Peptide related to human programmed cell death and DNA encoding it

A membrane protein related to human programmed cell death (PD-1) and DNA encoding the said protein is provided. PD-1 protein may be useful for the treatment of various infections, immunological depression or acceleration, or tumors etc.

FIELD OF THE INVENTION 
The present invention is directed to a novel peptide related to cell death 
and DNAs encoding the same. 
BACKGROUND OF THE INVENTION 
Developmentally and physiologically controlled cell death can be observed 
in almost all tissues of various species of animals. Such cell deaths are 
generally considered `programmed` and are distinguishable from 
`accidental` deaths that occur by pathological mechanisms. Most of the 
cells undergoing programmed death have been shown to require de novo 
synthesis of RNA and protein. 
These facts suggest that at least a few genes, if not specified ones, must 
be expressed to cause programmed cell death. 
The term "apoptosis", on the other hand, is used to describe the 
morphological characteristics of a class of cell death. In cells dying by 
apoptosis, chromatin condenses around the periphery of the nucleus, while 
mitochondria and other organelles are unaffected. A unique biochemical 
feature of apoptotic cells includes fragmentation of DNA into 
oligonucleosomal pieces. In mammals, apoptosis is often morphologically 
and biochemically associated with programmed cell death, but some of the 
cells undergoing programmed death apparently do not show the 
characteristic features of apoptosis. In addition, there are apoptotic 
cell deaths that can be induced in the absence of any protein synthesis. 
Thus, it is important to note that apoptosis is not synonymous with 
programmed cell death. 
It has recently become apparent that bcl-2, which is a oncogene, protects 
mortalized B cells from cell death, thus showing its importance to control 
cell death. 
Certain peptides that are related to programmed cell death have been 
reported, such as the Fas antigen (Itoh et al, Cell, 66:233 (1991)). 
Human Fas antigen is a polypeptide consisting of about 335 amino acids, 
having a signal peptide consisting of 16 hydrophobic amino acids 
N-terminal. Its mature protein is thought to have a structure divided into 
an extracellular domain (157 amino acids), a transmembrane region (17 
amino acids) and a cytoplasmic domain (145 amino acids). It is thought to 
function as a receptor to a factor (ligand) inducing cell death. 
SUMMARY OF THE INVENTION 
An object of the present invention is to find novel polypeptides that are 
alternatives to polypeptides represented by the Fas antigen. 
In this invention, a gene related to programmed cell death has been 
isolated, its nucleotide sequence determined and its amino acid sequence 
deduced. Thus, the present inventors have succeeded in isolating a novel 
polypeptide and the DNA encoding it. 
To isolate a gene related to programmed cell death in humans, mouse PD-1, 
which was obtained from mouse T cell hybridoma 2B 4.11 (Japanese Patent 
Kokai 5-336973), was used as a probe. 
There were no polypeptides having an amino acid sequence that is identical 
to or that has high homology to that of the polypeptide of the present 
invention, except for mouse PD-1, when amino acid sequences of the 
polypeptide identified in the present invention were searched by a 
computer program containing all known sequences in the data base of the 
National Biomedical Research Foundation. Needless to say, it was confirmed 
that the polypeptide of the present invention has no homology to the Fas 
antigen. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention is related to a polypeptide which is related to 
programmed cell death (abbreviated human PD-1 hereafter). 
The present invention is directed to a polypeptide having the amino acid 
shown in SEQ ID NO:1, in substantially purified form, a homologue thereof 
or a fragment of the sequence or homologue of a fragment, and DNA encoding 
such a polypeptide. More particularly, the present invention is related to 
DNA having the nucleotide sequences shown in SEQ ID NOs:2 or 3, and DNA 
having a fragment which is able to selectively hybridize to the nucleotide 
sequences shown in SEQ ID NOs:2 or 3. 
The present invention is related to: 
(1) a polypeptide having an amino acid sequence shown in SEQ ID NO:1, 
(2) a DNA molecule encoding the polypeptide described above in (1), 
(3) a DNA molecule having a nucleotide sequence shown in SEQ ID NO:2, and 
(4) a DNA molecule having a nucleotide sequence shown in SEQ ID NO:3. 
The polypeptide of SEQ ID NO:1 in substantially purified form will 
generally contain the polypeptide in a preparation in which more than 90%, 
e.g., 95%, 98% or 99% of the polypeptide in the preparation is that of SEQ 
ID NO:1. 
A polypeptide homologue of SEQ ID NO:1 will be generally at least 70%, 
preferably at least 80 or 90%, and more preferably at least 95%, 
homologous to the polypeptide of SEQ ID NO:1 over a region of at least 20, 
preferably at least 30, for instance 40, 60 or 100, or more contiguous 
amino acids. Such polypeptide homologues will be referred to below as a 
polypeptide according to the invention. 
Generally, fragments of SEQ ID NO:1 or its homologues will be at least 10, 
preferably at least 15, for example, 20, 25, 30, 40, 50 or 60 amino acids 
in length, and are also encompassed by the term "a polypeptide according 
to the invention" as used herein. 
DNA capable of selectively hybridizing to the DNA of SEQ ID NOs:2 or 3 will 
be generally at least 70%, preferably at least 80 or 90%, and more 
preferably at least 95%, homologous to the DNA of SEQ ID NOs:2 or 3 over a 
region of at least 20, preferably at least 30, for instance 40, 60 or 100 
or more contiguous nucleotides. Such DNA will be encompassed by the term 
"DNA according to the invention". 
Fragments of the DNA of SEQ ID NOs:2 or 3 will be at least 15, preferably 
at least 20, for example, 25, 30 or 40 nucleotides in length, and are also 
encompassed by the term "DNA according to the invention" as used herein. 
A further embodiment of the invention provides replication and expression 
vectors comprising DNA according to the invention. The vectors may be, for 
example, plasmid, virus or phage vectors provided with an origin of 
replication, optionally a promoter for the expression of the said DNA, and 
optionally a regulator of the promoter. The vector may contain one or more 
selectable marker genes, for example, an ampicillin resistance gene. The 
vector may be used in vitro, for example, for the production of RNA 
corresponding to the DNA, or used to transfect or transform a host cell. 
A further embodiment of the invention provides host cells transformed or 
transfected with the vectors for the replication and expression of DNA 
according to the invention, including DNA SEQ ID NOs:2 or 3 or the open 
reading frame thereof. The cells chosen are to be compatible with the 
vector and may, for example, be bacterial, yeast, insect or mammalian. 
A further embodiment of the invention provides a method of producing a 
polypeptide, which comprises culturing host cells of the present invention 
under conditions effective to express a polypeptide according to the 
invention. Preferably, in addition, such a method is carried out under 
conditions in which the polypeptide of the invention is expressed and then 
produced from the host cells. 
DNA according to the invention may also be inserted into the vectors 
described above, in an antisense orientation in order to provide for the 
production of antisense RNA. Antisense RNA may also be produced by 
synthetic means. Such antisense RNA may be used in a method for 
controlling the level of a polypeptide of the invention in a cell. 
The invention also provides monoclonal or polyclonal antibodies to a 
polypeptide according to the invention. The invention further provides a 
process for the production of monoclonal or polyclonal antibodies to the 
polypeptides of the invention. Monoclonal antibodies may be prepared by 
conventional hybridoma technology using a polypeptide of the invention or 
a fragment thereof, as an immunogen. Polyclonal antibodies may also be 
prepared by conventional means which comprise inoculating a host animal, 
for example, a rat or a rabbit, with a polypeptide of the invention and 
recovering immune serum. 
The present invention also provides pharmaceutical compositions containing 
a polypeptide of the invention, or an antibody thereof, in association 
with a pharmaceutically acceptable diluent and/or carrier. 
A polypeptide of the present invention includes one in which a part of its 
amino acid sequence is missing (e.g., a polypeptide containing only the 
sequence essential for biological activity from the amino acid sequence 
shown in SEQ ID NO:1), one in which a part of its amino acid sequence is 
replaced by other amino acids (e.g., those replaced by an amino acid 
having a similar property), and one in which other amino acids are added 
or inserted into a part of its amino acid sequence, as well as those 
having the amino acid sequence shown in SEQ ID NO:1. 
As is well-known, there are one to six codons that may encode an amino acid 
(for example, one codon for Methioine (Met), and six codons for leucine 
(Leu) are known). Accordingly, the DNA sequence can be changed, yet still 
encode the polypeptide having the same amino acid sequence. 
The DNA of the present invention, specified in (2) above includes the group 
of every nucleotide sequence encoding polypeptide (1) shown in SEQ ID 
NO:1. There is a probability that the production yield of a polypeptide 
can be improved by changing the nucleotide sequence. 
The DNA specified in (3) above, is an embodiment of the DNA shown in (2), 
and is the natural form of the sequence. 
The DNA shown in (4) above, indicates the sequence of the DNA specified in 
(3) having a non-translational region. 
The DNA of the present invention may be obtained by gene recombination, 
chemical synthesis or known methods by those skilled in the art. 
Human PD-1 includes a series of polypeptides that are different from the 
Fas antigen in structural features and occur commonly in mammals. That is, 
PD-1 of the present invention includes human PD-1 recited in the present 
invention and PD-1 of other mammals that have high homology (meaning an 
immunological equivalent that can be cross-reacted to human PD-1 antigen). 
The structural features of human PD-1 are as follows: 
Human PD-1 is predicted to be a membrane binding type protein consisting of 
288 amino acids. It contains two hydrophobic regions, one at the 
N-terminus and the other in the middle, which are likely to serve as a 
signal peptide and a transmembrane segment, respectively. 
Comparison of the N-terminal sequence of the PD-1 protein with typical 
signal peptide cleavage sites suggests that the signal peptide would be 
located from Met.sub.1 to Arg.sub.20. Thus, the predicted mature form of 
the PD-1 protein would contain 268 amino acids and consist of an 
extracellular domain (147 amino acids), a transmembrane region (27 amino 
acids) and a cytoplasmic domain (94 amino acids). Four potential 
N-glycosylation sites are found in the putative extracellular domain. 
Comparison of the amino acid sequence of the PD-1 protein with all 
sequences registered in the National Biomedical Research Foundation data 
base revealed that the extracellular domain of the PD-1 protein is 
homologous to some members of the immunoglobulin superfamily. 
Immunoglobulin domains have been classified into V, C1 and C2 sets based 
on conserved amino acid patterns and the number of antiparallel 
beta-strands. The 68 amino acid residues between two cysteine residues 
(Cys.sub.54 and Cys.sub.123) in PD-1 bear resemblance to a 
disulfide-linked immunoglobulin domain of the V-set sequences. In 
addition, all of the four amino acid residues characteristic of many V-set 
sequences are also conserved in PD-1 (Arg.sub.94, Phe.sub.95, Asp.sub.117 
and Gly.sub.119). 
The cytoplasmic domain of the predicted PD-1 protein contains a variant 
form of the consensus sequence 
(Asp/Glu-X8-Asp/Glu-X2-Tyr-X2-Leu/Ile-X7-Tyr-X2-Leu/Ile) found in the 
cytoplasmic tails of most of the polypeptides associated with antigen 
receptors and Fc receptors. It was recently shown that one signal unit of 
this consensus sequence is sufficient to transduce signals. 
It is thought that PD-1 of other mammals would be similar to human PD-1 in 
structural feature, whether or not the number or types of its amino acids 
would be different. 
DNA encoding human PD-1 of the present invention may be prepared by the 
following method. 
Once the nucleotide sequences shown in SEQ ID NOs:2 and 3 are determined, 
DNA of the present invention may be obtained by chemical synthesis, by the 
PCR method or by hybridization making use of a fragment of DNA of the 
present invention, as a probe. Furthermore, DNA of the present invention 
may be obtained in a desired amount by transforming a proper host with a 
DNA vector which has inserted therein a DNA of the present invention, 
followed by culturing the transformant. 
PD-1 polypeptides of the present invention (shown in SEQ ID NO:1) may be 
prepared by: 
(1) isolating and purifying from an organism or a cultured cell, 
(2) chemically synthesizing, or 
(3) using a biotechnological procedure, preferably, by the method described 
in (3). 
Examples of the expression systems that may be used when preparing a 
polypeptide by using a biotechnological procedure are, for example, the 
expression systems of bacteria, yeast, insects and mammalian cells. 
For example, expression in E. coli may be carried out by adding the 
initiation codon (ATG) to the 5' end of a DNA encoding a mature peptide, 
connecting the DNA thus obtained to the downstream end of a proper 
promoter (e.g., trp promoter, lac promoter, .lambda. PL promoter, T7 
promoter), and then inserting it into a vector (e.g., pBR322, pUC18, 
pUC19) that functions in an E. coli strain, to prepare an expression 
vector. 
Then, an E. coli strain (e.g., E. coli strain DH1, E. coli strain JM109, E. 
coli strain HB101) that is transformed with the expression vector thus 
obtained may be cultured in a proper medium to obtain the desired 
polypeptide. When a signal peptide of bacteria (e.g., signal peptide of 
pel B) is used, the desired polypeptide may be also be released in the 
periplasm. Furthermore, a fusion protein with another polypeptide may also 
be easily produced. 
Furthermore, expression in a mammalian cell may be carried out, for 
example, by inserting the total DNA encoding PD-1 downstream of a proper 
promoter (e.g., SV40 promoter, LTR promoter, metallothionein promoter) in 
a proper vector (e.g., retrovirus vector, papilloma virus vector, vaccinia 
virus vector, SV40 vector, etc.) to obtain an expression vector, and 
transforming a proper mammalian cell (e.g., monkey COS-7 cell, Chinese 
hamster CHO cell, mouse L cell) with the expression vector thus obtained, 
and then culturing the transformant in a proper medium to obtain a desired 
polypeptide in the culture medium. The polypeptide thus obtained may be 
isolated and purified by conventional biochemical methods. 
DNA encoding the PD-1 gene obtained by the present invention may be used as 
a probe for the isolation of PD-1 genes of other animals. 
cDNA having a nucleotide sequence shown in SEQ ID NO:3 may be prepared 
according to the following methods, that is: 
(i) isolating mRNA from a cell line which produces the polypeptide of the 
present invention (e.g., human esophageal cancer cell line), 
(ii) preparing first strand (single stranded DNA) from the mRNA thus 
obtained, followed by preparing a second strand (double stranded DNA) 
(synthesis of cDNA), 
(iii) inserting the cDNA thus obtained into a proper phage vector, 
(iv) transforming host cells with the recombinant DNA thus obtained 
(preparation of cDNA library), 
(v) screening the cDNA library thus obtained with cDNA of mouse PD-1 as a 
probe using plaque hybridization, 
(vi) preparing phage DNA from the positive clone obtained, subcloning the 
cDNA released into a plasmid vector, preparing a restriction enzyme map, 
and 
(vii) determining the sequence of each restriction enzyme fragment, and 
obtaining the full sequence of the complete length by combining them. 
Step (i) may be carried out in accordance with the method of Okayama et al 
(Methods in Enzymology, 154:3 (1987)) from a human cell line after 
stimulation by a proper stimulant (e.g., IL-1) or without stimulation. A 
cell which produces the polypeptide of the present invention is preferably 
the human cell line YTC3. 
Steps (ii), (iii) and (iv) are a series of steps for preparing a cDNA 
library, and may be carried out in accordance with the method of Gubler & 
Hoffman, (Gene, 25:263 (1983)) with a slight modification. As examples of 
the plasmid vector used in the step (iii), many plasmid vectors (e.g., 
pBR322, pBluescript II) and phage vectors (e.g., .lambda. gt10, .lambda. 
DASH II) are known, and phage vector .lambda. gt10 (43.3 kbp; Stratagene) 
may be preferably used. 
As the host cell used in step (iv), E. coli NM514 (Stratagene) may be 
preferably used. 
Steps (v) and (vi) may be carried out in accordance with the method 
described in Molecular Cloning (Sambrook et al, Cold Spring Harbor 
Laboratory Press (1989)). 
Step (vii) may be carried out in accordance with the method described in 
Molecular Cloning (Sambrook et al, supra). 
The sequencing in step (vii) may be carried out in accordance with the 
method of Maxam-Gilbert or the dideoxy termination method. 
It is necessary to determine whether or not the cDNA thus obtained codes 
for a complete or almost complete length. The confirmation may be carried 
out by a Northern analysis using said cDNA as a probe (see Molecular 
Cloning, supra). It is thought that a cDNA is almost full-length, if the 
length of the cDNA is almost the same as the length of the mRNA obtained 
in the hybridizing band. 
DNA or DNA fragments encoding the PD-1 gene may be used as a probe or 
primer for detection of a PD-1 gene and thereby, and may be utilized to 
investigate the relationship between said polypeptide and a protection 
mechanism in the living organism, an immunological function or a disease, 
like a tumor, or for the purpose of diagnosing diseases. 
The DNA of the present invention may be used as an important and essential 
template in preparing, by conventional gene recombination, the PD-1 
polypeptide, polypeptide fragment thereof or derivatives thereof that are 
expected to possess various uses. 
It is expected that the polypeptide, polypeptide fragments thereof or 
derived polypeptides thereof may be used for the treatment of infections, 
depression or acceleration of immunological functions or tumors. 
Further, polyclonal and monoclonal antibodies against the polypeptide or 
polypeptide fragments of the present invention can be prepared by 
conventional methods, and they can be used to quantitate said polypeptide 
in an organism, and thereby, may be used to investigate the relationship 
between said polypeptide and diseases, or for the purpose of diagnosing 
diseases. Said monoclonal antibody per se, a chimeric antibody against the 
human antibody, may be used as a treating agent. A polyclonal and 
monoclonal antibody thereof may be prepared by conventional methods by 
using the polypeptide or a fragment thereof as an antigen. 
The polypeptide of the present invention may be administered systemically 
or partially, usually by oral or parenteral administration, preferably 
orally, intravenously or intraventricularly. 
The dosage to be administered will vary depending upon age, body weight, 
symptoms, the desired therapeutic effect, the route of administration, and 
the duration of the treatment. In the human adult, the dosage per person 
per dose is generally between 100 .mu.g and 100 mg, by oral 
administration, up to several times per day, and between 10 .mu.g and 100 
mg, by parenteral administration up to several times per day. 
As mentioned above, the dose to be used depends upon various conditions. 
Therefore, there are cases in which dosages lower than or greater than the 
ranges specified above may be used. 
Administration of compounds of the present invention, may be as solid 
compositions, liquid compositions or other compositions for oral 
administration, or as injections, liniments or suppositories for 
parenteral administration. 
Solid compositions for oral administration include compressed tablets, 
pills, capsules, dispersible powders and granules. Capsules include soft 
capsules and hard capsules. 
In such compositions, one or more of the active compound(s) is or are 
admixed with at least one inert diluent (such as, lactose, mannitol, 
glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, 
polyvinylpyrrolidone, and magnesium metasilicate aluminate). The 
compositions may also comprise, as is normal practice, additional 
substances other than inert diluents, e.g., lubricating agents (such as, 
magnesium stearate), disintegrating agents (such as, cellulose calcium 
glycolate), stabilizing agents (such as, human serum albumin and lactose), 
and agents to assist dissolving (such as, arginine and asparaginic acid). 
The tablets or pills may, if desired, be coated with a film of gastric or 
enteric material (such as sugar, gelatin, hydroxypropyl cellulose or 
hydroxypropylmethyl cellulose phthalate), or be coated with more than two 
films. And further, coating may include containment within capsules of 
absorbable materials, such as gelatin. 
Liquid compositions for oral administration include 
pharmaceutically-acceptable emulsions, solutions, syrups and elixirs. In 
such compositions, one or more of the active compound(s) is or are 
contained in inert diluent(s) commonly used in the art (e.g., purified 
water, ethanol). Besides inert diluents, such compositions may also 
comprise adjuvants (such as, wetting agents and suspending agents), 
sweetening agents, flavoring agents, perfuming agents, and preserving 
agents. 
Other compositions for oral administration include spray compositions which 
may be prepared by known methods and which comprise one or more of the 
active compound(s). Spray compositions may comprise additional substances 
other than inert diluents, e.g., stabilizing agents (sodium sulfite), 
isotonic buffer (sodium chloride, sodium citrate and citric acid). For 
preparation of such spray compositions, for example, the method described 
in U.S. Pat. No. 2,868,691 or 3,095,355 (herein incorporated by reference 
in their entireties) may be used. 
Injections for parenteral administration include sterile aqueous or 
non-aqueous solutions, suspensions and emulsions. In such compositions, 
one or more active compound(s) is or are admixed with at least one inert 
aqueous diluent(s) (distilled water for injection, physiological salt 
solution) or inert non-aqueous diluents(s)(propylene glycol, polyethylene 
glycol, olive oil, ethanol, POLYSOLBATE 80.TM.). 
Injections may comprise additional agents other than inert diluents: e.g., 
preserving agents, wetting agents, emulsifying agents, dispersing agents, 
stabilizing agents (such as, human serum albumin and lactose), and 
assisting agents, such as agents to assist dissolving (arginine and 
asparaginic acid). 
They may be sterilized, for example, by filtration through a 
bacteria-retaining filter, by incorporation of sterilizing agents in the 
compositions or by irradiation. They may also be manufactured in the form 
of sterile solid compositions, for example, by freeze-drying, that can be 
dissolved in sterile water or some other sterile diluent for injection 
immediately before use. 
Other compositions for parenteral administration include liquids for 
external use, and endermic liniments (ointment), suppositories for rectal 
administration and pessaries which comprise one or more of the active 
compound(s) and may be prepared by known methods. 
The following examples are provided for illustrative purposes only, and are 
in no way intended to limit the scope of the present invention.

EXAMPLE 1 
Cell Culture 
Human cell lines (CESS, HPB-ALL, Jarkat, TC3, CCRF-CEM, JM and MOLT-4F) 
were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated 
fetal calf serum, 2.0 mM glutamine, 50 .mu.M 2-mercaptoethanol, 100 U/ml 
penicillin and 100 .mu.g/ml streptomycin. 
EXAMPLE 2 
Northern Blot Analysis 
Total RNA was prepared from the indicated cell lines by extraction 
according to the guanidium isothiocyanate method (see Molecular Cloning, 
supra), and poly (A)+RNA was isolated from total RNA by oligotex-dT.sup.30 
(Daiichi Chemical Co.). 3 .mu.g of poly (A)+ RNA was separated on a 1.2% 
formaldehyde-agarose gel, and transferred to a nylon membrane (Biodyne A, 
Japan Genetic). The filter was baked at 80.degree. C. for 2 hrs. Random 
priming was carried using to the EcoRI fragment (1 kb) containing the 
coding region of mouse PD-1, as a probe labeled with .sup.32 P. The 
specific activity of this probe was about 9.times.10.sup.8 d.p.m./.mu.g. 
Hybridization was carried out in 10.times.Denhardt's, 1.0M NaCl, 50 mM 
Tris (pH 7.5), 10 mM EDTA, 1.0% SDS and 1.0 mg/ml sonicated salmon sperm 
DNA at 65.degree. C. for 15 hrs. The filter was washed in 1.times.SSC, 
0.1% SDS at 65.degree. C. for 10 min. A hybridization signal (2.3 kb) was 
observed from the lymphocyte cell line YTC3 by autoradiography. 
EXAMPLE 3 
Construction of cDNA Library and Cloning of Human PD-1 cDNA 
The cDNA library was constructed with 5 .mu.g of poly (A)+RNA extracted 
from YTC3 cell lines by using Time Saver cDNA Synthesis Kit (Pharmacia). 
Synthesis of the first strand of cDNA was carried out with oligo dT 
primer. Double stranded cDNA, which was ligated to an EcoRI-NotI adapter, 
was cloned into .lambda. gt 10 vector, and packaged into phage (Gigapack 
II Gold, Stratagene). Phage was plated on a lawn of E. coli NM514. Phage 
DNA was transfected to duplicated library filters from each plate. The 
filters were baked at 80.degree. C. for 2 hrs and hybridized at 60.degree. 
C. for 15 hrs. The mouse PD-1 coding region (1 kb) excised with EcoRI from 
Bluescript SK plasmid vector (Stratagene) was used as a probe. The filter 
was washed with 1.times.SSC and 0.1% SDS at 60.degree. C. for 10 mins. 
51-Positive signals were observed from 1.2.times.10.sup.6 phages by 
autoradiography. These clones were purified. Further analysis was carried 
out, about 23 clones were picked, the longest cDNA insert observed was 2.1 
kb. This result coincided with the results of the Southern blot analysis. 
EXAMPLE 4 
Sequencing of DNA 
The cDNA inserts isolated from the human cDNA library were subcloned into 
Bluescript SK plasmid vectors (Stratagene), and sequenced by the 
dideoxynucleotide chain termination method (Sanger et al (1977)) using a 
modified T7 DNA polymerase (United States Biochemical) and 
[.alpha.-.sup.32 P]dCTP (3000 Ci/mmol, Amersham). The specific primer of 
Bluescript plasmid was used as a sequencing primer. Nucleotide sequencing 
was carried out by fully sequencing for both strands of the cDNA and the 
nucleotide sequence shown in SEQ ID NO:2 was obtained. The deduced peptide 
sequence (shown in SEQ ID NO:1) was determined from the nucleotide 
sequence. The total number of deduced amino acids is 288, which is the 
same as that of mouse PD-1. Homology between the two was found to be about 
60%. 
EXAMPLE 5 
Southern Blotting 
Genomic DNA was isolated from different kinds of animal cells by a 
conventional method (see Molecular Cloning, supra). DNAs were digested 
with EcoRI, BamHI or HindIII according to the 
manufacturer's-recommendation, and isolated by electrophoresis (100 V, 
0.8% agarose gel, TEA buffer). DNA fragments were washed with 0.25 N HCl 
for 10 min., denatured with 0.2 N NaOH/0.6M NaCl for 30 min., neutralized 
with 0.6M NaCl/0.2 M Tris (pH 7.5) for 1 hr., and transferred to nylon 
membranes (Bidyne A), which is standard Southern blot procedure. The 
filter were baked for 2 hrs. 
Random priming was carried with the EcoRI-StuI fragment (900 bp) containing 
the coding region of human PD-1, using .sup.32 P. The specific activity of 
this probe was about 9.times.108 d.p.m./.mu.g. Hybridization was carried 
out in 10.times.Denhardt's, 1.0M NaCl, 50 mM Tris (pH 7.5), 10 mM EDTA, 
1.0% SDS and 1.0 mg/ml sonicated salmon sperm DNA at 65.degree. C. for 10 
min. The filter was washed in 1.times.SSC, 0.1% SDS at 65.degree. C. for 
10 min. Only one band was detected by autoradiography, when the clone was 
cut with any enzyme. Thus, it was found that the human PD-1 gene exists as 
a single copy. 
Southern hybridization was carried out with genomic DNA of different kinds 
of animals under the same conditions (hybridization and washing) described 
in Example 2, using the EcoRI fragment (1 kb) containing the coding region 
of mouse PD-1 as a probe. Hybridization signals were detected only from 
genomic DNA of mouse and human, and were not detected from genomic DNA of 
Drosophila, Xenopus and rabbit. 
EXAMPLE 6 
Isolation of Genomic Clone of Human PD-1 
A genomic DNA library from an esophageal cancer cell line was constructed 
in the .lambda. DASH II vector via Sau3AI partial digestion and ligation 
into the BamHI site (obtained from Dr. Nishiyama, 1st Dept. of Pathology, 
School of Medicine, Kyoto University). The human PD-1 gene was isolated 
from this library by hybridization with the human PD-1, total cDNA was 
excised by EcoRI digestion from a Bluescript SK vector. The probe was 
labeled with .sup.32 P by random priming. Two positive clones was isolated 
and purified from 1.times.10.sup.6 phage plaques, digested by several 
restriction enzymes, and analyzed by Southern hybridization using the same 
probe. From CISS (chromosomal in situ suppression), it was found that the 
human PD-1 gene mapped on 2q37.3. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 288 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
MetGlnIleProGlnAlaProTrpProValValTrpAlaValLeuGln 
151015 
LeuGlyTrpArgProGlyTrpPheLeuAspSerProAspArgProTrp 
202530 
AsnProProThrPheSerProAlaLeuLeuValValThrGluGlyAsp 
354045 
AsnAlaThrPheThrCysSerPheSerAsnThrSerGluSerPheVal 
505560 
LeuAsnTrpTyrArgMetSerProSerAsnGlnThrAspLysLeuAla 
65707580 
AlaPheProGluAspArgSerGlnProGlyGlnAspCysArgPheArg 
859095 
ValThrGlnLeuProAsnGlyArgAspPheHisMetSerValValArg 
100105110 
AlaArgArgAsnAspSerGlyThrTyrLeuCysGlyAlaIleSerLeu 
115120125 
AlaProLysAlaGlnIleLysGluSerLeuArgAlaGluLeuArgVal 
130135140 
ThrGluArgArgAlaGluValProThrAlaHisProSerProSerPro 
145150155160 
ArgSerAlaGlyGlnPheGlnThrLeuValValGlyValValGlyGly 
165170175 
LeuLeuGlySerLeuValLeuLeuValTrpValLeuAlaValIleCys 
180185190 
SerArgAlaAlaArgGlyThrIleGlyAlaArgArgThrGlyGlnPro 
195200205 
LeuLysGluAspProSerAlaValProValPheSerValAspTyrGly 
210215220 
GluLeuAspPheGlnTrpArgGluLysThrProGluProProValPro 
225230235240 
CysValProGluGlnThrGluTyrAlaThrIleValPheProSerGly 
245250255 
MetGlyThrSerSerProAlaArgArgGlySerAlaAspGlyProArg 
260265270 
SerAlaGlnProLeuArgProGluAspGlyHisCysSerTrpProLeu 
275280285 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 864 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGG60 
CCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCC120 
CTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCG180 
GAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCC240 
GCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTG300 
CCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACC360 
TACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCA420 
GAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCC480 
AGGTCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGC540 
CTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATA600 
GGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCT660 
GTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCC720 
TGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTCCTAGCGGAATGGGCACCTCA780 
TCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAG840 
GATGGACACTGCTCTTGGCCCCTC864 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 921 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CACTCTGGTGGGGCTGCTCCAGGCATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGG60 
GCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGG120 
AACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTC180 
ACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCC240 
AGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGAC300 
TGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGG360 
GCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCG420 
CAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCC480 
ACAGCCCACCCCAGCCCCTCACCCAGGTCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGT540 
GTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGC600 
TCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGAC660 
CCCTCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAG720 
AAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTC780 
TTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGG840 
AGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTCTGACCGGCTTCC900 
TTGGCCACCAGTGTTCTGCAG921 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 921 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Homo sapiens 
(H) CELL LINE: YTC3 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 25..888 
(C) IDENTIFICATION METHOD: by similarity to some other pattern 
(ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- peptide 
(B) LOCATION: 25..84 
(C) IDENTIFICATION METHOD: by similarity with known sequence 
or to an established consensus 
(ix) FEATURE: 
(A) NAME/KEY: mat.sub.-- peptide 
(B) LOCATION: 85..888 
(C) IDENTIFICATION METHOD: by similarity with known sequence 
or to an established consensus 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CACTCTGGTGGGGCTGCTCCAGGCATGCAGATCCCACAGGCGCCCTGGCCA51 
MetGlnIleProGlnAlaProTrpPro 
20-15 
GTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTA99 
ValValTrpAlaValLeuGlnLeuGlyTrpArgProGlyTrpPheLeu 
10- 515 
GACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTG147 
AspSerProAspArgProTrpAsnProProThrPheSerProAlaLeu 
101520 
CTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCC195 
LeuValValThrGluGlyAspAsnAlaThrPheThrCysSerPheSer 
253035 
AACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGC243 
AsnThrSerGluSerPheValLeuAsnTrpTyrArgMetSerProSer 
404550 
AACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCC291 
AsnGlnThrAspLysLeuAlaAlaPheProGluAspArgSerGlnPro 
556065 
GGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGAC339 
GlyGlnAspCysArgPheArgValThrGlnLeuProAsnGlyArgAsp 
70758085 
TTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTAC387 
PheHisMetSerValValArgAlaArgArgAsnAspSerGlyThrTyr 
9095100 
CTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGC435 
LeuCysGlyAlaIleSerLeuAlaProLysAlaGlnIleLysGluSer 
105110115 
CTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACA483 
LeuArgAlaGluLeuArgValThrGluArgArgAlaGluValProThr 
120125130 
GCCCACCCCAGCCCCTCACCCAGGTCAGCCGGCCAGTTCCAAACCCTG531 
AlaHisProSerProSerProArgSerAlaGlyGlnPheGlnThrLeu 
135140145 
GTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTC579 
ValValGlyValValGlyGlyLeuLeuGlySerLeuValLeuLeuVal 
150155160165 
TGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGGA627 
TrpValLeuAlaValIleCysSerArgAlaAlaArgGlyThrIleGly 
170175180 
GCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCT675 
AlaArgArgThrGlyGlnProLeuLysGluAspProSerAlaValPro 
185190195 
GTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAG723 
ValPheSerValAspTyrGlyGluLeuAspPheGlnTrpArgGluLys 
200205210 
ACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCC771 
ThrProGluProProValProCysValProGluGlnThrGluTyrAla 
215220225 
ACCATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGG819 
ThrIleValPheProSerGlyMetGlyThrSerSerProAlaArgArg 
230235240245 
GGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGAT867 
GlySerAlaAspGlyProArgSerAlaGlnProLeuArgProGluAsp 
250255260 
GGACACTGCTCTTGGCCCCTCTGACCGGCTTCCTTGGCCACCAGTGTTCTG918 
GlyHisCysSerTrpProLeu 
265 
CAG921 
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