Patent Grant
7138276
1. Field of the Invention
This invention relates to a novel bioactive substance which suppresses differentiation of undifferentiated cells.
2. Description of Related Art
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.
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.
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).
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 growthfactor (TGF-β) has suppressive action for proliferation against various cells, but has no fixed actions against the hematopoietic stem cells or blood precursor cells.
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.).
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).
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).
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.
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.
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.
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).
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.
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.
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.
A principal object of the present invention is to provide a compound originated from novel factors which can suppress differentiation of undifferentiated cells.
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.
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).
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.
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.).
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.
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.
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.
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.
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.
The present invention is explained in detail in the following.
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 conventional 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.
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.
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′-untranslatedregion 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.
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.
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.
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.
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.
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 are 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.
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).
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).
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.
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.
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.
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.
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.
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.
We have prepared probes from about 50 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.
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 [3 2 P] γ-ATP and T4 polynucleotide kinase, or other labelling methods such as nick translation or primer extension method can be applied.
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.
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.
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.
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, λgt10 and λgt11, 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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 Bio-science 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.
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.
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 λ-phage and animal virus such as retrovirus and vaccinia virus.
Examples of promoters used in the present invention are any promoters suitable for corresponding to the host cells used in gene expression.
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.
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.
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.
As shown in Example 4, we have prepared expression vectors, which express extracellular proteins, as follow.
1) DNA encoding the amino acids from No. 1 to 1055 in amino acid sequence in the sequence listing, SEQ ID NO: 2.
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
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.
The expression vectors, which expresses full-length protein, can be prepared as follows.
4) DNA encoding amino acids from No. 1 to 1212 in the sequence listing, SEQ ID NO: 3 and
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 pMKITNeo 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.
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.
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.
The human Serrate-2 polypeptide can be isolated and purified from the above cultured mass, in general, by the following methods.
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.
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.
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.
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.
For that action, mainly an action suppressing cell differentiation will be expected, and actions such as stimulating tissue regeneration, etc. can be expected.
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.
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.
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.
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.
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 μg/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.
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.
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 μg/kg to 10 mg/kg.
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.
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.
As another method, multimerization method using chemical cross-linker can be mentioned. For example, dimethylsuberimidate dihydrochloride for cross-linking lysine residue, N-(γ-maleimidebutyryloxy) 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.
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.
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.
The following examples illustrate embodiments of the present invention, but are not to be construed as limiting.
Preparation of Human Serrate-1 Gene Probe
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.
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° 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/μl after purification, and used for PCR. Synthesis of oligonucleotide was performed by the same manner.
Amplification by PCR was performed as follows.
Human fetal brain originated cDNA mixture solution (QUICK-Clone cDNA, CLONTECH Inc., U.S.A.) 1 μl was used. 10× buffer solution [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2, 0.01% gelatin] 5 μl, dNTP Mixture (Takara Shuzo) 4 μl, sense primer DLTS1 (100 pmol/μl) 5 μl and antisense primer DLTA2 (100 pmol/μl) 5 μl which were specific to the above Serrate homologue and TaqDNA polymerase (AmpliTaq, Takara Shuzo., Japan, 5 U/μl) 0.2 μl were added thereto, and finally deionized water was added to set up total 50 μl. PCR was performed for 5 cycles of a cycle consisting of treatment at 95° C. for 45 seconds, at 42° C. for 45 seconds and 72° C. for 2 minutes, further 35 cycles of a cycle consisting of treatment at 95° C. for 45 seconds, at 50° C. for 45 seconds, and 72° C. for 2 minutes, and finally allowed to stand at 72° 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° 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.
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 μg/ml and allowed to stand at 37° 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° 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.
Cloning of Full Length Human Serrate-1 Gene
A screening of clones having full length cDNA was performed by hybridization from human placenta origin cDNA library (inserted cDNA in λgt-11, CLONTEC Inc., U.S.A.) in plaques corresponding to 1×106 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 3 2 P using the filter.
The DNA probe previously prepared was labeled with 3 2 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 μl and deionized water were added to DNA 25 ng to set up total volume of 33 μl, which was treated for 5 minutes in boiling water bath. Reaction buffer solution 10 μl containingd NTP, α-3 2 P-dCTP 5 μl and T4 DNA polynucleotide kinase solution 2 μl were added thereto, and treated at 37° 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.
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 μg/ml denatured by boiling (Sigma Co.) 10 μg/ml denatured by boiling water, shaken at 65° 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 3 2 P by the above mentioned method, and shaken at 55° C. for 16 hours to perform hybridization.
The filter was washed by immersing into SSPE solution containing 0.1% SDS, shaken at 55° C. twice, and further immersing into 10-fold dilution of SSPE solution containing 0.1% SDS four times at 55° 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.
The thus isolated phage clones were 22 clones. Phages of all of these clones was prepared to about 1×10 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.
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.
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.
Preparation of Probe by PCR
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, TO8853, R50026 and R45751. Hereinafter, a probe having gene sequence SEQ ID NO: 10 is designated as ¥1, a probe having gene sequence SEQ ID NO: 11 is designated as ¥2, and a probe having gene sequence SEQ ID NO: 12 is designated as ¥4.
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.
Amplification by PCR was performed as follows. Human fetal brain originated cDNA mixed solution (QUICK-Clone cDNA, CLONTECH Inc., U.S.A.) 1 μl was used. 10× buffer solution [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl 2, 0.01% gelatin] 5 μl, dNTP mixture (Takara Shuzo Co., Japan) 4 μl, the primers hereinbefore (20 pmol/μl) each 1 μl, and TaqDNA polymerase (AmpliTaq, Takara Shuzo Co., 5 U/μl) 0.2 μl were added thereto, and finally deionized water was added to set up total 50 μl. PCR was performed for 40 cycles of a cycle consisting of treatment at 95° C. for 1 minute, at 55° C. for 5 minutes and 72° C. for 3 minutes, and finally allowed to stand at 72° 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.
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° 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.
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 semi-solid medium plate of L-Broth (Takara Shuzo Co., Japan) containing ampicillin (Sigma Corp., U.S.A.) 50 μg/ml and allowed to stand at 37° 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° 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.
Cloning of Full Length Human Serrate-2 Gene
A screening of clones having full length cDNA was performed by hybridization from human fetal brain origin cDNA library (inserted cDNA in λgt-11, CLONTECH Inc.) in plaques corresponding 1×10 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 3 2 P by the method described in Referential example 2, screening was performed on each individually to obtain clones.
Isolated phage clones were: 2 clones from a case using ¥1 as a probe; 6 clones from a case using ¥2 as a probe; and 4 clones from a case using ¥4 as a probe. Clones isolated by ¥4 were all included in clones isolated from ¥2. All of the phages of these clones were prepared to about 1×10 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.
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.
The thus isolated clones in the second screening were six clones. Phages of all these clones were prepared about 1×10 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.
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.
As a result, DNA sequence in SEQ ID NO: 4, i.e. cDNA sequence encoding full length of human Serrate-2, was determined.
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 PCR was 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.
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.
Expression of Human Serrate-2 in Organs
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.
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.
Preparation of Expression Vector of Human Serrate-2
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.
1) Expression Vector of Secretory Extracellular Human Serrate-2
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 α promoter and Neomycin resistant gene, to prepare expression vector.
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.
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.
2) Expression Vector of FLAG Chimera Protein of Secretory Extracellular Human Serrate-2
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.
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.
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 enzymes Xho 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.
3) Expression Vector of IgG1Fc Chimera Protein of Secretoruy Extracellular Human Serrate-2
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.
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.
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.
About a 250 bp gene fragment was obtained 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 IgG1FC 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.
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.
4) Expression Vector of Full Length Human Serrate-2 Protein
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.
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 EcoRI and 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.
5) Expression Vector of FLAG Chimera Protein of Full Length Human Serrate-2
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.
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 3′ end (DNA sequence in SEQ ID NO: 24), termination codon and restriction enzyme Sal I site were attached in the 3′ end.
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.
Expression and Gene Transfer of the Human Serrate-2 Expression Vectors into Cells
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).
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 5×10 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 1×10 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 μl was collected in the cell for electroporation (0.4 mm), expression vector 20 μg was added, and allowed to stand in ice for 5 minutes. Electroporation was performed under the condition 3 μF, 450V twice, and during the two electroporations the cell mixture was allowed to 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° C. in 5% carbon dioxide incubator.
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.
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 μg/ml Aprotinin, 100 μM Leupeptin, 25 μM Pepstatin A and 1 mM PMSF] 500 μl, allowed to stand in ice for 20 minutes and centrifuged at 15000 rpm for 20 minutes to collect supernatant solution to obtain cell lysates.
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.).
The thus prepared filter was shaken overnight at 4° 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.
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.
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.
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.
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.
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.
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.
Purification of Secretory Extracellular Human Serrate-2 Chimera Proteins of Gene Transduction Cells
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.
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→column→peristaltic pump→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 μl of 0.5 M Tris-HCl (pH 9.5) previously added in each tube.
The eluate fraction, each 10 μl 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.
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.
Preparation of Antibodies Recognizing Human Serrate-2
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.
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 μg/mouse, immunized intracutaneously 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 polyethyleneglycol. Hybridoma was selected by HAT medium (Immunological and Biological Research 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.
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.
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×1 cm, containing gel 2 ml, was prepared.
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 μl for each fraction.
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.
Effects of Human Serrate-2 Protein on Colony Formation of Blood Undifferentiated Cells
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.
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).
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.
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° C. under 5% carbon dioxide, 5% oxygen, 90% nitrogen and 100% humidity. The formed blood colonies were counted under an invert microscope.
A medium used is α-medium (GIBCO-BRL Inc., U.S.A.), containing 2% deionized bovine serum albumin (BSA, Sigma, U.S.A.), 10 μg/ml human insulin (Sigma, U.S.A.), 200 μg/ml transferring (Sigma, U.S.A.), 10–5 M 2-mercaptoethanol (Nakarai Tesk Co., Japan), 160 μg/ml soybean lecithin (Sigma, U.S.A.), 96 μg/ml cholesterol (Sigma, U.S.A.) and 0.9% methylcellulose (Wako Pure Chemicals, Japan).
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 μg/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.
Conditions of cytokines are as follows. 100 ng/ml, human SCF, 10 ng/ml human IL-3, 100 ng/ml human IL-6, 2 U/ml Epo (Chugai Seiyaku Co., Japan) and 10 ng/ml human G-CSF (Chugai Seiyaku Co., Japan).
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.
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.
Effect of Human Serrate-2 on Blood Undifferentiated Cell LTC-IC in Liquid Culture
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.
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.
Media used in liquid culture medium were α-medium with 2% BSA added thereto, 10 μg/ml human insulin, 200 μg/ml transferrin, 40 μg/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 μg/ml was added 1 the above medium. In the control group, human IgG1 was added in the equal concentration.
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).
The bone marrow mononuclear cells, 1–2×10 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 μM (Upjohn Japan Co., Japan) in T-25 flask (Falcon Inc., U.S.A.) at 37° 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 2×10 4 cells/well and re-cultivation 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.
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° 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 α-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.
From the results shown in Table 2, human Serrate-2 has an action against LTC-IC and reduces its number.
Effect of Human Serrate-2 on Growth of Vascular Endothelial Cells
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 μg/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° C., under 5% carbon dioxide and 100% humidity for 3 days and the number of cells was calculated.
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.
Absorption at 540 nm was measured by using immuno reader (NJ-2000, Japan Intermed Inc., Japan).
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.
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.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 8-186220 | Jul 1996 | JP | national |
| 9-124063 | May 1997 | JP | national |
The present application is a divisional application of U.S. application Ser. No. 09/855,722, filed on May 16, 2001, now U.S. Pat. No. 6,638,741, which is a divisional application of U.S. application Ser. No. 09/214,278, filed on Jan. 26, 1999, now U.S. Pat. No. 6,291,210, which is a 371 of PCT/JP97/02414, filed Jul. 11, 1997.
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| 4603106 | Cerami et al. | Jul 1986 | A |
| 6004924 | Ish-Horowicz et al. | Dec 1999 | A |
| Number | Date | Country |
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| WO 9219734 | Nov 1992 | WO |
| WO 9602645 | Feb 1996 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20030022368 A1 | Jan 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09855722 | May 2001 | US |
| Child | 10219248 | US | |
| Parent | 09214278 | US | |
| Child | 09855722 | US |
