JP3454275B2 - Novel polypeptide associated with programmed cell death and DNA encoding the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polypeptide (PD-1) associated with programmed cell death and a DNA encoding the polypeptide.

[0002]

BACKGROUND OF THE INVENTION Developmentally and physiologically controlled cell death can be observed in almost any tissue of various animals. The death of such cells is commonly referred to as "programmed cell death" and is distinguished from "accidental cell death" caused by pathological mechanisms. Although it is not possible to uniformly define different types of programmed cell death in animals, most death programmed cells are depleted of new RNA or protein de novo to die. It has been shown that synthesis is required.

For example, postmortem moth (Manduca sexta) intersegmental muscle death, cell death due to tadpole tail metamorphosis, and fetal chick neuronal death are inhibition of protein synthesis such as actinomycin D and cyclohexaimide. Blocked by the agent. In addition, death of rat nerve cells induced by removal of nerve growth factor (NGF) or death of mouse thymocytes induced by glucocorticoid or endogenous superantigen is inhibited by actinomycin D or cyclohexaimide. There are also in vitro experiments showing that this is done.

All these facts indicate that some gene (possibly not a gene specific to cell death) must be expressed in order to cause programmed cell death. On the other hand, the term "apoptosis" is preferably used to describe the morphological characteristics of a class of cell death. In apoptotic cells, chromosomes aggregate around the nucleus, but mitochondria and other organelles do not change significantly.

Biochemically, in cells dying by apoptosis, the chromosome is irregularly divided into several nucleosome-unit DNA fragments. In mammals, most cells programmed for death appear to exhibit morphological and biochemical states of apoptosis, although some cells clearly do not. Furthermore, cell death due to apoptosis may be caused even without any protein synthesis. Thus, it would be important to understand that apoptosis is not a synonym for programmed cell death. Recently, it was revealed that bcl-2, which is one of the oncogenes, immortalizes B cells by preventing cell death, demonstrating the importance of cell death control.

[0006]

2. Description of the Related Art Up to now, several kinds of polypeptides related to programmed cell death have been reported. Fas antigen is known as a representative [Itoh, N. et a.
L., Cell, 66 , 233 (1991)]. Human Fas antigen is 33
A polypeptide consisting of 5 amino acids, with 1 at the N-terminus
A structure that has a signal peptide consisting of 6 hydrophobic amino acids, and the mature protein is divided into an extracellular region (157 amino acids), a transmembrane region (17 amino acids) and a cytoplasmic region (145 amino acids) from the N-terminal side. Is estimated to have. The Fas antigen is considered to function as a receptor for a factor (ligand) that induces cell death.

[0007]

OBJECT OF THE INVENTION The object of the present invention is to find a novel polypeptide, which is deeply involved in programmed cell death in mammals, and a DNA encoding the same, which is completely different from the known polypeptide represented by Fas antigen. And

In the present invention, a gene deeply involved in programmed cell death was isolated, its nucleotide sequence was determined, and its amino acid sequence was deduced. As a result, they succeeded in finding a completely novel polypeptide and DNA encoding the same, and completed the present invention.

In order to isolate genes that are deeply involved in programmed cell death, the inventors of the present invention have studied in vitro (in v
We selected four mouse cell lines as representative mammalian cells that readily undergo apoptotic death in the itro system. Similar to immature T lymphocytes, mouse T cell hybridoma, 2B4.11 dies when stimulated with the antigen it recognizes (I-E ^k + Hat cytochrome C) or when combined with ionomycin and PMA. . The mouse immature B cell line, WEHI-231, has its surface IgM
Is cross-linked by anti-IgM antibody, which induces death. Mouse lymphocytic / myeloid progenitor cell line, L
yD9 and a mouse cytotoxic T cell line, CTLL-2, are interleukin-3 (I) for survival and proliferation, respectively.
L-3) and interleukin-2 (IL-2) are required. These cell lines are then transformed from the culture medium into growth factors (I
Dies except L-3 or IL-2).

The present inventors first investigated whether new RNA synthesis was required for cell death of each cell line. It was confirmed that new RNA synthesis was required for death of 2B4.11 and LyD9, but not for death of WEHI-231 and CTLL-2. The pattern of death of 2B4.11 and LyD9 was found to have an interesting commonality, namely apoptotic death and the need for de novo RNA synthesis, and therefore a common biochemistry for these cell deaths. Using the subtractive hybridization technique, we hypothesized that there must be an alternative pathway and stimulated 2B4.11 cells and IL-3.
An attempt was made to identify a gene that is newly expressed in both LyD9 cells cultured in deficient medium.

Since the two cells are completely different not only in the method of inducing cell death but also in the nature of the cell line, the stimulated 2B4.11 cells and the Ly-3 deficient culture medium were cultured.
Genes activated in both D9 cells are thought to be deeply involved in the only phenomenon common to the two treated cell lines, programmed cell death.

The amino acid sequence of the polypeptide associated with programmed cell death identified in the present invention was compared with the amino acid sequences of known polypeptides registered in the database of National Biomedical Research Foundation. None had the same amino acid sequence. Of course, it was also confirmed that the polypeptide has no homology with Fas. Therefore, it was decided to name the novel polypeptide that is deeply involved in programmed cell death as PD-1, and to distinguish it from known polypeptides.

[0013]

The present invention relates to a polypeptide (hereinafter referred to as PD) which is deeply involved in programmed cell death in mammals.
Call it -1. ) Concerning. PD-1 contains a series of polypeptides (common to different mammals) that differ in their structural features from Fas. That is, it has a high homology with mouse PD-1, including mouse PD-1 whose amino acid sequence has been elucidated by the present invention (immunological equivalence such as cross-reaction with mouse PD-1 antibody). Other mammalian PD-1 (meaning sex), especially human PD-1 are included.

Further, the PD-1 of the present invention contains natural P
In addition to D-1, modified PD-1 is included. That is, a part of the amino acid sequence of natural PD-1 is deleted (for example, a polypeptide consisting of a mature protein portion lacking a signal peptide, or a mature protein consisting of only an essential portion for activity expression). Polypeptide), a part of which is replaced with another amino acid (for example, a part of which is substituted with an amino acid having similar physical properties), and a part of which another amino acid is added or inserted.

Among the PD-1 of the present invention, the structural characteristics of mouse PD-1 are as follows. Mouse PD-1 is 28
It is considered to be a membrane-bound protein consisting of 8 amino acids. It has two hydrophobic regions in the N-terminal part and the middle part, which are considered to correspond to the signal peptide and the cell transmembrane region, respectively. The N-terminal sequence of mouse PD-1 was labeled with a typical signal peptide cleavage site.
In comparison, the signal peptide of mouse PD-1 is estimated to be from Met at position 1 to Gln at position 20. Therefore, the mature protein part is 2 from the 21st Ser.
It consists of 268 amino acids up to the 88th Leu, and is considered to constitute an extracellular region (147 amino acids), a transmembrane region (27 amino acids) and a cytoplasmic region (94 amino acids). Further, there are possibilities that four N-glycosylation sites exist in the extracellular region.

When the amino acid sequence of mouse PD-1 was compared with all amino acid sequences registered in the database of National Biomedical Research Foundation, the extracellular region of mouse PD-1 was found to belong to the known immunoglobulin superfamily. It can be seen that it has homology with several kinds of polypeptides to which it belongs. Immunoglobulin domains are classified based on the conserved amino acid pattern and number of retrograde β-strands V, C1.
And C2 set. 54th Cys and 123
The 68 amino acid sequence flanked by the th Cys is similar to the disulfide linked V set sequence. In addition, four amino acid residues ( ⁹⁴ Arg, ⁹⁵ Phe, ¹¹⁷ A
sp and ¹¹⁹ Gly) are characteristic of many immunoglobulin V set sequences.

The cytoplasmic region has no apparent homology to any known sequence, but if examined carefully,
A common sequence (Asp / Glu-X7-Asp / Glu-X2-Tyr-X2-Leu-X7-Tyr-Leu) found in the tail of the cytoplasmic region of most polypeptides related to antigen receptors and Fc receptors. / Ile
It can be seen that the modified array of () is included. Recently,
One unit of this consensus sequence has been shown to be sufficient to transmit a signal. In mouse PD-1, although the number of non-conserved amino acids between Leu and the second Tyr is much higher than that of the consensus sequence, most conserved amino acid residues and spacer lengths are not consistent with the PD-1 polypeptide cytoplasm. Stored in the area. Although PD-1 of other mammals has some differences in the number and kinds of amino acids in the sequence, it is considered that the structural characteristics thereof are similar to those of mouse PD-1.

The DNA encoding the PD-1 polypeptide of the present invention can be prepared by the following method.
That is, mRNA extracted from cells that have undergone apoptosis by external stimuli (eg, blood cells, etc.)
To prepare a single-stranded cDNA. CDNA prepared
Is subtracted several times (preferably 2-3 times) by the excess amount of mRNA extracted from the same healthy cells. Remaining cDNA
Are labeled with ³² P by random priming to make the probe. CDN produced in this way
A probe was associated with cell death by apoptosis.
It is considered that RNA-derived cDNA is concentrated.

On the other hand, in order to construct a cDNA library, cDNA is prepared by using mRNA extracted from the stimulated same cells. The obtained single-stranded cDNA is converted into a double-stranded cDNA by a conventional method and introduced into a known vector (for example, λgt10). Next, this library is screened with the subtracted cDNA probe prepared above to obtain the clone of interest, that is, the P of the present invention.
The D-1 cDNA is obtained.

Also in the above method, the PDN cDN of the present invention is used.
If A is obtained, but there are other cells that undergo apoptosis, a gene that is commonly activated by cell death of two types of cells can be obtained in a more reliable manner. That is, mRNA extracted from one cell (provisionally referred to as cell a) that has undergone apoptosis by an external stimulus.
To prepare a single-stranded cDNA. CDNA prepared
Is subtracted several times (preferably 2-3 times) by the excess amount of mRNA extracted from the same healthy cells. Remaining cDNA
Are labeled with ³² P by random priming to make the probe.

On the other hand, in order to construct a cDNA library, single-stranded cD was prepared using mRNA extracted from the other cell dying by stimulation (probably cell b).
Create NA. The prepared cDNA was extracted several times (preferably 2-3 times) with an excess amount of mRNA extracted from healthy cells.
Times) will be deducted. The remaining cDNA is back-hybridized with an excess amount of mRNA extracted from the stimulated cells, converted into a double-stranded cDNA by a conventional method, and introduced into a known vector (for example, λgt10). The library is then screened using a subtracted cDNA probe previously made from cells a to yield the clone of interest, ie, a clone associated with cell death and common to the two cells.

However, even when one of the cells (eg, cell b) is in a healthy state, the target gene may be slightly expressed, and in that case, the deductible cDNA is used.
The step of preparing the A library is performed by extracting m from healthy cells of cells (eg, cell a) that are expressed only upon stimulation.
Should be deducted using RNA.

Once the base sequence of the DNA encoding PD-1 has been determined, it is then chemically synthesized.
Alternatively, by chemically synthesizing a fragment of the base sequence and hybridizing it with a probe, D of the present invention can be obtained.
NA can be obtained. Furthermore, the desired amount of the desired DNA of the present invention can be obtained by introducing the vector DNA containing the present DNA into a suitable host and allowing it to grow.

The method for obtaining the PD-1 polypeptide of the present invention includes (1) a method of purifying and isolating from living organisms or cultured cells, (2) a method of synthesizing peptides, or (3)
Examples of the method include a method of producing using a gene recombination technique, but the method described in (3) is industrially preferable.

The expression system (host-vector system) for producing a polypeptide using the gene recombination technique includes, for example, expression systems of bacteria, yeast, insect cells and mammalian cells.

For example, in the case of expression in Escherichia coli, a start codon (ATG) is added to the 5'end of the DNA encoding the mature protein portion, and the obtained DNA is used as an appropriate promoter (eg trp promoter, lac). A vector that is connected to the downstream of a promoter, a λP _L promoter, a T7 promoter, etc., and functions in E. coli (for example, p
BR322, pUC18, pUC19, etc.) to prepare an expression vector. Next, E. coli transformed with this expression vector (for example, E.ColiDH1, E.ColiJM1
09, E. Coli HB101 strain, etc.) can be cultured in an appropriate medium to obtain the desired polypeptide from the cells.

If a bacterial signal peptide (eg, pelB signal peptide) is used, the desired polypeptide can be secreted into the periplasm. In addition, fusion proteins with other polypeptides can be produced.

In the case of expressing in mammalian cells, for example, DNA encoding the full length of PD-1 is used as a suitable vector (eg, retrovirus vector, papillomavirus vector, vaccinia virus vector,
An expression vector is prepared by inserting it into the downstream of an appropriate promoter (for example, SV40 promoter, LTR promoter, metallothionein promoter, etc.) in SV40 system vector etc.).

Next, an appropriate mammalian cell (eg, monkey COS-7 cell, Chinese hamster CHO cell, mouse L cell) is transformed with the obtained expression vector, and the transformant is cultured in an appropriate medium. By,
The desired polypeptide is secreted into the culture medium.
The polypeptide obtained as described above can be isolated and purified by a general biochemical method.

The DNA encoding the PD-1 gene obtained by the present invention can be used as a probe to isolate the PD-1 gene of animals other than mouse including human. DNA encoding those PD-1 genes
Alternatively, the DNA fragment can be used as a probe or primer to detect the PD-1 gene, and thus can be used for studying the relationship between the gene and the biological defense function, immune function, or diseases such as tumors, or for diagnosing the disease.

These DNAs can be used as an essential template for producing PD-1 polypeptides, polypeptide fragments or derivatives thereof, which are expected to have great utility by the usual gene recombination methods. The polypeptide, polypeptide fragment or modified polypeptide thus produced is expected to be used as a therapeutic agent for infectious diseases, decreased or enhanced immune functions, tumors and the like.

Furthermore, since a polyclonal antibody and a monoclonal antibody can be prepared by a conventional method using the polypeptide of the present invention or a fragment thereof as an antigen, the relationship between the polypeptide and a disease can be determined by quantifying the polypeptide using them. It can be used for research or diagnosis of diseases. Further, the monoclonal antibody can be used as a therapeutic agent in the form as it is, in the form of a chimeric antibody with a human antibody or in a form converted to a human form.

[0033]

EXAMPLES The present invention will be described in more detail with reference to examples below, but these do not limit the scope of the present invention.

Example 1 Induction of Apoptosis Mouse 2B4.11 cells (Dr. JD Ashwell)
l, National Cancer Institute
donated by e.) is RPMI1640 culture solution (GIB
CO) 10% inactivated fetal bovine serum, 2 mM glutamine, 50 μM 2-mercaptoethanol, 100 U / m.
1 Penicillin and 100 μg / ml streptomycin were added to the cells (hereinafter referred to as complete culture medium) to culture the cells. Mouse LyD9 cells (Palacios et al., EM
BO J. , 6 , 3687 (1987)) was transformed with a mouse IL-3 expression vector into a complete culture solution.
63Ag myeloma cells (Karasuyama et al., E.
ur. J. Immunol. , 18 , 97 (1988)
The culture supernatant was added to 0.2% of the culture supernatant described in 1).

To induce apoptosis of 2B4.11 cells, 500 ng / ml ionomycin (manufactured by Calbiochem) and 10 ng / ml PMA (phorbol 12-myristate 13-acetate, manufactured by Calbiochem) were added to the complete culture medium at 37 ° C. It was performed by culturing in 5% CO _{2 for 2 to} 6 hours.

Induction of apoptosis of LyD9 cells was performed by depletion of growth factors. That is, the cells are
After washing three times with 640 culture medium, the cells were cultured in a complete culture medium containing no growth factor at 37 ° C. and 5% CO _{2 for} 4 to 12 hours.

Example 2: D as an index of apoptosis
Detection of NA fragmentation Test cells (5 × 10 ⁶ to 1 × 10 ⁷ cells) were collected by centrifugation and washed once with cold phosphate buffered saline (PBS ⁻ ). 600 μl of 10 mM EDTA, 0.2
It was dissolved in 10 mM Tris-HCl buffer (pH 7.5) containing 10% Triton X-100. After leaving it on ice for 10 minutes, use an Eppendorf centrifuge at 1400g at 4 ℃.
It was centrifuged for 10 minutes.

The supernatant (containing RNA and fragmented DNA but no intact chromatin) was phenol extracted and then phenol / chloroform: isoamyl alcohol (24: 1). A saline solution was added to the aqueous layer to 300 mM, and then twice the amount of ethanol was added to precipitate the nucleic acid. The precipitate was washed with 70% ethanol, air-dried, and then 10 mM containing 15 μl of 1 mM EDTA.
It was dissolved in Tris-HCl buffer (pH 7.5). 0.6 mg /
Add ml of RNase A and add 3
After reacting for 30 minutes at 7 ° C. to decompose RNA, Boyer buffer (50 mM Tris-HCl, 20 mM sodium acetate, 2 mM EDTA, 18 mM) was applied on a 2% agarose gel.
Electrophoresis was performed using mM NaCl, pH 8.05). DNA was detected by ethidium bromide staining.

The results are shown in FIGS. 1 and 2. In FIG. 1, lane 1 shows a pattern without stimulation, lane 2 shows a pattern when stimulated with ionomycin and PMA for 6 hours, and lanes 3 to 8 respectively show 0, 1, 2, 3 from stimulation.
The pattern when an RNA transcription inhibitor is added after 4 and 5 hours is shown. In FIG. 2, lane 1 is IL-3.
The pattern at the time of addition is shown, and the lane 2 shows the pattern at the time of depleting IL-3 and culturing for 12 hours.
7 shows the patterns when RNA transcription inhibitors were added 0, 2, 4, 6 and 8 hours after the depletion of IL-3, respectively.

The results show that in 2B4.11 cells, DNA fragmentation characteristic of apoptosis is observed when stimulated with ionomycin and PMA for 6 hours at the same time, and actinomycin which is an RNA transcription inhibitor is stimulated simultaneously with stimulation. It is shown that addition of D (200 ng / ml, manufactured by Sigma) is almost completely suppressed. This clearly shows that RNA dunovo synthesis is required for apoptosis of 2B4.11 cells. Further, from an experiment in which the actinomycin D addition timing was changed, it is shown that the mRNA synthesis required for cell death is completed by 3 hours after stimulation.

In the case of LyD9 cells, IL-3
It is shown that the same DNA fragmentation was observed after 12 hours of depletion and culture, and that IL-3 was completely suppressed by addition of actinomycin D at the same time as depletion of IL-3. However, from experiments in which the actinomycin D addition time was changed, the mR required for this cell death of LyD9 cells was determined.
NA synthesis appeared to be for a rather long time as compared to 2B4.11 cells.

Example 3: Preparation of subtracted cDNA library Ionomycin (500 ng / ml) and PMA (10 n
g / ml) and stimulated for 2 to 3 hours with 2B4.11.
From cells (approximately 5 × 10 ⁹ cells) to conventional method (Molecular
Cloning: Sambrook, J .; , Frit
sh, E .; F. , And Maniatis, T .; Author, C
old Spring Harbor Laborato
Poly (A) ⁺ RNA (300 μg) was obtained according to RY Press , published in 1989).

Poly (A) ⁺ RNA (40 μg) was used with an oligo dT primer and a 6-base random primer to actinomycin D and a trace amount of [α- ³² P] dCT.
First strand was synthesized by reverse transcriptase in the presence of P. Single-stranded cDNA obtained by hydrolyzing RNA template by adding 0.2 N sodium hydroxide at 68 ° C. for 30 minutes was extracted from LyD9 cells cultured in the presence of IL-3, and excess amount of Poly (A) ⁺ RNA ( 200 μg) and 5 m
Hybridization was performed at 68 ° C. for 26 hours in 0.5 M sodium phosphate buffer (pH 6.8) containing M EDTA and 0.2% SDS. Cot value of this reaction (corrected for the effect of high salt concentration, Britten et al., Meth. Enzy
m., 29 , 363-418 (1974)) is 7800 mol.sec.
/ L.

The reaction mixture was placed in a water jacket on a hydroxyapatite column (Biorad) equilibrated with 0.12 M sodium phosphate buffer (pH 6.8).
The single-stranded cDNA that did not hybridize with the cDNA / RNA heteroduplex was separated by passing it while keeping it at 0 ° C. This single-stranded cDNA was prepared by using the above-mentioned ionomycin and PM.
Poly (A) ⁺ extracted from 2B4.11 cells stimulated with A
RNA excess (200 μg) and 8000 mol · sec /
Back-hybridization was performed so that the Cot value was 1.
Double-stranded cDNA from the obtained cDNA / RNA heteroduplex
Conversion to NA and cloning into λgt10 was performed using Mole
It was performed according to the method described in cular Cloning (supra). Since about 90% of the total cDNA was subtracted by the above operation, it was judged that the cDNA clones that were considered to be cell death-specific were concentrated about 10-fold.

Example 4: Preparation of subtracted cDNA probe The methods of cDNA synthesis, hybridization and hydroxyapatite column chromatography were according to the method described in Example 3. That is, P extracted from LyD9 cells depleted of IL-3 and cultured for 4 to 6 hours.
First strand was synthesized from oly (A) ⁺ RNA (40 μg), and RNA template was hydrolyzed to obtain single strand c.
DNA was obtained. This is LyD9 cultured in the presence of IL-3.
Excess amount of Poly (A) ⁺ RNA extracted from cells (200μ
g) and hybridized so as to obtain a Cot value of 8000 mol · sec / l. Single-stranded cDNA that did not hybridize was separated by hydroxyapatite column chromatography.

Single-stranded cDNA (130 ng) obtained by repeating the cycle of hybridization and hydroxyapatite column chromatography again was Molecu.
According to the method described in larCloning (supra), a 6-base random primer was used to [α- ³² P] dCTP (3000 Ci
/ Mmol, manufactured by Amersham). A probe having a specific activity of about 4 × 10 ⁹ dpm / μg was obtained. In addition, about 96% of the total cDNA was subtracted by the above operation, and thus the subtraction probe specific to IL-3 depletion was concentrated about 25 times.

Example 5: Library screening From the cDNA library prepared in Example 3, 20 ×
About 1 × 10 ⁴ plaques on a 20 cm LB plate
Seed clones at plate density (total 2 x 10
⁴⁾ After 13 hours at 37 ° C., to produce a replica up transferred DNA to nitrocellulose filters.
The hybridization was carried out by using a hybridization buffer (5 × SSPE, 5 × Denhardt's solution, 120 μm) containing about 5 × 10 ⁸ dpm of the probe (prepared in Example 4).
g / ml salmon sperm DNA, 10% dextran sulfate,
0.3% sodium dodecyl sulfate) at 68 ° C. for 27 hours.

After the reaction, the filter was used in a 2 × SSC solution (1 × SSC: 0.15M NaCl, 0.015M sodium citrate) at 55 ° C. for 40 minutes, and then a 0.1 × SSC solution containing 0.1% sodium dodecylsulfate. 68 in
It was washed at 30 ° C. for 30 minutes. As a result of autoradiography, clones (120 clones) showing a strong signal were subjected to the following secondary screening.

That is, each phage clone was replated on an LB plate having a diameter of 10 cm (about 200 plaques / plate), and two replicas of a nitrocellulose filter were prepared. One filter group contains the above-mentioned labeled probe, and the other filter group contains IL-3.
The cDNA derived from LyD9 cells cultured in the presence was hybridized with a probe labeled in the same manner. After washing and autoradiography, four independent clones (that is, IL-3 depletion-specific clones) that hybridized only with the former probe and did not hybridize with the latter probe were obtained, and blue clone SK plasmid vector ( (Sold by Stratagene)
Subcloned into.

Although the insert sizes of these four clones were different, the restriction enzyme maps overlapped with each other and they hybridized even under very strong stringent conditions, so that they were considered to be derived from the same gene. . This gene was named PD-1.

Next, cloning was carried out from a λgt10 cDNA library (not subtracted) prepared from 2B4.11 cells stimulated with ionomycin and PMA using PD-1 as a probe, and a clone having the longest insert (PD- 1 cDNA # 7) was obtained. This was also subcloned into the Bluescript SK plasmid vector. The following analyzes are mainly PD-1 cDN
This was done using A # 7.

Example 6: Determination of nucleotide sequence The nucleotide sequence was determined by the dideoxy chain termination method (Sanger, F., et al., Proc. Nat. Acad. Sci. USA, 74 , 5463).
(1977)), modified T7 DNA polymerase (obtained from United States Biochemical) and [α-
³² P] dCTP (3000 Ci / mmol, manufactured by Amersham) was used.

The entire base sequence of PD-1 cDNA # 7 obtained and the amino acid sequence predicted from it are shown in Sequence Listings 1 to 4, and the restriction enzyme map is shown in FIG. It was speculated that the PD-1 gene encodes a polypeptide consisting of 288 amino acids including 27 amino acids that are considered to be a transmembrane site.

Example 7: Northern Blotting PD-1 gene-stimulated 2B4.11 cells and IL-3
In order to confirm that they were co-activated in depleted LyD9 cells, Northern blotting analysis was performed. Extraction of whole cell RNA, Poly (A) ⁺ RNA
The standard method described in Molecular Cloning (supra) was used for the purification and the Northern hybridization. PD-1 cDNA # 7 is used as a probe
PstI fragment (650b) present in the coding region of
p) with [α- ³² P] dC using random primers
The one labeled with TA was used. Also, as an internal standard,
EcoR of plasmid PMAβ-3′ut containing only 3 ′ non-coding region of mouse β-actin cDNA
The I fragment (300 bp) was labeled in the same manner as a probe.

The results are shown in FIG. In the figure, lane 1 shows the pattern of 2B4.11 cells without stimulation, lane 2 shows the pattern of 2B4.11 cells after stimulation, and lane 3
Shows the pattern of LyD9 cells after IL-3 containing culture, and lane 4 shows the pattern of LyD9 cells after IL-3 depleted culture. The result is 2B4.
Expression of PD-1 mRNA in 11 cells was enhanced very strongly (10-fold or more), and IL-3
It shows that it is specifically expressed in LyD9 cells after depletion.

Next, it was examined in which tissue of the body the PD-1 gene was expressed. A 5-week-old ICR mouse (purchased from Shizuoka Experimental Animal Center) was applied to a monoclonal antibody against mouse CD3 (ε) (Leo, O. et al., Proc. Nat. Aca).
BALB / injected with hybridoma 145-2C11 producing d.Sci.USA, 84 , 1374-1378 (1987)).
Defibrinated ascites 2 collected from C nu / nu mice
00 μl or 200 μl phosphate buffered saline was injected intraperitoneally. After a certain period of time, the mouse was sacrificed, and total RNA was extracted from each organ according to the method described in Molecular Cloning (supra), Poly (A) ⁺ RNA was purified, and Northern hybridization was performed. As the probe, the one described in this example was used.

The results are shown in FIGS. 5 and 6. In FIG. 5, lanes 1 to 7 show the expression of PD-1 mRNA in brain, heart, lung, thymus, spleen, liver and kidney, respectively. In FIG. 6, lanes 1 to 5 represent the thymus, and lanes 6 to 10
Represents the spleen, lanes 1 and 6 of the untreated group, lanes 2 and 7 8 hours after administration of PBS, lanes 3 and 8 of P.
Lanes 4 and 9 show the expression of PD-1 mRNA 12 hours after the administration of BS, 8 hours after the administration of anti-CD3 antibody, and lanes 5 and 10 show the expression of the PD-1 mRNA 12 hours after the administration of anti-CD3 antibody.

From FIG. 5, the expression of PD-1 mRNA is prominent in the thymus (thymocyte death is constantly occurring in the thymus), and is almost observed in the brain, heart, lung, spleen, liver and kidney. I know there isn't. Furthermore, from FIG. 6, PD-1 mRN was injected after 8 to 12 hours by injection of anti-CD3 monoclonal antibody known to enhance apoptotic cell death in mouse thymus.
It can be seen that the expression of A is enhanced (about 3-fold) in the thymus and, interestingly, also in the spleen.

[0059]

[Sequence list]

SEQ ID NO: 1 Sequence length: 288 Sequence type: Amino acid Topology: linear Sequence type: Protein Array Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln  1 5 10 15 Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp              20 25 30 Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala          35 40 45 Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met      50 55 60 Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala  65 70 75 80 Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln                  85 90 95 Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp             100 105 110 Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu         115 120 125 His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val     130 135 140 Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro 145 150 155 160 Lys Pro Glu Gly Arg Phe Gln Gly Met Val Ile Gly Ile Met Ser Ala                 165 170 175 Leu Val Gly Ile Pro Val Leu Leu Leu Leu Ala Trp Ala Leu Ala Val             180 185 190 Phe Cys Ser Thr Ser Met Ser Glu Ala Arg Gly Ala Gly Ser Lys Asp         195 200 205 Asp Thr Leu Lys Glu Glu Pro Ser Ala Ala Pro Val Pro Ser Val Ala     210 215 220 Tyr Glu Glu Leu Asp Phe Gln Gly Arg Glu Lys Thr Pro Glu Leu Pro 225 230 235 240 Thr Ala Cys Val His Thr Glu Tyr Ala Thr Ile Val Phe Thr Glu Gly                 245 250 255 Leu Gly Ala Ser Ala Met Gly Arg Arg Gly Ser Ala Asp Gly Leu Gln             260 265 270 Gly Pro Arg Pro Pro Arg His Glu Asp Gly His Cys Ser Trp Pro Leu         275 280 285

SEQ ID NO: 2 Sequence length: 864 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: cDNA to mRNA Array ATGTGGGTCC GGCAGGTACC CTGGTCATTC ACTTGGGCTG TGCTGCAGTT GAGCTGGCAA 60 TCAGGGTGGC TTCTAGAGGT CCCCAATGGG CCCTGGAGGT CCCTCACCTT CTACCCAGCC 120 TGGCTCACAG TGTCAGAGGG AGCAAATGCC ACCTTCACCT GCAGCTTGTC CAACTGGTCG 180 GAGGATCTTA TGCTGAACTG GAACCGCCTG AGTCCCAGCA ACCAGACTGA AAAACAGGCC 240 GCCTTCTGTA ATGGTTTGAG CCAACCCGTC CAGGATGCCC GCTTCCAGAT CATACAGCTG 300 CCCAACAGGC ATGACTTCCA CATGAACATC CTTGACACAC GGCGCAATGA CAGTGGCATC 360 TACCTCTGTG GGGCCATCTC CCTGCACCCC AAGGCAAAAA TCGAGGAGAG CCCTGGAGCA 420 GAGCTCGTGG TAACAGAGAG AATCCTGGAG ACCTCAACAA GATATCCCAG CCCCTCGCCC 480 AAACCAGAAG GCCGGTTTCA AGGCATGGTC ATTGGTATCA TGAGTGCCCT AGTGGGTATC 540 CCTGTATTGC TGCTGCTGGC CTGGGCCCTA GCTGTCTTCT GCTCAACAAG TATGTCAGAG 600 GCCAGAGGAG CTGGAAGCAA GGACGACACT CTGAAGGAGG AGCCTTCAGC AGCACCTGTC 660 CCTAGTGTGG CCTATGAGGA GCTGGACTTC CAGGGACGAG AGAAGACACC AGAGCTCCCT 720 ACCGCCTGTG TGCACACAGA ATATGCCACC ATTGTCTTCA CTGAAGGGCT GGGTGCCTCG 780 GCCATGGGAC GTAGGGGCTC AGCTGATGGC CTGCAGGGTC CTCGGCCTCC AAGACATGAG 840 GATGGACATT GTTCTTGGCC TCTT 864

SEQ ID NO: 3 Sequence length: 1973 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: cDNA to mRNA Array TGAGCAGCGG GGAGGAGGAA GAGGAGACTG CTACTGAAGG CGACACTGCC AGGGGCTCTG 60 GGCATGTGGG TCCGGCAGGT ACCCTGGTCA TTCACTTGGG CTGTGCTGCA GTTGAGCTGG 120 CAATCAGGGT GGCTTCTAGA GGTCCCCAAT GGGCCCTGGA GGTCCCTCAC CTTCTACCCA 180 GCCTGGCTCA CAGTGTCAGA GGGAGCAAAT GCCACCTTCA CCTGCAGCTT GTCCAACTGG 240 TCGGAGGATC TTATGCTGAA CTGGAACCGC CTGAGTCCCA GCAACCAGAC TGAAAAACAG 300 GCCGCCTTCT GTAATGGTTT GAGCCAACCC GTCCAGGATG CCCGCTTCCA GATCATACAG 360 CTGCCCAACA GGCATGACTT CCACATGAAC ATCCTTGACA CACGGCGCAA TGACAGTGGC 420 ATCTACCTCT GTGGGGCCAT CTCCCTGCAC CCCAAGGCAA AAATCGAGGA GAGCCCTGGA 480 GCAGAGCTCG TGGTAACAGA GAGAATCCTG GAGACCTCAA CAAGATATCC CAGCCCCTCG 540 CCCAAACCAG AAGGCCGGTT TCAAGGCATG GTCATTGGTA TCATGAGTGC CCTAGTGGGT 600 ATCCCTGTAT TGCTGCTGCT GGCCTGGGCC CTAGCTGTCT TCTGCTCAAC AAGTATGTCA 660 GAGGCCAGAG GAGCTGGAAG CAAGGACGAC ACTCTGAAGG AGGAGCCTTC AGCAGCACCT 720 GTCCCTAGTG TGGCCTATGA GGAGCTGGAC TTCCAGGGAC GAGAGAAGAC ACCAGAGCTC 780 CCTACCGCCT GTGTGCACAC AGAATATGCC ACCATTGTCT TCACTGAAGG GCTGGGTGCC 840 TCGGCCATGG GACGTAGGGG CTCAGCTGAT GGCCTGCAGG GTCCTCGGCC TCCAAGACAT 900 GAGGATGGAC ATTGTTCTTG GCCTCTTTGA CCAGATTCTT CAGCCATTAG CATGCTGCAG 960 ACCCTCCACA GAGAGCACCG GTCCGTCCCT CAGTCAAGAG GAGCATGCAG GCTACAGTTC 1020 AGCCAAGGCT CCCAGGGTCT GAGCTAGCTG GAGTGACAGC CCAGCGCCTG CACCAATTCC 1080 AGCACATGCA CTGTTGAGTG AGAGCTCACT TCAGGTTTAC CACAAGCTGG GAGCAGCAGG 1140 CTTCCCGGTT TCCTATTGTC ACAAGGTGCA GAGCTGGGGC CTAAGCCTAT GTCTCCTGAA 1200 TCCTACTGTT GGGCACTTCT AGGGACTTGA GACACTATAG CCAATGGCCT CTGTGGGTTC 1260 TGTGCCTGGA AATGGAGAGA TCTGAGTACA GCCTGCTTTG AATGGCCCTG TGAGGCAACC 1320 CCAAAGCAAG GGGGTCCAGG TATACTATGG GCCCAGCACC TAAAGCCACC CTTGGGAGAT 1380 GATACTCAGG TGGGAAATTC GTAGACTGGG GGACTGAACC AATCCCAAGA TCTGGAAAAG 1440 TTTTGATGAA GACTTGAAAA GCTCCTAGCT TCGGGGGTCT GGGAAGCATG AGCACTTACC 1500 AGGCAAAAGC TCCGTGAGCG TATCTGCTGT CCTTCTGCAT GCCCAGGTAC CTCAGTTTTT 1560 TTCAACAGCA AGGAAACTAG GGCAATAAAG GGAACCAGCA GAGCTAGAGC CACCCACACA 1620 TCCAGGGGGG CACTTGACTC TCCCTACTCC TCCTAGGAAC CAAAAGGACA AAGTCCATGT 1680 TGACAGCAGG GAAGGAAAGG GGGATATAAC CTTGACGCAA ACCAACACTG GGGTGTTAGA 1740 ATCTCCTCAT TCACTCTGTC CTGGAGTTGG GTTCTGGCTC TCCTTCACAC CTAGGACTCT 1800 GAAATGAGCA AGCACTTCAG ACAGTCAGGG TAGCAAGAGT CTAGCTGTCT GGTGGGCACC 1860 CAAAATGACC AGGGCTTAAG TCCCTTTCCT TTGGTTTAAG CCCGTTATAA TTAAATGGTA 1920 CCAAAAGCTT TAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAA 1973

SEQ ID NO: 4 Sequence length: 1973 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: cDNA to mRNA origin Organism name: Mus musculus Cell line: 2B4.11 Sequence features Characteristic symbol: CDS Location: 64..927 Method by which the characteristics were determined: P Characteristic symbol: sig peptide Location: 64..123 Method by which the characteristics were determined: S Characteristic symbol: mat peptide Location: 124..927 Method by which the characteristics were determined: S Array TGAGCAGCGG GGAGGAGGAA GAGGAGACTG CTACTGAAGG CGACACTGCC AGGGGCTCTG 60 GGC ATG TGG GTC CGG CAG GTA CCC TGG TCA TTC ACT TGG GCT GTG CTG 108     Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu     -20 -15 -10 CAG TTG AGC TGG CAA TCA GGG TGG CTT CTA GAG GTC CCC AAT GGG CCC 156 Gln Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro  -5 1 5 10 TGG AGG TCC CTC ACC TTC TAC CCA GCC TGG CTC ACA GTG TCA GAG GGA 204 Trp Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly              15 20 25 GCA AAT GCC ACC TTC ACC TGC AGC TTG TCC AAC TGG TCG GAG GAT CTT 252 Ala Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu          30 35 40 ATG CTG AAC TGG AAC CGC CTG AGT CCC AGC AAC CAG ACT GAA AAA CAG 300 Met Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln      45 50 55 GCC GCC TTC TGT AAT GGT TTG AGC CAA CCC GTC CAG GAT GCC CGC TTC 348 Ala Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe  60 65 70 75 CAG ATC ATA CAG CTG CCC AAC AGG CAT GAC TTC CAC ATG AAC ATC CTT 396 Gln Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu                  80 85 90 GAC ACA CGG CGC AAT GAC AGT GGC ATC TAC CTC TGT GGG GCC ATC TCC 444 Asp Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser              95 100 105 CTG CAC CCC AAG GCA AAA ATC GAG GAG AGC CCT GGA GCA GAG CTC GTG 492 Leu His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val         110 115 120 GTA ACA GAG AGA ATC CTG GAG ACC TCA ACA AGA TAT CCC AGC CCC TCG 540 Val Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser     125 130 135 CCC AAA CCA GAA GGC CGG TTT CAA GGC ATG GTC ATT GGT ATC ATG AGT 588 Pro Lys Pro Glu Gly Arg Phe Gln Gly Met Val Ile Gly Ile Met Ser 140 145 150 155 GCC CTA GTG GGT ATC CCT GTA TTG CTG CTG CTG GCC TGG GCC CTA GCT 636 Ala Leu Val Gly Ile Pro Val Leu Leu Leu Leu Ala Trp Ala Leu Ala                 160 165 170 GTC TTC TGC TCA ACA AGT ATG TCA GAG GCC AGA GGA GCT GGA AGC AAG 684 Val Phe Cys Ser Thr Ser Met Ser Glu Ala Arg Gly Ala Gly Ser Lys             175 180 185 GAC GAC ACT CTG AAG GAG GAG CCT TCA GCA GCA CCT GTC CCT AGT GTG 732 Asp Asp Thr Leu Lys Glu Glu Pro Ser Ala Ala Pro Val Pro Ser Val         190 195 200 GCC TAT GAG GAG CTG GAC TTC CAG GGA CGA GAG AAG ACA CCA GAG CTC 780 Ala Tyr Glu Glu Leu Asp Phe Gln Gly Arg Glu Lys Thr Pro Glu Leu     205 210 215 CCT ACC GCC TGT GTG CAC ACA GAA TAT GCC ACC ATT GTC TTC ACT GAA 828 Pro Thr Ala Cys Val His Thr Glu Tyr Ala Thr Ile Val Phe Thr Glu 220 225 230 235 GGG CTG GGT GCC TCG GCC ATG GGA CGT AGG GGC TCA GCT GAT GGC CTG 876 Gly Leu Gly Ala Ser Ala Met Gly Arg Arg Gly Ser Ala Asp Gly Leu                 240 245 250 CAG GGT CCT CGG CCT CCA AGA CAT GAG GAT GGA CAT TGT TCT TGG CCT 924 Gln Gly Pro Arg Pro Pro Arg His Glu Asp Gly His Cys Ser Trp Pro             255 260 265 CTT TGA CCAGATTCTT CAGCCATTAG CATGCTGCAG ACCCTCCACA GAGAGCACCG 980 Leu ***                                                                    GTCCGTCCCT CAGTCAAGAG GAGCATGCAG GCTACAGTTC AGCCAAGGCT CCCAGGGTCT 1040 GAGCTAGCTG GAGTGACAGC CCAGCGCCTG CACCAATTCC AGCACATGCA CTGTTGAGTG 1100 AGAGCTCACT TCAGGTTTAC CACAAGCTGG GAGCAGCAGG CTTCCCGGTT TCCTATTGTC 1160 ACAAGGTGCA GAGCTGGGGC CTAAGCCTAT GTCTCCTGAA TCCTACTGTT GGGCACTTCT 1220 AGGGACTTGA GACACTATAG CCAATGGCCT CTGTGGGTTC TGTGCCTGGA AATGGAGAGA 1280 TCTGAGTACA GCCTGCTTTG AATGGCCCTG TGAGGCAACC CCAAAGCAAG GGGGTCCAGG 1340 TATACTATGG GCCCAGCACC TAAAGCCACC CTTGGGAGAT GATACTCAGG TGGGAAATTC 1400 GTAGACTGGG GGACTGAACC AATCCCAAGA TCTGGAAAAG TTTTGATGAA GACTTGAAAA 1460 GCTCCTAGCT TCGGGGGTCT GGGAAGCATG AGCACTTACC AGGCAAAAGC TCCGTGAGCG 1520 TATCTGCTGT CCTTCTGCAT GCCCAGGTAC CTCAGTTTTT TTCAACAGCA AGGAAACTAG 1580 GGCAATAAAG GGAACCAGCA GAGCTAGAGC CACCCACACA TCCAGGGGGG CACTTGACTC 1640 TCCCTACTCC TCCTAGGAAC CAAAAGGACA AAGTCCATGT TGACAGCAGG GAAGGAAAGG 1700 GGGATATAAC CTTGACGCAA ACCAACACTG GGGTGTTAGA ATCTCCTCAT TCACTCTGTC 1760 CTGGAGTTGG GTTCTGGCTC TCCTTCACAC CTAGGACTCT GAAATGAGCA AGCACTTCAG 1820 ACAGTCAGGG TAGCAAGAGT CTAGCTGTCT GGTGGGCACC CAAAATGACC AGGGCTTAAG 1880 TCCCTTTCCT TTGGTTTAAG CCCGTTATAA TTAAATGGTA CCAAAAGCTT TAAAAAAAAA 1940 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAA 1973

A

[Brief description of drawings]

FIG. 1 is a diagram showing DNA fragmentation of 2B4.11 cells after stimulation, which was carried out in Example 2.

FIG. 2 L after IL-3 depletion culture performed in Example 2
It is a figure which shows the DNA fragmentation of yD9 cell.

FIG. 3 is a restriction map of mouse PD-1 gene.

FIG. 4 P in 2B4.11 cells after stimulation and LyD9 cells after IL-3 depleted culture performed in Example 7.
It is a figure which shows the expression of D-1 mRNA.

FIG. 5: PD-1 in each organ performed in Example 7
It is a figure which shows the expression of mRNA.

FIG. 6 shows the expression of PD-1 mRNA in thymus and spleen after administration of anti-CD3 antibody, which was carried out in Example 7.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Yasushi Ishida               97 Shiodobaba-cho, Jodo-ji, Sakyo-ku, Kyoto-shi, Kyoto Prefecture               Royal Center Building Kobayashi 403

Claims (2)

(57) [Claims] 1. A mouse PD-1 comprising the amino acid sequence represented by SEQ ID NO: 1. 2. A mouse PD-1 mature protein represented by amino acid sequence numbers 1 to 268 in SEQ ID NO: 4.