Note: Descriptions are shown in the official language in which they were submitted.
2051g7~
- 1 -
Our Ref.: IH-85
TITLE OF THE INVENTION
POLYPEPTIDE
BAC~CGROUND OF THE INVENTION
5 FIELD OF THE INVENTION-
The present invention is concerned with providing a
novel polypeptide which is a human TNF N-terminal mutein
and relates to the polypeptide itself, a pharmaceutical
composition containing the polypeptide, a process for
producing the polypeptide, a recombinant DNA sequence
encoding the polypeptide, a plasmid containing the
recombinant DNA sequence and a transformed microbial cell
having the plasmid.
DISCUSSION OF BACKGROUND
TNF (tumor necrosis factor alpha) is a
physiologically active compound, which was found to be
present in the serum of a mouse preliminarily transfected
with _acillus Calmette Guerin (BCG) and treated by
endotoxin, by Carswell et al. in 1975 (Proc. Natl. AcadO
Sci. USA, 72, 3666 (1975)). In 1984, cDNA of human TNF
was cloned by Pennica et al., and the entire primary
structure (amino acid sequence) of the human TNF protein
was clarified (Nature, 312, 724 (1984)). TNF has
specific antitumor effects such as a cytotoxic activity
against tumor cells and hemorrhagia necrosis or growth
inhibition against transplanted tumors. However, it has
been reported recently that side effects such as
205197~
- 2 -
hyperlipemia, hypotension or fever, are likely to result
from the administration of TNF. Accordingly, many
research and development effects are being made to obtain
a product superior in the activity as compared to TNF,
while exhibiting reduced side effects For example,
Japanese Vnexamined Patent Publications No. 40221/1986
(U.S. 4,650,674), No. 119692/1988 (U.S. 4,990,455) and
No. 277488/1989 (EP-A-340,333~ disclose deletion of a
certain specific amino acid residues in the human TNF
protein, or replacement of such an amino acid residues by
other amino acid residues or addition of other amino acid
residues, to provide a human TNF mutein.
However, it has not yet been possible to obtain a
human TNF mutein having fully satisfactory
pharmacological effects.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention
to provide a novel human TNF N-terminal mutein having
antitumor effects similar to human TNF or its mutein
while being free of side effects associated with TNF.
It is another object of the present invention to
provide a human TNF N-terminal mutein, having antitumor
effects similar to human TNF or a mutein thereof, which
is free of the side effect of promoting metastasis as
observed with human TNF or muteins thereof.
It is another object of the present invention to
provide a method for preparing such human TNF N-terminal
20~1~7~
muteins.
It is another object of the present invention to
provide pharmaceutical compositions containing such human
TNF N-terminal muteins.
It is another object of the present invention to
provide recombinant DNA sequences which encode such human
TNF N-terminal muteins.
It is another object of the present invention to
provide plasmids which contain such recombinant DNA
sequences.
It is another object of the present invention to
provide transformed microbial cells which have such
plasmids.
These and other objects, which will become apparent
during the following detailed description, have been
achieved by a polypeptide which is a tumor necrosis
factor polypeptide having an amino acid sequence
represented by the sequence from the 1st Ser to the 155th
Leu as shown by SEQ ID NO:l in the Sequence Listing, or a
mutein thereof, wherein the amino acid sequence of the
1st Ser to the 8th Asp of said SEQ ID NO:l or the
corresponding amino acid sequence of said mutein is
replaced by an amino acid sequence containing at least
one amino acid sequence of Arg-Gly-Asp and having from 3
to 16 amino acids.
The present inventors have found it possible to
obtain a novel human TNF N-terminal mutein polypeptide
2 ~ 7 ~
which has substantially the same antitumor activity as
human TNF or a mutein thereof but which does not
substantially promote tumor metastasis as observed with
human TNF or its mutein, by incorporating an amino acid
sequence of ~rg-Gly-Asp at a certain amino acid sequence
domain of the amino acid sequence of human TNE` or a
mutein thereof. The present invention has been
accomplished on the basis of this discovery.
RIEF DESCRIPTION OF THE DRAWINGS
10A more complete appreciation of the invention and
many of the attendant advantages thereof will be readily
obtained as the same becomes better understood by
reference to the following detailed description when
considered in connection with the accompanying drawings,
wherein
Figure 1 illustrates the process steps for cloning a
human TNF gene by ligation of synthetic oligonucleotides.
Figure 2 illustrates the process steps for cloning a
human TNF gene by ligation of synthetic oligonucleotides.
20Figure 3 illustrates the process steps for cloning an
expression plasmid vector.
Figure 4 illustrates the process steps for cloning a
human TNF expression vector.
Figure 5 illustrates the process steps for preparing
a human TNF mutein or N-terminal mutein gene by site
directed mutagenesis.
Figure 6 illustrates the process steps for preparing
20~197~
-- 5 --
a human TNF mutein or N-terminal mutein gene by site
directed mutagenesis.
Figure 7 illustrates the process steps for cloning a
human TNF N-terminal mutein expression vector by gene
recombination utilizing restriction endonuclease cleavage
sites.
Figure ~ illustrates the process steps for cloning a
human TNF N-terminal mutein expression vector by gene
recombination utilizing restriction endonuclease cleavage
sites.
Figure 9 illustrates the process steps for cloning an
expression plasmid vector.
Figure 10 illustrates the process steps for cloning
an expression plasmid vector.
Figure 11 illustrates the process steps for cloning
an expression plasmid vector.
Figure 12 illustrates the process steps for cloning a
human TNF N-terminal mutein expression vector by ~ene
recombination utilizing restriction endonuclease cleavage
sites.
Figure 13 illustrates the results of electrophoresis
of the purified samples of the human TNF polypeptide and
the human TNF N-terminal mutein polypeptide expressed by
Escherichia Coli.
Figure 14 illustrates the results of electrophoresis
of the purified samples of the human TNF N-terminal
mutein polypeptide expressed by Escherichia Coli.
2~975
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The tumor necrosis factor polypeptide having an amino
acid sequence represented by the sequence from the 1st
Ser to the 155th Leu as shown by SEQ ID NO:l in the
Sequence Listing represents human TNF.
In the present invention, the term "the mutein of
human TNF" and the term "human TNF or a mutein thereof"
include a mutein obtained by applying a single or
combined treatment of modifications such as addition,
deletion and replacement of one or more amino acids,
suitably to the amino acid sequence as shown by SEQ ID
NO:l in the Sequence Listing and having antitumor effects
similar to human TNF. Such muteins may be obtained by
recombinant DNA techniques. Accordingly, in the present
invention, the amino acid sequence of said mutein
corresponding to the amino acid sequence of from the 1st
Ser to the 8th Asp of SEQ ID NO:l, corresponds to the
amino acid sequence after modification, in a case where
the above amino acid sequence is modified in the mutein.
This modification includes deletion of one or more amino
acids and thus includes partial or entire deletion of the
amino acid sequence of from the 1st Ser to the 8th ASp.
Specifically, the human TNF mutein includes, for
example, the following, but the present invention is not
limited to such specific examples.
(1) As disclosed in Japanese Unexamined Patent
Publication No. 141999/1988 (U.S. 4,948,875):
2~97~
To the N-terminal, Val-Arg is added, and further,
29Arg-30Arg is changed to 29Asn-30Thr.
(2) As disclosed in Japanese Unexamined Patent
Publication No. 119692/1988 (U.S. 4,990,455):
(A) 32Asn is changed to 32Tyr, 32His 32Asp or 32Se
B) ll5Pro is changed to ll5Leu 115Ser llsAsp or
Gly .
(C) 1l7Tyr is changed to l17His.
(3) As disclosed in Japanese Unexamined Patent
Publication No. 277488/1989 (EP-A-340,333):
lSer to 3Asp are deleted, and Arg-Lys-Arg is added to
the N-terminal of 9Lys.
(4) As disclosed in Japanese Unexamined Patent
Publication No. 163094/1990 (WO-90-03395):
lSer to 3Asp are deleted, and Arg-Lys-Arg is added to
the N-terminal of 9Lys, and l54Ala is changed to l54Phe.
(5) As disclosed in Japanese Unexamined Patent
Publication No. 142493/1990 (WO-90-03395):
lSer to 3Asp are deleted, Arg-Lys-Arg is added to the
20 N-terminal of 9Lys, and l54Ala is changed to l54Trp.
(6) As disclosed in Japanese Unexamined Patent
Publication No. 270697/198~:
100Gln to l07Ala are deleted.
From lSer, from 2 to 8 amino acid residues are
deleted.
(7) As disclosed in Japanese Patent Application No.
193935/1990:
20~97~ `
68Pro is changed to 58Asp, 68Met or 68Tyr.
(8) As disclosed in Japanese Patent Application No.
311129/1990: l06Gly is deleted or replaced by another
amino acid.
~9) As disclosed in Japanese Patent Application No.
311130/1990: 29Arg is replaced by another amino acid.
Throughout the specification, symbols in the
following list are used to represent amino acids,
polypeptides, bases or their sequences.
Amino acids:
Symbols: Ala, Cys, Asp,
Meaning: alanine, cysteine, aspartic acid,
Symbols: Glu, Phe, Gly,
Meaning: glutamic acid, phenylalanine, glycine,
Symbols: His, Ile, Lys, Leu,
Meaning: histidine, isoleucine, Lysine, Leucine,
Symbols: Met, Asn, Pro,
Meaning: methionine, asparagine, proline,
Symbols: Gln, Arg, Ser, Thr,
Meaning: glutamine, arginine, serine, threonine,
Symbols: Val, Trp, Tyr
Meaning: valine, tryptophan, tyrosine
When used in a polypeptide sequence, these symbols
represent amino acid residues, rather than free amino
acids.
~051975
Bases:
Symbols: A, C, G, T, U
Meaning: adenine, cytosine, guanine, thymine, uracil
When used in a DNA sequence, these symbols represent
deoxyribonucleic acid residues rather than free molecules
such as adenosine phosphate or free bases such as
adenine.
Further, various abbreviations used in the
specification have the following meanings.
dATP: Deoxy adenosine triphosphate
dGTP: Deoxy guanosine triphosphate
dCTP: Deoxy cytidine triphosphate
TTP: Thymidine triphosphate
ATP: Adenosine triphosphate
SDS: Sodium dodecyl sulfate
BPB: Bromophenol Blue
DTT: Dithiothreitol
BSA: Bovine serum albumin
PMSF: Phenylmethylsulfonyl fluoride
EDTA: Ethylenediaminetetraacetic acid
CPG resin: Controlled-Pore Glass resin
In the polypeptide N-terminal mutein of the present
invention, the amino acid sequence containing at least
one amino acid sequence of Arg-Gly-Asp at a certain
position and from 3 to 16 amino acids, is optionally
determined taking the antitumor activity, the degree of
metastasis of cancer, the side effects, the applicability
205197~
-- 10 --
of the gene recombination technique, etc. into
consideration. The amino acid sequence containing from 3
to 16 amino acids, is preferably an amino acid sequence
containing from 3 to 11 amino acids, which has one or
more amino acid sequences of Arg-Gly-Asp. Specifically,
amino acid sequences shown by SEQ ID NOS:2 to 9 in the
Sequence Listing, and Arg-Gly-Asp may be mentioned.
Among them, SEQ ID NOS:2, 3, 5 and 6, and Arg-Gly-Asp are
particularly preferred.
The sequence of the present polypeptides, other than
the amino acid sequence replacing the sequence
corresponding to the 1st Ser to the 8th Asp, corresponds
substantially to the sequence from the 9th Lys to the
155th Leu in SEQ ID NO:l and may contain up to 15
additions, deletions or substitutions of amino acid
residues or combinations thereof. The sequence other
than the amino acid sequence containing at least one
amino acid sequence of Arg-Gly-Asp and from 3 to 16 amino
acids, of the polypeptide N-terminal mutein of the
present invention, is preferably an amino acid sequence
of human TNF represented by the sequence from the 9th Lys
to the 155th Leu of SEQ ID NO:l, or such an amino acid
sequence having the 29th Arg, the 68th Pro or the 106th
Gly deleted or replaced by another amino acid residue.
Such another amino acid residue is preferably Gln, Lys,
Asp, Val or Leu for the 29th Arg; it is preferably Asp or
Met for the 68th Pro; and it is preferably Trp, Pro, Ala,
205~975
-- 11 --
Asp or Arg for the 106th Gly.
Thus, the present polypeptide N-terminal mutein is
suitably from 135 to 178 amino acid residues long,
preferably 135 to 173 amino acid residues long.
The DNA for a tumor necrosis factor polypeptide
having a nucleotide sequence represented by the 1st T to
the 465th G as shown by SEQ ID NO:10 in the Sequence
Listing, is one form of DNA encoding the amino acid
sequence of human TNF.
The mutational DNA of a DNA encoding the amino acid
sequence of human TNF, is a DNA obtained by applying a
single or composite treatment of modifications such as
addition, deletion and replacement of one or more sets of
codons appropriately to the nucleotide sequence as shown
by SEQ ID NO:10 in the Sequence Listing and encoding the
amino acid sequence of the above-mentioned human TNF
mutein. Accordingly, such modifications are applicable
to the entire region of the nucleotide sequence
represented by the 1st T to the 465th G as shown by SEQ
ID NO:10. Namely, such modifications are applicable not
only to the nucleotide sequence of from the 1st T to the
24th C, but also to the nucleotide sequence of from the
25th A to the 465th G.
Now, the present invention will be described in
detail with reference to Examples. For the gene
manipulation involved in the present invention,
conventional methods and techniques disclosed in many
2~197~
references may be employed with suitable adjustments.
Some of such references will be listed below.
T. Maniatis et al., (1982): Molecular Cloninq, _
Laboratory Manual (hereinbelow, referred to simply as
Molecular Cloning), Cold Spring Harbor Laboratory, R. Wu
et al., (1983): Methods in Enzymoloqy, 100 and 101, R. Wu
et al., (1987): Methods in Enzymoloqyl 153, 154 and 155
The polypeptide of the present invention can be
produced by various methods, by means of various
techniques and equipments. A typical method for its
production will now be described.
(1) Designing of a human TNF gene
As mentioned above, the nucleotide sequence of a
human TNF gene and the amino acid sequence of the human
TNF have been clarified by Pennica et al., and the
nucleotide sequence is changed as the case requires to
design the human TNF gene as shown by SEQ ID NO:10. At
that time, it is preferred to select codons suitable for
the host cell (such as Escherichia coli), and it is
further preferred to provide restriction endonuclease
cleavage sites at appropriate positions to facilitate the
gene modification for the preparation of a mutein and to
facilitate the cloning by ligating DNA fragments as
described hereinafter. It is of course necessary to
provide a translation initiation codon (ATG) at the 5'-
side and a translation termination codon (TAA, TGA or
TAG) at the 3'-side of the human TNF gene respectively.
20~197~ -
- 13 -
Further, it is preferred to provide appropriate
restriction endonuclease cleavage sites upstream of the
translation initiation codon and downstream of the
translation termination codon, respectively, to improve
the applicability to a vector and to facilitate the
cloning.
Here, the preparation is based on the above-mentioned
DNA double-strands. The upper (coding or sense) strand
contains 474 bases (hereinafter referred to as a coding
474U strand) having 5'-ATG-3' joined in an upstream
direction to the 1st T in SEQ ID NO:10 and having 5'-
TAATGA-3' joined in a downstream direction to the 4b5th G
in SEQ ID NO:10. Accordingly, the lower strand
(hereinafter refered to as a complementary 474L strand)
contains 474 bases starting from T and ending at T, as
described below the upper strand, and is complementary to
the coding 474U strand.
The human TNF gene can be prepared by a method
wherein each of the upper and lower strands is divided
into a plurality of oligonucleotides, such a plurality of
oligonucleotides are chemically synthesized, and blocks
of such synthesiæed oligonucleotides are then
sequentially appropriately ligated. For example, in the
case of DNA double-strands prepared on the basis of the
DNA double-strands comprising the coding 474U strand and
the complementary 474L strand, each of the strands for
the human TNF gene is divided into ten oligonucleotides
2051975
each comprising about 50 bases, and a total of twenty
oligonucleotides are chemically synthesized.
As the method for such synthesis, a diester method
(H.G. Khorana, "Some Recent Developments in Chemistry of
Phosphate Esters of Biological Interest", John Wiley and
Sons, Inc., New York (1961)), a triester method (R. L.
Letsinqer et al., J. Am. Chem. Soc., 89, 4801 (1967)), or
a phosphite method (M. D. Matteucci et al., Tetrahedron
Lett., 21, 719 (1980)) may be mentioned. However, in
view of the operation efficiency, it is preferred to
employ a phosphite method using a completely automated
DNA synthesizer.
Synthesized oligonucleotides are then purified by a
conventional purification method such as high performance
chromatography using a reversed phase chromatocolumn or
electrophoresis using polyacrylamide gel. Thereafter,
the oligonucleotides are phosphorylated by means of,
e.g., T4 polynucleotide kinase, then annealed and ligated
by means of T4 DNA ligase. Here, the oligonucleotides
are divided into several blocks and sequentially ligated
so that the human TNF gene sequence will eventually be
obtained, followed by digestion with restriction
endonuclease or polishing (make blunt-end) with T4 DNA
polymerase, and the resulting DNA fragments are then
purified by electrophoresis. The obtained DNA fragments
are inserted into plasmid vectors such as pUC8, pUC9,
pUC18 and pUCl9 (J. Messinq et al., Gene, 19, 259
20~197S
(1982)), and the inserted plasmid vectors are introduced
into competent cells for cloning in accordance with a
conventional method. From the obtained clones, plasmid
DNAs are extracted and purified by a conventional method,
and they are examined to see whether or not the
nucleotide sequences of the DNA fragments inserted into
the vectors agree with the desired gene sequence. The
respective sections of the human TNF gene thus-obtained,
are then cut out from the plasmid vectors containing
them, by means of restriction endonucleases, then ligated
and inserted again into the above vector to obtain a
plasmid vector having the desired full length of the
human TNF gene. The plasmid vector thus-obtained is
digested by restriction endonucleases and separated and
purified by gel electrophoresis to obtain the desired
human TNF gene.
On the other hand, a method which comprises preparing
cDNA encoding human TNF from mRNA derived from human
cells expressing TNF and using such cDNA, may be used in
combination with the above described method, as the case
requires.
(2) Cloning of human TNF expression vector
The human TNF gene obtained in the above step (1) is
appropriately inserted into an expression vector to clone
a human TNF expression vector. The expression vector is
required to have a promoter and a SD (Shine-Dalgano)
sequence upstream of the translation initiation codon
20~197~
- 16 -
(ATG) and a terminator downstream of the translation
termination codon (TAA, TGA or TAG). As the promoter,
trp promoter, lac promoter, tac promoter, PL promoter, ~-
lactamase promoter, ~-amylase promoter, PH05 promoter, or
ADCI promoter may be suitably used, and as the
terminator, trp terminator, rrnB terminator or ADCI
terminator may be suitably used. Such an expression
vector is readily available among commercial products
such as pKK223-3 (Pharmacia), pPL-lambda (Pharmacia) and
pDR720 (Pharmacia). However, such commercial products
may further be improved for use to have better expression
properties or handling efficiency.
(3) Cloning of human TNF mutein or N-terminal mutein
expression vector
The following methods may, for example, be employed
for the preparation of DNA encoding the human TNF mutein
or N-terminal mutein polypeptide.
(A) In the same manner as the method described in the
above step (1) for designing the human TNF gene,
chemically synthesized oligonucleotides are appropriately
ligated to obtain such a mutant DNA. According to this
method, modification such as the replacement, addition or
deletion of a codon or codons encoding an amino acid, an
oligopeptide or a polypeptide, can freely be performed.
(B) q'he human TNF gene prepared in the above step (1)
is cut by appropriate restriction endonucleases to remove
a certain specific region in the gene, and then a
20~197~
- 17 -
synthetic oligonucleotide having a nucleotide sequence
with a mutation introduced (for example: a double-
stranded DNA fragment prepared by annealing the upper and
lower strands, and ligating them) or an appropriate other
gene is inserted. The modification can freely be
performed also by this method in the same manner as in
the above method (A).
(C) Introduction of a mutation is conducted by
extending the DNA strand by using, as a primer, a
synthesized oligonucleotide having a nucleotide sequence
with the mutation introduced [site directed mutagenesis
method (T. A. Kunkel et al., Methods in Enzymoloqy, 154,
367 (1987))]. This method is not suitable for addition
or insertion of a relatively long DNA chain exceeding ten
base pairs. However, other modifications can freely be
performed in the same manner as in the above method (A).
This method is suitable, particularly for replacing any
desired amino acids.
In the present invention, the site directed
mutagenesis method (C) and the method (B) of using the
fragments cut by restriction endonucleases, are usually
used in combination, as the case requires. Such a method
will be described below.
(i) By the site directed mutagenesis method, DNA
encoding a mutein or N-terminal mutein polypeptide is
prepared and suitably inserted into an expression vector.
Firstly, to conduct the site directed mutagenesis
205197~
- 18 -
method, template DNA is prepared. The human TNF gene
obtained in the above step (1) is ligated to a plasmid
vector (such as pUC118 or pUCll9) for the preparation of
a single-stranded plasmid DNA developed by Messinq et al.
(Methods in Enzymoloqy, 153, 3 (:Lg87)), and then
_
introduced into Escherichia coli. From the transformant
thereby obtained, a clone having the desired plasmid is
selected.
This plasmid is introduced into a dut- and ung~ E.
coli mutant (such as a CJ236 strain) to have uracil taken
in the gene, and the E. coli mutant is infected with a
recombinant helper phage such as M13K07 to obtain the
desired single stranded plasmid DNA.
On the other hand, an oligonucleotide primer with
from about 15 to 50 bases having the mutation
introduction site and the forward and backward base
sequences thereof according to the present invention, is
chemically synthesized. This primer and the uracil-
introduced single-stranded plasmid DNA obtained by the
previous step, are annealed and then double-stranded by
means of e.g. T4 DNA polymerase and T4 DNA ligase. The
double stranded plasmid is introduced into a ung+ E. coli
strain, to deactivate the DNA strand containing uracil as
a template and thereby to improve the frequency for
introduction of the mutation.
Using the above primer as a probe, colony
hybridization is conducted, whereupon a clone having a
20~197~
- 19 --
plasmid containing DNA encoding the desired mutein or N-
terminal mutein polypeptide, is selected from the
~btained transformants.
From the plasmid thus-obtained, a DNA fragment
encoding the human TNF mutein or N-terminal mutein
polypeptide, is cut out by restriction endonucleases and
inserted into an expression vector in the same manner as
in the case of the above step (2) to clone the desired
human TNF mutein or N-terminal mutein expression vector.
(ii) A section to which a mutation is to be
introduced, is cut out by suitable restriction
endonucleases and substituted by a DNA fragment prepared
by, e.g., chemical synthesis in accordance with the
modification design, to obtain an expression vector of
the mutein or N-terminal mutein polypeptide.
To modify the amino acid sequence in the vicinity of
the N-terminal of human TNF, it is preferred that there
exists suitable restriction endonuclease cleavage site in
the vicinity of the N-terminus. Therefore, a suitable
restriction endonuclease cleavage site is introduced near
the N-terminus of human TNF by the site directed
mutagenesis. It is further preferred to provide two
types of cleavage sites, i.e., this restriction
endonuclease cleavage site and a restriction endonuclease
cleavage site provided before or after the translation
initiation codon located upstream thereof.
On the other hand, in accordance with the
2 ~ 7 ~
- 2~ -
modification design of the present invention,
oligonucleoti.des corresponding to the upper (coding) and
lower (noncoding) strands are chemically synthesized in
the same manner as in the case oE the above step (1). It
is, of course, necessary to provide the above-mentioned
restriction endonuclease cleavage sites at both termini
in consideration oE the insertion into an expression
vector. Such oligonucleotides (the upper and lower
strands) are phosphorylated by means of, e.g., T4
polynucleotide kinase, followed by annealing, to obtain a
double-stranded DNA fragment. This double-stranded DNA
fragment is inserted and ligated by means of, e.g., T4
DNA ligase in the expression plasmid vector containing
the majority of human TNF digested by the above-mentioned
two types of restriction endonucleases (with the N-
terminal portion deleted), to obtain the desired
expression vector of the human TNF N-terminal mutein
having the amino acid sequence near the N-terminus
modified.
By using appropriate restriction endonuclease
cleavage sites in the gene encoding the mutein or N-
terminal mutein polypeptide, recombination among the
respective expression vectors may be conducted to produce
further expression vectors for novel mutein or N-terminal
mutein polypeptides.
Introduction of an expression vector into host cells
such as cells of a E. coli strain can be conducted by a
20~1~7S
- 21 -
conventional method, such as a method of employing
competent cells of an E. coli strain prepared by a
calcium chloride method (Molecular Cloninq, T. Maniatis
et al., (1982)). As such host cells, microbial cells
such as Escherichia coli, Bacillus or yeast can be used.
Among them, as Escherichia coli, mutants of a E. coli K-
12 strain such as JM83, JM103 and HB101, may be
mentioned.
(4) Preparation of human TNF N-terminal mutein
polypeptide
In the present invention, the transformed microbial
cells disclosed in the above step (3) are cultured,
whereby the desired human TNF N-terminal mutein
polypeptide will be produced and accumulated in the
culture, and the accumulated polypeptide is ther.
extracted and separated. As a method for culturing the
microbial cells, particularly cells of Escherichia coli,
a conventional method may be employed such as a method
wherein Escherichia coli is inoculated to a medium
containing nutrients requested by Escherichia coli, and
cultured in a large amount in a short period of time
usually by shaking or stirring the inoculated medium at a
temperature of from 32 to 37~C for from 12 to 24 hours.
As the medium, L-broth, M9 medium or M9CA medium (see the
above-mentioned Molecular Cloning) may, for example, be
used. If necessary, an antibiotic such as ampicillin may
be added, and in order to improve the efficiency of the
2~9~
- 22 -
promoter, it is possible to add a reagent such as
isopropyl-~-D-thiogalactopyranoside in the case where lac
promoter or tac promoter is used, or a reagent such as 3-
~-indole acrylic acid in the case where trp promoter is
used, at the initiation of culturing or during the
culturing.
The human TNF N-terminal mutein polypeptide of the
present invention is obtained usually by sonicating
microbial cells after culturing, in a suspended state in
Tris-buffer, followed by centrifugal separation to remove
debris. The product thus-obtained may further be
purified by treatment with nucleic acid endotoxin
removing agent, filtration by a filter, anion exchange
chromatography or any other conventional protein-
sepa~ation and purification method~
~ y applying the above-described methods
appropriately, the human TNF N-terminal mutein gene of
the present invention can be produced directly, or can be
produced after the preparation of the human TNF gene, or
can be produced after preparing the human TNF gene,
followed by preparation of its mutein gene.
The human TNF N-terminal mutein polypeptide of the
present invention has the same antitumor activity as
human TNF or a mutein thereof, while being free of the
metastasis-promoting side effect. ~hile it shows
antitumor activities at the same level as human TNF or
its mutein, it shows no substantial activity for
20~975
- 23 -
promoting metastasis of tumor which is observed ~ith
human TNF or its mutein. Therefore, it is effective as
an active ingredient for an antitumor agent or
pharmaceutical. Specific examples of the human TNF N-
terminal mutein polypeptide of the present inventioninclude, for example, F4168, F4415, F4416, F4417, F4418,
F4420, F4421, E`4113, F4137, F4601, F4602, F4607, F4608,
F4626, F4627, F4634, F4635, F4609, F4610, F4628, F4629,
F4638, F4639, F4611, F4612, F4613, F4614, F4615, F4642,
F4643, F4644, F4645 and F4646, which will be described
hereinafter. Among them, F4168, F4415, F4417, F4418,
F4420, F4601, F4609 and F4639 are preferred, and F4168
and F4418 are particularly preferred. To prepare its
pharmaceutical compositions containing the present
polypeptide, the present polypeptide can be formulated
into pharmaceutical compositions together with
pharmaceutically acceptable carriers or diluents. The
types of the pharmaceutical compositions of the present
invention include agents for external application, agents
for oral administration and agents for injection. The
pharmaceutical compositions are administered by
administration methods suitable for the respective
formulations.
As the administration method, injection is preferred.
The injection includes systemic injection and local
injection. The systemic injection includes intravenous
injection, subcutaneous injection, intramuscular
20~197~
- 24 -
injection and intradermal injection. The pharmaceutical
composition of the present invention may be applied to
any method. However, the system:ic injection is preferred
taking into consideration the applicability to various
cancer species. Further, Erom the nature of the drug,
intravenous injection is particularly preferred. The
cancer species include solid tumors such as colorectal
cancer, lung cancer, gastric cancer, pancreatic cancer
and melanoma, and leukemia. The pharmaceutical
composition of the present invention may be applied to
any cancer species. However, it is particularly
preferred to apply it to a solid tumor.
Having generally described the invention, a further
understanding can be obtained by reference to certain
specific examples, which are provided herein for purposes
of illustration only and are not intended to be limiting
unless otherwise specified.
EXAMPLES
EXAMPLE 1
Desiqninq of human TNF qene
On the basis of the amino acid sequence of the human
TNF structural gene already reported by Pennica et al. as
mentioned above, a nucleotide sequence of DNA double-
strands, i.e. coding 474U strand and complementary 474L
strand, in the DNA strand sequence of SEQ ID NO:10 in the
Sequential Listing and a nucleotide sequence of such DNA
double-strands with a certain modification, were designed
20~197~
- 25 -
for the convenience of gene cloning and mutein
preparation with respect to the nucleotide sequence of
the human TNF gene. Here, restriction endonuclease
cleavage sites were inserted at appropriate intervals.
Further, in order to facilitate the ligation to a plasmid
vector, a cleavage site by restriction endonuclease EcoR
I was provided upstream of a translation initiation codon
(ATG), and a cleavage site by restriction endonuclease
Hind III was provided downstream of a translation
termination codon (TAA and TGA).
EXAMPLE 2
Chemical synthesis of oliqonucleotides
The DNA designed in Example 1 was chemically
synthesized by a phosphite method by means of an
automatic DNA synthesizer (Model 381A, manufactured by
Applied Biosystems). After dividing the designed DNA
into twenty oligonucleotides of U-l to U-10 and L-l to L-
10 having nucleotide sequences as described hereinafter,
they were synthesized. Cleavage of the synthesized
oligonucleotides from CPG resin (sold by Funakoshi
Company) and removal of the protecting groups were
conducted in accordance with the Manual of Applied
Biosystems, Inc. The separation and purification of each
oligonucleotide was conducted by HPLC (high performance
liquid chromatography) using a reversed phase
chromatocolumn or by electrophoresis on a polyacrylamide
gel containing 7M urea (gel concentration: 10-20%).
20~97~
- 26 -
The nucleotide sequences of U-l to U-10 and L-l to L-
10 are as follows.
U-l: Comprises 27 bases and has a sequence of from
the 1st T to the l9th A of SEQ ID NO:10, and yet has a
sequence in which 5'-ATG-3' is joined upstream of the 1st
T and 5'-AATTC-3' is joined upstream thereof.
U-2: Comprises 50 bases and has a sequence of from
the 20th G to the 69th G of SEQ ID NO:10.
U-3: Comprises 49 bases and has a sequence of from
the 70th C to the 118th G of SEQ ID NO:10.
U-4: Comprises 50 bases and has a sequence of from
the ll9th A to the 168th C of SEQ ID NO:10.
U-5: Comprises 50 bases and has a sequence of from
the 169th T to the 218th T of SEQ ID NO:10.
U-6: Comprises 52 bases and has a sequence of from
the 219th C to the 270th C of SEQ ID NO:10.
U-7: Comprises 48 bases and has a sequence of from
the 271st C to the 318th C of SEQ ID NO:10.
U-8: Comprises 49 bases and has a sequence of from
the 319th G to the 367th C of SEQ ID NO:10.
U-9: Comprises 51 bases and has a sequence of from
the 368th A to the 418th C of SEQ ID NO:10.
U-10: Comprises 53 bases and has a sequence of from
the 419th T to the 465th G of SEQ ID NO:10, and further
has 5'-TAATGA-3' downstream thereof.
L-l to L-10 are for the lower strand (complementary
sequence) corresponding to the DNA strand of SEQ ID
20~L97~
- 27 -
NO: 10 .
L-l: Comprises 29 bases and has a sequence
complementary to the sequence of from the 25th A to the
1st T of SEQ ID NO:10, and yet has a sequence having 5'-
CATG-3' joined to the 3'-side of A complementary to the
1st T, of the upper strand.
L-2: Comprises 52 bases and has a sequence
complementary to the sequence of from the 77th G to the
26th A of SEQ ID NO:10.
L-3: Comprises 50 bases and has a sequence
complementary to the sequence of from the 127th G to the
78th G of SEQ ID NO:10.
L-4: Comprises 50 bases and has a sequence
complementary to the sequence of from the 177th G to the
128th A of SEQ ID NO:10.
L-5: Comprises 49 bases and has a sequence
complementary to the sequence of from the 226th C to the
178th G of SEQ ID NO:10.
L-6: Comprises 49 bases and has a sequence
complementary to the sequence of from the 275th T to the
227th A of SEQ ID NO:10.
L-7: Comprises 51 bases and has a sequence
complementary to the sequence of from the 326th C to the
276th C of SEQ ID NO:10.
L-8: Comprises 49 bases and has a sequence
complementary to the sequence of from the 375th G to the
327th C of SEQ ID NO:10.
20~75
- 28 -
L-9: Comprises 51 bases and has a sequence
complementary to the sequence of from the 426th T to the
376th A of SEQ ID NO:10.
L-10: Comprises 49 bases and has a sequence
complementary to the sequence of from the 465th G to the
427th G of SEQ ID NO:10, and yet has a sequence having
5'-TCATTA-3' joined to the 5'-side of C complementary to
the 465th G and having 5'-AGCT-3' joined to the 5'-side
thereof.
With respect to the HPLC methodr separation and
purification were conducted by eluting with an
acetonitrile-containing triethylamino acetic acid (100
mM) buffer (pH 7.0) by means of reversed chromatography
using a Nucleosil 5C18 column (~4.6 x 150 mm, sold by
Chemco Scientific Co., Ltd.). The elution was conducted
at a linear concentration gradient of acetonitrile of
from 5 to 35% (30 minutes), and a peak of about 15
minutes was recovered.
With respect to the polyacrylamide gel
electrophoresis, the respective synthesized
oligonucleotide samples were separated by
electrophoresis, and the band portion having the desired
size was cut out as a result of the observation of the
migration pattern by a UV-shadowing method, and the
polyacrylamide gel fragment was cut into a size of about
1 to 2 mm3, and about 2 me of an eluting buffer (0.5M
NH40Ac and 1 mM EDTA) was added, and the mixture was
20~97~
- 29 -
shaken overnight at 37C. The eluted buffer solutions
containing the respective oligonucleotides were
recovered, followed by phenol extraction (using a 50%
phenol/50% chloroform solution) and isobutanol
extraction, and ethanol precipitation operation to obtain
purified samples of the respective oligonucleotides.
With respect to some of the oligonucleotides thus-
synthesized and purified, it was confirmed by a Maxam-
Gilbert method (A. M. Maxam et al, Methods in Enzymoloqy,
65, 499 (1980)) that they had the desired nucleotide
sequences.
In the following gene recombination operations
(Examples 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12), the
reaction conditions, etc. of restriction endonucleases
and other related enzymes were mainly in accordance with
the method disclosed in the above-mentioned "Molecular
Cloning". Further, the above enæymes, etc. were
available mainly from I'akara Shuzo, and the manual of
Takara Shuzo was also used as a reference.
EXAMPLE 3
Cloninq of a human TNF qene by liqation of sYnthesized
oliqonucleotides
(1) Firstly, cloning of a human TNF gene was tried in
accordance with Figure 1. The synthesized
oligonucleotides obtained in Example 2 were divided into
three groups (U- and L-l to 4, U- and L-5 to 7 and U- and
L-8 to 10) for cloning. Namely, the 5'-terminus of each
2 ~ 7 5
- 30 -
(1-2 ~g) of oligonucleotides U-2, 3, 4, 6, 7, 9 and 10
and L-l, 2, 3, 5, 6, 8 and 9 was separately
phosphorylated by means o~ from 2 to 5 units of T4
polynucleotide kinase (Takara Shuzo). The
phosphorylation reaction was conducted at 37C for one
hour in 10 ~ll of an aqueous solution (50 mM Tris-HCl pH
7.6, 10 mM MgCl2, 0.1 mM Spermidine, 0.1 mM ~DTA, 10 mM
DTT and 1 mM ATP), and after the reaction, T4
polynucleotide kinase was deactivated by treatment at
70C for 10 minutes. Separately, 10 ~1 of aqueous
solutions of the same compositions as above which
contained from 1 to 2 ~g of oligonucleotides U-l, 5 and 8
and L-4, 7 and 10, respectively, were prepared. The
aqueous solutions of oligonucleotides with the same U-
and L-numbers (U-l to 10 and L-l to 10) were mixed,
respectively (20 ~1), and the respective mixtures were
boiled at 100C for 5 minutes, followed by gradual
cooling for annealing. Then, the obtained ten annealed
fragments (double stranded DNA fragments) were divided
into the above groups, and the fragments in each group
were added to an aqueous solution for a ligation reaction
(66 mM Tris-HCl pH 7.6, 6.S mM MgCl2, 10 mM DTT, 1 mM ATP
and 100 ~g/ml BSA) ~total amount: 120-160 ~1), and the
solution was heated to 40C and then gradually cooled for
annealing. Then, 700 units of T4 DNA ligase (Takara
Shuzo) was added thereto, and the ligation reaction was
conducted at 16C for 15 hours.
20~1~75
After completion of the reaction, the respective
reaction solutions were separated by polyacrylamide gel
electrophoresis (gel concentration: 6%). From the
results of observing the migration patterns by the
Ethidium Bromide Staining method, the band portions
having the desired sizes (176 bp, 150 bp and 153 bp) were
cut out, and the desired three DNA fragments were
recovered by an electro-elution method. Further, the
recovered samples were subjected to phenol extraction
(using a 50% phenol/50% chloroform solution) and
isobutanol extraction, followed by an ethanol
precipitation operation to purify the desired DNAs. The
three double stranded DNA fragments thus-purified, in
accordance with the above-mentioned method, were
phosphorylated respectively at their 5'-termini by means
of T4 polynucleotide kinase, and then they were mixed in
an aqueous solution for a ligation reaction, then
annealed at 40C and ligated by an addition of T4 DNA
ligase. The ligated DNA was recovered by an ethanol
precipitation operation and then dissolved in 50 ~1 of a
high salt buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10
mM MgCl2) containing 1 mM of DTT and 100 ~g/ml of BSA.
Then, a digestion reaction was conducted at 37C for two
hours by an addition of 15 units of restriction
endonuclease EcoR I (Takara Shuzo) and 15 units of
restriction endonuclease Hind III (Takara Shuzo). After
completion of the reaction, the desired DNA fragment
20~197~
(about 480 bp) was separated and purified by
polyacrylamide gel electrophoresis (gel concentration:
4%) in accordance with the above-mentioned method.
On the other hand, 5 ~g of plasmid vector pUC9
(obtained from Research Laboratory for Genetic
Information of Kyushu University) was digested with
restriction endonuclease EcoR I and Hind III in
accordance with the above method, and a DNA fragment of
about 2.7 Kbp was separated and purified by agarose gel
electrophoresis (gel concentration: 1%). This pUC9
fragment and the previously purified DNA fragment of
about 480 bp (containing the human TNF gene) were mixed
in 20 ~1 of a ligation reaction solution, and a ligation
reaction was conducted at 16C for 3 hours by an addition
of 350 units of T4 DNA ligase. Competent cells of E.
coli K-12 JM83 strain (obtained from Research Laboratory
for Genetic Information of Kyushu University) prepared by
a calcium chloride method (see Molecular Cloninq) were
transformed with the above-mentioned ligation reaction
mixture in accordance with a conventional method (see
Molecular Cloninq).
From the ampicillin resistant clone thus-obtained, a
plasmid was prepared by a conventional method, and it was
treated with restriction endonucleases (EcoR I and Hind
III) in accordance with the above-mentioned method, and
then its migration pattern was analyzed by agarose gel
electrophoresis to examine the insertion of the human TNF
205197~
- 33 -
gene into the pUC9 vector. As a result, insertion of the
gene of about 250 bp was confirmed. With respect to the
clone, the nucleotide sequence of the inserted gene was
examined by a dideoxy method (F. Sanqer, Science, 214,
1205 (1981)), whereby it was confirmed to be a gene
fragment having the nucleotide sequence of the desired
human TNF gene with a size of about 130 bp downstream
from the EcoR I site and a size of about 90 bp upstream
from the Hind III site. The clone and the plasmid were
designated as pUA41/JM83 and pUA41, respectively.
(2) Then, cloning of a human TNF gene was tried in
accordance with Figure 2.
In the nucleotide sequence of the human TNF gene, the
region which was not accomplished by the cloning in the
above step (1), was divided into two groups (U- and L-3
to 6 and U- and L-6 to 9), and in the same manner as in
the above step (1), the oligonucleotides U-4 to 6 and U-7
to 9 and L-3 to 5 and L-6 to 8 were 5'-phosphorylated,
then the two groups were annealed and ligated by means of
T4 DNA ligase. The products were separated and purified
by polyacrylamide gel electrophoresis (gel concentration:
6%) in accordance with the above-mentioned method, and
two DNA fragments thereby obtained (201 bp and 200 bp)
were respectively dissolved in 100 yl of aqueous
solutions containing 67 mM of Tris-HCl (pH 8.8), 6.7 mM
of MgCl2, 16.6 mM of (NH4)2SO4, 6.7 ~M of EDTA, 1 mM of
DTT, 200 ~g/ml of BSA and 330 ~M of each of
2 ~ 7 5
- 34 -
deoxyribonucleotide triphosphates (dATP, dGTP, dCTP and
TTP), and from 2 to 5 units of T4 DNA polymerase (Takara
Shuzo) was added. The mixture was reacted at 37C for 30
minutes to polish both termini of the DNA fragments.
After completion of the reaction, the mixture was treated
at 68C for 10 minutes to deactivate the T4 DNA
polymerase, and the desired two DNA fragments were
recovered by precipitation with ethanol.
On the other hand, 5 ~g of pUC9 vector was dissolved
in 50 ~1 of a medium salt buffer (10 mM Tris-HCl pH 7.5,
50 mM NaCl and 10 mM MgCl2) containing 1 mM of DTT and
100 ~g/ml of BSA, and 15 units of restriction
endonuclease Hinc II (Takara Shuzo) was added thereto.
The mixture was reacted at 37C for two hours, and then
the vector was recovered by precipitation with ethanol.
Into the cut and ring-opened pUC9 vector thus-obtained,
the previously polished and recovered two DNA fragments
were, respectively, inserted by means of T4 DNA ligase in
accordance with the above-mentioned method, whereby the
E. coli K-12 JM83 strain was transformed. With respect
to the respective clones thereby obtained, the nucleotide
sequences of the inserted DNA were examined in the same
manner as in the above step (1) to confirm that they were
the desired nucleotide sequences. These clones were
designated as pUA42/JM83 and pUA43/JM83, respectively,
and the plasmids were designated as pUA42 and pUA43,
respectively.
2 ~ 7 ~
- 35 -
The pUA41 obtained in the above step (1) was digested
with restriction endonuclease RcoR I (high salt buffer)
and Sac I (low salt buffer, manufactured by Takara Shuzo)
and restriction endonuclease Hae II (Takara Shuzo) and
Hind III (medium salt buffer); the pUA42 obtained in the
above step (2) was digested with restriction endonuclease
Sac I (low salt buffer) and Hpa I (KCl buffer,
manufactured by Takara Shuzo); and the pUA43 was digested
with restriction endonuclease Hpa I and Hae II (KCl
buffer), respectively, in accordance with the above-
mentioned method. With respect to the combination of the
low salt buffer (10 mM Tris-HCl pH 7. 5 and 10 mM MgCl2)
and the high salt buffer or the KCl buffer ( 20 mM Tris-
HCl pH 8.5, 100 mM KCl and 10 mM MgC12), the digestion
reaction was conducted twice separately, and the
precipitation operation with ethanol was conducted in
between the two reactions. EcoR I-Sac I DNA fragment
(127 bp) and Hae II-Hind III DNA fragment (80 bp) from
the pUA41, Sac I-Hpa I DNA fragment (147 bp) from the
pUA42 and Hpa I-~ae II DNA fragment (126 bp) from the
pUA43 were, respectively, separated and purified by
polyacrylamide gel electrophoresis (gel concentration:
6%) in accordance with the above-mentioned method.
On the other hand, 5 ~g of a pUCl9 plasmid vector
(obtained from Research Laboratory for Genetic
Information of Kyushu University) was digested with
restriction endonuclease EcoR I and Hind III in
20~197~
- 36 -
accordance with the above-mentioned method, and a DNA
fragment of about 2.7 Kbp was separated and purified by
agarose gel electrophoresis (gel concentration: 1%). As
illustrated (Figure 2), the previously purified four DNA
fragments were sequentially added and ligated thereto by
means of T4 DNA ligase in accordance with the above-
mentioned method, and finally inserted into the above-
purified pUCl9 vector (fragment of about 2.7 Kbp),
whereby the JM83 strain was transformed. With respect to
the plasmid contained in this transformant, the
nucleotide sequence of the inserted gene was examined in
accordance with the above-mentioned method, whereby it
was confirmed to be a clone having a plasmid vector
(about 3.2 Kbp) containing the desired human TNF gene of
full length (about 480 bp). This clone was designated as
pUA44/JM83, and the plasmid was designated as pUA44.
EXAMPLE 4
Clonin~ of a human TNF exPression vector
(1) The following improvements were applied to
expression vector pKK 223-3 (obtained from Pharmacia)
having a tac promoter so that the vector can more easily
be handled.
(A) To lower the molecular weight of the expression
vector.
(B) To make the cleavage site by restriction
endonuclease Bam~ I unique.
(C) To make the direction of the tac promoter
20~197~
opposite to the direction of the ampicillin resistant
gene.
The method is illustrated in Figure 3.
5 ~g of plasmid vector pBR 322 (obtained from
Research Laboratory for Genetic Information of Kyushu
University) was digested with restriction endonuclease
EcoR I and Hind III in accordance with the method of
Example 3, and both termini thereof were polished by
means of T4 DNA polymerase. In accordance with the
method of Example 3, a DNA fragment of about 4.4 Kbp was
separated and purified by agarose gel electrophoresis
(gel concentration: 1%). Then, lO0 ng of
nonphosphorylated Bgl II linker (a double-stranded DNA
fragment of lO bp containing a restriction endonuclease
Bgl II cleavage site, obtained from Takara ~huzo,
Catalogue No. 4721A, Takara Biotechnology Catalogue l991
Vol. l) was inserted and ligated to the ring-opened site
thereof by means of T4 DNA ligase. The plasmid contained
in the transformant obtained in the same manner as in
Example 3 was examined by the digestion with the
restriction endonucleases, whereby it was confirmed that
a clone having the desired plasmid pBR 9333 (about 4.4
Kbp) having the restriction endonuclease EcoR I and Hind
III cleavage sites deleted and having a restriction
endonuclease Bgl II cleavage site newly inserted, was
obtained.
Then, 5 ~g of plasmid pBR 9333 thus-obtained, was
2051~7~
- 38 -
dissolved in a high salt buffer, in accordance with the
method of Example 3, and digested with restriction
endonuclease Bgl II (Takara Shuzo) and Pvu II (Takara
Shuzo). Then, a DNA fragment of about 2.3 Kbp containing
a replication origin was separated and purified by
agarose gel electrophoresis (gel concentration: 1%). On
the other hand, 5 ~9 of expression vector pKK 223-3
(about 4.6 Kbp) was dissolved in a high salt buffer in
the same manner as above and digested with restriction
endonuclease BamH I (Takara Shuzo) and Sca I (Takara
Shuzo). The digestion reaction with the restriction
endonuclease BamH I was conducted by partial digestion
for a reaction time of from 5 to 30 minutes by reducing
the amount of the added enzyme to a level of about 1/2 of
a usual amount. After the digestion treatment, the
obtained DNA fragment of about 1.1 Kbp containing tac
promoter and rrnBTlT2 terminator was separated and
purified by agarose gel electrophoresis in the same
manner as above. The DNA fragment of about 1.1 Kbp was
inserted and ligated to the previously purified DNA
fragment of about 2.3 Kbp containing a replication
origin, by means of T4 DNA ligase in the same manner as
described above. The ligated DNA was introduced into
competent cells of an E. coli K-12 JM103 strain (obtained
from Research Laboratory for Genetic Information of
Kyushu University) prepared by a calcium chloride method,
in accordance with the method of Example 3. From the
2Q~7~
- 39 -
transformants thus--obtained, a clone having the desired
expression vector (about 3.4 Kbp) containing tac promoter
was selected, and this expression vector was designated
as pKK 101.
(2) Referring to Figure 4, the subsequent step will
be described.
Five ~lg of the expression vector pKK 101 obtained in
the above step (1) was digested with restriction
endonuclease EcoR I and Hind III, in accordance with the
above-mentioned method, and a DNA fragment of about 3.4
Kbp containing a replication origin and a transcriptional
regulation region, was separated and purified by agarose
gel electrophoresis (gel concentration: 1~). Likewise,
the plasmid pUA44 (about 3.2 Kbp) containing the human
TNF gene obtained in Example 3 was digested with
restriction endonuclease EcoR I and Hind III, whereupon a
DNA fragment of about 480 bp containing the entire region
of a human TNF gene was separated and purified by agarose
gel electrophoresis. This DNA fragment containing the
entire region of a human TNF gene was inserted and
ligated to the DNA fragment of about 3.4 Kbp previously
purified from the expression vector pKK 101, by means of
T4 DNA ligase in accordance with the above-mentioned
method. The ligated DNA was introduced into an E. coli
K-12 JM103 strain in accordance with the above-mentioned
method. From the transformants thus-obtained, a clone
having the desired human TNF expression vector (about 3.9
2~S197~
- 40 -
Kbp) was selected, and this expression vector was
designated as pKF 4102.
EXAMPLE 5
Cloninq of human TNF N-terminal mutein expression vector
pKF 4168
(1) The operation will be described with reference to
Figure 5.
The plasmid pUA44 obtained in Example 3 was digested
with restriction endonuclease EcoR I and Hind III, in
accordance with the above-mentioned method, and a DNA
fragment of the human TNF gene (about 480 bp containing
the entire region) was separated and purified by agarose
gel electrophoresis. On the other hand, plasmid vector
pUCll9 (obtained from Takara Shuzo) for the preparation
of a single-stranded plasmid DNA developed by Messinq et
al. (Methods in Enzymoloqy, 153, 3 (1987)) was likewise
digested with restriction endonuclease EcoR I and Hind
III, and a DNA fragment of about 3.2 Kbp containing an
intergenic (IG) region was separated and purified by
agarose gel electrophoresis. By the presence of this IG
region (intergenic region of M13 phage DNA), the plasmid
pUCll9 will, after being infected to a helper phage
M13K07, preferentially become a single-stranded DNA and
will be enclosed by phage particles and will be
discharged out of the microbial cells. The DNA fragment
of about 480 bp ,purified as above and containing the
entire region of the human TNF gene and the pUCll9
2~51~7~
- 41 -
fragment of about 3.2 Kbp containing the IG region were
ligated by means of T4 DNA ligase in accordance with the
above-mentioned method, and the ligated DNA was
introduced into an E. coli K-12 JM83 strain in accordance
with the method of Example 3. From the transformants
thus-obtained, a clone having the desired plasmid (about
3.7 Kbp) was selected. This clone was designated as
pUCll9-hTNF/JM83, and the plasmid was designated as
pUCll9-hTNF.
The plasmid pUC119-hTNF thus-obtained was introduced
into competent cells of an E. coli CJ236 strain ~dut ,
ung~) prepared by a calcium chloride method, in
accordance with the method of Example 3, to incorporate
uracil into the DNA of the plasmid. The CJ236 strain has
a mutation (dut ) in the enzyme dUTPase (deoxyuridine
triphosphate-phosphatase) gene, and a competing reaction
is thereby caused, whereby it is possible to produce a
DNA having uracil partially incorporated instead of
thymine. Further, due to the ung~ mutation, it lacks the
enzyme uracil N-glycosylase, whereby it is possible to
maintain uracil in the DNA. Such CJ236 strain was
obtained from Bio-Rad. The clone (pUCll9-hTNF/CJ236)
obtained by such introduction was, after infected with
helper phage M13K07 (obtained from Takara Shuzo),
cultured in a 2 x YT broth (1.6% tryptone, 1% yeast
extract and 0.5% NaCl pH 7.6) containing 100 ~g/ml of
ampicillin, 70 ~g/ml of Kanamycin and 30 ~g/ml of
2 ~ 7 ~
- 42 -
chloramphenicol, whereby the des:ired single-stranded
plasmid DNA (about 3.7 K bases) having uracil introduced
was discharged out of the cells in a form enclosed by
phage particles. The discharged phage particles were
recovered from the supernatant of the broth, and the
desired single-stranded plasmid DNA was prepared in
accordance with a method for preparing a single-stranded
phage DNA.
(2) The operation will be described with reference to
Figure 6.
Primer 4168 was designed to introduce a mutation to
the human TNF gene using a coding strand oligonucleotide.
Primer 4168 is an oligonucleotide comprising 12 bases
with a nucleotide sequence of from the 10th C to the 21st
T as shown by SEQ ID NO:10 wherein the 13-15th 5'-ACC-3'
is replaced by 5'-GGC-3' and the 16-18th 5'~CCG-3' is
replaced by 5'-GAT-3'.
The chemical synthesis and purification of this
oligonucleotide was conducted in accordance with the
method of Example 2.
The site-directed mutagenesis of the human TNF gene
was conducted in accordance with the Bio-Rad system
(Muta-GeneTM in vitro mutagenesis kit). Namely, about
0.5 ~g of the primer prepared as above and having the 5'-
terminus phosphorylated with T4 polynucleotide kinase inaccordance with the above-mentioned method and about 200
ng of the previously prepared uracil-introduced single-
20~1~7~
- 43 -
stranded plasmid DNA (pUCll9-hTNF) were annealed in 10 ~1
of an annealing buffer (20 mM Tris-HCl pH 7.4, 2 mM MgCl2
and 50 mM NaCl) (i.e. heated at 70C followed by gradual
cooling). After completion of the annealing, a 1/10
volume of 10 x synthesis buffer (5 mM deoxyribonucleotide
triphosphates (dATP, dGTP, dCTP and TTP), 10 mM ATP, 100
mM Tris-HCl pH 7.4, 50 mM MgCl2 and 20 mM DTT) was added
thereto, and a double-stranding reaction (37C, 90
minutes) was conducted by means of 1 unit of T4 DNA
polymerase and from 2 to 4 units of T4 DNA ligase. About
8 volumes of TE buffer (10 mM Tris-HCl pH 7.5 and 1 mM
EDTA) was added and the mixture was freezed to stop the
reaction. This reaction mixture was applied to competent
cells of the E. coli K-12 TGl strain (ung+, obtained from
Amersham) prepared by a calcium chloride method, in
accordance with the method of Example 3, to introduce the
double-stranded DNA.
By the introduction of the hetero double-stranded DNA
into the ung+ strain, the uracil-containing DNA strand as
the template was deactivated and not replicated.
(Therefore, the mutation frequency becomes as high as
more than 50%.) From the transformants thus-obtained, a
clone having a plasmid (about 3.7 Kbp) containing the
desired mutein DNA was selected by means of colony
hybridization method employing as a probe the primer used
for the introduction of mutation. With respect to the
selected clone, the nucleotide sequence around the
20~1975
mutation-introduced site of the plasmid was examined by
the dideoxy method (F. Sanger: as mentioned above) to
confirm that it was modified to the mutein DNA as
designed. This plasmid was designated as pUCll9-F4168.
(3) The desired human TNF N-terminal mutein gene
obtained by the introduction of mutation was inserted
into expression vector pKK 101 having tac promoter in
accordance with the method of cloning the human TNF
expression vector of Example 4, to obtain a human TNF N-
terminal mutein expression vector. The human TNF N-
terminal mutein gene (about 480 bp) was separated and
purified after digestion of the plasmid pUCll9-F4168
(about 3.7 Kbp) obtained above with restriction
endonuclease EcoR I and Hind III in accordance with the
above-mentioned method. The desired human TNF N-terminal
mutein expression vector (pKF 4168) was obtained by using
as host an E. coli K-12 JM103 in the same manner as in
the case of the human TNF expression vector (pKF 4102).
The N-terminal mutein expression vector induces the
expression to produce a novel physiologically active
polypeptide having the following replacement, in E. coli
cells.
Vector pKF 4]68: coding for polypeptide F4168 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is substituted by the amino
acid sequence shown by SEQ ID NO:2.
EXAMPLE 6
20~1~75
- 45 -
Cloninq of human TNF N-termina _mutein expressior~ vectors
EKF 4415, pKF 4416, pKF 4417 and pKF 4418
(1) As in Example 5 (2), except that primer 4415,
4416, 4417 or 4418 was used instead of primer 4168 to
obtain a plasmid (about 3.7 Kbp) containing the desired
mutein DNA. After confirming by the dideoxy method that
the DNA was modified to the mutein DNA as designed, such
plasmid was designated as pUCll9-F4415, pUCll9-F4416,
pUCll9-F4417 or pUCll9-F4418, respectively.
Primers 4415, 4416, 4417 and 4418 ùsed here were the
following oligonucleotides, and their chemical synthesis
and purification were conducted in accordance with the
method of Example 2.
Primer 4415: an oligonucleotide comprising 24 bases
having the nucleotide sequence of from the 7th T to the
30th T as shown by SEQ ID NO:10 wherein the 16-18th 5'-
CCG-3' is replaced by 5'-CGT-3' and the 19-21st 5'-AGT-3'
is replaced by 5'-GGT-3'.
Primer 4416: an oligonucleotide comprising 27 bases
having the nucleotide sequence of from the 1st T to the
18th G as shown by SEQ ID NO:10 wherein 5'-CGTGGTGAT-3'
is inserted between the 9th T and the 10th C.
Primer 4417: an oligonucleotide comprising 27 bases
having the nucleotide sequence of from the 10th C to the
27th G as shown by SEQ ID NO:10 wherein 5'-CGTGGTGAT-3'
is inserted between the 18th G and the l9th A.
Primer 4418: an oligonucleotide comprising 27 bases
20~197~
- 46 -
having the l-9th nucleotide sequence as shown by SEQ ID
NO:10 wherein 5'-CGTGGTGAT-3' is added to the 5'-side of
the 1st T and 5'-GAATTCATG-3' is added to the 5'-side
thereof.
(2) The desired human TNF N-terminal mutein gene
obtained by the introduction of mutation was inserted
into expression vector pKK 101 having tac promoter in
accordance with the method of cloning a human TNF
expression vector of Example 4 to obtain a human TNF N-
terminal mutein expression vector. The human TNF N-
terminal mutein gene (about 480 bp) was separa~ed and
purified after the digestion of the plasmid pUCll9-F4415,
pUCll9-F4416, pUCll9-F4417 or pUCll9-F4418 (about 3.7
Kbp) obtained above, with restriction endonucleases EcoR
I and Hind III in accordance with the above-mentioned
method. The desired human TNF N-terminal mutein
expression vector pKF 4415, pKF 4416, pKF 4417 or pKF
4418 was obtained by using as the host an E. coli K-12
JM103 strain in the same manner as in the case of the
human TNF expression vector (pKF 4102).
The above N-terminal mutein expression vector induces
the expression of a novel physiologically active
polypeptide having the following replacement in E. coli
cells.
Vector pKF 4415: coding for polypeptide F4415 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
2051~7~
- 47 -
acid sequence shown by SEQ ID NO:3.
Vector pKF 4416: coding for polypeptide F4416 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:4.
Vector pKF 4417: coding for polypeptide F4417 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:5.
Vector pKF 4418: coding for polypeptide F4418 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the l-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:6.
EXAMPLE 7
Cloninq of human TNF N-terminal mutein expression vector
pKF 4420
(1) An oligonucleotide having 16 bases of from the
5th C to the 20th G as shown by SEQ ID NO:10 wherein the
13th A is replaced by G, was designed and designated as
primer 4104. The chemical synthesis and purification oE
this oligonucleotide were conducted in accordance with
the method of Example 2. As in Example 5 (2), except
this primer 4104 was used instead of primer 4168 to
obtain a plasmid (about 3.7 Kbp) containing the desired
mutein DNA. The nucleotide sequence around the mutation-
introduced site was examined by the dideoxy method to
confirm that the DNA was modified to the mutant DNA as
20~g75
- 48 -
designed, and this plasmid was designated as pUCll9-
F4104.
(2) As in Example 5 (1), pUC119-F4104 was used
instead of pUCll9--hTNF to obtain the desired single-
stranded plasmid DNA. Further, an oligonucleotide having21 bases of from the 46th G to the 66th G as shown by S~Q
ID NO:10 wherein the 56th A is replaced by G, was
designed and designated as primer 4226. The chemical
synthesis and purification of this oligonucleotide were
conducted in accordance with the method of Example 2.
Then, using the single-stranded plasmid DNA of
pUCll9-F4104 and primer 4226 prepared above, the site
directed mutagenesis of a human TNF mutein gene was
conducted in accordance with the method of Example 5 (2)
to obtain a plasmid (about 3.7 Kbp) containing the
desired mutein DNA. It was confirmed by the dideoxy
method that the DNA was modified to the mutein DNA as
designed, and this plasmid was designated as pUCll9-
F4226.
(3) Using the plasmid pUCll9-F4226 having two
restriction endonuclease Xho I cleavage sites obtained in
Example 7 (2), an expression vector (designated as pKF
4420) of the N-terminal substituted mutein polypeptide
(designated as F4420) was cloned by ligating a chemically
synthesized oligonucleotide (double-stranded by
annealing) to the N-side of the formed restriction
endonuclease Xho I cleavage site. The cloning method
20~1~75
- 49 -
will be described with reference to Figure 7.
Five ~g of plasmid pUCll9-F4226 (about 3.7 Kbp) was
dissolved in a high salt buffer, in accordance with the
method of Example 3, and digested with restriction
endonuclease Xho I (Takara Shuzo) and Hind III, whereupon
a DNA fragment of about 420 bp containing F4226 gene
having the N-terminal portion deleted, was separated and
purified by agarose gel electrophoresis (gel
concentration: 1~). On the other hand, an upper (coding)
strand (U-4920) and the lower (noncoding) strand (L-4420)
of an oligonucleotide encoding an oligopeptide designed
for the purpose of substitution at the N-terminal portion
of E4226, were chemically synthesized and purified in
accordance with the method of Example 2. The upper
strand U-4420 is an oligonucleotide having the nucleotide
sequence as shown by SEQ ID NO:ll. The lower strand L-
4420 is a strand having a sequence complementary to the
upper strand U-4420, but this lower strand does not have
a sequence portion complementary to the l-4th 5'-AATT-3'
of the upper strand U-4420. On the other hand, it is an
oligonucleotide having a nucleotide sequence having 5'-
TCGA-3' added to the 5'-side of the 5'-terminal G of the
lower strand. The two oligonucleotides (each 1 ~g)
obtained after purification were phosphorylated at their
5'-termini with T4 polynucleotide kinase in accordance
with the method of Example 3, followed by annealing to
obtain a double stranded DNA fragment (46 bp). On the
20~137~
- 50 -
other hand, the plasmid vector pECK 101 was digested with
restriction endonuclease EcoR I and Hind III in
accordance with the method of Example 4, whereupon a DNA
fragment of about 3.4 Kbp containing a replication origin
and a transcriptional regulation region, was separated
and purified.
The three DNA fragments obtained by the above method
were ligated with T4 DNA ligase, in accordance with the
method of Example 3, and ligated DNA was introduced into
competent cells of E. coli K-12 JM103 strain. From the
transformants thus-obtained, a clone having the desired
F4420 expression vector pKF 4420 (about 3.9 Kbp) was
selected by confirming the digestion pattern with
restriction endonuclease EcoR I, Xho I and Hind III and
by examining the nucleotide sequence around the N-
terminal substituted site of the N-terminal mutein
polypeptide.
The N-terminal mutein expression vector cloned in
accordance with the above method, induces the expression
Of a novel physiologically active polypeptide having the
following replacement in E. coli cells.
Vector pKF 4420: coding for polypeptide F4420 having
the amino acid sequence as shown by SEQ ID NO:l in the
Sequence Listing wherein the 1-8th amino acid sequence is
replaced by the amino acid sequence Arg-Gly-Asp.
EXAMPLE 8
Cloninq of human TNF N-terminal mutein expression vectors
205197~
- 51 -
pKF 4421 and pKF 4137
Using the plasnlid pUCll9-F4104 having restriction
endonuclease Xho I cleavage site obtained in Example 7
(1), a chemically synthesized oligonucleotide (double-
stranded by annealing) was ligated to the N-side of the
restriction endonuclease Xho I cleavage site, whereby
expression vectors (designated as pKF 4421 and pKF 4137)
of the N-terminal substituted mutein polypeptides
(designated as F4421 and F4137) were cloned.
Five ~g of plasmid pUCll9-F4104 (about 3.7 Kbp) was
dissolved in a high salt buffer, in accordance with the
method of Example 3, and digested with restriction
endonuclease Xho I and Hind III, whereupon a DNA fragment
of about 460 bp containing F4104 gene having the N-
1~ terminal portion deleted, was separated and purified by
agarose gel electrophoresis (gel concentration: 1%). On
the other hand, the upper (coding) strand (U-4421 or U-
4137) and the lower (noncoding) strand (L-4421 or L-4137)
of an oligonucleotide encoding an oligopeptide designed
for the purpose of substitution to the N-terminal portion
of F4104 were chemically synthesized and purified in
accordance with the method of Example 2. The upper
strand U-4421 is an oligonucleotide having the sequence
shown by SEQ ID NO:12 in the Sequence Listing. Whereas,
the lower strand L-4421 is a strand having a sequence
complementary to the upper strand U-4421, but has no
sequence portion complementary to the l-4th 5'-AATT-3' of
20~97~
- 52 -
the upper strand, and it is an oligonucleotide having a
sequence in which 5'-TCGA-3' is added to the 5'-side of
the 5'-terminal G of the lower strand. Further, the
upper strand U-4137 is an oligonucleotide having the
sequence shown by SEQ ID NO:13 in the Sequence Listing.
The lower strand L-4137 is a strand having a sequence
complementary to the upper strand U-4137, but has no
sequence portion complementary to the l-4th 5'-AATT-3' of
the upper strand U-4137, and it is an oligonucleotide
having a sequence wherein 5'-TCGA-3' is added to the 5'-
side of the 5'-terminal G of the lower strand. The
respective upper and lower two oligonucleotides ~each 1
~g) obtained after the purification were phosphorylated
at the 5'-termini by means of T4 polynucleotide kinase,
followed by annealing to obtain a double stranded DNA
fragment (34 bp).
On the other hand, plasmid vector pKK 101 was
digested with restriction endonuclease EcoR I and Hind
III in accordance with the method of Example 4, and a DNA
fragment of about 3.4 Kbp containing a replication origin
and a transcriptional regulation region was separated and
purified.
The three DNA fragments obtained by the above method
were ligated by means of T4 DNA ligase, in accordance
with the method of Example 3, and the ligated DNA was
introduced into competent cells of E. coli K-12 ~M103
strain. From the transformants thus-obtained, a clone
20~1~7~
having the desired F4421 expression vector pKF 4421
(about 3.9 Kbp) or F4137 expression vector pKF 4137
(about 3.9 Kbp) was selected by confirming the digestion
patterns with restriction endonuclease EcoR I, Xho I and
Hind III and by examining the nucleotide sequence around
the N-terminal substituted site of the N-terminal mutein
polypeptide.
The N-terminal mutein expression vector thus-cloned
by the above method induces the expression of a novel
physiologically active polypeptide having the following
replacement in E. coli cells.
Vector pKF 4421: codinq for polypeptide F4421 having
the amino acid sequence as shown by SEQ ID NO:l in the
Sequence Listing wherein the 1-8th amino acid sequence is
replaced by the amino acid sequence shown by SEQ ID NO:7.
Vector pKF 4137: coding for polypeptide F4137 having
the amino acid sequence as shown by SEQ ID NO:l in the
Sequence Listing wherein the 1-8th amino acid sequence is
replaced by the amino acid sequence shown by SEQ ID NO:8.
EXAMPLE 9
Cloninq of human TNF N-terminal mutein expression vector
pKF 4113
A plasmid vector (designated as pSK407) having trp
promoter as the promoter, two translation initiation
signals (SD sequence) arranged in tandem and rrnBTlT2
terminator as the terminator, was cloned in accordance
with the method shown in Figures 9 to 11. Five ~g of
20~75
- 54 -
plasmid vector pGF101 (about 4.3 Kbp, obtained from Osaka
University, Facalty of Pharmaceutical Science) was
dissolved in a medium salt buffer in accordance with the
above-mentioned method and digested by the addition of 10
units of restriction endonuclease Cla I (Takara Shuzo).
The cut DNA fragment was polished by means of T4 DNA
polymerase in accordance with the method of Example 3,
and after the ethanol precipitation operation, it was
dissolved in a high salt buffer having NaCl added to a
NaCl concentration of 175 mM and digested by an addition
of 15 units of restriction endonuclease Sal I (Takara
Shuzo). A DNA fragment of about 4.0 Kbp was separated
and purified by agarose gel electrophoresis (gel
concentration: 1~) in accordance with the method of
Example 3.
On the other hand, 5 ~g of plasmid pMC1403 (about 9.9
Kbp, obtained from Institute for Virus Research, Kyoto
University) developed by Casadaban et al. (J. Bacteriol.,
143, 971 (1980)), was digested with restriction
endonuclease EcoR I in accordance with the above-
mentioned method, followed by polishing and digestion
with restriction endonuclease Sal I in the same manner as
above, whereupon a DNA fragment of about 6.2 Kbp was
separated and purified by agarose gel electrophoresis.
In accordance with the method of Example 3, this DNA
fragment was ligated to the previously purified DNA
fragment by means of T4 DNA ligase, and the ligated DNA
20~1~7~
- 55 -
was introduced into competent cells of an E. coli K-12
HB101 strain (obtained from Institute for Virus Research,
Kyoto University) prepared by a calcium chloride method.
From the transformants thereby obtained, a clone having
the desired plasmid vector (designated as pSK101) of
about 10.2 Kbp containing trp promoter and restriction
endonuclease EcoR I and BamH I cleavage sites, was
selected.
In each of two tubes, 5 ~g of the plasmid vector
pSK101 obtained above was dissolved in a high salt buffer
in accordance with the above-mentioned method and
digested by an addition of restriction endonuclease EcoR
I and Sal I, and restriction endonuclease BamH I and Sal
I, respectively, whereupon DNA fragments of about 4.0 Kbp
and about 6.2 Kbp were, respectively, separated and
purified by agarose gel electrophoresis. On the other
hand, the upper (coding) and lower (noncoding) strands
(each 100 ng) of a portable translation initiation site-
oligonucleotide (which is referred to simply as SD-ATG
linker and which is an oligonucleotide comprising 21
bases containing a translation initiation signal SD
sequence and a translation initiation codon ATG, obtained
from Pharmacia, Catalogue No. 27-4878-01 and 27-4898-01
Pharmacia Molecular Biologicals, 1985 May) were
phosphorylated at their 5'-termini and annealed to obtain
a double stranded DNA f~agment (21 bp).
The three DNA fragments obtained above were ligated
2 ~ 7 ~
- 56 -
by means of T4 DNA ligase, in accordance with the above-
mentioned method, and the ligated DNA was introduced into
competent cells of an E. coli K-12 HB101 strain. From
the transformants thereby obtained, a clone having the
desired plasmid vector (designated as pSK211) of about
10.2 Kbp having the SD-ATG linker inserted downstream of
the trp promoter and immediately upstream of the
restriction endonuclease BamH I cleavage site, was
selected. This plasmid vector has two SD sequences
arranged in tandem downstream of the trp promoter.
The following operation was conducted for the purpose
of eliminating the restriction endonuclease Sal I
cleavage site of this plasmid vector and introducing a
new restriction endonuclease Hind III cleavage site in
its vicinity. Five ~g of plasmid vector pSK211 was
digested with restriction endonuclease Sal I in
accordance with the above-mentioned method and then
polished by means of T4 DNA polymerase. The DNA fragment
recovered by an ethanol precipitation operation was
digested with restriction endonuclease BamH I, whereupon
a DNA fragment of about 4.0 Kbp was separated and
purified by agarose gel electrophoresis. On the other
hand, 5 ~g of plasmid vector pGFK 503-1 (about 5.7 Kbp)
cloned by recombination in the same manner as the above-
mentioned cloning of pSK211, using plasmid vector pKK223-3 having the tac promoter and SD-ATG linker, etc.,
was digested with restriction endonuclease Hind III in
20~1 97~
accordance with the above-mentioned method and then
polished by means of T4 DNA polymerase. The DNA fragment
recovered by an ethanol precipitation operation, was
digested with restriction endonuclease BamH I, and a DNA
fragment of about 1~l Kbp was separated and purified by
agarose gel electrophoresis.
The two DNA fragments purified above, were ligated by
means of T4 DNA ligase, in accordance with the above-
mentioned method, and the ligated DNA was introduced into
competent cells of an E. coli K-12 HB101 strain. From
the transformants thereby obtained, a clone having the
desired plasmid vector (designated as pSK301) of about
5.1 Kbp, containing the trp promoter, having the
restriction endonuclease Sal I cleavage site deleted, and
having a Hind III cleavage site formed anew, was
selected.
Five ~g of this plasmid vector pSK301 was dissolved
in a low salt buffer in accordance with the above-
mentioned method, and the digestion reaction was
conducted by an addition of 5 units of restriction
endonuclease Bal I (Takara Shuzo). After completion of
the reaction, the NaCl concentration was adjusted to 50
mM, and digestion with restriction endonuclease Hind III
was conducted, whereupon a DNA segment of about 4.3 Kbp
was separated and purified by agarose gel
electrophoresis. On the other hand, 5 ~g of plasmid
vector pKK 223-3 was dissolved in a high salt buffer in
20~1~7~
- 58 -
accordance with the above-mentioned method and digested
with restriction endonuclease Hind III and Sca I,
whereupon a DNA fragment of about 0.8 Kbp was separated
and purified by agarose gel electrophoresis.
The two DNA fragments purified above, were ligated by
means of T4 DNA ligase, in accordance with the above-
mentioned method, and the ligated DNA was introduced into
competent cells of an E. coli K-12 HB101 strain. From
the transformants thereby obtained, a clone having the
desired plasmid vector pSK407 (about 5.1 Kbp) having the
rrnBTlT2 terminator located downstream of the trp
promoter, was selected.
Then, two oligonucleotides (U-4113 and L-4113) were
designed.
U-4113 (SEQ ID NO:14 in the Sequence Listing, number
of bases: 32)
L-4113 (number of bases: 32) which is complementary
to U-4113, but does not have a nucleotide sequence
complementary to the l-4th 5'-GATC-3' of SEQ ID NO:14,
and on the other hand, has a nucleotide sequence in which
5'-TCGA-3' is added to the 5'-side of G complementary to
the 32nd C of SEQ ID NO:14.
The oligonucleotides (1 ~g each) obtained by the
chemical synthesis and purification in accordance with
the method of Example 2 were phosphorylated at their 5'-
termini by means of T4 polynucleotide kinase, followed by
annealing, in accordance with the method of Example 3, to
205197~
- 59 -
obtain a double-stranded DNA fragment (32 bp). On the
other hand, 5 ~g of plasmid vector pSK407 (about 5.1 Kbp)
having the trp promoter and rrnBTlT2 terminater, etc.,
was dissolved in a high salt buffer in accordance with
the method of Example 3 and digested with restriction
endonuclease BamH I and Hind III, whereupon a DNA
fragment of about 4.0 Kbp containing a replication origin
and a transcriptional regulation region, etc. was
separated and purified by agarose gel electrophoresis
(gel concentration: 1%). On the other hand, 5 ~g of
plasmid pUCll9-E4104 (about 3.7 Kbp) obtained in Example
7 (1), was digested with restriction endonuclease Xho I
and Hind III in the same manner as in Example 8,
whereupon a DNA fragment of about 460 bp containing the
F4104 gene having the N-terminal portion deleted, was
separated and purified (Figure 12).
The three DNA fragments obtained in accordance with
the above methods, were ligated by means of T4 DNA
ligase, in accordance with the method of Example 3, and
the ligated DNA was introduced into competent cells of an
E. coli K-12 HB101 strain. From the transformants
thereby obtained, a clone having the desired F4113
expression vector (designated as pKF 4113) of about 4.5
Kbp, was selected by confirming the digestion pattern
with restriction endonuclease samH I, Xho I and Hind III
and by examining the nucleotide sequence around the N-
terminal addition site of the N-terminal mutein
2~5~97~
- 60 -
polypeptide.
The N-terminal mutein expression vector cloned in
accordance with the above method induces the expression
of a novel physiologically active polypeptide having the
following replacement in E. coli cells.
Vector pKF 4113: coding for polypeptide F4113 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:9.
EXAMPLE 10
Cloninq of human TNF N-terminal mutein expression vectors
pKF 4601 and pKF 4602
(1) Primers 4291 and 4292 were designed. These
primers are oligonucleotides comprising 21 bases having a
nucleotide sequence of from the 193th C to the 213th T of
SEQ ID NO:10 wherein the 202-204th 5'-CCA-3' is replaced
by 5'-GAT-3' in the case of primer 4291 and by 5'-ATG-3'
in the case of primer 4292.
The chemical synthesis and purification of these
oligonucleotides were conducted in accordance with the
method of Example 2. Using such primer 4291 or 4292, a
plasmid (about 3.7 Kbp) containing the desired mutant DNA
was obtained in accordance with the method of Example 5
(1) and (2). It was confirmed by the dideoxy method that
the DNA was modified to the mutein DNA as designed, and
this plasmid was designated as pUCll9-F4291 or pUCll9-
F4292. The desired human TNF mutein gene obtained by the
205197~
- 61 -
introduction of mutation, was inserted into expression
vector pKK 101 having tac promoter in accordance with the
method of cloning a human TNF expression vector of
Example 4, to obtain a human TNF mutein expression
vector. The human TNF mutein gene (about 480 bp) was
separated and purified after digestion of the plasmid
pUCll9-F4291 or pUCll9-F4292 (about 3.7 Kbp) obtained
above with restriction endonuclease EcoR I and Hind III
in accordance with the above-mentioned method. The
desired human TNF mutein expression vector pKF 4291 or
pKF 4292 was obtained by using as the host an E. coli K-
12 JM103 strain in the same manner as in the case of the
human TNF expression vector (pKF 4102).
(2) The operation will be described with reference to
Figure 8. Five ~g of the human TNF N-terminal mutein
expression vector pKF 4168 (about 3.9 Kbp) obtained in
Example 5 was dissolved in a medium salt buffer, in
accordance with the method of Example 3, and digested
with restriction endonuclease Sac I and Hind III,
whereupon a DNA fragment of about 3.5 Kbp containing a
replication origin and a transcriptional regulation
region, was separated and purified by agarose gel
electrophoresis (gel concentration: 1%). On the other
hand, 5 ~g of the human TNF mutein expression vector pKF
4291 or pKF 4292 (about 3.9 Kbp) obtained in the above
step (1) was digested with restriction endonuclease Sac I
and Hind III in the same manner as above, whereupon a DNA
205~975
- 62 -
fragment of about 360 bp containing a human TNF mutein
gene having the N-terminal portion deleted, was separated
and purified.
The two DNA fragments obtained by the above methods,
were ligated by means of T4 DNA ligase, in accordance
with the method of Example 3, and the ligated DNA was
introduced into competent cells of an E. coli K-12 JM103
strain. From the transformants thus-obtained, a clone
having the desired F4601 or F4602 expression vector pKF
4601 or pKF 4602 (about 3.9 Kbp) was selected by
confirming the digestion pattern with restriction
endonuclease Sac I and Hind III and by examining the
nucleotide sequence around the replaced sites derived
from the mutein polypeptide.
The N-terminal mutein expression vector cloned in
accordance with the above method, induces the expression
of a novel physiologically active polypeptide having the
following replacements in E. coli cells.
Vector pKF 4501: coding for polypeptide F4601 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the l-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:2, and the 68th Pro is
replaced by Asp.
Vector pKF 4602: coding for polypeptide F4602 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:2, and the 68th Pro is
20~1975
- 63 -
replaced by Met.
Host E. coll cells having the above expression
vectors produce polypeptides showing antitumor
activities. This was confirmed by a test in which a
supernatant fraction obtained after the centrifugal
separation of a suspension of sonicated E. coli cells,
was tested in accordance with the method of Example 14
given hereinafter.
EXAMPLE 11
Cloninq of human TNF N-terminal mutein expression vectors
pKF 4607, pKF 4608, pKF 4626, pKF 4627, pKF 4634, pKF
4635, pKF 4609, pKF 4610, pKF 4628, pKF 4629, pKF 4638
and pKF 4639
(1) Primers 4268, 4150, 4222, 4267, 4123 and 4223
were designed. Primer 4268 is an oligonucleotide
comprising 30 bases having a nucleotide sequence of from
the 301st A to the 333rd C of SEQ ID NO:10 wherein the
316-318th 5'-GGC-3' is deleted. On the other hand, other
primers are oligonucleotides having 21 bases having a
nucleotide sequence of from the 307th A to 327th C of SEC
ID NO: 10 wherein the 316-318th 5'-GGC-3' is replaced by
5'-TGG-3' in the case of primer 4150, by 5'-CCC-3' in the
case of primer 4222, by 5'-GCT-3' in the case of primer
4267, by 5'-GAC-3' in the case of primer 4123 and by 5'-
CGC-3' in the case of primer 4223.
The chemical synthesis and purification of these
oligonucleotides were conducted in accordance with the
20~1975
- 64 -
method of Example 2. Using each of these primers, a
plasmid (about 3.7 Kbp) containing the desired mutant DNA
was obtained in accordance with the method of Example 5
(1) and (2). It was confirmed by the dideoxy method that
the DNA was modified to the mutein DNA as designed, and
this plasmid was designated as pUC119-F4268, pUCll9-
F4150, pUCll9-F4222, pUCll9-F4267, pUC119-F4123 or
pUC119-F4223. The desired human TNF mutein gene obtained
by the introduction of mutation, was inserted into
expression vector pKK 101 having tac promoter in
accordance with the method of cloning a human TNF
expression vector of Example 4, to obtain a human TNF
mutein expression vector. The human TNF mutein gene
(about 480 bp) was separated and purified after digestion
Of the plasmid (about 3.7 Kbp) obtained above with
restriction endonuclease EcoR I and Hind III in
accordance with the above-mentioned method. The desired
human TNF mutein expression vector pKF 4268, pECF 4150,
pKF` 4222, pKF 4267, pKF 4123 or pKF 4223 (about 3.9 Kbp)
was obtained by using as the host an E. coli K-12 JM103
strain in the same manner as in the case of the human TNF
expression vector (pKF 4102).
(2) In accordance with the method of Example 10 (2),
5 ~g of the human TNF N-terminal mutein expression vector
pKF 4168 (about 3.9 Kbp) obtained in Example 5 was
digested with restriction endonuclease Sac I and Hind
III, whereupon a DNA fragment of about 3.5 Kbp containing
20~1 975
- 65 -
a replication origin and a transcriptional regulation
region, was separated and purified.
On the other hand, 5 ~g of the human TNF mutein
expression vector pKF 4268, pKF 4150, pKF 4222, pKF 4267,
pKF 4123 or pKF 4223 obtained in the above step (1) was
digested with restriction endonuclease Sac I and Hind III
in the same manner as above, whereupon a DNA fragment of
about 360 bp containing a human TNF mutein gene having
the N-terminal portion deleted, was separated and
purified.
The two DNA fragments obtained by the above methods,
were ligated by means of T4 DNA ligase, in accordance
with the method of Example 3, and the ligated DNA was
introduced into competent cells of an E. coli K-12 JM103
strain. From the transformants thus-obtained, a clone
having the desired expression vector pKF 4607, pKF 4608,
pKF 4626, pKF 4627, pKF 4634 or pKF 4635 (about 3.9 Kbp)
for F4607, F4608, F4626, F4627, F4634 or F4635 was
selected by confirming the digestion pattern with
restriction endonuclease Sac I and Hind III and by
examining the nucleotide sequence around the replaced
sites derived from the mutein polypeptide.
The N-terminal mutein expression vector cloned in
accordance with the above method, induces the expression
of a novel physiologically active polypeptide having the
following replacements in E. coli cells.
Vector pKF 4607: coding for polypeptide F4607 having
7 ~
- 66 -
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:2, and the 106th Gly is
deleted.
Vector pKF 4608: coding for polypeptide F4608 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:2, and the 106th Gly is
replaced by Trp.
Vector pKF 4626: coding for polypeptide F4626 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:2, and the 106th Gly is
replaced by Pro.
Vector pKF 4627: coding for polypeptide F4627 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the l-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:2, and the 106th Gly is
replaced by Ala.
Vector pKF 4634: coding for polypeptide F4634 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:2, and the 106th Gly is
replaced by Asp.
Vector pKF 4635: coding for polypeptide F4635 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
20~197~
- 67 -
acid sequence shown by SEQ ID NO:2, and the 106th Gly is
replaced by Arg.
Host E coli cells having the above expression
-
vectors produce polypeptides showing antitumor
activities. This was confirmed by a test in which a
supernatant fraction obtained after the centrifugal
separation of a suspension of sonicated E. coli cells,
was tested in accordance with the method of Example 14
given hereinafter.
(3) Five ~g of the human TNF N-terminal mutein
expression vector pKF 4418 (about 3.9 Kbp) obtained in
Example 6 was dissolved in a medium salt buffer, in
accordance with the method of Example 3, and digested
with restriction endonuclease Sac I and Hind III,
whereupon a DNA fragment of about 3.5 Kbp containing a
replication origin and a transcriptional regulation
region, was separated and purified by agarose gel
electrophoresis (gel concentration: 1%). On the other
hand, 5 ~g of the human TNF mutein expression vector pKF
4268, pKF 4150, pKF 4222, pKF 4267, pKF 4123 or pKF 42Z3
obtained in the above step (1) was digested with
restriction endonuclease Sac I and Hind III in the same
manner as above, whereupon a DNA fragment of about 360 bp
containing a human TNF mutein gene having the N-terminal
portion deleted, was separated and purified.
The two DNA fragments obtained by the above methods,
were ligated by means of T4 DNA ligase, in accordance
2051975
- 68 -
with the method of Example 3, and the ligated DNA was
introduced into competent cells of an E. coli K-12 JM103
strain. E'rom the transformants thus~obtained, a clone
having the desired expression vector pKF 4609, pKF 4610,
pKF 4628, pKF 4629, pKF 4638 or pKF 4639 (about 3.9 Kbp)
for F4609, F4610, F4628, F4629, F4638 or F4639 was
selected by confirming the digestion pattern with
restriction endonuclease Sac I and Hind III and by
examining the nucleotide sequence around the replaced
sites derived from the mutein polypeptide.
The N-terminal mutein expression vector cloned in
accordance with the above method, induces the expression
of a novel physiologically active polypeptide having the
following replacements in E. coli cells.
Vector pKF 4609: coding for polypeptide F4609 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the l-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:6, and the 106th Gly is
dele'ed.
Vector pKF 4610: coding for polypeptide F4610 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:6, and the 106th Gly is
replaced by Trp.
Vector pKF 4628: coding for polypeptide F4628 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the l-8th amino acid sequence is replaced by the amino
7 S
- 69 -
acid sequence shown by SEQ ID NO:6, and the 106tn Gly is
replaced by Pro.
Vector pKF 4629: coding for polypeptide F4629 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:6, and the 106th Gly is
replaced by Ala.
Vector pKF 4638: coding for polypeptide F4638 having
the amino acid sequence as shown by SEQ ID NO:1 wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:6, and the 106th Gly is
replaced by Asp.
Vector pKF 4639: coding for polypeptide F4639 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:6, and the 106th Gly is
replaced by Arg.
Host E. coli cells having the above expression
vectors produce polypeptides showing antitumor
acti~ities. This was confirmed by a test in which a
supernatant fraction obtained after the centrifugal
separation of a suspension of sonicated E. coli cells,
was tested in accordance with the method of Example 14
given hereinafter.
EXAMPLE 12
Cloninq of human TNF N-terminal mutein expression vectors
pKF 4611, pKF 4612, pKF 4613, pKF 4614, pKF 4615, pKE`
20~1975
- 70 -
4642, pKF 4643, pKE' 4644, pKE 4645 and pKF 4646
(1) Primers 4134, 4391, 4392, 4409 and 4410 were
desiyned. These primers are oligonucleotides comprising
21 bases having a nucleotide sequence of from the 76th T
to the 96th T of SEQ ID NO:10 wherein the 85-87th 5'-CGC-
3' is replaced by 5'-CAA-3' in the case of primer 4134,
by 5'-AAG-3' in the case of primer 4391, by 5'-GAC-3' in
the case of primer 4392, by 5'-GTC-3' in the case of
primer 4409 and by 5'-CTC-3' in the case of primer 4410.
The chemical synthesis and purification of these
oligonucleotides were conducted in accordance with the
method of Example 2.
(2) A desired single-stranded plasmid DNA was
prepared by using pUCll9-F4168 obtained in Example 5
instead of pUCll9-hTNF in Example 5 (1). Then, using
this uracil-containing single-stranded plasmid DNA of
pUCll9-F4168 and primer 4134, 4391, 4392, 4409 or 4410
purified in the above step (1), the site-directed
mutagenesis was conducted in accordance with the method
Of Example 5 (2) to obtain a plasmid (about 3.7 Kbp)
containing DNA having the desired mutation introduced.
It was confirmed by the dideoxy method that the mutation
was introduced as designed, and this plasmid was
designated as pUCll9-F4611, pUCll9-F4612, pUCll9-F4613,
pUCll9-F4614 or pUCll9-F4615. The desired human TNF N-
terminal mutein gene obtained by the introduct;on of
mutation, was inserted into expression vector pKK 101
2051975
having tac promoter in accordance with the method of
cloning a human TNF expression vector of Example 4, to
obtain a human TNF N-terminal mutein expression vector.
The human TNF N-terminal mutein gene (about 480 bp) was
separated and purified after digestion of the pla~mid
pUCll9-F4611, pUCll9-F4612, pUCll9-F4613, pUCll9-F4614 or
pUCll9-F4615 (about 3.7 Kbp) obtained above with
restriction endonuclease EcoR I and Hind III in
accordance with the above-mentioned method. The desired
human TNF N-terminal mutein expression vector pKF 4611,
pKF 4612, pKF 4613, pKF 4614 or pKF 4615 was obtained by
using as the host an E. coli K-12 JM103 strain in the
same manner as in the case of the human TNF expression
vector (pKF 4102).
The N-terminal mutein expression vector cloned in
accordance with the above method, induces the expression
of a novel physiologically active polypeptide having the
following replacements in E. coli cells.
Vector pKF 4611: coding for polypeptide F4611 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the l-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:2, and the 29th Arg is
replaced by Gln.
Vector pI~F 4612: coding for polypeptide F4612 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:2, and the 29th Arg is
20~1975
- 72 -
replaced by Lys.
Vector pKF 4613: coding for polypeptide F4613 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the l-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:2, and the 29th Arg is
replaced by Asp.
Vector pKF 4614: coding for polypeptide F4614 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the l-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:2, and the 29th Arg is
replaced by Val.
Veetor pKF 4615: coding for polypeptide F4615 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino aeid sequenee is replaeed by the amino
aeid sequenee shown by SEQ ID NO:2, and the 29th Arg is
replaeed by Leu.
Host E. coli eells having the above expression
veetors produee polypeptides showing antitumor
aetivities. This was eonfirmed by a test in whieh a
supernatant fraetion obtained after the eentrifugal
separation of a suspension of sonieated E. eoli eells,
was tested in aceordance with the method of Example 14
given hereinafter.
(3) A uracil-eontaining single-stranded plasmid DNA
was prepared using pUC119-F4418 obtained in Example 6
instead of pUCll9-F4168 in the above step (2), and the
site-directed mutagenesis was conducted in the same
2~51975
- 73 -
manner as in the above step (2) to obtain a plasmid
containing DNA having the desirecl mutation introduced,
i.e. p~C119-F4642, pUCll9-F4643, pUCll9-F4644, pUCll9-
F4645 or pUC119-F4646 (about 3.7 Kbp). Using the plasmid
containing the desired human TNF N-terminal mutein gene
(about 480 bp) obtained as above, the desired human TNF
N-terminal mutein expression vector pKF 4642, pKF 4643,
pKF 4644, pKF 4645 or pKF 4646 (about 3.9 Kbp) was cloned
in the same manner as in the above step (2). In this
operation, an E. coli K-12 JM103 strain was used as the
host.
The N-terminal mutein expression vector cloned in
accordance with the above method, induces the expression
of a novel physiologically active polypeptide having the
following replacements in E. coli cells.
Vector pKF 4642: coding for polypeptide F4642 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the 1-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:6, and the 29th Arg is
replaced by Gln.
Vector pKF 4643: coding for polypeptide F4643 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the l-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:6, and the 29th Arg is
replaced by Lys.
Vector pKF 4644: coding for polypeptide F4644 having
the amino acid sequence as shown by SEQ ID NO:l wherein
~0~1~75
- 74 -
the l-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:6, and the 29th Arg is
replaced by Asp.
Vector pKF 4645: coding for polypeptide F4645 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the l-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:6, and the 29th Arg is
replaced by Val.
Vector pKF 4646: coding for polypeptide F4646 having
the amino acid sequence as shown by SEQ ID NO:l wherein
the l-8th amino acid sequence is replaced by the amino
acid sequence shown by SEQ ID NO:6, and the 29th Arg is
replaced by Leu.
Host E. coli cells having the above expression
vectors produce polypeptides showing antitumor
activities. This was confirmed by a test in which a
supernatant fraction obtained after the cen-trifugal
separation of a suspension of sonicated E. coli cells,
was tested in accordance with the method of Example 14
given hereinafter.
EXAMPLE 13
Expression of human TNF Polypeptide and human TNF N-
terminal mutein polypeptide by E. coli and purification
E. coli K-12 JM103 strains having the human TNF
expression vector (pKF 4102) obtained in Example 4 and
the human TNF N-terminal mutein expression vectors (pKE'
4168, pKF 4415, pKF 4416, pKF 4417, pKF 4418, pKF 4420,
2051~7~
- 75 -
pKF 4421, pKF 4137~ pKF 4601, pKF 4609 and pKF 4639)
obtained in E~amples 5, 6, 7, 8, 10 and 11 were,
respectively, inoculated to 20 ml of a M9 culture medium
(0.6% Na2HPO4, 0.3~ KH2PO4, 0.05% NaCl, 0.1% NH4Cl, 2 mM
MgSO4, 0.2% glucose and 0.1 mM CaCl2) containing from 25
to 50 ~g/ml of ampicillin and 0.001% of vitamin Bl and
cultured with shaking at 37C for 18 hours. Twenty ml of
each cultured solution was added to 1 e of the above
culture medium, followed by cultivation with shaking at
37C for from 2 to 3 hours. Then, isopropyl-~-D-
thiogalactopyranoside (IPTG) was added so that the final
concentration would be 1 mM, and the culturing was
continued with shaking for a further 18 hours at 37C.
An E. coli K-12 HB101 strain having the human TNF N-
terminal mutein expression vector (pKF 4113) obtained inExample 9, was inoculated to 20 ml of a M9C~ culture
medium (0.6% Na2HPO4, 0.3~ KH2PO4, 0.05% NaCl, 0.1% NH4Cl,
2 mM MgSO4, 0.2% glucose, 0.1 mM CaC12 and 0.2% casamino
acid) containing from 25 to 50 ~g/ml of ampicillin,
0.001% of vitamin Bl and 5 ~g/ml of tryptophan, and
cultured at 37~C for 18 hours. Twenty ml of this
cultured solution was added to 1 e of the above culture
medium not containing 5 ~g/ml of tryptophan, and the
culturing was conducted with shaking at 37C for 18
hours.
The E. coli cells were recovered by a centrifugal
separation, then the cells were washed by means of a TP
20~7~
- 76 -
buffer (10 mM Tris-HCl pH 8.0 and 100 ~M PMSF). Af~er
washing, the cells were suspended in the TP buffer in an
amount of 10 volume (ml) per wet weight (g) of the cells,
and the suspension was sonicated by means of a sonicator
(Ultrasonic Proccesor, Model W-225 (Heat Systems,
Ultrasonics, Inc.)). The obtained suspension was
subjected to centrifugal separation, and the debris was
removed, and the supernatant fraction was recovered. The
purification process subsequent to this sonicating
treatment, was conducted mainly at a low temperature (0
to 4C).
This supernatant was filtered by a 0.45 ~m filter and
then fractionated by anion exchange chromatography
~column size: ~ 2.5 x 1.5 cm, flow rate: 0.5 ml/min)
using Sepabeads FP-DA13 (Mitsubishi Chemical Industries
Ltd.), whereupon an active fraction was recovered through
identifying by SDS-polyacrylamide gel electrophoresis
which will be described hereinafter and by determining
the presence or absence of the activity in accordance
with the method of Example 14 using L929 cells. For
elution, TP buffer containing NaCl was employed, and the
NaCl concentration was stepwise increased from 0.05M to
O.lM, 0.2M and 0.5M. The human TNF N-terminal mutein
F4639 was eluted with 0.05M NaCl. The human TNF (1) was
eluted with O.lM NaCl, the human TNF (2) and the human
TNF N-terminal muteins F4415, F4417, F4418, F4420, F4601
and F4609 were eluted with 0.2M NaCl, and the human TNF
2~197~
- 77 -
N-terminal mutein F4168 was eluted with 0.5M NaCl.
Further, in order to remove endotoxin, etc. derived from
E. coli cells, treatment with nucleic acid endotoxin
removing agent C-9 (manufactured by Kurita Water Industry
Ltd. and sold by Dainippon Pharmaceutical Co., Ltd.) was
conducted in accordance with the attached manual. Thus,
partially purified samples of the human TNF polypeptides
and the human TNF N-terminal mutein polypeptides were
obtained.
Using these samples, evaluation of the antitumor
activities and evaluation of the effects to experimental
lung metastasis were conducted in Examples 14, 15 and 16.
With respect to the N-terminal mutein polypeptides F4416,
F4421 and F4137, no active fraction was obtained probably
because they are unstable in the host E. coli K-12 JM103
strain used, and likewise with respect to F4113, no
active fraction was obtained probably because it is
unstable in the host E. coll K-12 HB101 strain used.
However, it may be possible to obtain the active
fractions by using other host cells.
The above samples were filtered by a 0.20 ~m filter,
and then subjected to anion exchange chromatography using
Mono Q (registered trademark) tHR 10/10 and HR 5/5)
prepacked column (manufactured by Pharmacia LKB
Biotechnology) under the control by a FPLC system and
eluted with TPQ buffer (20 mM Tris-HCl pH 8.0 and 10 ~M
PMSF) containing NaCl, whereupon an active fraction was
20~197~
- 78 -
recovered through identifying by SDS-polyacrylamide gel
electrophoresis which will be described hereinafter and
by determining the presence or absence of the activity in
accordance with the method of Example 14 employing L929
cells. The procedure of elution was conducted in
accordance with the following program under the control
by a FPLC system. The third step was repeated a ~ew
times until a single band was obtained by the SDS-
polyacrylamide gel electrophoresis, in order to increase
the purity.
First step: Mono Q (registered trademark) (HR 10/10)
column was used (flow rate: 4 ml/min)
0-5 minutes: 0-0.2M NaCl linear gradient in
concentration
5-16 minutes: 0.2M NaCl
16-20 minutes: 0.2-0.5M NaC1 linear gradient in
concentration
20-24 minutes: 0.5M NaC1
The fractionation results (NaC1 concentration and
retention time) of the active fractions in accordance
with the above method were as follows: 0.2M, 6.6 minutes
in the case of human TNF (1); 0.2M, 5.6 minutes in the
case of human TNF (2); 0.2M, 6.4 minutes in the case of
human TNF N-terminal mutein F4168; 0.2M, 7.8 minutes in
the case of human TNF N-terminal mutein F4415; 0.2M, 5.4
minutes in the case of human TNF N-terminal mutein F4417;
0.2M, 5.4 minutes in the case of human TNF N-terminal
20~975
- 79 -
mutein F4418; 0.18M, 4.1 minutes in the case of human T~F
N-terminal mutein F4420; 0.2M, 7.0 minutes in the case of
human TNF N-terminal mutein F4601; 0.2M, 5.0 minutes in
the case of human TNF N-terminal mutein F4609; and 0.14M,
3.1 minutes in the case of human TNF N-terminal mutein
F4639.
Second step: Mono Q (registered trademark) (HR 5/5)
column was used (flow rate: 1 ml/min)
0-2.5 minutes: 0-0.2M NaCl linear ~radient in
concentration
2.5-8 minutes: 0.2M NaCl
8-10 minutes: 0.2-0.5M NaCl linear gradient in
concentration
10-12 minutes: 0.5M NaCl
The fractionation results (NaC1 concentration and
retention time) of the active fractions in accordance
with the above method were as follows: 0.2M, 3.7 minutes
in the case of human TNF (1); 0.2M, 4.1 minutes in the
case of human TNF (2); 0.2M, 3.9 minutes in the case of
human TNF N-terminal mutein F4168; 0.2M, 6.0 minutes in
the case of human TNF N-terminal mutein F4415; 0.2M, 3.9
minutes in the case of human TNF N-terminal mutein F4417;
0.2M, 3.3 minutes in the case of human TNF N-terminal
mutein F4418; 0.2M, 3.2 minutes in the case of human TNF
N-terminal mutein F4420; 0.2M, 4.9 minutes in the case of
human TNF N-terminal mutein F4601; 0.2M, 3.2 minutes in
the case of human TNF N-terminal mutein F4609; and flow
20~1~7~
- 80 -
through in the case of human TNF N-terminal mutein F4639.
Third step: Mono Q (registered trademark) (HR 5/5) column
was used (flow rate: 1 ml/min)
0-6 minutes: 0-0.15M NaCl linear gradient in
concentration
6-11 minutes: 0.15-0.2M NaCl linear gradient in
concentration
11-13 minutes: 0.2-0.5M NaCl linear gradient in
concentration
13-15 minutes: 0.5M NaCl
The fractionation results (NaCl concentration and
retention time) of the active fractions in accordance
with the above method were as follows: 0.16M, 6.5 minutes
in the case of human TNF (1); 0.16M, 6.5 minutes in the
case of human TNF (2); 0.17M, 6.7 minutes in the case of
human TNF N-terminal mutein F4168; 0.16M, 6.4 minutes in
the case of human TNF N terminal mutein F4415; 0.16M, 6.5
minutes in the case of human TNF N-terminal mutein F4417;
0.16M, 6.2 minutes in the case of human TNF N-terminal
mutein F4418; 0.15M, 5.6 minutes in the case of human TNF
N-terminal mutein F4420; O.l9M, 9.8 minutes in the case
of human TNF N-terminal mutein F4601; 0.15M, 5.7 minutes
in the case of human TNF N-terminal mutein F4609; and
flow through in the case of human TNF N-terminal mutein
F4639
Thus, purified samples of human TNF polypeptides and
human TNF N-terminal mutein polypeptides were obtained.
20~97~
- 81 -
Using these samples, evaluation of the antitumor
activities was conducted in the following Example 14.
During the above purification and with respect to the
purified samples, SDS-polyacrylamide gel electrophoresis
was conducted to confirm the expression and purification
of the human TNF polypeptides and the human TNF N-
terminal mutein polypeptides. Each sample was added in a
Laemmli's sample buffer (62.5 mM Tris--HCl pH 6.8, 2% SDS,
0.01% BPB and 10% glycerol) containing 10 mM of DTT, and
electrophoresis was conducted in accordance with the
method of Laemmli (Nature, 227, 680 (1970)) using a 15%
separation gel. After completion of the electrophoresis,
the protein in the separation gel was confirmed by
staining it with Coomassie Brilliant Blue. Some of the
results are shown in Figures 13 and 14. The expression
quantities by the respective expression vectors for the
human TNF polypeptides and the human TNF N-terminal
mutein polypeptides for which active fractions were
recovered, were substantially equal, and when the
expression efficiency was calculated by subjecting the
obtained stained gels to a chromatoscanner (CS-920 Model,
manufactured by Shimadzu Corporation), it was found to be
about 20% of the total cell proteins of E. coli.
Further, each of the finally purified samples showed a
single band in the obtained stained gel. From the
migration positions, the molecular weights of the human
TNF polypeptides and the human TNF N-terminal mutein
2~5~97~
- 82 -
polypeptides were calculated and shown in Table 1.
As one of the physical properties of proteins,
isoelectric points of the human TNF polypeptides and the
human TNF N-terminal mutein polypeptides were measured by
an isoelectric focusing method using Ampholine
(manufactured by l,KB), and the results thereby obtained
are shown in Table 1.
~0~1~75
- 83 -
Table 1
Table 1 (1) Isoelectric points ancl molecular weights of
human TNF polypeptide and human TNF N-terminal mutein
polypeptide
Polypeptide Isoelectric Molecular weight (Kd)
point
Human TNF 5.6 17.0
(1)
F4168 5.3 17.0
Table 1 (2) Isoelectric points and molecular weights of
human TNF polypeptide and human TNF N-terminal mutein
polypeptides
Polypeptide Isoelectric Molecular weight (Kd)
point
Human TNF 5.6 17.0
(2)
F4415 6.5 17.0
F4417 5.6 17.0
2G F4418 5.6 17.5
F4420 6.5 16.5
F4601 5.2 17.0
F4609 5.6 17.5
F4639 6.4 17.5
20~197~
- 84 -
Example 14
Evaluation of antitumor activities in vitro
With respect to the partially purified samples and
finally purified samples of the human TNF polypeptides
and the human TNF N-terminal mutein polypeptides obtained
in Example 13, cell lytic activities against fibroblast
cells (L929 (ATCC CCLl) derived from connective tissues
of mouse), were determined in accordance with the method
of Aqqarwal et al. (J. Biol. Chem., 260, 2345 (1985)).
Namely, L929 cells were seeded on a 96-well microplate
for tissue culture (manufactured by Corning) in an amount
of 3 x 104 cells/0.1 ml/well and cultured overnight at
37C in the presence of 5% carbon dioxide gas. As the
culture medium, Dulbecco's modified Eagle's minimum
essential medium (DME medium, manufactured by Sigma)
containing 10% fetal calf serum, was used. On the next
day, the medium was changed to the above culture medium
having Actinomycin D added to a final concentration of 1
~g/ml. Each sample diluted stepwisely by this culture
medium, was applied to the respective wells (total amount
of the medium: 0.1 ml). After culturing for further 20
hours, live cells attached to the plate were stained with
a 0.5% Crystal Violet solution (0.5% Crystal Violet/20%
methanol) (at room temperature for 15 minutes). The
plate having stained cells was thoroughly washed with a
phosphate buffer PBS (10 mM Na-K phosphates pH 7.4, 0.8%
NaCl and 0.02~ KCl) containing 1 mM of CaCl2 and 1 mM of
20~1975
- 85 -
MgCl2, and then Crystal Violet remained on the plate was
extracted with 0.1 ml of a O.OlN ~Cl solution containing
30% ethanol, and then the absorbance (492 nm) was
measured by an EIA reader (Model 2550, manufactured by
Bio-Rad). This absorbance corresponds to the number of
live cells. Therefore, the final dilution ratio of a
sample at a well showing the absorbance corresponding to
a 50% value of the absrobance of the well non-treated by
the sample was obtained, and the reciprccal number of
this dilution ratio of the sample is defined as the
number of units per 1 ml of the sample (units/ml).
To calculate the specific activities
(units/mg-protein) of the respective samples from the
cell lytic activities (units/ml) against L929 obtained in
accordance with the above method, the quantitative
analysis of the proteins of the respective samples was
conducted. The quantitative analysis was conducted in
accordance with a Bradford method (Anal. Biochem , 72,
248 (1976)), whereby the protein concentration (mg/ml)
was obtained by using bovin serum albumin (sSA) as the
standard sample. From these results, the specific
activities were calculated with respect to the human TNF
polypeptide samples and the human TNF N-terminal mutein
polypeptide samples and are shown in Table 2.
20~197~
- 86 -
Table 2
Table 2 (1) Cell lytic activities against L929 of human
TNF polypeptide and human TNF N-terminal mutein
polypeptide
Specific activity (units/mq-protein)
Polypeptide Partially purified Finally purified
sample sample
Human TNF 2.7 x 107 8.0 x 107
(1)
F4168 4.0 x 106 3.2 x 107
Table 2 (2) Cell lytic activities against L929 of human
TNF polypeptide and human TNF N-terminal mutein
polypeptides
Specific activity (units/mq-protein)
Polypeptide Partially purified Finally purified
sample sample
Human TNF 1.2 x 107 2.0 x 107
(2)
F4415 3.5 x 106 1. 7 x 107
F4417 1.2 x 107 3.3 x 107
F4418 1.3 x 107 2.1 x 107
F4420 3.0 x 106 2.3 x 107
F4601 1.4 x 1o6 2.5 x 106
F4609 3.0 x 107
F4639 3.0 x 107
2~51~7~
- 87 -
From the above Table 2, it is apparent that the human
TNF N-terminal mutein polypeptides of the present
invention have cell lytic activities, against fibroblast
cells L929 derived from connective tissues of mouse,
similar to that of the human TNF.
EXAMPLE 15
Evaluation of antitumor activities in vivo
With respect to the partially purified samples of the
human TNF polypeptides and the human TNF N-terminal
mutein polypeptides obtained in Example 13, the antitumor
activities against a Meth A fibrosarcoma were determined.
The test was conducted in such a manner that 1 x 106
cells/0.2 ml of Meth A fibrosarcoma cells (obtained from
Sasaki Institutes, Sasaki Foundation) suspended in a
saline were subcutaneously transplanted to the side part
of the back of a BALB/C mouse (male, five weeks old,
Charles River), and eight days later, after confirming
that the tumor diameter reached a level of from 6 to 10
mm, a sample (0.2 ml/mouse) diluted stepwise with a
saline was intravenously administered in the tail vein.
The lethal dose was taken as the maximum dose, and
samples of various doses were prepared by stepwise
dilution.
For about two weeks after the administration,
observation of tumor growth, etc. was continued. With
respect to the tumor growth, the tumor volume
205~97~
- 88 -
ab2/2:
a = long diameter of the tumor
~ b = short diameter of the tumor J
was measured, and the tumor volume ratio of the tumor
volume after administration to the tumor volume on the
day of administration of the sample (O day) was obtained,
whereby the number of days from O da~ when the tumor
volume ratio became 2 or 5 (D2 or D5) was calculated.
Then, the ratio to the control group (to which the saline
was administered) was calculated, and the D2% control
value and the D5% control value were obtained. The
larger the values, the lower the tumor growing ability,
i.e., the higher the antitumor activities.
The results obtained in the above manner are shown in
Tables 3 and 4.
2 ~ 7 ~
- ~39-
~ ~ ~o ~ .~00
O O In ~ O U~ a~
~ 3 ~ ~ ~
a) ~ ~
Z ~
O~!
C OC ~ ~_ o r~ o
O ~ l o N U'')
E~ ~ ) Ll') t`-) Ll~ 1~ N
~ E~ o\
al N
~ _
o\ N O ~-- O O
Q~ _ _
o
z E
~ ~ ~ u~
30 ~ ~ ~
~: Q
a~ ~ _
¢ ~ ,_
a ~ u~ u~ G ~ q~
O ~ O O O O O
U~ ~ 0~ r~ l ~
a) ~ x )< x x x
c ~ o o o In o
~ ~ C N ~1 117 N ~1
0~
~ V
~ ~ ~ z
aJ a) I ~a ~ co
Q ~ o aJ :~ ~ ~r
~ ~ ~ ~ ~ _ ~ _
E~
2051~75
- 90 -
_
~,
~o
__C o co a~ ~ ~ a~ c~ ~r In r) ~r m Cl~ ~ Lr ~ C~ CO
O ~ r~ r~ ~ r~ ~D co r~ m co N
C ~V ~" N ~1 ~1 ~ ~1 ~1 ~1 ~I r-l N ~ ~1 ~1 ~1 ~I N ~1 ~1
Z
Z
g O
C ~ C 00 ~ O r~ m ~ r~ r~ ~ r~
O O ~ r~ ~r ~ o co r~ n r~ ~ ~ n
S ~ 0\o ~ u~
a~
ra
,~ ,~ r~ o o r~ o o ~ o o r~ o o r~ o o ~ o o
~o\
V ~0
P~ _
o
E~
Z~ ~_
C ~ O ~ ~ ~ ,1 O
E~ O ~ r~ r~ n r~ ~ r~
R ~ ~ In ~ ~ ~ ~ ~ ~1
v ~: a~ ,_
~ ~ O a) ~ ~ In In ~r Ul ~r ~ U- m er ~ ~ ~ ~r ~r ~
o ~ n u~ O O O O O O O O O O O O O O O O O O
U~ ~ o ~, ~ ~ ~ ~ ~ ~, ~ ~ ~ ~. ~ ~,
~ V ~ X X X X X X X X X X X X X X X X X X
~ C v o o o o o o o o u~ o o o o In o o In o
¦> _ ~ ) ~1 ~1 Irl ~ In N ~ ~1 11-1 11~ ~ ~1 11~ ~ ~1
O ~r~
~; V
~C
0~ ~ E~
C I ,~ C In r~ ~D o
~ ~ ~ P~ ~ ,_
Q ~ O ~ ~
~ ~ ~ ~ m --
E~ ~ _ _ _
7 ~
- 91 -
From Tables 3 and 4, it is evident that the human TNF
N-terminal mutein polypeptides of the present invention
have antitumor activities, against Meth A fibrosarcoma
transplanted to mouse, similar to that of the human TNF.
5 EXAMPLE 16
Effects aqainst lunq metastasis of tumor
By cloning B16F10 mouse melanoma cells (obtained from
School of Medicine, Chiba University) by the following
method, a clone showing a high level of metastasis to the
lung was produced. Namely, 2 x 104 cells/0.2 ml of
B16F10 cells suspended in an Eagle's minimum essential
medium (MEM medium, manufactured by Nissui Pharmaceutical
Co., Ltd.), were injected into the tail vein of a C57BL/6
NCrj mouse (female, six weeks old, Charles River), and 14
days later, the lung was taken out. After washing the
lung with a DME culture medium, one of metastatic nodules
having a diameter of about 1 mm formed on the surface of
the lung was sucked by a 26 G injection needle-attached
syringe (manufactured by Termo) and suspended in 1 ml of
the DME medium and cultured on a DME medium-containing
soft agar containing 10% of fetal calf serum at 37C in
the presence of 5% carbon dioxide gas to form a colony.
This colony formation method was conducted in accordance
with the method disclosed in "Tissue Culturing
Techniques" (p. 35-36, 1984, compiled by Japan Tissue
Culture Association, Asakura Shoten). Ten to twelve days
later, when the colony diameter reached a level of from 1
20~197a
- 92 -
to 2 mm, the colony was sucked by a Pasteur pipet and
cultured in a DME medium containing 10% of fetal calf
serum. After the culturing, the proliferated cells were
adjusted to a cell concentration of 2 x 10~ cells/0.2 ml
again by using the MEM medium and injected into the tail
vein of a C57BL/6 NCrj mouse in the same manner as above.
Fourteen days later, the lung was taken out, and one of
metastatic nodules was isolated and cultured in the same
manner as above. Such operation was repeated five times
to obtain a clone (designated as B16F10/L5) resulting in
a high level of metastasis to the lung.
The B16F10/L5 cells in a logarithmic phase cultured
in the DME medium containing 10% of fetal calf serum by
means of a dish for tissue culture having a diameter of
10 cm (Coning (registered trademark), manufactured by
Iwaki Glass Co., Ltd.), were washed once with a phosphate
buffer PBS in a state attached to the dish, and 5 ml of
the MEM medium was added, followed by pipetting to obtain
a cell suspension. The cells were collected by
centrifugal separation and again suspended in 2 ml of the
MEM medium. On the other hand, the partially purified
samples of the human TNF polypeptides and the human TNF
N-terminal mutein polypeptides obtained in Example 13
were diluted with the MEM medium to prescribed
concentrations (the doses at which the antitumor effects
were observed in the antitumor test in Example 15, were
used as the doses for this test).
20~1~7~
- 93 -
To this solution, the previously prepared B16~10/L5
cell suspension was added to prepare a cell suspension
containing 2 x 104 cells/0.2 ml of each partially
purified sample solution. This cell suspension (0.2
ml/mouse) was injected into the tail vein of a C57BL/6
NCrj mouse (female, six weeks old). To the control
group, only L16F10/L5 cells were injected. Fourteen days
after the injection, the lung was taken out, and the
number of metastatic nodules on the lung surface was
counted.
The results obtained in the above manner are shown in
Tables 5 and 6.
~0~1975
- 94 -
_ __.
.~
~ ~a _ ~ ,_
v ~ ~ a ~ ~ ~
~: o +l o ,1~ r~
a) - +l +l +l
~ L~ ~1 C~ Ln ~
a tu ~ . . .
.,, Z ~ ra ~
:~ __ _
v ~ 1~- ~
v a oI x x
o ~
._
O ~D
m ~ ~:
V _ V
E~ ~
2051~7~
- 95 -
o o _ = ^ô~ ~ C o
U~ N ~ ~ O r~~1 ~ ~fl ~1 ~ ~
.~ ~ ~ l I I I ~ I ~ `I a) u~ v
Il~ E~ ul ~, ~ co(~I ~1 I r` I t- ~1 1 C,) ~ C
(~ ti- r-l C C N U~~ ~ _ _ _ _ _ _ r-l C)
C ~ ~ ~ ~ ~ ~ ~
~ u~ ~- ~o ~ ~ ~ ~ o a~ r In ~ ~ ~
C O +I C N ~ ~ ~ 1`~1 ~1 ~I C~) Z O ,~
:~ ~ +1 +1 +1 +1 +1 -1-1 +1 +1 +1 +1 ~ V 3
.~ ~ ~1 u~ o r~o Isl o O ~ ~ ra
aJ ~ .. .... .... ~S C C~
U~ ~ ~ ~IJ ~ N 01 r~) OQ ~0 ~ ) O ,~
¢ '1:1 N ~ ~ r~ 1 N ~) ~1 ~1
O :> ~
~ ~ U) g
Q~ ~ c~ 3 'o
I ~ r~ ~D I ~ ~D
,~ O o~ oo ~ t- ~ ~ 7 ~ C O V
0~ E~ ~ ,~ ,~
c a) ~ ,~
O
, Q u~ u7 u~ ~ m .r In n ~
:~ o o o o o o o o 3 ~ :>
,~ O ,~ ,~ ~
~C ~ I X X X X XI X X X U~r~ ,,
.~ U~ o oooo oo o ~ ~O
~ ,~ ,~,~ In ~ C
Z _ o~ J~ C a~
C
Z q) ~
C ~
a) N ~ O ~ C
~0 E~ ~ Z 4Z.,1 q)
~ ~4 r~ ro E-l ~ '-I ~ ~a C ~
~ ) r-l ~ ~I C1~'1 t~ 00 0 ~ C r-l ~ ~ p~ ~1) (I) U~
>~ ~ ~ ~ ~ (~ O 1 4 u~
C E~r ~ c E~ ~ l~ ¢ E-~
O o ~ r O
~nP~ _ c~ c~ 4
r~
Q E4~ 1~ Z ,~ _ v v
2~51975
- 96 -
From Tables 5 and 6, it is evident that while the
human TNF polypeptide and the human TNF mutein
polypeptide have activities to promote experimental lung
metastasis, the human TNF N-terminal mutein polypeptide
of the present invention, having an Arg-Gly-Asp sequence
introduced in the vicinity of the N-terminus, does not
promote the experimental lung metastasis.
According to the present invention, a novel human TNF
N-terminal mutein is provided which has substantially the
same antitumor activities as the human TNF or a mutein
thereof and which, on the other hand, does not have an
activity to promote tumor metastasis observed with the
human TNF or its mutein, by replacing, on the human TNF
or its mutein, the amino acid sequence of from the 1st
Ser to the 8th Asp of SEQ ID NO:l in the Sequence Listing
or the corresponding amino acid sequence of the human TNF
mutein with an amino acid sequence containing at least
one amino acid sequence of Arg-Gly-Asp and having from 3
to 16 amino acids.
Obviously, numerous modifications and variations of
the present invention are possible in light of the above
teachings. It is therefore to be understood that within
the scope of the appended claims, the invention may be
practiced otherwise than as specifically described
herein.
2051~75
SEQUENCE l,ISTING
SEQ ID NO:l:
SEQUENCE CHARACTERISTICS:
LENGTH: 155 amino acids
TYPE: amino acid
TOPOLOGY: linear
MOLEC~LE TYPE: protein
PUBLICATION :[NF`ORMATION:
A~THORS: Pennica, et al
JOURNAL: Nature
VOLUME: 312
PAGES: 724-
DATE: 1984
SEQUENCE DESCRIPTION: SEQ ID NO:l:
Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala
1 5 10 15
Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn
Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val
Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly
Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg
Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys
Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp
100 105 110
Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp
115 120 125
Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu
130 135 140
Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala l,eu
145 150 155
SEQ ID NO:2:
SEQ~ENCE CHARACTERISTICS:
LENGTH: 8 amino acids
TYPE: amino acid
TOPOLOGY: linear
MOLEC~LE TYPE: peptide
- 9S - 2~51 9 7~
SEQ~ENCE DESCRIPTION: SEQ ID NO:2:
Ser Ser Ser Arg Gly Asp Ser Asp
1 5
SEQ ID NO:3:
SEQUENCE CHARACTERISTICS:
LENGT~: 8 amino acids
TYPE: amino acid
TOPOLOGY: linear
MOLEC~LE TYPE: peptide
SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ser Ser Ser Arg Thr Arg Gly Asp
1 5
SEQ ID NO:4:
SEQUENCE CHARACTERISTICS:
LENGTH: 11 amino acids
TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
SEQUENCE DESCRIPTION: SEQ ID NO:4:
Ser Ser Ser Arg Gly Asp Arg Thr Pro Ser Asp
1 5 10
SEQ ID NO:5:
SEQUENCE CHARACTERISTICS:
LENGTH: 11 amino acids
TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ser Ser Ser Arg Thr Pro Arg Gly Asp Ser Asp
1 5 10
SEQ ID NO:6:
SEQUENCE CHARACTERISTICS:
LENGTH: 11 amino acids
TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
99 ~ ~5197a
SEQUENCE DEscRIeTIoN: SEQ ID NO:6:
Arg Gly Asp Ser Ser Ser Arg Thr Pro Ser Asp
1 5 10
SEQ ID NO:7:
SEQUENCE CHARACTERISTICS:
LENGTH: 14 amino acids
TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ser Ser Arg Gly Asp Arg Gly Asp Ser Arg Ala Pro Ser Asp
1 5 10
SEQ ID NO:8:
SEQVENCE CHARACTERISTICS:
LENGTH: 14 amino acids
TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptlde
SEQUENCE DESCRIPTION: SEQ ID NO:8:
Gly Arg Gly Asp Ser Pro Ser Ser Ser Arg Ala Pro Ser Asp
1 5 10
SEQ ID NO:9:
SEQUENCE CHARACTERISTICS:
LENGTH: 16 amino acids
TYPE: amino acid
TOPOLOGY: linear
MOLECULE TYPE: peptide
SEQUENCE DESCRIPTION: SEQ ID NO:9:
Asp Pro Gly Arg Gly Asp Ser Pro Ser Ser Ser Arg Ala Pro Ser Asp
1 5 10 15
SEQ ID NO:10:
SEQUENCE CHARACTERISTICS:
LENGTH: 465 bases
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
- loo - 2 ~5 ~ ~ 7~
MOLEC~LE TY~E: DNA (genomic)
SEQ~ENCE DESCRIPTION: SEQ ID NO:10:
TCATCTTCTC GAACCCCGAG TGACAAGCCT GTAGCCCATG TTGTAGCAAA CCCTCAAGCT 60
GAAGGGCAGC TCCAGTGGCT GAACCGCCGG GCC'AATGCCC TCCTGGCTAA TGGAGTGGAG 120
CTCAGAGATA ACCAACTAGT GGTGCCATCA GAGGGCCTGT ACCTGATCTA CTCTCAGGTC 180
CTCTTCAAGG GTCAAGGCTG CCCATCCACC CATGTGCTCC TCACCCACAC CATCAGCCGC 240
ATCGCCGTCT CCTACCAGAC CAAGGTTAAC CTCCTCTCTG CTATTAAGAG CCCCTGCCAG 300
AGGGAGACCC CCGAGGGCGC AGAGGCCAAG CCCTGGTATG AGCCCATCTA TCTGGGAGGG 360
GTCTTTCAAC TGGAGAAGGG TGACCGACTC AGCGCTGAGA TCAATCGGCC CGACTATCTC 420
GACTTTGCCG AGTCTGGGCA GGTCTACTTT GGGATCATTG CCCTG 465
SEQ ID NO:ll:
SEQUENCE CHARACTERISTICS:
LENGTH: 46 bases
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: DNA (genomic)
SEQUENCE DESCRIPTION: SEQ ID NO:ll:
AATTCATGCG AGGTGACAAG CCTGTAGCCC ATGTTGTAGC AAACCC 46
SEQ ID NO:12:
SEQUENCE CHARACTERISTICS:
LENGTH: 34 bases
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: DNA (genomic)
SEQUENCE DESCRIPTION: SEQ ID NO:12:
AATTCATGTC ATCTCGAGGT GACAGAGGCG ATTC 34
SEQ ID NO:13:
SEQUENCE CHARACTERISTICS:
LENGTH: 34 bases
TYPE: nucleic acid
STRANDEDNESS: single
TOPOLOGY: linear
MOLECULE TYPE: DNA (genomic)
- 101- 205197~
SEQ~ENCE DESCRlPTlON: SEQ ID NO:13:
AATTCATGGG GCGCGGAGAT TCTCCCTCAT CTI'C 34
SEQ ID NO:L4:
SEQ~ENCE C~IARACTERISTICS:
LENGTH: 32 bases
TYPE: nucleic acid
STRANDEDNESS: single
TOPOI,OGY: linear
MOLECULE TYPE: DNA (genomic)
SEQ~ENCE DESCRIPTION: SEQ ID NO:14:
GATCCCGGGC GCGGAGATTC TCCCTCATCT TC 32
