK-10. In additional embodiments of
the invention, nucleotide sequences which selectively hybridize to
the MCK-10 nucleotide sequence shown in FIGS. 1A, 1B and 1C (SEQ ID
NO: 1) may also be used to express gene products with MCK-10
activity. Hereinafter all such variants of the MCK-10 nucleotide
sequence will be referred to as the MCK-10 DNA sequence.
[0040] In a specific embodiment described herein, the human MCK-10
gene was isolated by performing a polymerase chain reaction (PCR)
in combination with two degenerate oligonucleotide primer pools
that were designed on the basis of highly conserved sequences
within the kinase domain of receptor tyrosine kinases corresponding
to the amino acid sequence HRDLAA (sense primer) and SDVWS/FY
(antisense primer) (Hanks et al., 1988). As a template cDNA
synthesized by reverse transcription of poly-A RNA from the human
mammary carcinoma cell line MCF7, was used. A novel RTK, designated
MCK-10 (mammary carcinoma kinase 10) was identified that within the
tyrosine kinase domain exhibited extensive sequence similarity to
the insulin receptor family. The PCR fragment was used to screen a
lambda gt11 library of human fetal brain cDNA (Clontech). Several
overlapping clones were identified. The composite of these cDNA
clones is depicted in FIGS. 1A, 1B and 1C. Furthermore, screening
of a human placental library yielded two cDNA clones, MCK-10-1 and
MCK-10-2, which encoded the entire MCK-10 protein but contained a
shorter 5' untranslated region starting at position 278 of the
MCK-10 sequence (FIGS. 1A, 1B and 1C). Sequences analysis of the
two clones revealed complete identity with the exception of 111
additional nucleotides within the juxtamembrane domain, between
nucleotides 1832 and 1943. One of the clones isolated from the
human fetal brain library contained an additional 18 nucleotides in
the tyrosine kinase domain. These sequences were in-frame with the
MCK-10 open reading frame and did not contain any stop codons. The
MCK-10 splice isoforms have been designated MCK-10-1 (with the
additional 111 bp), MCK-10-2 (without any insertions), MCK-10-3
(with the additional 111 bp and 18 bp), and MCK-10-4 (with the
additional 18 bp) (FIG. 2).
[0041] As shown in FIGS. 1A, 1B, and 1C and FIGS. 3A, 3B, 3C and
3D, MCK-10 have all of the characteristics of a receptor PTK: the
initiation codon is followed by a stretch of essentially
hydrophobic amino acids, which may serve as a signal peptide. Amino
acids 417-439 are also hydrophobic in nature, with the
characteristics of a transmembrane region. The extracellular domain
encompasses 4 consensus N-glycosylation sites (AsnXSer/Thr) and 7
cysteine residues. The extracellular region is shorter than that of
the insulin receptor family and shows no homology to other receptor
tyrosine kinases, but contains near the N-terminus the consensus
sequences for the discoidin I like family (Poole et al. 1981, J.
Mol. Biol. 153: 273-289), which are located as tandem repeats in
MGP and BA46, two milk fat globule membrane proteins (Stubbs et al.
1990, Proc. Natl. Acad. Sci. USA, 87, 8417-8421, Larocca et al.
1991, Cancer Res. 51: 4994-4998), in the light chains of factor V
(Kane et al. 1986, Proc. Natl. Acad. Sci. USA, 83: 6800-6804) and
VIII (Toole et al. 1984, Nature 312: 342-347), and in the A5
protein (Takagi et al. 1987, Dev. Biol., 122: 90-100)
[0042] The protein backbone of MCK-10-1 and MCK-10-2 proreceptors,
with predicted molecular weights of 101.13 and 97.17 kD,
respectively, can thus be subdivided into a 34.31 kD .alpha.
subunit and 66.84 or 62.88 kD .beta.-subunits that contain the
tyrosine kinase homology and alternative splice sites.
[0043] The consensus sequence for the ATP-binding motif is located
at positions 617-627. When compared with other kinases, the ATP
binding domain is with 176 amino acids (including the additional 37
amino acids) further from the transmembrane domain than any other
tyrosine kinase. The additional 37 amino acids are located in the
long and proline/glycine-rich juxtamembrane region and contain an
NPAY sequence (where A can be exchanged for any amino acid), which
is found in cytoplasmic domains of several cell surface proteins,
including RTKs of the EGF and insulin receptor families (Chen et
al. 1990, J. Biol: Chem., 265: 3116-3123). This consensus motif is
followed by the sequence TYAXPXXXPG, which is repeated downstream
in MCK-10 in the juxtamembrane domain at positions 585-595.
Recently it has been shown that this motif is deleted in the
cytoplasmic juxtamembrane region of the activin receptor,
serine/threonine kinase, resulting in reduced ligand binding
affinity (Attisano et al. 1992,Cell, 68: 97-108).
[0044] In comparison with other RTKs, the catalytic domain shows
the highest homology to the TrkA receptor. The YY-motifs (position
802/803) and the tyrosine at position 798, representing putative
autophosphorylation sites, characterize MCK-10 as a member of the
insulin receptor family. Finally, MCK-10 shares homology with the
Trk kinases with their characteristic short carboxyl-terminal tail
of 9 amino acids.
[0045] To determine whether the additional 111 nucleotides present
in MCK-10-1 and -3 were ubiquitously expressed or expressed only in
specific human tissues, a PCR analysis on different human cDNAs
using oligonucleotide primers corresponding to sequences flanking
the insertion site was carried out. Parallel PCR amplifications
were performed on plasmid DNAs of MCK-10-1/MCK-10-2 as controls.
Expression of both isoforms were identified in brain, pancreas,
placenta, colon, and kidney, and in the cell lines Caki 2 (kidney
ca), SW 48 (colon ca), and HBL100 and T47D (breast ca). The PCR
products were subcloned into the Bluescript vector to confirm the
nucleotide sequence.
[0046] Using a hybridization probe comprising the 5' 1694 bp cDNA
fragment of MCK-10 (excluding the 111 bp insert), which encompasses
the extracellular, transmembrane, and juxtamembrane domains, the
MCK-10 gene revealed the existence of multiple transcript sizes
with a major form of 4.2 kb. The highest expression of MCK-10 mRNA
was detected in lung, intermediate levels were found in kidney,
colon, stomach, placenta and brain, low levels in pancreas, and no
MCK-10 mRNA was detected in liver (FIG. 5A). FIG. 5B illustrates
the levels of expression of MCK-10 in a variety of breast cancer
cell lines and FIG. 5C presents the levels of MCK-10 expression in
different tumor cell lines. A summary of the expression patterns of
MCK-10 in different cell lines is presented in TABLE 1.
TABLE-US-00001 TABLE 1 MCK-10 EXPRESSION IN DIFFERENT CELL LINES
BREAST CANCER CELL LINES BT-474 + T-47D ++++ BT-20 +++ MDA-MB-453
++ MDA-MB-468 ++ MDA-MB-435 ++ MDA-MB-175 ++++ MDA-MB-231 ++ HBL
100 + SK-BR-3 + MCF-7 ++ LUNG CANCER CELL LINES WI-38 + WI-26 +
MELANOMA CELL LINES SK-Mel-3 + Wm 266-4 + HS 294T ++ COLON CANCER
CELL LINES Caco-2 +++ -SNU-C2B +++ SW48 ++ KIDNEY CANCER CELL LINE
CAKI-2 +++ EPIDERMOID CANCER CELL LINE A431 ++ OTHER CANCERS
rhabdomyosarcoma ++ Ewing sarcoma ++ glioblastoma ++ neuroblastoma
- hepatoblastoma + HEMAPOIETIC CELL LINES EB3 - CEM - MOLT4 - DAUDI
- RAJI - MEG01 - KG1 - K562 -
[0047] In situ hybridization analysis with the 5' 1865 bp of
MCK-10-2 indicated that MCK-10 was expressed specifically in
epithelial cells of various tissues including: [0048] cuboidal
epithelial cells lining the distal kidney tubulus (FIG. 7) [0049]
columnar epithelial cells lining the large bowel tract [0050] deep
layer of epithelial cells lining the stomach [0051] epithelial
cells lining the mammary ducts [0052] islet cells of the pancreas
(FIG. 13 and FIG. 14) [0053] epithelial cells of the thyroid gland,
which produces thyroid hormones No detectable MCK-10 expression was
observed in connective tissues, endothelial cells, adipocytes,
muscle cells, or hemopoietic cells.
[0054] MCK-10 expression was also detected in all tumors
investigated which included: [0055] adenocarcinoma of the colon
(FIG. 16 and FIG. 17) [0056] adenocarcinoma of the stomach [0057]
adenocarcinoma of the lung [0058] infiltrating ductal carcinoma of
the breast [0059] cystadenoma of the ovary [0060] multi endocrine
tumor of the pancreas [0061] carcinoid tumor of the pancreas [0062]
tubular cells of renal cell carcinoma [0063] transitional cell
carcinoma (a malignant epithelial tumor of the bladder) [0064]
meningiothelial tumor (FIG. 18) [0065] medulloblastoma with
hyperchromatic atypical nuclei and spare cytoplasm (MCK-10
expression is only seen in cells with well developed cytoplasm)
(FIG. 20 and FIG. 20) [0066] glioblastoma (a tumor of the
neuroepithelial tissue) (FIG. 19)
[0067] The in situ hybridization experiments revealed the highest
expression of MCK-10 in malignant cells of the ductal breast
carcinoma, in the tumor cells of a multi-endocrine tumor, and in
the tumor cells of a transitional cell carcinoma of the
bladder.
5.2 The CCK-2 Coding Sequence
[0068] The present invention also relates to other members of the
MCK-10 family of receptor kinases. Members of the MCK-10 family are
defined herein as those DNA sequences capable of hybridizing to
MCK-10 DNA sequences as presented in FIGS. 1A, 1B and 1C. Such
receptors may demonstrate 80% homology at the amino acid level in
substantial stretches of DNA sequences. In addition, such receptors
can be defined as those receptors containing an intracellular
tyrosine kinase domain and a discoidin I sequence located near the
amino-terminal end of the protein. The discoidin I domain is
defined as that region of MCK-10 located between amino acid 31-185
as presented in FIG. 1.
[0069] In a specific embodiment of the invention described herein,
an additional member of the MCK-10 family of receptor tyrosine
kinases was cloned and characterized. The nucleotide coding
sequence and deduced amino acid sequence of the novel receptor
tyrosine kinase, herein referred to as CCK-2, is presented in FIGS.
3A, 3B, 3C and 3D. In accordance with the invention, any nucleotide
sequence which encodes the amino acid sequence of the CCK-2 gene
product can be used to generate recombinant molecules which direct
the expression of CCK-2. In additional, embodiments of the
invention, nucleotide sequences which selectively hybridize to the
CCK-2 nucleotide sequence as shown in FIGS. 3A, 3B, 3C and 3D (SEQ.
ID NO: 2) may also be used to express gene products with CCK-2
activity.
[0070] Analysis of the CCK-2 sequence revealed significant homology
to the extracellular, transmembrane and intracellular region of the
MCK-10 receptor indicating that it was a member of the MCK-10
family of receptors. The shared homology between CCK-2 and MCK-10
is depicted in FIGS. 4A and 4B.
5.3. Expression of MCK-10 Receptor and Generation of Cell Lines
That Express MCK-10
[0071] For clarity of discussion the expression of receptors and
generation of cell lines expressing receptors are described by way
of example for the MCK-10 gene. However, the principles may be
analogously applied to expression and generation of cell lines
expressing spliced isoforms of MCK-10 or to other members of the
MCK-10 family of receptors, such as CCK-2.
[0072] In-accordance with the invention, MCK-10 nucleotide
sequences which encode MCK-10, peptide fragments of MCK-10, MCK-10
fusion proteins or functional equivalents thereof may be used to
generate recombinant DNA molecules that direct the expression of
MCK-10 protein or a functionally equivalent thereof, in appropriate
host cells. Alternatively, nucleotide sequences which hybridize to
portions of the MCK-10 sequence may also be used in nucleic acid
hybridization assays, Southern and Northern blot analyses, etc.
[0073] Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode substantially the same or a functionally
equivalent amino acid sequence, may be used in the practice of the
invention for the cloning and expression of the MCK-10 protein.
Such DNA sequences include those which are capable of hybridizing
to the human MCK-10 sequence under stringent conditions.
[0074] Altered DNA sequences which may be used in accordance with
the invention include deletions, additions or substitutions of
different nucleotide residues resulting in a sequence that encodes
the same or a functionally equivalent gene product. These
alterations would in all likelihood be in regions of MCK-10 that do
not constitute functionally conserved regions such as the discordin
I domain or the tyrosine kinase domain. In contrast, alterations,
such as deletions, additions or substitutions of nucleotide
residues in functionally conserved MCK-10 regions would possibly
result in a nonfunctional MCK-10 receptor. The gene product itself
may contain deletions, additions or substitutions of amino acid
residues within the MCK-10 sequence, which result in a silent
change thus producing a functionally equivalent MCK-10. Such amino
acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipatic nature of the residues involved. For example,
negatively charged amino acids include aspartic acid and glutamic
acid; positively charged amino acids include lysine and arginine;
amino acids with uncharged polar head groups having similar
hydrophilicity values include the following: leucine, isoleucine,
valine; glycine, alanine; asparagine, glutamine; serine, threonine;
phenylalanine, tyrosine.
[0075] The DNA sequences of the invention may be engineered in
order to alter the MCK-10 coding sequence for a variety of ends
including but not limited to alterations which modify processing
and expression of the gene product. For example, mutations may be
introduced using techniques which are well known in the art, e.g.
site-directed mutagenesis, to insert new restriction sites, to
alter glycosylation patterns, phosphorylation, etc. For example, in
certain expression systems such as yeast, host cells may over
glycosylate the gene product. When using such expression systems it
may be preferable to alter the MCK-10 coding sequence to eliminate
any N-linked glycosylation site.
[0076] In another embodiment of the invention, the MCK-10 or a
modified MCK-10 sequence may be ligated to a heterologous sequence
to encode a fusion protein. For example, for screening of peptide
libraries it may be useful to encode a chimeric MCK-10 protein
expressing a heterologous epitope that is recognized by a
commercially available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the MCK-10
sequence and the heterologous protein sequence, so that the MCK-10
may be cleaved away from the heterologous moiety.
[0077] In an alternate embodiment of the invention, the coding
sequence of MCK-10 could be synthesized in whole or in part, using
chemical methods well known in the art. See, for example,
Caruthers, et al., 1980, Nuc. Acids Res. Symp. Ser. 7:215-233; Crea
and Horn, 180, Nuc. Acids Res. 9(10):2331; Matteucci and Caruthers,
1980, Tetrahedron Letters 21:719; and Chow and Kempe, 1981, Nuc.
Acids Res. 9(12):2807-2817. Alternatively, the protein itself could
be produced using chemical methods to synthesize the MCK-10 amino
acid sequence in whole or in part. For example, peptides can be
synthesized by solid phase techniques, cleaved from the resin, and
purified by preparative high performance liquid chromatography.
(E.g., see Creighton, 1983, Proteins Structures And Molecular
Principles, W.H. Freeman and Co., N.Y. pp. 50-60). The composition
of the synthetic peptides may be confirmed by amino acid analysis
or sequencing (e.g., the Edman degradation procedure; see
Creighton, 1983, Proteins, Structures and Molecular Principles,
W.H. Freeman and Co., N.Y., pp. 34-49.
[0078] In order to express a biologically active MCK-10, the
nucleotide sequence coding for MCK-10, or a functional equivalent,
is inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence. The MCK-10 gene
products as well as host cells or cell lines transfected or
transformed with recombinant MCK-10 expression vectors can be used
for a variety of purposes. These include but are not limited to
generating antibodies (i.e., monoclonal or polyclonal) that bind to
the receptor, including those that competitively inhibit binding of
MCK-10 ligand and "neutralize" activity of MCK-10 and the screening
and selection of drugs that act via the MCK-10 receptor; etc.
5.3.1. Expression Systems
[0079] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing the MCK-10
coding sequence and appropriate transcriptional/translational
control signals. These methods include in vitro recombinant DNA
techniques, synthetic techniques and in vivo recombination/genetic
recombination. See, for example, the techniques described in
Maniatis et al., 1989, Molecular Cloning A Laboratory Manual, Cold
Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current
Protocols in Molecular Biology, Greene Publishing Associates and
Wiley Interscience, N.Y.
[0080] A variety of host-expression vector systems may be utilized
to express the MCK-10 coding sequence. These include but are not
limited to microorganisms such as bacteria transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing the MCK-10 coding sequence; yeast transformed
with recombinant yeast expression vectors containing the MCK-10
coding sequence; insect cell systems infected with recombinant
virus expression vectors (e.g., baculovirus) containing the MCK-10
coding sequence; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing the MCK-10 coding
sequence; or animal cell systems The expression elements of these
systems vary in their strength and specificities. Depending on the
host/vector system utilized, any of a number of suitable
transcription and translation elements, including constitutive and
inducible promoters, may be used in the expression vector. For
example, when cloning in bacterial systems, inducible promoters
such as pL of bacteriophage .lamda., plac, ptrp, ptac (ptrp-lac
hybrid promoter) and the like may be used; when cloning in insect
cell systems, promoters such as the baculovirus polyhedrin promoter
may be used; when cloning in plant cell systems, promoters derived
from the genome of plant cells (e.g., heat shock promoters; the
promoter for the small subunit of RUBISCO; the promoter for the
chlorophyll a/b binding protein) or from plant viruses (e.g., the
35S RNA promoter of CaMV; the coat protein promoter of TPV) may be
used; when cloning in mammalian cell systems, promoters derived
from the genome of mammalian cells (e.g., metallothionein promoter)
or from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter) may be used; when generating cell
lines that contain multiple copies of the MCK-10 DNA, SV40-, BPV-
and EBV-based vectors may be used with an appropriate selectable
marker.
[0081] In bacterial systems a number of expression vectors may be
advantageously selected depending upon the use intended for the
MCK-10 expressed. For example, when large quantities of MCK-10 are
to be produced for the generation of antibodies or to screen
peptide libraries, vectors which direct the expression of high
levels of fusion protein products that are readily purified may be
desirable. Such vectors include but are not limited to the E. coli
expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in
which the MCK-10 coding sequence may be ligated into the vector in
frame with the lac Z coding region so that a hybrid AS-lac Z
protein is produced; pIN vectors (Inouye & Inouye, 1985,
Nucleic acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J.
Biol. Chem. 264:5503-5509); and the like. pGEX vectors may also be
used to express foreign polypeptides as fusion proteins with
glutathione S-transferase (GST). In general, such fusion proteins
are soluble and can easily be purified from lysed cells by
adsorption to glutathione-agarose beads followed by elution in the
presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned polypeptide of interest can be released from the GST
moiety.
[0082] In yeast, a number of vectors containing constitutive or
inducible promoters may be used. For a review see, Current
Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al.,
Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et
al., 1987, Expression and Secretion Vectors for Yeast, in Methods
in Enzymology, Eds. Wu & Grossman, 1987, Acad. Press, N.Y.,
Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL
Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene
Expression in Yeast, Methods in Enzymology, Eds. Berger &
Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular
Biology of the Yeast Saccharomyces, 1982, Eds. Strathern et al.,
Cold Spring Harbor Press, Vols. I and II.
[0083] In cases where plant expression vectors are used, the
expression of the MCK-10 coding sequence may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
RNA and 19S RNA promoters of CaMV (Brisson et al., 1984, Nature
310:511-514), or the coat protein promoter of TMV (Takamatsu et
al., 1987, EMBO J. 6:307-311) may be used; alternatively, plant
promoters such as the small subunit of RUBISCO (Coruzzi et al.,
1984, EMBO J. 3:1671-1680; Broglie et al., 1984, Science
224:838-843); or heat shock promoters, e.g., soybean hsp17.5-E or
hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may be
used. These constructs can be introduced into plant cells using Ti
plasmids, Ri plasmids, plant virus vectors, direct DNA
transformation, microinjection, electroporation, etc. For reviews
of such techniques see, for example, Weissbach & Weissbach,
1988, Methods for Plant Molecular Biology, Academic Press, NY,
Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant
Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.
[0084] An alternative expression system which could be used to
express MCK-10 is an insect system. In one such system, Autographa
californica nuclear polyhidrosis virus (AcNPV) is used as a vector
to express foreign genes. The virus grows in Spodoptera frugiperda
cells. The MCK-10 coding sequence may be cloned into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter). Successful insertion of the MCK-10 coding sequence will
result in inactivation of the polyhedrin gene and production of
non-occluded recombinant virus (i.e., virus lacking the
proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugirerda
cells in which the inserted gene is expressed. (E.g., see Smith et
al., 1983, J. Viol. 46:584; Smith, U.S. Pat. No. 4,215,051).
[0085] In mammalian host cells, a number of viral based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the MCK-10 coding sequence may be ligated to an
adenovirus transcription/translation control complex, e.g., the
late promoter and tripartite leader sequence. This chimeric gene
may then be inserted in the adenovirus genome by in vitro or in
vivo recombination. Insertion in a non-essential region of the
viral genome (e.g., region E1 or E3) will result in a recombinant
virus that is viable and capable of expressing MCK-10 in infected
hosts. (E.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci.
(USA) 81:3655-3659). Alternatively, the vaccinia 7.5K promoter may
be used. (See, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci.
(USA) 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864;
Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931).
[0086] Specific initiation signals may also be required for
efficient translation of inserted MCK-10 coding sequences. These
signals include the ATG initiation codon and adjacent sequences. In
cases where the entire MCK-10 gene, including its own initiation
codon and adjacent sequences, is inserted into the appropriate
expression vector, no additional translational control signals may
be needed. However, in cases where only a portion of the MCK-10
coding sequence is inserted, exogenous translational control
signals, including the ATG initiation codon, must be provided.
Furthermore, the initiation codon must be in phase with the reading
frame of the MCK-10 coding sequence to ensure translation of the
entire insert. These exogenous translational control signals and
initiation codons can be of a variety of origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of appropriate transcription enhancer elements,
transcription terminators, etc. (see Bittner et al., 1987, Methods
in Enzymol. 153:516-544).
[0087] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. The presence of four consensus N-glycosylation sites in
the MCK-10 extracellular domain support that proper modification
may be important for MCK-10 function. Different host cells have
characteristic and specific mechanisms for the post-translational
processing and modification of proteins. Appropriate cells lines or
host systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERO, BHK, HeLa,
COS, MDCK, 293, WI38, etc.
[0088] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the MCK-10 may be engineered. Rather than
using expression vectors which contain viral origins of
replication, host cells can be transformed with the MCK-10 DNA
controlled by appropriate expression control elements (e.g.,
promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of foreign DNA, engineered cells may be allowed to
grow for 1-2 days in an enriched media, and then are switched to a
selective media. The selectable marker in the recombinant plasmid
confers resistance to the selection and allows cells to stably
integrate the plasmid into their chromosomes and grow to form foci
which in turn can be cloned and expanded into cell lines. This
method may advantageously be used to engineer cell lines which
express the MCK-10 on the cell surface. Such engineered cell lines
are particularly useful in screening for drugs that affect
MCK-10.
[0089] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for dhfr, which confers resistance to
methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567;
O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt,
which confers resistance to mycophenolic acid (Mulligan & Berg,
1981), Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers
resistance to the aminoglycoside G-418 (Colberre-Garapin, et al.,
1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to
hygromycin (Santerre, et al., 1984, Gene 30:147) genes. Recently,
additional selectable genes have been described, namely trpB, which
allows cells to utilize indole in place of tryptophan; hisD, which
allows cells to utilize histinol in place of histidine (Hartman
& Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); and ODC
(ornithine decarboxylase) which confers resistance to the ornithine
decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO
(McConlogue L., 1987, In: Current Communications in Molecular
Biology, Cold Spring Harbor Laboratory ed.).
5.3.2. Identification of Transfectants or Transformants That
Express the MCK-10
[0090] The host cells which contain the coding sequence and which
express the biologically active gene product may be identified by
at least four general approaches; (a) DNA-DNA or DNA-RNA
hybridization; (b) the presence or absence of "marker" gene
functions; (c) assessing the level of transcription as measured by
the expression of MCK-10 mRNA transcripts in the host cell; and (d)
detection of the gene product as measured by immunoassay or by its
biological activity.
[0091] In the first approach, the presence of the MCK-10 coding
sequence inserted in the expression vector can be detected by
DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide
sequences that are homologous to the MCK-10 coding sequence,
respectively, or portions or derivatives thereof.
[0092] In the second approach, the recombinant expression
vector/host system can be identified and selected based upon the
presence or absence of certain "marker" gene functions (e.g.,
thymidine kinase activity, resistance to antibiotics, resistance to
methotrexate, transformation phenotype, occlusion body formation in
baculovirus, etc.). For example, if the MCK-10 coding sequence is
inserted within a marker gene sequence of the vector, recombinants
containing the MCK-10 coding sequence can be identified by the
absence of the marker gene function. Alternatively, a marker gene
can be placed in tandem with the MCK-10 sequence under the control
of the same or different promoter used to control the expression of
the MCK-10 coding sequence. Expression of the marker in response to
induction or selection indicates expression of the MCK-10 coding
sequence.
[0093] In the third approach, transcriptional activity for the
MCK-10 coding region can be assessed by hybridization assays. For
example, RNA can be isolated and analyzed by Northern blot using a
probe homologous to the MCK-10 coding sequence or particular
portions thereof. Alternatively, total nucleic acids of the host
cell may be extracted and assayed for hybridization to such
probes.
[0094] In the fourth approach, the expression of the MCK-10 protein
product can be assessed immunologically, for example by Western
blots, immunoassays such as radioimmuno-precipitation,
enzyme-linked immunoassays and the like.
5.4. Uses of the MCK-10 Receptor and Engineered Cell Lines
[0095] For clarity of discussion the uses of the expressed
receptors and engineered cell lines expressing the receptors is
described by way of example for MCK-10. The described uses may be
equally applied to expression of MCK-10 spliced isoforms or
additional members of the MCK-10 gene family such as CCK-2.
[0096] In an embodiment of the invention the MCK-10 receptor and/or
cell lines that express the MCK-10 receptor may be used to screen
for antibodies, peptides, or other ligands that act as agonists or
antagonists of the MCK-10 receptor. For example, anti-MCK-10
antibodies may be used to inhibit MCK-10 function. Alternatively,
screening of peptide libraries with recombinantly expressed soluble
MCK-10 protein or cell lines expressing MCK-10 protein may be
useful for identification of therapeutic molecules that function by
inhibiting the biological activity of MCK-10. The uses of the
MCK-10 receptor and engineered cell lines, described in the
subsections below, may be employed equally well for MCK-10 family
of receptor tyrosine kinases.
[0097] In an embodiment of the invention, engineered cell lines
which express the entire MCK-10 coding region or its ligand binding
domain may be utilized to screen and identify ligand antagonists as
well as agonists. Synthetic compounds, natural products, and other
sources of potentially biologically active materials can be
screened in a number of ways.
5.4.1. Screening of Peptide Library with MCK-10 Protein or
Engineered Cell Lines
[0098] Random peptide libraries consisting of all possible
combinations of amino acids attached to a solid phase support may
be used to identify peptides that are able to bind to the ligand
binding site of a given receptor or other functional domains of a
receptor such as kinase domains (Lam, K. S. et al., 1991, Nature
354: 82-84). The screening of peptide libraries may have
therapeutic value in the discovery of pharmaceutical agents that
act to inhibit the biological activity of receptors through their
interactions with the given receptor.
[0099] Identification of molecules that are able to bind to the
MCK-10 may be accomplished by screening a peptide library with
recombinant soluble MCK-10 protein. Methods for expression and
purification of MCK-10 are described in Section 5.2.1 and may be
used to express recombinant full length MCK-10 or fragments of
MCK-10 depending on the functional domains of interest. For
example, the kinase and extracellular ligand binding domains of
MCK-10 may be separately expressed and used to screen peptide
libraries.
[0100] To identify and isolate the peptide/solid phase support that
interacts and forms a complex with MCK-10, it is necessary to label
or "tag" the MCK-10 molecule. The MCK-10 protein may be conjugated
to enzymes such as alkaline phosphatase or horseradish peroxidase
or to other reagents such as fluorescent labels which may include
fluorescein isothyiocynate (FITC), phycoerythrin (PE) or rhodamine.
Conjugation of any given label, to MCK-10, may be performed using
techniques that are routine in the art.
[0101] Alternatively, MCK-10 expression vectors may be engineered
to express a chimeric MCK-10 protein containing an epitope for
which a commercially available antibody exist. The epitope specific
antibody may be tagged using methods well known in the art
including labeling with enzymes, fluorescent dyes or colored or
magnetic beads.
[0102] The "tagged" MCK-10 conjugate is incubated with the random
peptide library for 30 minutes to one hour at 22.degree. C. to
allow complex formation between MCK-10 and peptide species within
the library. The library is then washed to remove any unbound
MCK-10 protein. If MCK-10 has been conjugated to alkaline
phosphatase or horseradish peroxidase the whole library is poured
into a petri dish containing substrates for either alkaline
phosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoyl
phosphate (BCIP) or 3,3',4,4''-diamnobenzidine (DAB), respectively.
After incubating for several minutes, the peptide/solid
phase-MCK-10 complex changes color, and can be easily identified
and isolated physically under a dissecting microscope with a
micromanipulator. If a fluorescent tagged MCK-10 molecule has been
used, complexes may be isolated by fluorescent activated sorting.
If a chimeric MCK-10 protein expressing a heterologous epitope has
been used, detection of the peptide/MCK-10 complex may be
accomplished by using a labeled epitope specific antibody. Once
isolated, the identity of the peptide attached to the solid phase
support may be determined by peptide sequencing.
[0103] In addition to using soluble MCK-10 molecules, in another
embodiment, it is possible to detect peptides that bind to cell
surface receptors using intact cells. The use of intact cells is
preferred for use with receptors that are multi-subunits or labile
or with receptors that require the lipid domain of the cell
membrane to be functional. Methods for generating cell lines
expressing MCK-10 are described in Sections 5.2.1. and 5.2.2. The
cells used in this technique may be either live or fixed cells. The
cells will be incubated with the random peptide library and will
bind to certain peptides in the library to form a "rosette" between
the target cells and the relevant solid phase support/peptide. The
rosette can thereafter be isolated by differential centrifugation
or removed physically under a dissecting microscope.
[0104] As an alternative to whole cell assays for membrane bound
receptors or receptors that require the lipid domain of the cell
membrane to be functional, the receptor molecules can be
reconstituted into liposomes where label or "tag" can be
attached.
5.4.2. Antibody Production and Screening
[0105] Various procedures known in the art may be used for the
production of antibodies to epitopes of the recombinantly produced
MCK-10 receptor. Such antibodies include but are not limited to
polyclonal, monoclonal, chimeric, single chain, Fab fragments and
fragments produced by an Fab expression library. Neutralizing
antibodies i.e., those which compete for the ligand binding site of
the receptor are especially preferred for diagnostics and
therapeutics.
[0106] Monoclonal antibodies that bind MCK-10 may be radioactively
labeled allowing one to follow their location and distribution in
the body after injection. Radioactivity tagged antibodies may be
used as a non-invasive diagnostic tool for imaging de novo cells of
tumors and metastases.
[0107] Immunotoxins may also be designed which target cytotoxic
agents to specific sites in the body. For example, high affinity
MCK-10 specific monoclonal antibodies may be covalently complexed
to bacterial or plant toxins, such as diphtheria toxin, abrin or
ricin. A general method of preparation of antibody/hybrid molecules
may involve use of thiol-crosslinking reagents such as SPDP, which
attack the primary amino groups on the antibody and by disulfide
exchange, attach the toxin to the antibody. The hybrid antibodies
may be used to specifically eliminate MCK-10 expressing tumor
cells.
[0108] For the production of antibodies, various host animals may
be immunized by injection with the MCK-10 protein including but not
limited to rabbits, mice, rats, etc. Various adjuvants may be used
to increase the immunological response, depending on the host
species, including but not limited to Freund's (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum.
[0109] Monoclonal antibodies to MCK-10 may be prepared by using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include but are not
limited to the hybridoma technique originally described by Kohler
and Milstein, (Nature, 1975, 256:495-497), the human B-cell
hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72;
Cote et al., 1983, Proc. Natl. Acad. Sci., 80:2026-2030) and the
EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition,
techniques developed for the production of "chimeric antibodies"
(Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855;
Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985,
Nature, 314:452-454) by splicing the genes from a mouse antibody
molecule of appropriate antigen specificity together with genes
from a human antibody molecule of appropriate biological activity
can be used. Alternatively, techniques described for the production
of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted
to produce MCK-10-specific single chain antibodies.
[0110] Antibody fragments which contain specific binding sites of
MCK-10 may be generated by known techniques. For example, such
fragments include but are not limited to: the F(ab').sub.2
fragments which can be produced by pepsin digestion of the antibody
molecule and the Fab fragments which can be generated by reducing
the disulfide bridges of the F(ab').sub.2 fragments. Alternatively,
Fab expression libraries may be constructed (Huse et al., 1989,
Science, 246:1275-1281) to allow rapid and easy identification of
monoclonal Fab fragments with the desired specificity to
MCK-10.
5.5. Uses of MCK-10 Coding Sequence
[0111] The MCK-10 coding sequence may be used for diagnostic
purposes for detection of MCK-10 expression. Included in the scope
of the invention are oligoribonucleotide sequences, that include
antisense RNA and DNA molecules and ribozymes that function to
inhibit translation of MCK-10. In addition, mutated forms of
MCK-10, having a dominant negative effect, may be expressed in
targeted cell populations to inhibit the activity of endogenously
expressed MCK-10. The uses described below may be equally well
adapted for MCK-10 spliced isoform coding sequences and sequences
encoding additional members of the MCK-10 family of receptors, such
as CCK-2.
5.5.1. Use of MCK-10 Coding Sequence in Diagnostics and
Therapeutics
[0112] The MCK-10 DNA may have a number of uses for the diagnosis
of diseases resulting from aberrant expression of MCK-10. For
example, the MCK-10 DNA sequence may be used in hybridization
assays of biopsies or autopsies to diagnose abnormalities of MCK-10
expression; e.g., Southern or Northern analysis, including in situ
hybridization assays.
[0113] Also within the scope of the invention are
oligo-ribonucleotide sequences, that include anti-sense RNA and DNA
molecules and ribozymes that function to inhibit the translation of
MCK-10 mRNA. Anti-sense RNA and DNA molecules act to directly block
the translation of mRNA by binding to targeted mRNA and preventing
protein translation. In regard to antisense DNA,
oligodeoxyribonucleotides derived from the translation initiation
site, e.g., between -10 and +10 regions of the MCK-10 nucleotide
sequence, are preferred.
[0114] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. The mechanism of ribozyme action
involves sequence specific hybridization of the ribozyme molecule
to complementary target RNA, followed by a endonucleolytic
cleavage. Within the scope of the invention are engineered
hammerhead motif ribozyme molecules that specifically and
efficiently catalyze endonucleolytic cleavage of MCK-10 RNA
sequences.
[0115] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences, GUA,
GUU and GUC. Once identified, short RNA sequences of between 15 and
20 ribonucleotides corresponding to the region of the target gene
containing the cleavage site may be evaluated for predicted
structural features such as secondary structure that may render the
oligo-nucleotide sequence unsuitable. The suitability of candidate
targets may also be evaluated by testing their accessibility to
hybridization with complementary oligonucleotides, using
ribonuclease protection assays.
[0116] Both anti-sense RNA and DNA molecules and ribozymes of the
invention may be prepared by any method known in the art for the
synthesis of RNA molecules. These include techniques for chemically
synthesizing oligodeoxyribonucleotides well known in the art such
as for example solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in
vivo transcription of DNA sequences encoding the antisense RNA
molecule. Such DNA sequences may be incorporated into a wide
variety of vectors which incorporate suitable RNA polymerase
promoters such as the T7 or SP6 polymerase promoters.
Alternatively, antisense cDNA constructs that synthesize antisense
RNA constitutively or inducibly, depending on the promoter used,
can be introduced stably into cell lines.
[0117] Various modifications to the DNA molecules may be introduced
as a means of increasing intracellular stability and half-life.
Possible modifications include but are not limited to the addition
of flanking sequences of ribo- or deoxy- nucleotides to the 5'
and/or 3' ends of the molecule or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the
oligodeoxyribonucleotide backbone.
5.5.2. Use of Dominant Negative MCK-10 Mutants in Gene Therapy
[0118] Receptor dimerization induced by ligands, is thought to
provide an allosteric regulatory signal that functions to couple
ligand binding to stimulation of kinase activity. Defective
receptors can function as dominant negative mutations by
suppressing the activation and response of normal receptors by
formation of unproductive heterodimers. Therefore, defective
receptors can be engineered into recombinant viral vectors and used
in gene therapy in individuals that inappropriately express
MCK-10.
[0119] In an embodiment of the invention, mutant forms of the
MCK-10 molecule having a dominant negative effect may be identified
by expression in selected cells. Deletion or missense mutants of
MCK-10 that retain the ability to form dimers with wild type MCK-10
protein but cannot function in signal transduction may be used to
inhibit the biological activity of the endogenous wild type MCK-10.
For example, the cytoplasmic kinase domain of MCK-10 may be deleted
resulting in a truncated MCK-10 molecule that is still able to
undergo dimerization with endogenous wild type receptors but unable
to transduce a signal.
[0120] Recombinant viruses may be engineered to express dominant
negative forms of MCK-10 which may be used to inhibit the activity
of the wild type endogenous MCK-10. These viruses may be used
therapeutically for treatment of diseases resulting from aberrant
expression or activity of MCK-10, such as cancers.
[0121] Expression vectors derived from viruses such as
retroviruses, vaccinia virus, adeno-associated virus, herpes
viruses, or bovine papilloma virus, may be used for delivery of
recombinant MCK-10 into the targeted cell population. Methods which
are well known to those skilled in the art can be used to construct
those recombinant viral vectors containing MCK-10 coding sequence.
See, for example, the techniques described in Maniatis et al.,
1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor
Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in
Molecular Biology, Greene Publishing Associates and Wiley
Interscience, N.Y. Alternatively, recombinant MCK-10 molecules can
be reconstituted into liposomes for delivery to target cells.
6. EXAMPLES
Cloning and Characterization of MCK-10
[0122] The subsection below describes the isolation and
characterization of a cDNA clones encoding the novel receptor
tyrosine kinase designated MCK-10 and differentially spliced
isoforms of MCK-10.
6.1. Materials and Methods
6.1.1. cDNA Cloning and Characterization of MCK-10
[0123] Confluent plates of the human breast cancer cell line MCF7
(American Type Culture Collection HTB22) were lysed by treatment
with guanidinium-thiocyanate according to Chirgwin et al. (1979,
Biochemistry 18:5294-5299). Total RNA was isolated by CsC1-gradient
centrifugation. First-strand cDNA was synthesized from 20 pg total
RNA with avian myeloblastosis virus (AMV) reverse transcriptase
(Boehringer Mannheim).
[0124] cDNA was used in a polymerase chain reaction under standard
conditions (PCR Technology-Principles and Applications for DNA
Amplifications, H. E. Erlich, ed., Stockton Press, New York 1989).
The following ool of primers were used for the amplification:
Sense Primer
[0125] corresponding to the amino acid sequence HRDLAA EcoRI
TABLE-US-00002 5' GGAATTCC CAC AGN GAC TTN GCN GCN AG 3' T C A T C
A A C
Antisense Primer
[0126] corresponding to the amino acid sequence SDVWS F/Y
TABLE-US-00003 EcoRI 3' TCN GAC GTN TGG ACN TTC CCTTAAGG 5' G G TG
CAT
[0127] Thirty-five PCR cycles were carried out using 8 .mu.g (0.8
.mu.g) of the pooled primers. (Annealing 55.degree. C., 1 min;
Extension 72.degree. C., 2 min; Denaturation 94.degree. C., 1 min).
The reaction product was subjected to polyacrylamide gel
electrophoresis. Fragments of the expected size (.sup.-210 bp) were
isolated, digested with the restriction enzyme EcoRI, and subcloned
into the pBluescript vector (Stratagene) using standard techniques
(Current Protocols in Molecular Biology, eds. F. M. Ausubel et al.,
John Wiley & Sons, New York, 1988).
[0128] The recombinant plasmids were transformed into the competent
E. coli strain designated 298.
[0129] The subcloned PCR products were sequenced by the method of
Sanger et al. (Proc. Natl. Acad. Sci. USA 74, 5463-5467) using
Sequenase (United States Biochemical, Cleveland, Ohio 44111 USA).
one clone, designated MCK-10 was identified as novel RTK.
6.1.2. Full-Length cDNA Cloning
[0130] The partial cDNA sequence of the new MCK-10 RTK, which was
identified by PCR, was used to screen a .lamda.gt11 library from
human fetal brain cDNA (Clontech) (complexity of 1.times.10.sup.10
recombinant phages). One million independent phage clones were
plated and transferred to nitrocellulose filters following standard
procedures (Sambrook, H. J., Molecular Cloning, Cold Spring Harbor
Laboratory Press, USA, 1989). The filters were hybridized to the
EcoRI/EcoRI fragment of clone MCK-10, which had been radioactively
labeled using 50 .mu.Ci [.alpha..sup.32P]ATP and the random-primed
DNA labeling kit (Boehringer Mannheim). The longest cDNA insert (8)
of .about.3500 bp was digested with the restriction enzymes
EcoRI/SacI to obtain a 5' end probe of 250 bp. This probe was used
to rescreen the human fetal brain library and several overlapping
clones were isolated. The composite of the cDNA clones are shown in
FIGS. 1A, 1B and 1C. Some of the clones had a deletion of 6 amino
acids at position 2315 in the MCK-10 sequence.
[0131] The 1.75 million independent phage clones of a human
placenta library, .lamda.ZAP were plated and screened with the 5'
end probe (EcoRI/SacI) of clone 8. Two clones were full-length with
a shorter 5' end starting at position 278 of the nucleotide
sequence shown in FIGS. 1A, 1B and 1C. Subcloning of positive
bacteriophages clones into pBluescript vector was done by the in
vivo excision protocol (Stratagene).
[0132] The composite cDNA sequence and the predicted amino acid
sequence of MCK-10 are shown in FIGS. 1A, 1B, and 1C. Different
cDNA sequence variations of MCK-10 is presented in FIG. 2.
6.1.3. Northern Blot Analysis of MCK-10
[0133] Total RNA was isolated from the following human tissues:
lung, pancreas, stomach, kidney, spleen, liver, colon and placenta.
RNA was also isolated from various breast cancer cell lines and
cell lines of tumor origin.
[0134] PolyA.sup.+ RNA was isolated on an oligo (dT) column (Aviv
and Leder, 1972, Proc. Natl. Acad. Sci. USA 69, 1408-1412). The RNA
was separated on an agarose gel containing 2.2M formaldehyde and
blotted on a nitrocellulose filter (Schleicher and Schuell). 3
.mu.g of poly A.sup.+ RNA was loaded per lane. The filter was
hybridized with a .sup.32P-labeled EcoRI/EcoRI DNA fragment
obtained by PCR. Subsequently, the filter was exposed to x-ray film
at -70.degree. C. with an intensifying screen. The results are
depicted in FIGS. 5A, 5B and 5C.
6.1.4. Generation of MCK-10 Specific Antibodies
[0135] Antisera was generated against synthetic peptides
corresponding to the amino acid sequence of MCK-10. .alpha.MCK-10-N
antisera was generated against the following N-terminal peptide
located between amino acids 26-42: [0136]
H-F-D-P-A-K-D-C-R-Y-A-L-G-M-Q-D-R-T-I. .alpha.MCK-10-c antisera was
generated against the following C-terminal peptide located between
amino acids 902-919 [0137] R-P-P-F-S-Q-L-H-R-F-L-A-E-D-A-L-N-T-V.
.alpha.MCK-10-.beta. antisera was generated against the following
peptide near the processing site of .beta.-subunit of MCK-10
located between amino acids 309-322: [0138]
P-A-M-A-W-E-G-E-P-M-R-H-N-L. .alpha.MCK-10-C2 antisera was
generated against the C-terminal peptide located between amino
acids 893-909: [0139] C-W-S-R-E-S-E-Q-R-P-P-F-S-Q-L-H-R.
[0140] Peptides were coupled to keyhole limpet 30 hemocyanin and
injected with Freunds adjuvant into Chinchilla rabbits. After the
second boost, the rabbits were bled and the antisera were tested in
immunoprecipitations using lysates of 293 cells transiently
overexpressing MCK-10-1 and MCK-10-2.
[0141] The samples were loaded on a 7.5% polyacrylamide gel and
after electrophoresis transferred onto a nitrocellulose filter
(Schleicher and Schuell). The blot was probed with the different
antibodies as above and developed using the ECL Western blotting
detection system according the manufacturer's instructions (Cat no.
RPN 2108 Amersham International, UK).
6.1.5. In Situ Hybridization
[0142] The 5' located cDNA fragment corresponding to nucleotides
278-1983 of clone MCK-10, excluding the 111 base pair insert, were
subcloned in the bluescript SK+ (Stratagene). For in situ
hybridization, a single-strand antisense DNA probe was prepared as
described by Schnurch and Risau (Development 1991, 111, 1143-1154).
The plasmid was linearized at the 3' end of the cDNA and a sense
transcript was synthesized using SP6 RNA polymerase (Boehringer).
The DNA was degraded using DNase (RNase-free preparation,
Boehringer Mannheim). With the transcript, a random-primed cDNA
synthesis with .alpha.-.sup.35S ATP (Amersham) was performed by
reverse transcription with MMLV reverse transcriptase (BRL). To
obtain small cDNA fragments of about 100 bp in average, suitable
for in situ hybridization, a high excess of primer was used.
Subsequently, the RNA transcript was partially hydrolyzed in 100 nM
NaOH for 20 min at 70.degree. C., and the probe was neutralized
with the same amount of HCL and purified with a Sephadex-G50
column. After ethanol precipitation the probe was dissolved at a
final specific activity of 5.times.10.sup.5 cpm. For control
hybridization, a sense probe was prepared using the same
method.
[0143] Sectioning, postfixation was essentially performed according
to Hogan et al. (1986, Manipulating the Mouse Embryo: A Laboratory
Manual, New York: Cold Spring Harbor Laboratory Press). 10 .mu.m
thick sections were cut at -18.degree. C. on a Leitz cryostat. For
hybridization treatment, no incubation with 0.2M HCL for removing
the basic proteins was performed. Sections were incubated with the
.sup.35S-cDNA probe (5.times.10.sup.4 cpm/.mu.l) at 52.degree. C.
in a buffer containing 50% formamide, 300 mM NaCl, 10 mM Tris-HCL,
10 mM NaPO.sub.4 (pH 6.8), 5 mM EDTA, 2% Ficoll 400, 0.2%
polyvinylpyrrolidone, 0.02% BSA, 10 mg/ml yeast RNA, 10% dextran
sulfate, and 10 mM DTT. Posthybridization washing was performed at
high stringency (50% formamide, 300 mM NaCl, 10 mM Tris-HCL, 10 mM
NaPO.sub.4 (pH6.8), 5 mM EDTA, 10 mMDTT at 52.degree. C.). For
autoradiography, slides were created with Kodak NTB2 film emulsion
and exposed for eight days. After developing, the sections were
counterstained with toluidine blue.
6.2. Results
6.2.1. Characterization of MCK-10 Clone
[0144] To identify novel receptor tyrosine kinases (RTKs) that are
expressed in mammary carcinoma cell lines, we used the polymerase
chain reaction in combination with two degenerate oligonucleotide
primer pools based on highly conserved sequences within the kinase
domain of RTKS, corresponding to the amino acid sequence HRDLAA
(sense primer) and SDVWS/FY (antisense primer) (Hanks et al. 1988,
Science 241, 42-52), in conjunction with cDNA synthesized by
reverse transcription of poly A RNA from the human mammary
carcinoma cell line MCF7. We identified a novel RTK, designated
MCK-10 (mammary carcinoma kinase 10), that within the tyrosine
kinase domain exhibited extensive sequence similarity to the
insulin receptor family. The PCR fragment was used to screen a
lambda gt11 library of human fetal brain cDNA (Clontech). Several
overlapping clones were identified and their composite sequence is
shown in FIGS. 1A, 1B and 1C. Furthermore, screening of a human
placenta library yielded two CDNA clones which encoded the entire
MCK-10 protein but whose 5' nucleotide sequence began at nucleotide
278 in the sequence shown in FIG. 1. Sequence analysis of the two
clones revealed complete identity with the exception of 111
additional nucleotides within the juxtamembrane domain, between
nucleotides 1832 and 1943. One of the clones isolated from the
human fetal brain library contained an additional 18 nucleotides in
the tyrosine kinase domain. These sequences were in-frame with the
MCK-10 open reading frame and did not contain any stop codons. We
designated these MCK-10 splice isoforms MCK-10-1 (with the
additional 111 bp, MCK-10-2 (without any insertions), MCK-10-3
(with the additional 111 bp and 18 bp), and MCK-10-4 (with the
additional 18 bp). This new receptor tyrosine kinase was recently
described by Johnson et al. (1993, Proc. Natl. Acad. Sci. USA, 90
5677-5681) as DDR.
[0145] As shown in FIG. 1, MCK-10 has all of the characteristics of
a receptor PTK: the initiation codon is followed by a stretch of
essentially hydrophobic amino acids, which may serve as a signal
peptide. Amino acids 417-439 are also hydrophobic in nature, with
the characteristics of a transmembrane region. The extracellular
domain encompasses 4 consensus N-glycosylation sites (AsnXSer/Thr)
and 7 cysteine residues. The extracellular region is shorter than
that of the insulin receptor family and shows no homology to other
receptor tyrosine kinases, but contains near the N-terminus the
consensus sequences for the discoidin 1 like family (Poole et al.
1981, J. Mol. Biol. 153, 273-289), which are located as tandem
repeats in MGP and BA46, two milk fat globule membrane proteins
(Stubbs et al. 1990, proc. Natl. Acad. Sci. USA, 87, 8417-8421,
Larocca et al. 1991, Cancer Res. 51, 4994-4998), in the light
chains of factor V (Kane et al. 1986, Proc. Natl. Acad. Sci. USA,
83, 6800-6804) and VIII (Toole et al. 1984, Nature, 312, 342-347),
and in the A5 protein (Takagi et al. 1987, Dev. Biol., 122,
90-100).
[0146] The protein backbone of MCK-10-1 and MCK-10-2 proreceptors,
with predicted molecular weights of 101.13 and 97.17 kD,
respectively, can thus be subdivided into a 34.31 kD .alpha.
subunit and 66.84 kD .beta.-subunits that contain the tyrosine
kinase homology and alternative splice sites.
[0147] The consensus sequence for the ATP-binding motif is located
at positions 617-627. When compared with other kinases, the ATP
binding domain is 176,amino acids (including the additional 37
amino acids) further from the transmembrane domain than any other
tyrosine kinase. The additional 37 amino acids are located in the
long and proline/glycine-rich juxtamembrane region and contain an
NPAY sequence (where A can be exchanged for any amino acid), which
is found in cytoplasmic domains of several cell surface proteins,
including RTKs of the EGF and insulin receptor families (Chen et
al. 1990, J. Biol. Chem., 265, 3116-3123). This consensus motif is
followed by the sequence TYAXPXXXPG, which is repeated downstream
in MCK-10 in the juxtamembrane domain at positions 585-595.
Recently it has been shown that this motif is deleted in the
cytoplasmic juxtamembrane region of the activin receptor, a
serine/threonine kinase, resulting in reduced ligand binding
affinity (Attisano et al. 1992, Cell, 68, 97-108).
[0148] In comparison with other RTKs, the catalytic domain shows
the highest homology to the TrkA receptor. The yy-motifs (position
802/803) and the tyrosine at position 798, representing putative
autophosphorylation sites, characterize MCK-10 as a member of the
insulin receptor family. Finally, MCK-10 shares with the Trk
kinases their characteristic short caraboxy-terminal tail of 9
amino acids.
[0149] To determine whether the additional 111 nucleotides present
in MCK-10-1 and -3 were ubiquitously expressed or expressed only in
specific human tissues, we performed PCR on different human cDNAs
using oligonucleotide primers corresponding to sequences flanking
the insertion site. Parallel PCR amplifications were performed on
plasmid DNAs of MCK-10-1/MCK-10-2 as controls. Expression of both
isoforms was identified in brain, pancreas, placenta, colon, and
kidney, and in the cell lines Caki 2 (kidney ca), SW 48 (colon ca),
and HBL100 and T47D (breast ca). The PCR products were subcloned
into the Bluescript vector to confirm the nucleotide sequence.
6.2.2. Northern Blot Analysis: Expression of MCK-10 in Various
Human Tissues and Cell Lines
[0150] Using as a hybridization probe a 5' 1694 bp cDNA fragment of
MCK-10 (excluding the 111 base pair insert), which encompasses the
extracellular, transmembrane, and juxtamembrane domains, the MCK-10
gene revealed the existence of multiple transcript sizes with a
major form of 4.2 kb. The highest expression of MCK-10 mRNA was
detected in lung, intermediate levels were found in kidney, colon,
stomach, placenta, and brain, low levels in pancreas, and no MCK-10
mRNA was detected in liver (FIG. 5A). MCK-10 mRNA was also detected
in a variety of different tumor cell lines as depicted in FIG. 5B
and FIG. 5C. Northern blot analysis-with the GAPDH gene was carried
out as a control.
6.2.3. In Situ Hybridization
[0151] To determine which cells in the different human tissues
contain MCK-10 transcripts, in situ hybridization of various human
tissues and of tissues of different tumors were carried out.
Hybridization analyses with the 5' 1694 bp of MCK-10 (excluding the
111 base pair insert) indicated that MCK-10 expression was
specifically detected in epithelial cells of various tissues:
[0152] cuboidal epithelial cells lining the distal kidney tubulus
[0153] columnar epithelial cells lining the large bowl tract [0154]
deep layer of epithelial cells lining the stomach [0155] epithelial
cells lining the mammary ducts [0156] islet cells of the pancreas
[0157] epithelial cells of the thyroid gland, which produces
thyroid hormones
[0158] No detectable MCK-10 expression was observed in connective
tissues, endothelial cells, adipocytes, muscle cells, or
hemapoletic cells.
[0159] MCK-10 expression was detected in all tumors investigated:
[0160] adenocarcinoma of the colon [0161] adenocarcinoma of the
stomach [0162] adenocarcinoma of the lung [0163] infiltrating
ductal carcinoma of the breast [0164] cystadenoma of the ovary
[0165] multi endocrine tumor of the pancreas [0166] carcinoid tumor
of the pancreas [0167] tubular cells of renal cell carcinoma [0168]
transitional cell carcinoma (a malignant epithelial tumor of the
bladder) [0169] meninglothelial tumor [0170] medulloblastoma with
hyperchromatic atypical nuclei and spare cytoplasm (MCK-10
expression is only seen in cells with well developed cytoplasm)
[0171] glioblastoma (a tumor of the neuroepithelial tissue)
[0172] These in situ hybridization experiments revealed the highest
expression of MCK-10 in malignant cells of the ductal breast
carcinoma, in the tumor cells of a multi endocrine tumor, and in
the tumor cells of a transitional cell carcinoma of the bladder.
The in situ hybridization results are depicted in FIGS. 7-21.
6.2.4. Transient Overexpression of MCK-10 in 293 Cells
[0173] To analyze the MCK-10 protein in detail, we used the 293
cell system for transient overexpression. The cDNAs of MCK-10-1 and
MCK-10-2 were cloned into an expression vector. Cells were
transfected in duplicate with the two splice variants or a control
plasmid and starved overnight. One part was incubated prior to
lysis with 1 mM sodium-orthovanadate for 90 min. This agent is
known to be a potent inhibitor of phosphotyrosine phosphatases,
thereby enhancing the tyrosine phosphorylation of cellular
protein.
[0174] The precursor and the .beta.-subunit of MCK-10 showed strong
tyrosine phosphorylation after orthovanadate treatment, (FIG. 4A,
left panel). Surprisingly, the MCK-10-1, containing the 37 amino
acid insertion, exhibited lower kinase activity than MCK-10-2.
Reprobing the same blot with a peptide antibody raised against the
MCK-10 C-terminus revealed equal amounts of expressed receptor and
a slight shift of MCK-10-1 precursor and .beta.-subunit due to the
additional 37 amino acids of the insertion (FIG. 4A, right
panel).
[0175] We further analyzed the N-linked glycosylation of the splice
variants. Transfected cells were treated overnight with
tunicamycin, which inhibits the maturation of proteins by
glycosylation. Two affinity purified antibodies raised against
peptide sequence of MCK-10 N- and C-terminus, respectively, were
used for subsequent immunoprecipitations. Both antibodies
precipitated the predicted 101 kD or 97 kD polypeptides from
tunicamycin-treated cells (FIG. 4B). Interestingly, the size of the
fully glycosylated forms of MCK-10-1 and MCK-10-2 suggested that
the latter was more extensively glycosylated than the putative
alternative splice form. This data indicates that the 37 amino acid
insertion of MCK-10-1 influences its posttranslational modification
which may influence ligand.
7. EXAMPLES
Cloning and Characterization of CCK-2
[0176] The following subsection describes methods for isolation and
characterization of the CCK-2 gene, an additional member of the
MCK-10 receptor tyrosine kinase gene family.
7.1. Materials and Methods
7.1.1. cDNA Cloning and Characterization of CCK-2
[0177] cDNA was synthesized using avian myeloblastosis virus
reverse transcriptase and 5 .mu.g of poly A.sup.+ RNA prepared from
tissue of a primary colonic adenocarcinoma, sigmoid colon,
moderately well differentiated grade II, staging pT3, pN1, removed
from a 69 year old white female of blood type O, RH positive. The
patient had not received therapy.
[0178] The tissue was minced and lysed by treatment with
guanidinium-thiocyanate according to Chirgwin, J. M. et al. (1979,
Biochemistry 18:5294-5299). Total RNA was isolated by guanidinium
thiocyanate-phenol-chloroform extraction (Chomczyrski et al. 1987,
Anal. Biochem. 162:156-159). Poly A.sup.+ RNA was isolated on an
oligo-dT column (Aviv and Leder, 1972, Proc. Natl. Acad. Sci. USA
69:1408-1412).
[0179] One tenth of the cDNA was subjected to the polymerase chain
reaction using standard conditions (PCR Technology--Principles and
Applications for DNA Amplifications, H. E. Erlich, ed. Stockton
Press, New York, 1989) and the same pool of primers used for
amplification of MCK-10 (See, Section 6.1.1., lines 4-16).
Thirty-five cycles were carried out (Annealing 55.degree. C., 1
min; Extension 72.degree. C., 2 min: Denaturation 94.degree. C., 1
min.). The reaction products were subjected to polyacrylamide gel
electrophoresis. Fragments of the expected size were isolated,
digested with the restriction enzyme EcoRI, and subcloned into
pBluescript vector (Stratagene) using standard techniques (Current
Protocols in Molecules Biology, eds. M. Ausubel et al., John Wiley
& Sons, New York, 1988). The subcloned PCR products were
sequenced by the method of Sanger et al. (Proc. Natl. Acad. Sci.
USA 74, 5463-5467) using T7-Polymerase (Boehringer Mannheim).
[0180] The CCK-2 PCR fragment was used to screen a human placenta
library in lambda ZAP. The longest cDNA insert .sup.-1300 bp was
digested with the restriction enzymes EcoRI/Ncol to obtain a 5' end
probe of 200 bp. Rescreening of the human placenta library yielded
in a cDNA clone which encoded the entire CCK-2 protein (subcloning
of positive bacteriophages clones into pBluescript vector was done
by the in vivo excision protocol (Stratagene)). The DNA sequence
and the deduced amino acid sequence of CCK-2 is shown in FIG.
3.
7.2. Results
7.2.1. Cloning and Characterization of CCK-2
[0181] An additional member of the MCK-10 receptor tyrosine kinase
family was identified using a polymerase chain reaction and CDNA
prepared from colonic adenocarcinoma RNA. The nucleotide sequence
of the novel receptor, designated CCK-2, is presented in FIGS. 3A
and 3B. Analysis of the CCK-2, nucleotide sequence and encoded
amino acid sequence indicated significant homology with MCK-10
throughout the extracellular, transmembrane and intracellular
region of the MCK-10 receptor. The regions of homology between
CCK-2 and MCK-10 extend into the N-terminus consensus sequence for
the discoidin I like family of proteins. (Poole et al. 1981, J.
Mol. Biol. 153, 273-289). The homology between CCK-2 and MCK-10 is
diagramed in FIGS. 4A and 4B.
8. DEPOSIT OF MICROORGANISMS
[0182] The following organisms were deposited with the American
Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville,
Md. 20852. TABLE-US-00004 Strain Designation Containing Accession
No. CCK-2 pCCK-2 69468 MCK-10-1 pMCK-10-1 69464 MCK-10-2 pMCK-10-2
69465 MCK-10-3 pMCK-10-3 69466 MCK-10-4 pMCK-10-4 69467
[0183] The present invention is not to be limited in scope by the
exemplified embodiments or deposited organisms which are intended
as illustrations of single aspects of the invention, and any
clones, DNA or amino acid sequences which are functionally
equivalent are within the scope of the invention. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying drawings. Such modifications
are intended to fall within the scope of the appended claims.
[0184] It is also to be understood that all base pair sizes given
for nucleotides are approximate and are used for purposes of
description.
Sequence CWU 1
1
* * * * *