U.S. patent application number 10/219247 was filed with the patent office on 2003-02-13 for differentiation-suppressive polypeptide.
This patent application is currently assigned to ASAHI KASEI KOGYO KABUSHIKI KAISHA. Invention is credited to Itoh, Akira, Sakano, Seiji.
Application Number | 20030032781 10/219247 |
Document ID | / |
Family ID | 26460815 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030032781 |
Kind Code |
A1 |
Sakano, Seiji ; et
al. |
February 13, 2003 |
Differentiation-suppressive polypeptide
Abstract
A novel human serrate-2 polypeptide consisting of a polypeptide
containing the amino acid sequence described in SEQ ID NO:1 in the
Sequence Listing and having the effect of regulating the
differentiation of undifferentiated cells involving stem cells; its
gene; a process for producing the same; and an antibody
specifically recognizing the polypeptide.
Inventors: |
Sakano, Seiji; (Shizuoka,
JP) ; Itoh, Akira; (Shizuoka, JP) |
Correspondence
Address: |
YOUNG & THOMPSON
745 SOUTH 23RD STREET 2ND FLOOR
ARLINGTON
VA
22202
|
Assignee: |
ASAHI KASEI KOGYO KABUSHIKI
KAISHA
Osaka
JP
|
Family ID: |
26460815 |
Appl. No.: |
10/219247 |
Filed: |
August 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10219247 |
Aug 16, 2002 |
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09855722 |
May 16, 2001 |
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09855722 |
May 16, 2001 |
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09214278 |
Jan 26, 1999 |
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6291210 |
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09214278 |
Jan 26, 1999 |
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PCT/JP97/02414 |
Jul 11, 1997 |
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Current U.S.
Class: |
530/388.1 |
Current CPC
Class: |
C07K 14/47 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
530/388.1 |
International
Class: |
C07K 016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 16, 1996 |
JP |
8-186220 |
May 14, 1997 |
JP |
9-124063 |
Claims
1. An antibody specifically recognizing a polypeptide having any
one of SEQ ID NOs. 1 to 3.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a novel bioactive substance which
suppresses differentiation of undifferentiated cells.
[0003] 2. Description of Related Art
[0004] Human blood and lymph contain various types of cells and
each cell plays important roles. For example, erythrocytes carry
oxygen; platelets have hemostatic action; and lymphocytes prevent
infection. These various cells originate from hematopoietic stem
cells in the bone marrow. Recently, it has been clarified that the
hematopoietic stem cells are differentiated to various blood cells,
osteoclasts and mast cells by stimulation of various cytokines in
vivo and environmental factors. In the cytokines, there have been
found, for example, erythropoietin (EPO) for differentiation to
erythrocytes; granulocyte colony stimulating factor (G-CSF) for
differentiation to leukocytes; and platelet growth factor (mpl
ligand) for differentiation to megakaryocytes which are platelet
producing cells; and the former two examples have already been
clinically applied.
[0005] The differentiated blood cells are generally classified into
two groups consisting of blood precursor cells which are destined
to differentiate to specific blood series and hematopoietic stem
cells which have differentiation ability to all series and
self-replication activity. The blood precursor cells can be
identified by various colony assays; however, an identification
method for the hematopoietic stem cells have not been established.
In these cells, stem cell factor (SCF), interleukin-3 (IL-3),
granulocyte-macrophage colony stimulating factor (GM-CSF),
interleukin-6 (IL-6), interleukin-1 (IL-1), granulocyte colony
stimulating factor (G-CSF) and oncostatin M have been reported to
stimulate cell differentiation and proliferation.
[0006] Trials for expansion of hematopoietic stem cells in vitro
have been conducted in order to replace bone marrow transplantation
for applying hematopoietic stem cell transplantation therapy or
gene therapy. However, when the hematopoietic stem cells are
cultured in the presence of the above mentioned cytokines,
multi-differentiation activities and self-replication activities,
which are originally in the position of the hematopoietic stem
cells, gradually disappeared and are changed to the blood cell
precursors which only differentiate to specific series after 5
weeks of cultivation, and multi-differentiation activity, which is
one of the specific features of the hematopoietic stem cells, is
lost (Wanger et al. Blood 86, 512-523, 1995).
[0007] For proliferation of the blood precursor cells, a single
cytokine is not sufficient, but rather the synergistic action of
several cytokines is important. Consequently, in order to
proliferate the hematopoietic stem cells while maintaining the
specific features of the hematopoietic stem cells, it is necessary
to add cytokines which suppress differentiation together with the
cytokines which proliferate and differentiate the undifferentiated
blood cells. In general, many cytokines which stimulate
proliferation or differentiation of cells are known, but few
cytokines which suppress cell differentiation are known. For
example, leukemia inhibitory factor (LIF) has an action of
proliferation of mouse embryonic stem cells without
differentiation, but it has no action against the hematopoietic
stem cells or blood precursor cells. Transforming growth factor
(TGF-.beta.) has suppressive action for proliferation against
various cells, but has no fixed actions against the hematopoietic
stem cells or blood precursor cells.
[0008] Not only blood cells but also undifferentiated cells,
especially stem cells, are thought to be involved in tissue
regeneration. These regeneration of tissues and proliferation of
undifferentiated cells in each tissue can be applied in various
known ways (Katsutoshi Yoshizato, Regenration--a mechanism of
regeneration, 1966, Yodosha Publ. Co.).
[0009] Notch is a receptor type membrane protein involved in
regulation of nerve cell differentiation found in Drosophia.
Homologues of Notch are found in various invertebrates and
vertebrates including nematoda (Lin-12), Xenopus laevis (Xotch),
mouse (Motch) and human (TAN-1).
[0010] Ligands of Notch in Drosophila are known. These are
Drosophila Delta (Delta) and Drosophila Serrate (Serrate). Notch
ligand homologues are found in various animals similar to those of
Notch receptors (Artavanis-Tsakonas et al., Science 268, 225-232,
1995).
[0011] Human Notch homologue, TAN-1 is found widely in the tissues
in vivo (Ellisen et al., Cell 66, 649-661, 1991). Two Notch
analogous molecules other than TAN-1 have been reported
(Artavanis-Tsakonas et al., Science 268, 225-232, 1995). Expression
of TAN-1 was also observed in CD34 positive cells in blood cells by
PCR (Polymerase Chain Reaction) (Milner et al., Blood 83,
2057-2062, 1994). However, in relation to humans, gene cloning of
human Delta and human Serrate, which are thought to be Notch
ligand, has not been reported.
[0012] In Drosophila Notch, binding with the ligand was studied and
investigated in detail, and it was found that the Notch can be
bound to the ligand with Ca++ at the binding region, which is a
repeated amino acid sequence No. 11 and No. 12 in the amino acid
sequence repeat of Epidermal Growth Factor (EGF) (Fehon et al.,
Cell 61, 523-534, 1990, Rebay et al., ibid. 67, 687-699, 1991 and
Japan. Patent PCT Unexam. Publ. 7-503123). EGF-like repeated
sequences are conserved in Notch homologues of other species.
Consequently, the same mechanism in binding with ligand is
assumed.
[0013] An amino acid sequence which is called DSL
(Delta-Serrate-Lag-2) near the amino acid terminal, and EGF-like
repeated sequence like in the receptor are conserved in the ligand
(Artavanis-Tsakonas et al., Science 268, 225-232, 1995). EGF-like
sequence has been found in thrombo-modulin (Jackman et al., Proc.
Natl. Acad. Sci. USA 83, 8834-8838, 1986), low density lipoprotein
(LDL) receptor (Russell et al., Cell 37, 577-585, 1984), and blood
coagulating factor (Furie et al., Cell 53, 505-518,1988), and is
thought to play important roles in extracellular coagulation and
adhesion.
[0014] The vertebrate homologues of the cloned Drosophila Delta
were found in chicken (C-Delta-1) and Xenopus laevis (X-Delta-1),
and it has been reported that X-Delta-1 had acted through Xotch in
the generation of the protoneuron (Henrique et al., Nature 375,
787-790, 1995 and Chitnis et al., ibid. 375, 761-766, 1995).
[0015] A vertebrate homologue of Drosophila Serrate was found in
rat as rat Jagged (Jagged)(Lindsell et al., Cell 80, 909-917,
1995). According to Lindsell et al., mRNA of the rat Jagged is
detected in the spinal cord of fetal rats. As a result of
cocultivation of a myoblast cell line that is forced to over
express rat Notch with a rat Jagged expression cell line,
suppression of differentiation of the myoblast cell line is found.
However, the rat Jagged has no action against the myoblast cell
line without forced expression of the rat Notch.
[0016] Considering the above reports, Notch and ligand thereof may
be involved in differentiation regulation of the nerve cells;
however, except for some myoblast cells, their actions against
cells including blood cells, especially primary cells, are
unknown.
[0017] As mentioned above, concerning undifferentiated cells,
proliferation while maintaining their specificities has not been
performed. Major reasons are that factors suppressing
differentiation of the undifferentiated cells have not been
sufficiently identified.
SUMMARY AND OBJECTS OF THE INVENTION
[0018] A principal object of the present invention is to provide a
compound originated from novel factors which can suppress
differentiation of undifferentiated cells.
[0019] We have set up a hypothesis that the Notch and its ligand
have an action of differential regulation not only for neurogenic
cells but also for various undifferentiated cells. However, in case
of clinical application in humans, prior known different species
such as chicken or Xenopus laevos type Notch ligand have
species-previously specific problems and anti-genicities.
Consequently, to obtain prior unknown human Notch ligand is
essentially required. We had an idea that ligands of the human
Notch (TAN-1 etc.), which are a human Delta homologue (hereinafter
designated as human Delta) and human Serrate homologue (hereinafter
designated as human Serrate), may be found. Also we had an idea
that these findings may be a candidate for drugs useful for
differential regulation of the undifferentiated cells. We have
tried to discover the same.
[0020] In order to discover human Notch ligands, we have analyzed
amino acid sequences which are conserved in animals other than
humans, and tried to discover genes by PCR using mixed primers of
the corresponding DNA sequence. As a result of extensive studies,
we have succeeded in isolation of cDNAs coding amino acid sequences
of two new molecules, novel human Delta-1 and novel human
Serrate-1, and have prepared protein expression systems having
various forms using these cDNAs. Also we have established a
purification method of the proteins which were purified and
isolated, and already filed a patent application therefor
(International Publication WO 97/19172).
[0021] Furthermore, we have tried to discover Drosophila Delta and
Serrate analogous molecules other than human Delta and human
Serrate (hereinafter designated as human Delta-1 and human
Serrate-1, respectively) of the above patent application in
vertebrates.
[0022] We have tried to search on the data base of genetic
sequences. Namely, based on the human Serrate-1 genetic sequence
(amino acids sequence in SEQ ID NO: 5) which was at first
discovered by us, we have found several numbers of gene fragments
(length with 200-350 bp) with highhomology from EST (Expressed
Sequence Tag), which is a data of gene fragments of random human
cDNA sequence in the gene sequence data base GenBank, using a gene
sequence search software Genetyx/CD (Software Development Co.).
[0023] These short length gene fragments were cloned by PCR, and
these gene fragments were used as probes to try cloning of the
longer length gene fragments from human fetal cDNA libraries. The
thus isolated longer gene fragments, of which the genetic sequences
were determined, were again compared with genetic sequence of human
Serrate-1. As a result, a gene, which has relatively high homology
with human Serrate-1, is identified and is designated as human
Serrate-2. The full length of Serrate-2 gene was isolated
successfully.
[0024] Furthermore, expression vectors of the said cloned Serrate-2
were constructed. A purification method of these proteins was
established and the said protein was purified and isolated.
Antibodies against human Serrate-2 are prepared using the said
human Serrate-2, and a purification method of the said antibodies
was established, then the activity against undifferentiated blood
cells was confirmed. The present invention was completed
accordingly.
[0025] The present invention relates to a polypeptide comprising
amino acid sequence of SEQ ID NO: 1, 2 or 3 of the sequence listing
and the polypeptide having differentiation suppressive action
against undifferentiated cells. Furthermore, the undifferentiated
cells are undifferentiated cells except for those of the brain and
nervous system or muscular system cells, and in which the
undifferentiated cells are undifferentiated blood cells. The
present invention also relates to polypeptides having growth
inhibitory action against vascular endothelial cells, a
pharmaceutical composition containing the said polypeptides, a cell
culture medium containing the said polypeptides and a cell culture
medium in which the cells are undifferentiated blood cells.
[0026] The present invention furthermore relates to a DNA coding a
polypeptide comprising amino acid sequence of SEQ ID NO: 1, 2 or 3
of the sequence listing, the DNA having DNA sequence 90-731 DNA
sequence 90-3254, or DNA sequence 90-3725 of SEQ ID NO: 4 of the
sequence listing. The present invention still further relates to a
recombinant DNA made by ligating a DNA coding a polypeptide
comprising amino acid sequence of SEQ ID NO: 1, 2 or 3 and a vector
DNA which can express in the host cell and a cell comprising
transformed by the recombinant DNA.
[0027] The present invention also relates to a process for
production of polypeptides by culturing cells and isolating the
thus produced compounds, and an antibody specifically recognizing
the polypeptide having the amino acid sequence of SEQ ID NO. 1, 2
or 3 of the sequencing listing.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0028] The present invention is explained in detail in the
following.
[0029] Preparation of cDNA necessary for gene manipulation,
expression analysis by Northern blotting, screening by
hybridization, preparation of recombinant DNA, determination of DNA
base sequence and preparation of cDNA library, all of which are
series of molecular biological experiments, can be performed
according to a description of the conven-tional textbook for the
experiments. The above conventional textbook of the experiments is,
for example, Maniatis et al. ed. Molecular Cloning, A laboratory
manual, 1989, Eds., Sambrook, J., Fritsch, E. F. and Maniatis, T.,
Cold Spring Harbor Laboratory Press.
[0030] A novel compound of the present invention has at least
polypeptides in the sequence listing SEQ ID NO: 1-3. Mutants and
alleles which naturally occur in nature are included in the
polypeptide of the present invention unless the polypeptides of the
sequence listing, SEQ ID NO: 1, 2 or 3 lose their properties.
Modification and substitution of amino acids are described in
detail in the patent application by the name of Bennet et al.
(National Unexam. Publ. WO 96/2645) and can be performed according
to the description thereof. A modified polypeptide in the present
invention means the modified polypeptide prepared by these amino
acid replacements and is defined as amino acid sequences having
identity of more than 90% in its amino acid sequence.
[0031] A DNA sequence coding polypeptides of the sequence listing,
SEQ ID NO: 1-3 is shown in the sequence listing, SEQ ID NO: 4 as
well as its amino acid sequence. In these DNA sequences, even if an
amino acid level mutation is not generated, naturally isolated
chromosomal DNA or cDNA thereof may have a possibility to mutate in
the DNA base sequence as a result of degeneracy of the genetic code
without changing amino acid sequence coded by the DNA. A
5'-untranslated region and 3'-untranslated region are not involved
in amino acid sequence determination of the polypeptide, so the DNA
sequences of these regions are easily mutated. The base sequence
obtained by these degeneracies of the genetic codes is included in
the DNA of the present invention.
[0032] Undifferentiated cells in the present invention are defined
as cells which can grow by specific stimulation, and cells which
can be differentiated to cells having specific functions as a
result of specific stimulations. These include undifferentiated
cells of the skin tissues, undifferentiated blood cells and nervous
systems, undifferentiated cells of the muscular systems and
undifferentiated blood cells. These cells include those having
self-replication activity which are called stem cells, and those
having an ability to generate the cells of these lines. The
differentiation-suppressive action means suppressive action for
autonomous or heteronomous differentiation of the undifferentiated
cells, and is an action for maintaining the undifferentiated
condition. The brain and nervous undifferentiated cells can be
defined as cells having an ability to differentiate to the cells of
the brain or nerve having specific functions by specific
stimulation. The undifferentiated cells of the muscular systems can
be defined as cells having an ability to differentiate to the
muscular cells having specific functions by specific stimulation.
The blood undifferentiated cells in the present invention can be
defined as cell groups consisting of the blood precursor cells
which are differentiated to the specific blood series identified by
blood colony assay, and hematopoietic stem cells having
differentiation to every series and self-replication
activities.
[0033] In the sequence listing, the amino acid sequence in SEQ ID
NO: 1 is a sequence of the active center of the present invention
of human Serrate-2 minus the signal peptide, and corresponds to an
amino acid No. 1 to 217 in SEQ ID NO: 3 of the mature full length
amino acid sequence of human Serrate-2 of the present invention.
The amino acid sequence in SEQ ID NO:2 is amino acid sequence of
extracellular domain of the present invention of human Serrate-2
minus the signal peptide, and corresponds to an amino acid No. 1 to
1058 in SEQ ID NO: 3 of the mature full length amino acid sequence
of human Serrate-2 of the present invention. The amino acid
sequence of SEQ ID NO: 3 is the mature full length amino acid
sequence of the human Serrate-2 of the present invention. The
sequence of SEQ ID NOS: 4 and 5 is total amino acid sequence of
human Serrate-2 of the present invention and cDNA coding the same.
The sequence of SEQ ID NOS: 6 and 7 is total amino acid sequence of
human Serrate-1 used in the present invention and cDNA coding the
same.
[0034] The left and right ends of the amino acid sequences in the
sequence listings indicate amino terminal (hereinafter designates
as N-terminal) and carboxyl terminal (hereinafter designates as
C-terminal), respectively, and the left and right ends of the
nucleotide sequences are 5'-terminal and 3'-terminal,
respectively.
[0035] Cloning of human Notch ligand gene can be performed by the
following method. During the evolution of the organisms, a part of
the amino acid sequences of the human Notch ligand is conserved.
DNA sequence corresponding to the conserved amino acid sequence is
designed, and is used as a primer of RT-PCR (Reverse Transcription
Polymerase Chain Reaction), then a PCR template of human origin is
amplituded by PCR reaction, whereby fragments of human Notch ligand
can be obtained. Furthermore, RT-PCR primer is prepared by applying
the known DNA sequence information of the Notch ligand homologue of
the organisms other than humans, and the known gene fragments can
be possibly obtained from a PCR template of the said organisms.
[0036] In order to perform PCR for obtaining fragments of human
Notch ligand, PCR for DSL sequence was considered, but a large
number of combinations of DNA sequence corresponding to amino acid
sequence conserved in this region can be expected, and a design for
PCR is difficult. As a result, PCR of the EGF-like sequence has to
be selected. As explained above, since EGF-like sequence is
conserved in a large number of molecules, to obtain the fragments
and identification a r e extremely difficult. We have designed and
prepared about 50 PCR primer sets, for example the primer set of
the sequence shown in Referential example 1, and PCR was performed
with these primer sets by using PCR template of cDNA prepared from
poly A+RNA of various tissues of human origin, and more than 10 PCR
products from each tissue were subcloned, as well as performing
sequencing for more than 500 types. A clone having a desired
sequence of human Serrate-1 could be identified.
[0037] Namely, as shown in Reference example 1, the obtained PCR
product is cloned in the cloning vector, transforming the host
cells by using recombinant plasmid which contains the PCR product,
culturing the host cells containing the recombinant plasmid on a
large scale, purifying and isolating the recombinant plasmid,
checking the DNA sequence of PCR product which is inserted into the
cloning vector, and trying to obtain the gene fragment which may
have a sequence of human Serrate-1 by comparing with the sequence
of the known Serrate homologue of other species. We have succeeded
in discovering a the gene fragment which contains a part of cDNA of
human Serate-1, the same sequence of DNA sequence from 1272 to 1737
described in the sequence listing, SEQ ID NO: 6. As shown in
Referential example 2, using the thus obtained human Serrate-1 gene
fragment, full length cDNA is obtained from human cDNA library. We
have already filed a patent application with these inventions (WO
97/19172).
[0038] In the present invention, there may be possibly to exist
ligands other than this human Serrate-1 molecule, gene fragments
showing high homology in relation to the ligand to the gene
sequence coding amino acid sequence of human Serrate-1 molecule,
i.e. DNA sequence from 409 to 4062 in the sequence listing SEQ ID
NO: 6, are screened in the data base of DNA sequence. Screening was
performed by using gene sequence search software Genetyx/CD
(software Development Co.) on the DNA/fragments of random human
cDNA sequence data base EST (Expressed Sequence Tag) of Genbank
(release 91, 1995).
[0039] Recently, DNA sequencing technology has progressed, and
analysis of total geneomic DNA and full cDNA sequence of humans,
nematoda, Arabidopsisthaliana Heynh, etc. were tried by random
sequencing of genomic DNA and cDNA (Genome Directory, Nature, 377,
3S, 1995). In the human cDNA, EST project of TIGR (The Institute
for Genomic Research), EST project of Washington Univ.--Merck, STS
project of Colorad Univ. joined in these projects. Partial base
sequences of cDNA provided by these organizations are registered in
DNA database of Genbank and EMBL and are disclosed. According to
Genbank release 91 of October, 1995, cumulative registered numbers
of EST clone are about 330,000 clones with average length 346
bp.
[0040] Based on data in these databases, gene sequences or amino
acid sequences of known or namely cloned novel molecules are
searched by homology search. Possibility of existence of the
analogues or similar family molecules of these molecules can be
known and the sequence information of the partial DNA sequence can
be obtained.
[0041] For analysis, commercially available analysis software can
be used, or analysis software attached to the database, for example
BLAST of Genbank can be used. By using these, analysis can be
performed by accessing to National Center of Biotechnology
Information, U.S.A., Institute of Chemistry, Kyoto Univ., Japan,
through WWW (world wide webb) or E-mail.
[0042] Gene sequence information of gene fragments (about 200-350
bp) with high similarity to the target gene can be obtained through
these operations. The information of the obtained gene fragment
includes general gene sequence information together with clone name
of the gene and organs or tissues in which the gene was extracted.
In the information on DNA sequence, this is essentially raw data
obtained by DNA sequence, including unknown DNA sequences with
marked "n", and incorrect DNA sequence information. Consequently,
this DNA sequence information is not always exact.
[0043] From this gene information, DNA sequences without unknown
residues N are thought to be highly probable DNA sequence
information. Further the most probable DNA sequences within these
DNA sequences are compared and DNA fragments having significant
homology are identified in this region (in case of a gene with 200
bases, similarity of DNA sequence above 40% is preferable). The
thus identified DNA fragment can be obtained from Genom System
Inc., U.S.A. etc., if the name of the clone is known; however,
because of knowing disclosed origin of organs, it can be also
isolated by PCR from cDNA of commercially available expression
organs.
[0044] The thus found gene information is partial information, and
unless total information is obtained, a full length amino acid
sequence, which may be encoded by the said partial sequence of
gene, is not always analogous similar molecule used in the original
homology search. Exact information about the molecule cannot be
shown only by that information. As shown below, we have prepared a
number of probes having homology and performed cloning by plaque
hybridization; however, most DNA fragments did not code the desired
molecules. Consequently, this technique may be theoretically
possible but is not easy technology.
[0045] We have prepared probes from about SO DNA fragments which
showed similarity with human Serrate-1 cDNA by PCR. Finally, as
shown in the following, cloning was performed by library screening
technique. As a result of determining the DNA sequence, a gene
isolated by using 3 types of DNA probes having sequence of SEQ ID
NOS: 10, 11 and 12, which were prepared based on DNA sequence
clones registered in Genbank (Reg. No. T08853, R50026 and R46751)
as described in Example 1, is a DNA fragment coding human Serrate-2
molecule.
[0046] Furthermore, using the thus isolated cDNA fragment as a
probe and screening a cDNA library of the expression organs, then
the longer gene having DNA sequence or full length cDNA gene can be
screened. The full length cloning can be made by isotope labelling
and non-isotope labelling with the partial cloning gene, and
screening the library by hybridization or other method. Isotope
labelling can be performed by, for example, terminal labelling by
using [32 P] .gamma.-ATP and T4 polynucleotide kinase, or other
labelling methods such as nick translation or primer extension
method can be applied.
[0047] Furthermore, cloning of the full length gene or longer gene
fragments can be performed by methods for extension of gene
sequence with 5'-RACE or 3'-RACE method without using a library. In
other methods, human origin cDNA library is ligated into the
expression vector, expressing by COS-7 or other cells, the ligated
molecule is searched using receptor Notch protein and the objective
gene is screened by expression cloning to isolate cDNA of the
ligand. In the expression cloning, a cell sorter fractionation
method which is applied with binding with polypeptide containing
amino acid sequence of prior known 4 Notches such as TAN-1, and a
detection method by film emulsion using radioisotope can be
mentioned.
[0048] In this specification, a method for obtaining genes of human
Serrate-2 is explained, and various methods clearly shown in this
invention including PCR, by which clonings of human Delta-1 and
human Serrate-1 were performed, can be applied for obtaining new
Notch ligand family molecules which have never been cloned. For
example, the conserved domains are found by comparison with amino
acid sequence or DNA sequence of human Serrate-1 or human
Serrate-2, and cloning thereof is performed after applying PCR, and
also cloning can be performed by searching EST based on human
Delta-1 or human Serrate-2. These cloned new Notch ligand family
molecules can be used as the same human Serrate-2 shown in the
present invention, for example by full length cloning, preparating
expression vector, preparation of transformed cells, protein
production, antibody production or screening the bioactive
substances, and differentiation suppressive action for cells can be
expected.
[0049] As shown in Example 2, these three gene fragments are
labelled with radioisotope to prepare hybridization probes,
screened using cDNA of human fetal brain origin as the screening
library, whereupon the DNA sequence of the thus obtained clones is
determined, and found to be highly similar with human Serrate-1 in
full length of DNA nucleotide sequence. In these screenings, a full
length cDNA sequence encoding a full length amino acids sequence
cannot be cloned. A further DNA probe is prepared based on the
cloned DNA sequence, and again screening is performed, but the full
length gene cannot be identified. Finally, gene cloning containing
the translation initiation Met codon is performed by 5-RACE method,
the DNA sequence is determined and finally we succeeded in cloning
of cDNA encoding full length of gene sequence of human Serrate-2.
The thus cloned cDNA was ligated as shown in Example 2 and cDNA
encoding the full length of the said human Serrate-2 can be
obtained.
[0050] Examples of plasmids integrated with cDNA are, for example,
E. coli originated pBR322, pUC18, pUC19, pUC118 and pUC119 (Takara
Shuzo Co., Japan), but other plasmids can be used if they replicate
and proliferate in the host cells. Examples of phage vectors
integrated with cDNA are, for example, .lambda. gt10 and .lambda.
gt11, but other vectors can be used if they can grow in the host
cells. The thus obtained plasmids are transduced into suitable host
cells such as genus Escherichia and genus Bacillus using calcium
chloride method. Examples of the above genus Escherichia are
Escherichia coli K12HB101, MC1061, LE392, JM109. Examples of the
above genus Bacillus is Bacillus subtilis MI114. Phage vector can
be introduced into the proliferated E. coli by the in vitro
packaging method (Proc.Natl.Acad. Sci., 71: 2442, 1978).
[0051] The cloned full length DNA sequence was compared with the
database (Genbank release 93, 1996), and it was found that the
total sequence is a novel sequence, although there are partially
the previously mentioned three EST clones and several EST clone
data as non-identical partial sequences other than those three EST
clones.
[0052] Furthermore, the said amino acid sequence of human
Serrate-2, i.e. amino acid sequences in SEQ ID NO: 1, 2 and 3, were
compared with the database of the prior known amino acid sequence
(SWISS-PROT, release 32, 1995 and Genbank CDS, release 93, 1996),
and found that there are no identical amino acid sequences and that
these are novel sequences. According to a comparison in amino acid
sequence of human Serrate-1 and Serrate homologue of the other
organisms, the homologies with human Serrate-1, Drosophila Serrate,
and rat jagged are 53.1%, 34.3%, and 52.3%, respectively. The
substance of the present invention is different from these
substances and is a novel substance having new amino acid sequences
and is first discovered by the present inventors.
[0053] The amino acid sequence was analyzed in hydrophilic part and
hydrophobic part according to a method by Kyte-Doolittle (J. Mol.
Biol., 157: 105, 1982). Results indicate that in the amino acid
sequence listed in SEQ ID NO: 5, amino acid sequence of a precursor
of full length gene consists of 1238 amino acids residue from -26
to 1212 in amino acid sequence, and the signal peptide domain is
estimated to correspond amino acid sequence of 26 amino acids
residue from No. -26 methionine to No. -1 proline; extracellular
domain: 1055 amino acids residue from No. 1 methionine to No. 1055
glycine; transmembrane domain: 24 amino acids residue from No. 1056
leucine to No. 1079 tryptophane; and intracellualr domain: region
from No. 1080 threonine to No. 1212 glutamate. These domains are
the estimated domain construction from the amino acid sequence, and
the actual form may differ from the above structure, and the
constituent amino acids of each domain hereinabove defined may
change 5 to 10 amino acids per sequence.
[0054] In the amino terminal (N-terminal), as shown in Example 6,
identification of N-terminal amino acid sequence of the purified
ligand polypeptides EXS2Fc and EXS2FLAG of the present invention
was performed and found that it was methionine of No. 1 in SEQ ID
NO: 1-3. Consequently, signal peptide is at least from -26
methionine to -1 proline in SEQ ID NO: 5.
[0055] The family molecules of Notch ligand in relation to
extracelluar domain have evolutionally conserved common sequence,
i.e. DSL sequence and repeated EGF-like sequence. As a result of
comparison with amino acid sequence of human Serrate-2 and human
Serrate-1, the conserved sequence is estimated from amino acid
sequence Serrate-2. Namely, DSL sequence corresponds to the 43
amino acid residue from No. 72 cysteine to No. 214 cysteine of the
amino acid sequence in the sequence listing, SEQ ID NO: 5.
[0056] EGF-like sequence exists with 16 repeats wherein, in the
amino acid sequence in the sequence listing, SEQ ID NO: 5, the
first EGf-like sequence from No. 217 cysteine to No. 247 cysteine;
the second EGF-like sequence from No. 250 cysteine to No. 278
cysteine; the third EGF-like sequence from No. 285 cysteine to No.
318 cysteine; the fourth EGF-like sequence from No. 325 cysteine to
No. 356 cysteine; the fifth EGF-like sequence from No. 363 cysteine
to No. 394 cysteine; the sixth EGF-like sequence from No. 401
cysteine to No. 432 cysteine; the seventh EGF-like sequence from
No. 439 cysteine to No. 469 cysteine; the eighth EGF-like sequence
from No. 476 cysteine to No. 507 cysteine; the ninth EGF-like
sequence from No. 514 cysteine to No. 545 cysteine; the 10th
EGF-like sequence from No. 563 cysteine to No. 607 cysteine; the
11th EGF-like sequence from No. 614 cysteine to No. 645 cysteine;
the 12th EGF-like sequence from No. 652 cysteine to No. 683
cysteine; the 13th EGF-like sequence from No. 690 cysteine to No.
721 cysteine; the 14th EGF-like sequence from No. 729 cysteine to
No. 760 cysteine; the 15th EGF-like sequence from No. 767 cysteine
to No. 798 cysteine; and the 16th EGF-like sequence from No. 805
cysteine to No. 836 cysteine.
[0057] On these cysteine residues, there are 2 cysteine residues
between the 9th EGF-like sequence and the 10th EGF-like sequence.
Also there are 6 cysteine residues to the direction of N-terminal
of DSL sequence and 16cysteine residues to the direction of
C-terminal in the 16th EGF-like sequence. These cysteine residues
including EGF-like sequence are conserved in almost the same
position of the human Serrate-1.
[0058] A part for sugar chain attachment is estimated from amino
acid sequence of the human Serrate-2 as No. 127, 544, 593, 726 and
1032 asparagine residue in the sequence listing, SEQ ID NO: 3 as a
possible binding site of N-glycoside bonding for
N-acetyl-D-glycosamine. O-glycoside bond of
N-acetyl-D-galactosamine is estimated to be a serine or threonine
residue rich part. Protein bound with sugar chain is generally
thought to be stable in vivo and to have strong physiological
activity. Consequently, in the amino acid sequence of polypeptide
having sequence of the sequence listing, SEQ ID NO: 1, 2 or 3,
polypeptides having N-glucoside or O-glucoside bond with sugar
chain of N-acetyl-D-glucosamine or N-acetyl-D-galactosamine is
included in the present invention.
[0059] As a result of studies on binding of Drosophila Notch and
its ligand, amino acid region necessary for binding with ligand of
Drosophila Notch with the Notch is from N-terminal to DSL sequence
of the mature protein, in which signal peptide is removed (Japan.
Pat. PCT Unexam. Publ. No. 7-503121). Furthermore, as a result of
the similar studies, a study using nematoda by Fitzgerald and
Greenwald (Development, 121, 4275-4282, 1995) clearly indicated
that full length of Notch ligand-like molecule APX-1 from amino
terminal to DSL domain was sufficient length for activation of
Notch-like receptor. These facts indicate that a domain necessary
for expression of ligand action of human Serrate-2 molecule is a
novel amino acid sequence of the sequence listing, SEQ ID NO:
1.
[0060] Northern blotting can be performed by using DNA encoding a
part or all of gene sequence in the sequence listing, SEQ ID NO: 4.
Consequently, a method for detection of expression of these genes
can be achieved by applying with hybridization or PCR by using
complementary nucleic acids of above 12mer or above 16mer,
preferably above 18mer having nucleic acid sequence of a part of
sequence in the sequence listing SEQ ID NO: 4, i.e. antisense DNA
or antisense RNA, its methylated, methylphosphated, deaminated, or
thiophosphated derivatives. By the same method, detection of
homologues of the gene of other organisms such as mice or gene
cloning can be achieved.
[0061] Further cloning of genes in the genome including humans can
be made. Using these genes cloned by such like methods, further
detailed functions of the human Serrate-2 of the present invention
can be identified. For example, using the modern gene manipulation
techniques, every method including transgenic mouse, gene targeting
mouse or double knockout mouse in which genes relating to the gene
of the present invention are inactivated, can be applied. If
abnormalities in the genome of the present gene is found,
application to gene diagnosis and gene therapy can be made.
[0062] As described in Example 3, an expression in normal human
tissues is observed in many tissues, and length of the expressed
mRNA is one type of the mRNA with about 5 kb length. This means
tjat detecting the expression of mRNA of the said molecule can be
applied for diagnosis or detection of malignant tumors in the part
of normal organs in which expression of these mRNA cannot be
observed. Furthermore, by referring to patterns of the expressed
organs, use of human Serrate-2, for which concrete use is not
indicated in the present invention, can be found.
[0063] A transformant in which vector pUCSR-2, which contains cDNA
coding total amino acid sequence of human Serrate-2 of the present
invention, is transformed into E. coli JM109, has been deposited in
the National Institute of Bioscience and Human-Technology, Agency
of Industrial Science and Technology, MITI, of 1-1-3, Higashi,
Tsukuba-shi, Ibaragi-ken, Japan. as E. coli: JM109-pUCsr-2. Date of
deposit was Oct. 28, 1996, and deposition No. is FERM BP-5727.
[0064] Expression and purification of various forms of human
Serrate-2 using cDNA coding amino acid sequence of human Serrate-2
isolated by the above methods are known in the literature
(Kriegler, Gene Transfer and Expression-A Laboratory Manual
Stockton Press, 1990 and Yokota et al. Biomanual Series 4, Gene
transfer and expression and analysis, Yodosha Co., 1994). A cDNA
coding the amino acid sequence of the isolated said humanSerrate-2
is ligated to preferred expression vector and is produced in the
host cells of eukaryotic cells such as animal cells and insect
cells or prokaryotic cells such as bacteria.
[0065] In the expression of the molecule of the present invention,
DNA encoding a polypeptide of the present invention may have the
translation initiation condon in 5'-terminal and translation
termination codon in 3'-terminal. These translation initiation
codon and translation termination codon can be added by using
preferred synthetic DNA adapter. Furthermore, for expression of the
said DNA, promoter is linked upstream of the DNA sequence. Examples
of vector are plasmid originated from Bacillus, plasmid originated
from yeast or bacteriophage such as .lambda.-phage and animal virus
such as retrovirus and vaccinia virus.
[0066] Examples of promoters used in the present invention are any
promoters suitable for corresponding to the host cells used in gene
expression.
[0067] In case that the host cell in the transformation is genus
Escherichia, tac-promoter, trp-promoter and lac-promoter are
preferred, and in case of host of genus Bacillus, SPO1 promoter and
SPO2 promoter are preferred, and in case of host of yeast, PGK
promoter, GAP promoter and ADH promoter are preferred.
[0068] In case that the host cell is animal cells, a promoter
originated from SV40, promoter of retrovirus, metallothionein
promoter and heatshock promoter can be applied.
[0069] Expression of the polypeptide of the present invention can
be effected by using only DNA encoding the amino acid sequence of
the sequence listing, SEQ ID NO: 1, 2 or 3. However, a protein
having an additional specific function can be produced by using
DNA, to which is added cDNA encoding the known antigen epitope for
easier detection of the produced polypeptide or to which is added
cDNA encoding the immunoglobulin Fc for forming a multimer of the
said human Serrate-2.
[0070] As shown in Example 4, we have prepared expression vectors,
which express extracellular proteins, as follow.
[0071] 1) DNA encoding the amino acids from No. 1 to 1055 in amino
acid sequence in the sequence listing, SEQ ID NO: 2.
[0072] 2) DNA encoding chimera protein to which added polypeptide
having 8 amino acid, i.e. an amino acid sequence consisting of Asp
Tyr Lys Asp Asp Asp Asp Lys (hereinafter designates FLAG sequence,
the sequence listing, SEQ ID NO: 25), in the C-terminal of the
amino acids from No. 1 to 1055 in amino acid sequence in the
sequence listing, SEQ ID NO: 2, and
[0073] 3) DNA encoding chimera protein to which is added Fc
sequence below the hinge region of human IgG1 (refer to
International Patent Unexam. Publ. WO 94/02053) in the C-terminal
of the amino acids from No. 1 to 1055 in amino acid sequence in the
sequence listing, SEQ ID NO: 2, and to have dimer structure by
disulfide bond in the hinge region, are ligated individually with
the expression vector pMKITNeo (Maruyama et al., Japan Molecular
Biology Soc. Meeting Preliminary lecture record, obtainable from
Dr. Maruyama in Tokyo Medical and Dental College) to prepare
extracellular expression vectors of human Serrate-1.
[0074] The expression vectors, which expresses full-length protein,
can be prepared as follows.
[0075] 4) DNA encoding amino acids from No. 1 to 1212 in the
sequence listing, SEQ ID NO: 3 and
[0076] 5) DNA encoding chimera protein to which is added
polypeptide having FLAG sequence in the C-terminal of amino acids
from No. 1 to 1212 in the sequence listing, SEQ ID NO: 3 re ligated
individually with the expression vector pMKIT-Neo to prepare the
full-length expression vector of human Serrate-2. The
transformation is prepared by using expression plasmid containing
DNA encoding the thus constructed said human Serrate-2.
[0077] Examples of the host are genus Escherichia, genus Bacillus,
Yeast and animal cells. Examples of animal cells are simian cell
COS-7 and Vero, Chinese hamster cell CHO and silk worm cell
SF9.
[0078] As shown in Example 5, the above 5 type expression vectors
are transduced individually; the human Serrate-2 is expressed in
COS-7 cell (obtainable from the Institute of Physical and Chemical
Research, Cell Development Bank, RCB0539); and the transformants
which were transformed by these expression plasmids, can be
obtained. Furthermore, human Serrate-2 polypeptide can be produced
by culturing the transformants under suitable culture conditions in
medium by known culture methods.
[0079] The human Serrate-2 polypeptide can be isolated and purified
from the above cultured mass, in general, by the following
methods.
[0080] For extraction of the substance from cultured microbial
cells or cells, microbial cells or cells are collected by known
method such as centrifugation after the cultivation, suspended in
siutable buffer solution, whereafter the microbial cells or cells
are disrupted by means of ultrasonication, lysozyme and/or
freeze-thawing and a crude extract of human Serrate-2 protein is
collected by centrifugation or filtration. The buffer solution may
contain protein-denaturing agents such as urea and guanidine
hydrochloride or surface active agents such as Triton X-100. In
case of secretion in the cultured solution, the cultured mass is
separated by known methods such as centrifugation to separate from
microbial cells or cells, and the supernatant solution is
collected.
[0081] The thus obtained human Serrate-2, which are contained in
the cell extracts or cell supernatants, can be purified by known
protein purification methods. During the purification process, for
confirmation of existence of the protein, in case of the fused
protein of the above FLAG and human IgGFc, it can be detected by
immunoassay using antibody against known antigen epitope and can be
purified. In case the fused protein is not expressed as such, the
antibody against human Serrate-2 in Example 7 can be used for
detection.
[0082] A more useful purification method is an affinity
chromatography using antibody. Antibodies used in this case are
antibodies described in Example 7. For fused protein, antibodies
against other than human Serrate-2 are used, for example antibody
against FLAG in the case of FLAG, and protein G or protein A in the
case of human IgGFc as shown in Example 6.
[0083] Physiological functions of the thus purified human Serrate-2
protein or human Serrate-2 can be identified by various assay
methods, for example, physiological activity assaying using cell
lines and animals such as mice and rats, assay methods of
intracellular signal transduction based on molecular biological
means and binding with Notch receptor etc.
[0084] For that action, mainly an action suppressing cell
differentiation will be expected, and actions such as stimulating
tissue regeneration, etc. can be expected.
[0085] Namely, we have found that, as shown in Example 8, in the
umbilical cord blood derived blood undifferentiated cells in which
CD34 positive cell fraction is concentrated, polypeptides of the
present invention have suppressive action of colony forming action
against blood undifferentiated cells, which shows colony formation
in the presence of cytokines.
[0086] Furthermore, as shown in Example 9, we have found that as a
result of adding IgG1 chimera protein of human Serrate-2 to the
liquid culture in the presence of cytokines, the human Serrate-2
had activities for significantly decreasing LTC-IC (Long-Term
Culture-Initiating Cells) counts, which were positioned with most
undifferentiated blood stem cells in the human blood
undifferentiated cells.
[0087] These results indicate that the human Serrate-2 suppresses
differentiation of blood undifferentiated cells, and these actions
spread from blood stem cells to colony forming cells. Furthermore,
pharmaceuticals containing the polypeptide of the present invention
have action for protection and release of the bone marrow
suppressive action, which is observed in adverse effects of
antitumor agents.
[0088] Furthermore, as shown in example 10, we have studied on an
action against vascular cells, for which an action of the molecules
of the present invention has never been known except for blood
cells, and found that the molecules of the present invention have
an action to suppress growth of the human vascular endothelia
cells. Consequently, the present invention includes growth
suppressive agents for vascular cells and therapeutic agents for
disease (refer to Folkman and Klagsbrun, SCIENCE 235, 442-447,
1987), which effect is expected by suppressing vascularization,
containing polypeptides having amino acid sequence of SEQ ID NO:
1-3. The molecules of the present invention can be used for
treatment of these diseases.
[0089] In pharmaceutical use, polypeptides of the present invention
having above form is lyophilized with adding preferable stabilizing
agents such as human serum albumin, and is used in dissolved or
suspended condition with distilled water for injection when it is
in use. For example, preparation for injection or infusion at the
concentration of 0.1-1000 .mu.g/ml may be provided. A mixture of
the compound of the present invention 1 mg/ml and human serum
albumine 5 mg/ml divided in a vial could maintain activity of the
said compound for long term. For culturing and activating cells in
vitro, lyophilized preparations or liquid preparations of the
polypeptide of the present invention are prepared and are added to
the medium or immobilized in the vessel for culture. Toxicity of
the polypeptide of the present invention was tested. Any
polypeptide, 10 mg/kg was administered intraperitoneally in mice,
but no death of mice was observed.
[0090] In vitro physiological activity of the polypeptide of the
present invention can be evaluated by administering to disease
model mice or its resembled disease in rats or monkeys, and
examining recovery of physical and physiological functions and
abnormal findings. For example, in case of searching abnormality in
relation to hemopoietic cells, bone marrow suppressive model mice
are prepared by administering 5-FU series of antitumor agents, and
bone marrow cell counts, peripheral blood cell counts and
physiological functions are examined in the administered group or
the non administered group of mice. Furthermore, in case of
searching in vitro cultivation and growth of hemopoietic
undifferentiated cells including hemopoietic stem cells, the bone
marrow cells of mice are cultured in the groups with or without
addition of the compound of the present invention, and the cell
cultures are transferred into the lethal dose irradiated mice.
Result of recovery is observed with the indications of survival
rate and variation of blood counts. These results can be
extrapolated to the humans, and accordingly useful effective data
for evaluation of the pharmacological activities of the compound of
the present invention can be obtained.
[0091] Applications of the compound of the present invention for
pharmaceuticals include diseases with abnormal differentiation of
cells, for example leukemia and malignant tumors. These are a cell
therapy, which is performed by culturing human derived cells in
vitro while maintaining their original functions or adding new
functions, and a therapy, which is performed by regenerating
without damage the functions originally existing in the tissues by
administering the compound of the present invention under the
regeneration after tissue injury. Amount of administration may
differ in the type of preparation and ranges from 10 .mu.g g/kg to
10 mg/kg.
[0092] Further strong physiological activity can be achieved by
expression forming a multimer of the polypeptide of the present
invention. Human Serrate-2 having multimer structure can be
produced by a method of expressing chimera protein with human IgG
Fc region as described in the example and expressing the multimer
having disulfide bond with hinge region of the antibody, or a
method expressing chimera protein, in which antibody recognition
region is expressed in the C-terminal or N-terminal, and reacting
with the polypeptide containing extracellular part of the thus
expressed said human Serrate and the antibody which recognize
specifically the antibody recognition region in the C-terminal or
N-terminal.
[0093] Among other methods, a method in which the fused protein
bound with only the hinge region of the antibody is expressed and
the dimer is formed by constructing with disulfide bond, can be
mentioned. A multimer of human Serrate-2 having higher specific
activity than the dimer can be obtained. The said multimer is
constructed by fused protein which is prepared for expressing the
peptide in the C-terminal, N-terminal or other region. The protein
is prepared by forming a disulfide bond without affecting any ether
activities of the human Serrate-2. The multimer structure can also
be expressed by arranging one or more peptides containing SEQ ID
NO: 1 or 2, with genetic engineering method in series or in
parallel. Other known methods for providing multimer structure
having dimer or more can be applied. Accordingly, the present
invention includes any polypeptides containing SEQ ID NO: 1 or 2 in
a dimer or higher structure prepared by genetic engineering
techniques.
[0094] As another method, multimerization method using chemical
cross-linker can be mentioned. For example, dimethylsuberimidate
dihydrochloride for cross-linking lysine residue,
N-(.gamma.-maleimidebut- yryloxy) succinimide for cross-linking
thiol group of cysteine residue and glutaraldehyde for
cross-linking between amino groups can be mentioned. A multimer
with dimer or higher structure can be synthesized by applying these
cross-linking reactions. Accordingly, the present invention
includes any polypeptides containing SEQ ID NO: 1 or 2 in the form
of dimer or higher structure prepared by chemical cross-linking
agents.
[0095] In application of medical care in which cells are
proliferated and activated in vitro and are returned to the body,
human Serrate-2 of the form hereinabove can be added directly in
the medium, but immobilization can also be made. Immobilization
method includes applying amino group or carboxyl group in the human
Serrate-2, using suitable spacers or the abovementioned
cross-linkers, and the ligand can be covalently bound to the
culture vessels. Accordingly, the present invention includes any
polypeptides containing SEQ ID NO: 1 or 2 in the form existing on a
solid surface. The human Serrate-2 molecule binds specifically with
receptor, a Notch receptor molecule. For example, expression of
Notch receptor can be detected by using fused protein with above
extracellular region of the human Serrate-2 and human IgGFc. Notch
is known to be involved in some types of leukemia (Elissen et al.,
Cell 66, 649-661, 1991). Accordingly, the polypeptides having SEQ
ID NO:1 or 2 can be used for diagnostic reagents for in vitro or in
vivo.
[0096] Antibody specifically recognizing the said human Serrate-2
can be prepared as shown in Example 7. Also it can be prepared by
various methods described in the literature (Antibodies a
laboratory manual, E. Harlow et al., Cold Spring Harbor
Laboratory), and by recombinant antibody expressed in cells using
immunoglobulin gene isolated by a method of gene cloning. These
antibodies can be used for purification of human Serrate-2. Namely,
detection and measurement of the human Serrate-2 of the present
invention can be performed by using antibody, which specifically
recognizes the humanSerrate-2 shown in Example 7, and can be
applied as diagnostic agent for diseases such as malignant tumor
accompanied with abnormal cell differentiation.
EXAMPLES
[0097] The following examples illustrate embodiments of the present
invent-ion, but are not to be construed as limiting.
Referential Example 1
[0098] Preparation of Human Serrate-1 Gene Probe
[0099] A mixed primer corresponding to amino acid sequence
conserved in Drosophila Serrate and rat jagged, i.e. sense primer
(SEQ ID NO: 8) and antisense primer (SEQ ID NO: 9), were used. The
signals used in these sequence show each equivalent mixture: i.e.
S: C and G, Y: T and C, W: T and A, K: G and T, R: A and G, N: C,
G, T and A.
[0100] A synthetic oligonucleotide was prepared by using automatic
DNA synthesizer with the principal immobilized method. The
automatic DNA synthesizer used was 391PCR-MATE of Applied
Biosystems Inc., U.S.A. Nucleotide, carrier immobilized with
3'-nucleotide, solution and reagents are used according to the
instructions by the same corporation. Oligonucleotide was isolated
from the carrier after finishing the designated coupling reaction
and treating the oligonucleotide carrier, from which protective
group of 5'-terminal was removed, with concentrated liquid ammonia
at room temperature for one hour. For removing protective groups of
nucleic acid and phosphoric acid, the reactant soluion containing
nucleic acid was allowed to stand in the concentrated ammonium
solution in the sealed vial at 55.degree. C. for over 14 hours.
Each oligonucleotide, from which the carrier and protective groups
were removed, was purified by using OPC cartridge of the Applied
Biosystems Inc., and detritylated by using 2% trifluoroacetic acid.
Primer was dissolved in deionized water to set final concentration
100 pmol/.mu.l after purification, and used for PCR. Synthesis of
oligonucleotide was performed by the same manner.
[0101] Amplification by PCR was performed as follows.
[0102] Human fetal brain originated cDNA mixture solution
(QUICK-Clone cDNA, CLONTECH Inc., U.S.A.) 1 .mu.l was used.
10.times. buffer solution [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15
mM MgCl 2 , 0.01 % gelatin] 5 .mu.l, dNTP Mixture (Takara Shuzo) 4
.mu.l, sense primer DLTS1 (100 pmol/.mu.l) 5 .mu.l and antisense
primer DLTA2 (100 pmol/.mu.l) 5 .mu.l which were specific to the
above Serrate homologue and TaqDNA polymerase (AmpliTaq, Takara
Shuzo., Japan, 5 U/.mu.l) 0.2 .mu.l were added thereto, and finally
deionized water was added to set up total 50 .mu.l. PCR was
performed for 5 cycles of a cycle consisting of treatment at
95.degree. C. for 45 seconds, at 42.degree. C. for 45 seconds and
72.degree. C. for 2 minutes, further 35 cycles of a cycle
consisting of treatment at 95.degree. C. for 45 seconds, at
50.degree. C. for 45 seconds, and 72.degree. C. for 2 minutes, and
finally allowed to stand at 72.degree. C. for 7 minutes. A part of
the PCR products was subjected to 2% agarose gel electrophoresis,
stained with ethidium bromide (Nippon Gene Co., Japan), and
observed under ultraviolet light to confirm amplification of about
500 bp cDNA. The total amount of the thus obtained PCR product was
subjected to electrophoresis with 2% agarose gel prepared with low
melting point agarose (GIBCO BRL Inc., U.S.A.), stained with
ethidium bromide, cutting out about 500 bp bands under the UV
light, adding distilled water of the same volume as the gel,
heating at 65.degree. C. for 10 minutes, and completely dissolving
the gel. The dissolved gel was centrifuged at 15000 rpm for 5
minutes to separate supernatant solution after adding an equal
volume of TE saturated phenol (Nippon Gene Co., Japan) and the same
separation operation was performed after adding TE saturated
phenol:chloroform (1:1) solution and chloroform. DNA was recovered
from the final solution by ethanol precipitation.
[0103] A vector, pCRII vector (Invitrogen Inc., U.S.A., hereinafter
designated as pCRII) was used. The vector and the above DNA in a
molar ratio of 1:3 were mixed and DNA was ligated into the vector
by using T4 DNA ligase (Invitrogen Inc., U.S.A.). The pCRII, to
which DNA was integrated, was subjected to gene transduction into
E. coli one shot competent cells (Invitrogen Inc., U.S.A.) and was
spread on the semi-solidmedium plate of L-Broth (Takara Shuzo Co.,
Japan) containing ampicillin (Sigma Corp., U.S.A.) 50 .mu.g/ml and
allowed to stand at 37.degree. C. for about 12 hours. The visible
colonies were randomly selected, inoculated in the L-Broth liquid
medium 2 ml containing same concentration of ampicillin and shake
cultured at 37.degree. C. for about 18 hours. The cultured
bacterial cells were recovered and the plasmid was separated by
using Wizard Miniprep (Promega Inc., U.S.A.) according to the
attached explanation sheet. The plasmid was digested by restriction
enzyme EcoRI. Integration of the said PCR product was confirmed by
incision of about 400 bp DNA. The base sequence of the incorporated
DNA in the confirmed clone was determined by fluorescent DNA
sequencer (Model 373S, Applied System Inc., U.S.A.). The gene
cloning gene fragment was compared with amino acid sequence of
Notch ligand molecule, i.e. Drosophila Serrate and rat Jagged, and
significant analogous sequence was found, then the sequence was
confirmed as cDNA fragment coding human Serrate-1.
Referential Example 2
[0104] Cloning of Full Length Human Serrate-1 Gene
[0105] A screening of clones having full length cDNA was performed
by hybridization from human placenta origin cDNA library (inserted
cDNA in .lambda. gt-11, CLONTEC Inc., U.S.A.) in plaques
corresponding to 1.times.106 plaques. Generated plaques were
transcribed to nylon filter (Hybond N+: Amersham Inc., U.S.A.). The
transcribed nylon filter was subjected to alkaline treatment [allow
to stand for 7 minutes on a filter paper permeated with a mixture
of 1.5 M NaCl and 0.5 M NaOH], followed by twice neutralizing
treatments [allow to stand for 3 minutes on a filter paper
permeated with a mixture of 1.5 M NaCl, 0.5 M Tris-HCl (pH 7.2) and
1 mM EDTA]. Subsequently, the nylon filter was shaken for 5 minutes
in 2-fold concentrated SSPE solution [0.36 M NaCl, 0.02 M sodium
phosphate (pH 7.7) and 2 mM EDTA], washed, and air-dried. Then the
nylon filter was allowed to stand for 20 minutes on a filter paper,
which was permeated with 0.4 M NaOH, and was shaken for 5 minutes
with 5-fold concentrated SSPE solution and was washed, then again
air-dried. Screening was conducted in the human Serrate-1 probe
labeled with radioisotope 32 P using the filter.
[0106] The DNA probe previously prepared was labeled with 32 P as
follows. A DNA fragment was cut out by EcoRI from PCR II, to which
purified PCR product by human Serrate primers (about 500 bp) was
inserted and DNA fragments were isolated from low melting point
agarose gel. The thus obtained DNA fragment was labeled by DNA
labeling kit (Megaprime DNA labeling system: Amersham, U.S.A.). The
primer solution 5 .mu.l and deionized water were added to DNA 25 ng
to set up total volume of 33 .mu.l , which was treated for 5
minutes in boiling water bath. Reaction buffer solution 10 .mu.l
containingd NTP, .alpha.-32 P-dCTP 5 .mu.l and T4 DNA
polynucleotide kinase solution 2 .mu.l were added thereto, and
treated at 37.degree. C. for 10 minutes in water bath.
Subsequently, the mixture was purified by Sephadex column (Quick
Spin Column Sephadex G-50: Boehringer Mannheim Inc., Germany), then
treated for 5 minutes in boiling water bath and ice-cooled for 2
minutes for use.
[0107] Hybridization was performed as follows. The prepared filter
hereinabove was immersed into prehybridization solution consisting
of SSPE solution, in which final concentration of each component is
set at 5-fold concentration, 5-fold concentration of Denhardt's
solution (Wako Purechemicals), 0.5% SDS (sodium dodecyl sulfate)
and salmon sperm (Sigma Co.) 10 .mu.g/ml denatured by boiling
(Sigma Co.) 10 .mu.g/ml denatured by boiling water, shaken at
65.degree. C. for 2 hours, then the filter was immersed into the
hybridization solution, which was the same composition as the above
prehybridization solution, containing the probe labeled with 32 P
by the above mentioned method, and shaken at 55.degree. C. for 16
hours to perform hybridization.
[0108] The filter was washed by immersing into SSPE solution
containing 0.1% SDS, shaken at 55.degree. C. twice, and further
immersing into 10-fold dilution of SSPE solution containing 0.1%
SDS four times at S5.degree. C. The washed filter was treated with
autoradiography using a sensitized screen. Clones of strongly
exposed part were collected and plaques obtained were again spread
and screened by the same method hereinbefore to separate a complete
single clone.
[0109] The thus isolated phage clones were 22 clones. Phages of all
of these clones was prepared to about 1.times.10 9 pfu, whereafter
the phage DNA was purified, digested by restriction enzyme EcoRI
and inserted into pBluescript (Stratagene Inc., U.S.A.), which was
digested by EcoRI in the same way. DNA sequences of both ends of
these clones were analyzed by DNA sequencer. Two clones of S16 and
S20 were the clone containing DNA sequence from No. 1 to 1873 in
the sequence listing, SEQ ID NO: 6. Clones S5 and S16 were the
clone containing DNA sequence from No. 990 to 4005 in the sequence
listing, SEQ ID NO: 6. The deletion mutant of these clones were
prepared by using kilosequence deletion kit (Takara Shuzo Co.)
according to a description of the attached paper. The full-length
cDNA base sequence encoding a polypeptide of the present invention
was determined using the DNA sequencer (Applied Biosystem Inc.)
from both direction of 5'-direction and 3'-direction.
[0110] As a result, about 100 bp in an area coding C-terminal amino
acid sequence were found to be not cloned, accordingly cloning of
full-length gene was performed by using GIBCO-BRL, 3' RACE system
kit according to the attached manual. Namely, cDNA cloning was
performed by human origin poly A+RNA (CLONTECH Corp.) to
3'-direction and gene sequence was determined.
[0111] The thus cloned three gene fragments in a plasmid containing
in a full-length DNA sequence of SEQ ID NO: 6 are inserted by
applying restriction enzyme Bgl 2 site at DNA sequence No. 1293 in
sequence ID NO: 6 and AccI site at No. 3943, between EcoRI and XbaI
of multicloning site in pUC18 to prepare pUCSR-1. The sequence of
this gene together with aminoacid sequence is shown in SEQ ID NO:
6.
Example 1
[0112] Preparation of Probe by PCR
[0113] Gene probes used for screening, i.e. gene described in SEQ
ID NO: 10, 11 and 12, were obtained as follows. These sequences
correspond to Genbank registered number, T08853, R50026 and R45751.
Hereinafter, a probe having gene sequence SEQ ID NO: 10 is
designated as .Yen.1, a probe having gene sequence SEQ ID NO: 11 is
designated as .Yen.2, and a probe having gene sequence SEQ ID NO:
12 is designated as .Yen.4.
[0114] Namely, a gene SEQ ID NO: 10 was isolated by PCR using
primers of oligonucleotide having SEQ ID NO: 13 and 14; a gene SEQ
ID NO: 11 was isolated by PCR using primers of oligonucleotide
having SEQ ID NO: 15 and 16; and a gene SEQ ID NO: 12 was isolated
by PCR using primers of oligonucleotide having SEQ ID NO: 17 and
18.
[0115] Amplification by PCR was performed as follows. Human fetal
brain originated cDNA mixed solution (QUICK-Clone cDNA, CLONTECH
Inc., U.S.A.) 1 .mu.l was used. 10.times. buffer solution [500 mM
KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2, 0.01% gelatin] 5
.mu.l, dNTP mixture (Takara Shuzo Co., Japan) 4 .mu.l , the primers
hereinbefore (20 pmol/.mu.l) each 1 .mu.l, and TaqDNA polymerase
(AmpliTaq, Takara Shuzo Co., 5U/.mu.l) 0.2 .mu.l were added
thereto, and finally deionized water was added to set up total 50
.mu.l . PCR was performed for 40 cycles of a cycle consisting of
treatment at 95.degree. C. for 1 minute, at 55.degree. C. for 5
minutes and 72.degree. C. for 3 minutes, and finally allowed to
stand at 72.degree. C. for 7 minutes. A part of the PCR products
was subjected to 2% agarose gel electrophoresis, stained with
ethidium bromide (Nippon Gene Co., Japan), and observed under
ultraviolet light to confirm amplification of objective size
gene.
[0116] The total amount of the thus obtained PCR product was
subjected to electrophoresis with 2% agarose gel prepared with low
melting point agarose (GIBCO BRL Inc., U.S.A.), stained with
ethidium bromide, cutting out each band under the UV light, adding
distilled water of the equal volume of the gel, heating at
65.degree. C. for 10 minutes, and completely dissolving the gel.
The dissolved gel was centrifuged at 15000 rpm for 5 minutes to
separate supernatant solution after adding equal volume of TE
saturated phenol (Nippon Gene Co., Japan) and the same separation
operation was performed after adding TE saturated phenol:chloroform
(1:1) solution and chloroform. DNA was recovered from the final
solution by ethanol precipitation.
[0117] A vector, pCRII vector (Invitrogen Inc., U.S.A., hereinafter
designated as pCRII) was used. The vector and the above DNA in a
molar ratio 1:3 were mixed and DNA was ligated into the vector by
using T4 DNAligase (Invitrogen Inc., U.S.A.). The PCRII, to which
DNA was integrated, was subjected to gene transduction in E. coli
one shot competent cells (Invitrogen Inc., U.S.A.) and was spread
on the semisolid medium plate of L-Broth (Takara Shuzo Co., Japan)
containing ampicillin (Sigma Corp., U.S.A.) 50 .mu.g/ml and allowed
to stand at 37.degree. C. for about 12 hours. The visible colonies
were randomly selected, inoculated in the L-Broth liquid medium 2
ml containing the same concentration of ampicillin, and shake
cultured at 37.degree. C. for about 18 hours. The cultured
bacterial cells were recovered and the plasmid was separated by
using Wizard Miniprep (Promega Inc., U.S.A.) according to the
attached explanation sheet. The plasmid was digested by restriction
enzyme EcoRI. Integration of the said PCR product was confirmed by
incision of objective size DNA. The base sequence of the
incorporated DNA in the confirmed clone was determined by
fluorescent DNA sequencer (Model 373S3, Applied System Inc.,
U.S.A.). The sequence was compared with DNA sequence registered in
Genbank hereinbefore. Isolation of gene having DNA sequences SEQ ID
NO: 10, 11 and 12 was confirmed.
Example 2
[0118] Cloning of Full Length Human Serrate-2 Gene
[0119] A screening of clones having full length cDNA was performed
by hybridization from human fetal brain origin cDNA library
(inserted cDNA in .lambda. gt-11, CLONTECH Inc.) in plaques
corresponding 1.times.10 6 plaques. Appeared plaques were fixed
with alkali by the same method as described in Referential example
2. Using these filter and three types of probes, which were
isolated in Example 1 and labeled with 32 P by the method described
in Referential example 2, screening was performed on each
individually to obtain clones.
[0120] Isolated phage clones were: 2 clones from a case using Y 1
as a probe; 6 clones from a case using .Yen.2 as a probe; and 4
clones from a case using .Yen.4 as a probe. Clones isolated by
.Yen.4 were all included in clones isolated from .Yen.2. All of the
phages of these clones were prepared to about 1.times.10 9 pfu, and
phage DNA was purified by using Wizard Lambda preps (Promega Inc.)
according to the attached explanation sheet, and digested by EcoRI,
then incorporated into pBlusecript (Stratagene Inc.) or pUC18
(Pharmacia Inc.) digested by EcoRI.
[0121] The full DNA sequences of these clones were determined by
DNA sequencer as same as Referential example 2, and a part of the
identical sequence was compared. Result indicates that: #5 clone
was a clone containing DNA sequence from No. 484 to 2025 in
sequence ID NO: 4; #21 clone was a clone containing DNA sequence
from No. 1882 to 3537 in SEQ ID NO: 4; and # 86 clone was a clone
containing DNA sequence from No. 2455 to 3955 in SEQ ID NO: 4.
Remaining clones were those having only short insert consisting of
a part identical with sequences of these clones. This result
indicated that, as a result of comparison with amino acid sequences
of human Serrate-1, the area encoding the N-terminal amino acid
sequence was not cloned. Consequently, a probe having DNA sequence
of SEQ ID NO: 19 was prepared and a screening of the second time
was performed for cloning the cDNA in 5'-region. A probe was
prepared as same as described in Example 1,namely PCR primers
having DNA sequence of SEQ ID NO: 20 and 21 were subjected to PCR
using #5 clone as a template. Library used was prepared as same as
the first screening and conditions were performed by the same
method as before.
[0122] The thus isolated clones in the second screening were six
clones. Phages of all these clones were prepared about 1.times.10 9
pfu, purified by using Wizard Lambda Preps (Promega Inc.) according
to the attached explanation sheet, digested by EcoRI and inserted
into pUC 18 which was also digested with EcoRI. The full DNA
sequences of these clones were determined by DNA sequence as same
as in Referential example 2. As a result of comparison with a part
of identical sequence, S43-1 clone was considered to contain the
most 5'-direction. This clone was a clone containing DNA sequence
from No. 38 to 1538 in SEQ ID NO: 4. The remaining clones have only
short inserts consisting of a part identical with sequence
determined already in the other clone or isolated in the first
time.
[0123] Although a sequence of ATG, which codes translation
initiation codon methionine, could not be found in the second
screening clones, further cloning of cDNA sequence for 5'-direction
was performed by 5'RACE method. 5' RACE was performed by using
5'RACE system kit (GIBCO-BRL Inc.) according to the attached
manual. A cloning of cDNA with 5'-direction in the gene using human
heart origin poly A+RNA (CLONTECH Inc.) was performed, and a gene
sequence from DNA sequence No. 1 to No. 37 in SEQ ID NO: 4 was
determined.
[0124] As a result, DNA sequence in SEQ ID NO: 4, i.e. cDNA
sequence encoding full length of human Serrate-2, was
determined.
[0125] In order to prepare cDNA encoding the full-length gene, and
to obtain cDNA of the 5'-terminal, which could ligate with other
clones, the following PCRwas performed for cloning. Namely, using
the oligonucleotide having DNA sequence in SEQ ID NO: 22 and the
oligonucleotide having DNA sequence in SEQ ID NO: 23, PCR was
performed using S43-1 clone as a template according to a method
described in Example 1. Similarly, it was subcloned to pCRII and
gene sequence was determined to prepare the clone having DNA
sequence from No. 1 to No. 503 in SEQ ID NO: 4. This clone is
designated as S2-5.
[0126] Among the above clones, i.e. gene S2-5, S43-1, #5, #21 and
#86, S2-5 and S43-1 were applied in the restriction enzyme Spl I
site at DNA sequence No. 217 in SEQ ID NO: 4; S43-1 and #5 were the
same as in Kpn I site at No. 1453; #5 and #21 were the same as in
Sac I site at No. 2016;and # 21 and # 86 were the same as in BamHI
site at No. 2991, and finally DNA having DNA sequence SEQ ID NO: 4
was inserted between EcoRI site and Hind III site of multi cloning
site of pUC 18 to prepare pUCSR-2.
Example 3
[0127] Expression of Human Serrate-2 in Organs
[0128] In order to examine expression of mRNA of human Serrate-2,
using filters, which was previously transcribed with mRNA, i.e.
Human Multiple Tissue Northern Blot, Human Multiple Tissue Northern
Blot II, Human Multiple Tissue Northern Blot III and Human Fetal
Multiple Tissue Northern-Blot II (CLONTECH Inc.), 3 2 P labeling
was performed by the previous method using DNA labeling kit (Mega
Prime DNA lebeling system: Amersham Inc.) hereinbefore mentioned,
with a probe DNA having SEQ ID NO: 19 described in Example 2, and
expression was examined with performing hybridization according to
description of instruction for use attached to the above
filters.
[0129] As a result, length of expressed mRNA was about 5 kb. Strong
expressions in human adult tissues were observed in heart, skeletal
muscle, thyroid gland, spinal cord and trachea; clear expression
was observed in pancreas, prostate, testis, small intestine and
adrenal gland, very weak expression was observed in brain,
placenta, kidney, thymus, ovary, stomach and lymph node, and no
expression was observed in lung, liver, spleen, colon, peripheral
lympocytes and bone marrow. In the human fetal tissues, strong
expression was observed in the fetal lung, clear expression: fetal
brain and fetal kidney and no expression in fetal liver.
Example 4
[0130] Preparation of Expression Vector of Human Serrate-2
[0131] Using the gene consisting of DNA sequence described in the
sequence listing, SEQ ID NO: 4, expression vectors of human
Serrate-2 and its chimera protein mentioned in the following 1) to
5) were prepared.
[0132] 1) Expression Vector of Secretory Extracellular Human
Serrate-2
[0133] The cDNA coding polypeptide of amino acid sequence from No.
1 to 1055in the sequence listing, SEQ ID NO: 2 was ligated with
expression vector pMKITNeo (Maruyama et al., Preliminary Paper,
Japan Molecular Biology Soc.1991, obtainable from Prof. Maruyama,
Tokyo Medical and Dental Univ.), which has a SR .alpha. promoter
and Neomycin resistant gene, to prepare expression vector.
[0134] Namely, vector pUCSR-2 containing DNA sequence in SEQ ID NO:
4 was used as template and oligonucleotide having sequence in SEQ
ID NO: 26 and oligonucleotide having sequence in SEQ ID NO: 27 were
used as primer, and PCR was performed according to a method
described hereinbefore. The PCR product was ligated into cloning
vector pCRII, whereupon the gene sequence of the PCR product was
determined to prepare DNA having gene sequence from No. 2986 to
3254 of DNA sequence in SEQ ID NO: 4, in which termination codon
and restriction enzyme Sal I site were attached in the 3' end.
[0135] About a 3 kbp gene fragment was obtained by digesting the
pUCSR-2 with restriction enzyme EcoRI and BamHI, about a 250 bp
gene fragment was obtained by digesting the pCRII vector with
restriction BamHI and Sal I, the vector of which contained the
above PCR product as an insert, and about a 4.3 kb gene fragment
was obtained by digesting the pMKITNeo with restriction enzymes
EcoRI and XhoI, and these 3 gene fragments were simultaneously
ligated to obtain the expression vector containing gene fragment of
DNA sequence from No. 1 to 3254 in SEQ ID NO: 4. Secretory
extracellular human Serrate-2 protein (hereinafter designated this
protein as EXS2) expression vector pMEXS2 was obtained.
[0136] 2) Expression Vector of FLAG Chimera Protein of Secretory
Extracellular Human Serrate-2
[0137] The cDNA coding chimera protein, to which cDNA coding FLAG
sequence (SEQ ID NO: 24) was added to the C-terminal of polypeptide
from No. 1 to 1055 of amino acid sequence in the sequence listing,
SEQ ID NO: 2 was ligated to the expression vector pMKINeo to
prepare the expression vector.
[0138] Namely, vector pUCSR-2 containing DNA sequence in SEQ ID NO:
4 was used as template and oligonucleotide having sequence in SEQ
ID NO: 26 and oligonucleotide having sequence in SEQ ID NO: 28 were
used as primer, and PCR was performed according to a method
described hereinbefore. The PCR product was ligated to cloning
vector pCRII, and the gene sequence of the PCR product was
determined to prepare DNA having gene sequence from No. 2986 to
3254 of DNA sequence in SEQ ID NO: 4, in which DNA sequence coding
FLAG sequence in the 3' end (DNA sequence in SEQ ID NO: 24),
termination codon and restriction enzyme Sal I site were attached
in the 3' end.
[0139] About a 3 kbp gene fragment was obtained by digesting the
pUCSR-2 with restriction enzyme EcoRI and BamHI, about a 300 bp
gene fragment was obtained by digesting the pCRII vector with
restriction enzymes BamHI and Sal I, the vector of which contained
the above PCR product as an insert, and about a 4.3 kb gene
fragment was obtained by digesting the pMKITneo with restriction
enzymes EcoRI and XhoI, and these 3 gene fragments were
simultaneously ligated (though recognition sequence of restriction
enzymesXho I and Sal I is different, they can be ligated due to
complementary terminal gene sequence) to obtain the expression
vector containing gene fragment of DNA sequence from No. 1 to 3254
in SEQ ID NO: 4 and gene fragment encoding FLAG sequence. Secretory
extracellular human Serrate-2 FLAG chimera protein (hereinafter
designated this protein as EXS2FLAG) expression vector pMEXS2FLAG
was obtained.
[0140] 3) Expression Vector of IgG1Fc Chimera Protein of Secretoruy
Extracellular Human Serrate-2
[0141] A cDNA coding chimera protein, to which cDNA coding amino
acid sequence of Fc region below the hinge part of human IgG1 was
added to the C-terminal of polypeptide having amino acid sequence
from No. 1 to 1055 of in the sequence listing, SEQ ID NO: 2, was
ligated to the expression vector pMKINeo to prepare the expression
vector. Peparation of fused protein with immunoglobulin Fc protein
was performed according to the method of Zettlmeissl et al.
(Zettlmeissl et al., DNA cell Biol., 9, 347-354, 1990). A gene
using genome DNA with intron was applied and the said gene was
prepared by using PCR.
[0142] Human genomic DNA was used as a template. Oligonucleotide of
the sequence in the sequence listing, SEQ ID NO: 31 with
restriction enzyme BamHI site, and oligonucleotide of the sequence
in the sequence listing, SEQ ID NO: 32 with restriction enzyme XbaI
site were used as primer. PCR of gene sequence encoding human IgG1F
was performed using the primers and human genomic DNA as template.
About 1.4 kbp band was purified, treated by restriction enzyme
BamHI and XbaI (Takara Shuzo Co., Japan), and genes were ligated to
pBluescript, which was similarly treated by restriction enzyme, by
using T4 DNA ligase to prepare subcloning. Later, the plasmid DNA
was purified and sequenced to confirm gene sequence, then the said
gene sequence was confirmed as genome DNA in the hinge region of
heavy chain of the human IgG1. (The sequence is referred to Kabat
et al., Sequence of Immunological Interest, NIH Publication No.
91-3242, 1991). Hereinafter this plasmid is designated as
pBShIgFc.
[0143] A vector pUCSR-2 having DNA sequence in SEQ ID NO: 4 was
used as template and oligonucleotide having sequence in SEQ ID NO:
26 and oligonucleotide having sequence in SEQ ID NO: 29 were used
as primer, and PCR was performed according to a method described
hereinbefore. The PCR product was ligated to cloning vector pCRII,
and the gene sequence of the PCR product was determined to prepare
DNA having gene sequence from No. 2986 to 3254 of DNA sequence in
SEQ ID NO: 4 in which restriction Bgl 2 site was attached in the 3'
end.
[0144] About a 250 bp gene fragment wasobtained by digesting the
pCRII vector, which contained the above PCR product as an insert,
with restriction enzymes EcoRI and BamHI containing a gene encoding
the human IgGlFC as an insert were ligated. In this case, BamHI and
Bgl 2 sites can be ligated due to complementary of the digested
terminal gene sequence. Further this part cannot be digested by
these restriction enzymes.
[0145] About a 1.5 kbp gene fragment was obtained by digesting this
vector with restriction enzyme BamHI and Not 1, about a 3 kbp gene
fragment was obtained by digesting pUCSR-2 with restriction enzymes
EcoRI and BamHI, and about a 4.3 kb gene fragment was obtained by
digesting pMKITneo with restriction enzyme EcoRI and Not 1, and
these 3 gene fragments were simultaneously ligated (though
restriction enzymes Xho I and Sal I have different recognition
sequence, they can be ligated due to complementary terminal gene
sequence). An expression vector containing gene fragment from DNA
sequence No. 1 to 3254 in SEQ ID NO: 4 and a gene fragment coding
human IgG1Fc, expression vector pMEXS2Fc of Ig chimera protein of
secretory extracellular humanSerrate-2 (hereinafter this protein is
designated as EXS2Fc), was obtained.
[0146] 4) Expression Vector of Full Length Human Serrate-2
Protein
[0147] The cDNA coding polypeptide from No. 1 to 1212 of amino acid
sequencein the sequence listing, SEQ ID NO: 3, was ligated to the
expression vector pMKITNeo to prepare the expression vector.
[0148] Namely, about 4 kbp gene fragment, which was cut out by
digesting pUCSR-2 with restriction enzymes EcoRI and Hind III, was
ligated into pBluescript, which was digested with the same
restriction enzymes. Subsequently, about 4 kbp gene fragment cut
from this vector by digesting with EcoRI and XhoI was ligated with
about 4.3 kb gene fragment obtained by digesting the expression
vector pMKITneo with restriction enzymes EcoRIand Not 1 to prepare
expression vector containing gene fragment of DNA sequence from No.
1 to 3955 in SEQ ID NO: 4. Full length human Serrate-2 protein
(hereinafter this protein is designated as FS2) expression vector
pMFS2 was obtained.
[0149] 5) Expression Vector of FLAG Chimera Protein of Full Length
Human Serrate-2
[0150] The cDNA coding chimera protein, to which cDNA coding FLAG
sequence (SEQ ID NO: 24) was added to the C-terminal of polypeptide
from No. 1 to 1212 of amino acid sequence in the sequence listing,
SEQ ID NO: 3, was ligated to the expression vector pMKITNeo to
prepare the expression vector.
[0151] Namely, vector pUCSR-2 having DNA sequence in SEQ ID NO: 4
was used as template and oligonucleotide having sequence in SEQ ID
NO: 26 and oligonucleotide having sequence in SEQ ID NO: 30 were
used as primer, and PCR was performed according to a method
described hereinbefore. The PCR product was ligated to cloning
vector pCRII, and the gene sequence of the PCR product was
determined to prepare DNA having gene sequence from No. 2986 to
3725 of DNA sequence in SEQ ID NO: 4, in which DNA sequence coding
FLAG sequence in the 31 end (DNA sequence in SEQ ID NO: 24),
termination codon and restriction enzyme Sal I site were attached
in the 3' end.
[0152] About a 3 kbp gene fragment was obtained by digesting the
pUCSR-2 with restriction enzymes EcoRI and BamHI, about a 700 bp
gene fragment was obtained by digesting the pCRII vector by
restriction enzymes BamHI and SalI, the vector of which contained
the above PCR product vector of which contained the above PCR
product as an insert, and about a 4.3 kb gene fragment was obtained
by digesting the pMKITneo with restriction enzymes EcoRI and XhoI,
and these 3 gene fragments were simultaneously ligated (though
recognition sequence of restriction enzymes XhoI and SalI is
different, they can be ligated due to complementary terminal gene
sequence) to obtain the expression vector containing gene fragment
of DNA sequence from No. 1 to 3725 in SEQ ID NO: 4 and gene
fragment coding FLAG sequence. Full length human Serrate-2 FLAG
chimera protein (hereinafter designated this protein as FS2FLAG)
expression vector pMFS2FLAG was obtained.
Example 5
[0153] Expression and Gene Transfer of the Human Serrate-2
Expression Vectors Into Cells
[0154] The expression vectors prepared in Example 4 were gene
transduced into COS-7 cells (obtained from RIKEN Cell Bank,
Physical and Chemical Research Institute, Japan, RCB0539).
[0155] Cell culture before transduction was performed by culturing
in D-MEM (Dulbecco modified Eagle's medium, GIBCO-BRL Inc., U.S.A.)
10% FCS. On the day before gene transduction, medium of cells was
changed to set cell counts 5.times.10 7 cells/ml and cultured
overnight. On the day of gene transduction, cells were sedimented
by centrifugation, centrifugally washed twice with PBS (-) and
prepared to 1.times.10 7 cells/ml in PBS (-), 1 mM MgCl 2 and gene
transfer was performed by electroporation using gene transduction
device Gene-pulsar (Bio-Rad Inc., U.S.A.). The above cell
suspension 500 .mu.l was collected in the cell for electropora-tion
(0.4 mm), expression vector 20 .mu.g was added, and allowed to
stand in ice for 5 minutes. Electroporation was performed under the
condition 3 .mu.F, 450V twice, and during the two electroporations
the cell mixture was allowedto stand at room temperature for 1
minute. After 5 minutes in ice, cells were spread in the culture
medium, diameter 10 cm previously added with 10 ml of the medium
described hereinbefore, and cultured at 37.degree. C. in 5% carbon
dioxide incubator.
[0156] The next day, the culture supernatant solution was removed,
the cells adhered to the dish were washed twice with PBS (-) 10 ml
and serum-free D-MEM 10 ml was added and cultured for 4 days. In
case of gene transduction into expression vectors pMEXS2,
pMEXS2FLAG and pMEXS2Fc, culture supernatant solution was recovered
and was replaced the buffer to PBS (-) by Centricon 30 (Amicon
Inc., U.S.A.) and simultaneously the solution was concentrated to
10-fold to obtain cell culture supernatant solution.
[0157] In case of gene transduction of pMSF2 and pMFS2FLAG, after 4
days culture, cells were washed with PBS (-) 10 ml. Cells were
scraped using cell scraper (Corster Corp.), PBS (-) 10 ml was added
again, centrifuged at 1500 rpm for 5 minutes and washed. Cell
precipitates were suspended in the cell lysis buffer [50 mM Hepes
(pH 7.5), 1% Triton X-100, 10% glycerol, 4 mM EDTA, 50 .mu.g/ml
Aprotinin, 100 .mu.M Leupeptin, 25 .mu.M Pepstatin A and 1 mM PMSF]
500 .mu.l, allowed to stand in ice for 20 minutes and centrifuged
at 15000 rpm for 20 minutes to collect supernatant solution to
obtain cell lysates.
[0158] Using these samples, expression of FLAG chimera and
immunoglobulin chimera proteins were detected by Western blotting.
Namely, concentrated cultured supernatants or cell lysates were
subjectd to SDS-PAGE using an electrophoresis tank and
polyacrylamide gel for SDS-PAGE (gradient gel 5-15%) (ACI Japan
Inc.) according to manufacturer's construction. Samples were
prepared by treatment in boiling water for 5 minutes with
2-mercapto-ethanol (2-ME) for reduction, and non-reduced condition
without taking the above treatment. As a marker, Rainbow Marker
(high molecular weight, Amersham Inc.) was used. Sample buffer
solution and electrophoresis buffer were prepared with reference to
the attached leaflet. When the SDS-PAGE was finished, acrylamide
gel was transcribed to PVDF membrane filter (BioRad Inc., U.S.A.)
using the Mini Trans Blot Cell (BioRad Inc.).
[0159] The thus prepared filter was shaken overnight at 4.degree.
C. in Blockace (Dainippon Pharm. Co., Japan), TBS-T [20 mM Tris,
137 mM NaCl (pH 7.6) and 0.1% Tween 20] to effect blocking.
According to the explanation of the attached leaflet of ELC Western
blotting detection system (Amersham Inc., U.S.A.); in case that
protein was FLAG chimera, anti-FLAG M2 mouse monoclonal antibody
(Eastman Kodak, U.S.A.) was used as primary antibody, and
peroxidase labeled anti-mouse Ig sheep antibodies (Amersham Inc.,
U.S.A.) as a secondary antibody, were reacted. In case of human
IgG1Fc chimera, peroxidase labeled anti-human Ig sheep antibodies
(Amersham Inc., U.S.A.) were reacted. Reaction time for antibodies
was 1 hour at room temperature, and at an interval of each
reaction, washing was performed by shaking in TBS-T at room
temperature for 10 minutes for three times. After the final
washing, the filter was immersed in the reaction solution of
ELC-Western blotting detection system (Amersham Inc., U.S.A.) for 1
minute, and wrapped in polyvinylidene chloride wrap for exposure to
X-ray film.
[0160] As a result, the bands showing protein obtained by
transduction of expression vector pMEXS2FLAG were detected from COS
supernatant about 135 kD by anti-FLAGM2 antibody; and production of
objective protein EXS2FLAG was confirmed, and transduced cells by
expression vector pMEXS2FLAG. Molecular weight changes depending on
reduction treatment at the SDS-PAGE were not observed. About 20 kD
of sugar chains was added to the molecules as a result of comparing
with a molecular weight estimated by amino acid sequence.
[0161] Furthermore, a band having molecular weight about 165 kD was
detected from supernatant solution of COS cells, to which the
expression vector pMEXS2Fc was gene transduced, on SDS-PAGE by
anti-human Ig sheep antibody under reducing conditions. A band
having molecular weight about 330 kD was detected under
non-reducing conditions. These results indicated that objective
protein EXS2Fc was produced, and consequently transformed cells by
the expression vector pMEXS2Fc could be obtained. As the molecular
weight of EXS2Fc under reducing conditions is about half of that
under non-reducing conditions, the EXS2Fc is estimated to have a
construction of dimer through disulfide bond. Furthermore, the
molecular weight of the band is about 40 kD larger than that
calculated from amino acid sequence. This indicates addition of
sugar chain to the molecule.
[0162] Furthermore, a band having molecular weight about 150 kD was
detected from extract of COS cells, to which the expression vector
pMFS2FLAG was gene transduced, on SDS-PAGE by anti-FLAG M2 antibody
under reducing conditions. The results indicated that the objective
protein FS2FLAG was produced, and consequently cells transformed by
expression vector pMFS2FLAG, were obtained. As the molecular weight
of FS2FLAG of the band is about 20 kD larger than that calculated
by amino acid sequence, sugar chains may be added to the
extracellular region.
[0163] As for a protein other than chimera protein, detection was
conducted by using anti-human Serrate-2 mouse monoclonal antibody
and anti-human Serrate-2 rabbit polyclonal antibody, which were
described in Example 7, as primary antibodies in the Western
blotting. Also as secondary antibody, anti-mouse Ig sheep antibody
(Amersham Inc.) or peroxidase labeled rabbit Ig sheep antibody
(Amersham Inc.) were used.
[0164] As a result, a band having molecular weight about 135 kD was
detected in the supernatant of COS cells, to which the expression
vector pMEXS2 wasgene transduced. This indicated that objective
protein EXS2 was produced, and cells transformed by an expression
vector pMEXS2 could be obtained. No changes of molecular weight
were observed caused by reduction treatment on SDS-PAGE. A band
having molecular weight about 150 kD was detected under reducing
conditions from COS cell extract, to which the expression vector
pMFS2 was gene transduced. These results indicated that objective
protein FS2 was produced, and consequently cells transformed by the
expression vector pMFS2 could be obtained. Furthermore, in every
case, molecular weight of the band is about 20 kD larger than that
calculated from amino acid sequence. This indicates addition of
sugar chains to the molecule of the extracellualr region.
[0165] In the control experiments, cell lysate and cultured
supernatant solution of COS-7 cells, to which pMKITNeo vector was
transformed, were tested. No bands reacted with anti-FLAG antibody,
anti-human Ig antibody or anti-human Serrate-2 antibody could be
detected.
Example 6
[0166] Purification of Secretory Extracellular Human Serrate-2
Chimera Proteins of Gene Transduction Cells
[0167] Cultured supernatant of COS-7 cells transformed by the
expression vector pMEXS2FLAG or pMEXS2Fc by a method in Example 5,
were prepared in large scale, and chimera protein, i.e. EXS2FLAG or
EXS2Fc, was purified by affinity column chromatography.
[0168] In case of EXS2FLAG, 2 liters of the cultured supernatant
obtained by the method in Example 5 was passed through a column
packed with Anti-FLAG M2 Affinity Gel (Eastman Kodak, U.S.A.). The
chimera protein was absorbed in a column by a reaction of affinity
of anti-FLAG antibody of the gel and FLAG sequence of the chimera
protein. An inner diameter 10 mm, disposable column (BioRad Inc.,
U.S.A.) was used with packing the above gel 5 ml. A circulation
system consisting of medium bottle.fwdarw.column.fwdarw.peristaltic
pump.fwdarw.medium bottle was set up. The circulation was run by a
flow 1 ml/min. for 72 hours. Thereafter the column was washed with
PBS (-) 35 ml and eluted by 0.5 M Tris-glycine (pH 3.0) 50 ml. The
eluate of 25 fractions, each 2 ml, was collected into the tube
(Farcon Inc.2063),and each fraction was neutralized by 200 .mu.l of
0.5 M Tris-HCl (pH 9.5) previously added in each tube.
[0169] The eluate fraction, each 10 .mu.l of the EXS2FLAG which was
purified by the above method was subjected to reduction treatment
described in Example 5. SDS-PAGE electrophoresis by 5-10% gradient
polyacrylamide gel was performed. After finishing the
electrophoresis, silver staining was conducted by using Wako silver
stain kit II according to the explanation of the attached leaflet.
Fractions from No. 4 to 8 showed detectable bands in EXS2FLAG. The
size is identical with the result of Western blotting of anti-FLAG
antibody obtained in Example 5. Therefore, purified EXS2FLAG was
obtained.
[0170] In the EXS2Fc, two liters of the cultured supernatant
solution was absorbed in Protein A Sepharose column (Pharmacia
Inc., Sweden) according to the same method as above to collect the
eluate fractions. Using a part of eluate as same as in EXS2FLAG, a
determination of the eluate fraction, identification of the size
and detection of the purity were performed by SDS-PAGE
electrophoresis and silver staining in reducing conditions.
Therefore, the eluate fractions from No. 4 to 15 were detected as
bands. The molecular weight thereof is identical with the result of
Example 5. Therefore, purified EXS2Fc was obtained.
Example 7
[0171] Preparation of Antibodies Recognizing Human Serrate-2
[0172] EXS2FLAG, purified by the method in Example 6, was used as
immunogen, and rabbits were immunized. After assaying antibody
titer, whole blood was collected and serum was obtained. Anti-human
Serrate-2 rabbit polyclonal antibody were purified by using
Econopack serum IgG purification kit (BioRad Inc., U.S.A.) with
reference to the attached explanation leaflet.
[0173] EXS2FLAG purified by a method described in Example 6 was
used as immunogens, and mouse monoclonal antibodies were prepared
according to the explanation of the textbook. The purified HSFLAG
was administered in Balb/c mice (Nippon SLC Co., Japan), 10
.mu.g/mouse, immunized intracutane-ously and subcutaneously. After
second immunization, increased serum titer was confirmed by
collecting blood ophthalmologically, and the third immunization was
performed. Subsequently, the spleen of mice was collected and fused
with mouse myeloma cells P3X63Ag8 (ATCC TIB9) using
polyethylene-glycol. Hybridoma was selected by HAT medium
(Immunological and BiologicalResearch Institute, Japan), and the
hybridoma strains, which produced antibody specifically recognizing
extracellular region of human Serrate in the medium, were isolated
by enzyme immunoassay. The hybridoma strains producing mouse
monoclonal antibody, which specifically recognized human Serrate-2,
were established.
[0174] Anti-human Serrate-2 monoclonal antibody was purified and
prepared by using Mab Traq GII (Pharmacia Inc., Sweden) and
according to the explanation of the leaflet, from the supernatant
of the thus established hybridoma.
[0175] Affinity column chromatography was performed by using the
monoclonal antibody. Preparation of the affinity column was
performed according to the explanation attached to the CNBr
activated Sephadex 4B (Pharmacia Inc., Sweden). Coupling efficiency
was 99.6%. A column, 2 cm.times.1 cm, containing gel 2 ml, was
prepared.
[0176] A supernatant of the cultured cells, which contained EXS2,
was passed through the column. The supernatant solution was passed
at 20 ml/hr, subsequently PBS (-) 15 ml was passed at the same flow
rate and washed the column. Finally, the products were eluted by a
mixture of 0.1 M sodium acetate and 0.5 M NaCl (pH 4.0). The
eluate, each 1 ml fraction was collected, and was neutralized by
adding 1 M Tris-HCl (pH 9.1) 200 .mu.l for each fraction.
[0177] SDS-PAGE of purified protein was conducted under reducing
conditions according to the method described in Example 5, followed
by silver staining and Western blotting to estimate molecular
weight. A band of about 140 kD was detected. Consequently, Western
blotting can be made by using the said monoclonal antibodies and
human Serrate-2 can be purified by the affinity columns.
Example 8
[0178] Effects of Human Serrate-2 Protein on Colony Formation of
Blood Undifferentiated Cells
[0179] In order to observe physiological action of human Serrate-2
on blood undifferentiated cells, CD34 positive cells were cultured
in a serum-free semi solid medium in the presence of EXS2Fc and
known cytokines, and the number of colony forming cells were
observed.
[0180] CD34 positive cells of human umbilical cord blood or human
normal bone marrow blood were isolated from the mononuclear cells,
which were treated by silica solution (Immunological and Biological
Research Institute, Japan) according to the attached explanation
leaflet and fractionated from the low density cellular fraction
(<1.077 g/ml) by densitometric centrifugation of Ficoll pack
(Pharmacia Inc., Sweden).
[0181] Separation of CD34 positive cells was performed by using
Dynabeads M-45 CD34 or DETACH a BEADS CD34 (Dynal Inc., Norway) and
according to the attached explanation leaflets. After separation,
the purity was measured as follows. Cells were stained by FITC
labeled CD34 antibody HPCA2 (Beckton-Deckinson Inc., U.S.A.) and
examined by flow-cytometer (FACS Calibur, Beckton-Deckinson.,
U.S.A.). Purity above 85% was confirmed for use.
[0182] The thus isolated CD34 positive cells were suspended
homogeneously to form 400 cells/ml of the medium hereinbelow, and
spread in a 35 mm dish (Falcon Inc., U.S.A.), then cultured for 2
weeks in a carbon dioxide incubator at 37.degree. C. under 5%
carbon dioxide, 5% oxygen, 90% nitrogen and 100% humidity. The
formed blood colonies were counted under an invert microscope.
[0183] A medium used is .alpha.-medium (GIBCO-BRL Inc., U.S.A.),
containing 2% deionized bovine serum albumin (BSA, Sigma, U.S.A.),
10 .mu.g/ml human insulin (Sigma, U.S.A.), 200 .mu.g/ml
transferring (Sigma, U.S.A.), 10-5 M 2-mercaptoethanol (Nakarai
Tesk Co., Japan), 160 .mu.g/ml soybean lecithin (Sigma, U.S.A.), 96
.mu.g/ml cholesterol (Sigma, U.S.A.) and 0.9% methylcellulose (Wako
Pure Chemicals, Japan).
[0184] To the above medium, under the following conditions of
cytokines, human Serrate-2 extracellular Ig chimera protein
(EXS2Fc) was added to the final concentration of 1 .mu.g/ml. For
control, human IgG1 (Athens Research and Technology Inc., U.S.A.)
was added with the same concentration in order to observe the
effect of IgGFc region.
[0185] Conditions of cytokines are as follows.
[0186] 100 ng/ml, human SCF, 10 ng/ml human IL-3, 100 ng/ml human
IL-6, 2U/ml Epo (Chugai Seiyaku Co., Japan) and 10 ng/ml human
G-CSF (Chugai Seiyaku Co., Japan).
[0187] Results are shown in Table 1. Number of colonies/400 CD34+
cells are shown in mean of n=3. Four different origin human
umbilical cord blood CD34 positive cells were used.
1 TABLE 1 EXS2Fc not added EXS2Fc added Experiment 1 30.1 12.8
Experiment 2 48.3. 40.7 Experiment 3 38.2. 28.1 Experiment 4 50.9.
37.1
[0188] As shown in Table 1, the human Serrate-2 has an action
against four different origin umbilical cord blood CD34 positive
cells. Therefore, human Serrate-2 of the present invention has
suppressive action for differentiation of blood undifferentiated
cells including blood cells.
Example 9
[0189] Effect of Human Serrate-2 on Blood Undifferentiated Cell
LTC-IC in Liquid Culture
[0190] In order to observe physiological action of human Serrate-2
on the blood undifferentiated cells, umbilical cord blood positive
cells were cultured for two weeks in the serum-free liquid medium
in the presence of EXS2Fc and known cytokines, and the numbers of
LTC-IC, which was thought to represent most undifferentiated cells
at present, were observed.
[0191] The umbilical cord blood mononuclear CD34 positive cells,
100000 to 20000 cells, separated by a method described in Example 8
were cultured in the following medium for 2 weeks. Numbers of
LTC-IC in 3 experimental groups, which include a group before
cultivation, a group of EXS2Fc and a control group, were
determined.
[0192] Media used in liquid culture medium were .alpha.-medium with
2% BSA added thereto, 10 .mu.g/ml human insulin, 200 .mu.g/ml
transferrin, 40 .mu.g/ml low density lipoprotein, 10-5 M
2-mercaptoethanol, further containing 100 ng/ml human SCF, 10 ng/ml
human IL-3, and 100 ng/ml IL-6. EXS2Fc 1 .mu.g/ml was added 1 the
above medium. In the control group, human IgG1 was added in the
equal concentration.
[0193] Preparation of human bone marrow stromal cell layer used for
LTC-IC, and quantitative assay of frequency of LTC-IC by a limit
dilution were performed according to a method of Sutherland et al.
(Blood, 74, 1563-, 1989 and Proc. Natl. Acad. Sci. USA, 87, 3584-,
1990).
[0194] The bone marrow mononuclear cells, 1-2.times.10 7 cells,
obtained in Example 8 before the separation and without the liquid
silica treatment, were cultured in LTC medium (MyceloCult, Stem
Cell Technologies Inc., Canada) 5 ml containing hydrocortisone 1
.mu.M (Upjohn Japan Co., Japan) in T-25 flask (Falcon Inc., U.S.A.)
at 37.degree. C. under 5% carbon dioxide and 100% humidity in the
carbon dioxide incubator. Culture was conducted until the adhesive
cell layers of the stromal cell formation spread more than 80% of
the bottom area of the culture flask. Detachment of the cell layer
was performed by treating with EDTA solution (Cosmobio Co., Japan).
Cells were plated in the 96 well plate (Beckton-Deckinson Inc.,
U.S.A.), about 2.times.10 4 cells/well and recultivation was
continued in the same medium. X-ray, 15 Gy, 250 KV peak was
irradiated to the reconstituted stromal cell layer. Growth of
stromal cells was stopped and blood cells in the stromal cells were
removed. The thus prepared stromal cells were used as stromal cell
layer for the experiments.
[0195] In the assay of LTC-IC, cell counts in each group were
adjusted within the ranges of 25-400 cells/well for CD34 positive
cells before the cultivation, and 625-20000 cells/well for the
cells after the cultivation, and cells were diluted for a six
step-dilution within these ranges. Each dilution step of cells was
cocultured with the above stromal cell layer in the 96 well plate,
for 16 wells of one dilution step. Culture was performed in the
same medium as used in stromal formation, at 37.degree. C., 5%
carbon dioxide and 100% humidity in the carbon dioxide gas
incubator for 5 weeks. Cells in suspension and in attachment after
cultivation were recovered in each well. Collected cells were
transferred to the semi-solid culture medium consisting of
.alpha.-medium containing 0.9% methylcellulose, 30% fetal calf
serum (FCS, ICN Biochemical Japan), 1% BSA, 10-5 M
2-mercaptoethanol, 100 ng/ml human SCF, 10 ng/ml human IL-3, 100
ng/ml human IL-6, 2 U/ml Epo and 10 ng/ml human G-CSF. After 2
weeks of cultivation, colony forming cells were detected in the
same way as described in Example 8, and numbers of wells in which
colony forming cells were found, were detected. Incidence of LTC-IC
was calculated according to the method of Taswell et al. (J.
Immunol. 126, 1614-, 1981) based on the above data. Results are
shown in Table 2.
2TABLE 2 Before cultivation EXS2Fc not added EXS2Fc added Total
number of cells 19475 1210000 930000 Number of LTC-IC 185 23 5
[0196] From the results shown in Table 2, human Serrate-2 has an
action against LTC-IC and reduces its number.
Example 10
[0197] Effect of Human Serrate-2 on Growth of Vascular Endothelial
Cells
[0198] The vascular endothelial cells used were passage cultures of
four generations of normal human aortic endothelial cells and
normal human pulmonary arterial endothelial cells (Kurabo Inc.,
Japan). Cells were plated 500 cells/well in 96 well plate for
tissue culture (Falcon Inc., U.S.A.) in the tertiary passage
culture, and cultured in a medium with low serum level for growth
of vascular endothelial cells (HuMedia-EG2, Kurabo Inc., Japan)
containing human recombinant EGF (KuraboInc., Japan) 100 ng/ml and
human recombinant EGF-B 5 ng/ml. Human Serrate-2 extracellular
chimera protein (EXS2Fc) was added to the final concentration of 1
.mu.g/ml. For control, human IgG1 (Athens Research and Technology
Inc., U.S.A.) was added with the same concentration in order to
observe effect of IgGFc region. A control experiment was conducted
without adding protein except for HuMedia-EG2. Culture was
performed at 37.degree. C., under 5% carbon dioxide and 100%
humidity for 3 days and the number of cells was calculated.
[0199] Vascular endothelial cell counts were performed by using NR
reagent set (Kurabo Inc., Japan). The method was developed by
Borenfeund and Puerner (Journal of Tissue Culture Methods, 9(1),
7-9, 1984), i.e. the neutral red method which applied that vital
stain pigment neutral red (3-amino-7-dimetylamino-2-methylphenazine
hydrochloride) passed through plasma membrane of living cells and
was accumulated in lysosome.
[0200] Absorption at 540 nm was measured by using immuno reader
(NJ-2000, Japan Intermed Inc., Japan).
[0201] Results showed that in case of aortic endothelial cells,
absorption in the control group was at an optical density (OD)
0.21.+-.0.02, which is almost the same level of human IgG1 added
group 0.20.+-.0.01, and in EXS2Fc containing group, it was
0.10.+-.0.02 which was significantly smaller than the control.
[0202] In case of pulmonary arterial endothelial cells, the control
group showed 0.15.+-.0.01 and the human IgG1 containing group
showed almost same level 0.16.+-.0.02, whereas EXS2Fc added group
shows significantly low level of 0.07.+-.0.02. This result
indicated that EXS2Fc suppresses growth of vascular endothelial
cells.
[0203] Human Serrate-2 of the present invention therefore has an
action for regulating differentiation of undifferentiated cells,
and can be used as a novel regulating agent for differentiation of
cells.
Sequence CWU 0
0
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