JPH05271290A - Polypeptide - Google Patents

Abstract

PURPOSE:To provide a new transformed human TNF having antitumor action similar to that of human TNF or its conversion product and free from promoting action on cancer metastasis in contrast with human TNF. CONSTITUTION:The objective polypeptide is a tumor necrosis factor polypeptide or its conversion product having an amino acid sequence expressed by the amino acids from the 1st Ser to the 155th Leu of formula provided that the amino acid sequence from the 1st Ser to the 8th Asp or the corresponding amino acid sequence of the conversion product contains at least one cell-adhesive peptide sequence of laminin and is substituted with an amino acid sequence containing 5 to 30 amino acids. The present invention also relates to a conversion product of the polypeptide, a plasmid, a transformant, their production process, a pharmaceutical composition and a DNA.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel polypeptide as a human TNF transformant, the polypeptide itself, a pharmaceutical composition containing the same, a method for producing the same, a recombinant DNA, a plasmid, and a trait thereof. Converted microbial cells, etc.

[0002]

2. Description of the Related Art TNF (Tumor Necrosis Factor) was previously reported in 1975 by Carswell et al. In Bacillus C.
It is a bioactive substance found to be present in the serum of endotoxin-treated mice infected with almette Guerin (BCG) [Proc. Nat
l. Acad. Sci. USA 72 3666 (197)
5)], in 1984 by Pennica et al., On human TN.
The cDNA of F was cloned and the entire primary structure (amino acid sequence) of the human TNF protein was elucidated [Natur.
re 312, 724 (1984)]. TNF has a specific antitumor effect such as cytotoxic activity on tumor cells, hemorrhagic necrosis on transplanted tumors, and suppression of proliferation, but recently it has been reported that side effects such as hyperlipidemia, hypotension, fever, etc. may occur. Research and development are being conducted to find out more superior drug efficacy and side effects. For example, in Japanese Patent Laid-Open Nos. 61-40221, 63-119692, and 1-277488, a specific amino acid in human TNF protein is deleted, replaced with another amino acid, or added by a genetic engineering technique. Human TNF
We provide a converter.

[0003]

However, a human TNF converter having a sufficiently satisfactory pharmacological action has not yet been obtained. The object of the present invention is to provide a novel human TNF transformant having the same antitumor effect as that of human TNF or its transformant, while not exhibiting the cancer metastasis-promoting effect observed therein, and a polypeptide related thereto. It is intended to provide a method for producing them, a plasmid, a microbial cell, etc., and an application.

[0004]

SUMMARY OF THE INVENTION The present invention will be described in brief. The present invention provides a tumor necrosis factor polypolynucleotide having an amino acid sequence represented by Ser at position 1 to Leu at position 155 shown in SEQ ID NO: 1. In the peptide or its transformant, the As to the 8th As to the 1st Ser of the above-mentioned SEQ ID NO: 1
The amino acid sequence up to p or the corresponding amino acid sequence of the above-mentioned transformant contains at least one laminin cell-adhesive peptide sequence and is substituted with an amino acid sequence containing 5 to 30 amino acids. To the polypeptide. The present invention also provides a recombinant plasmid containing DNA encoding the polypeptide, a microbial cell transformed with the recombinant plasmid,
The method for producing a polypeptide using the microbial cell, the pharmaceutical composition, and the polypeptide in which methionine is bound to the N-terminal of the amino acid sequence are further described. In the DNA having the represented nucleotide sequence or the mutation-introduced DNA thereof,
The base sequence from the 1st to 3rd TCA to the 22nd to 24th GAC of SEQ ID NO: 7 or the base sequence of the mutation-introduced DNA corresponding thereto contains at least one laminin cell-adhesive peptide sequence, and 5 DNAs substituted with a base sequence encoding an amino acid sequence containing 30 to 30 amino acids.

The present inventors have made an amino acid sequence having a cell-adhesive peptide sequence of laminin present in a certain amino acid sequence region of the amino acid sequence of human TNF or its transformant. Anti-tumor activity is similar to that of human TN
It was found that a novel human TNF convert polypeptide, which hardly promotes the metastasis of cancer while F or the converter thereof can be obtained, was completed, and the invention was completed based on the finding. 1 from the first Ser shown in SEQ ID NO: 1 in the sequence listing
Tumor necrosis factor polypeptide having an amino acid sequence represented by the 55th Leu means human TNF.

In the present invention, the human TNF converter is a modification of addition, deletion or substitution of one or two or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, either alone or in combination. Human TNF
It has an antitumor effect similar to. Therefore,
Regarding the human TNF converter in the present invention, the amino acid sequence corresponding to the amino acid sequence from Ser at position 1 to Asp at position 8 in SEQ ID NO: 1 of human TNF means human TNF.
If the amino acid sequence itself from Ser to Asp is not modified in the transformant, it corresponds to it, and if the amino acid sequence is modified, the modified amino acid sequence corresponds to it. Since this modification includes deletion of amino acids, it also includes partial or complete deletion of the amino acid sequence from the 1st Ser to the 8th Asp.

Specific examples of the human TNF converter include the following, but the present invention does not exclude any other compounds.

(1) Described in JP-A-63-141999: Val-Arg is added to the N-terminal, and ²⁹ A is further added.
rg- ³⁰ Arg converted to ²⁹ Asn- ³⁰ Thr. (2) Described in JP-A-63-119692: (A) ³² Asn is ³² Tyr, ³² His, ³² As
Converted to p or ³² Ser, respectively. (B) ¹¹⁵ Pro to ¹¹⁵ Leu, ¹¹⁵ Ser,
Those converted to ¹¹⁵ Asp or ¹¹⁵ Gly, respectively. (C) ¹¹⁷ Tyr converted to ¹¹⁷ His. (3) JP-A-1-277488 discloses: ¹ Ser
~ ⁸ Asp is deleted, and Arg-Ly is added to the N-terminal of ⁹ Lys.
s-Arg added. (4) Described in JP-A-2-163094: ¹ Ser
~ ⁸ Asp is deleted, and Arg-Ly is added to the N-terminal of ⁹ Lys.
¹⁵⁴ Ala was further converted to ¹⁵⁴ Phe by adding s-Arg. (5) Described in JP-A-2-142493: ¹ Ser
~ ⁸ Asp is deleted, and Arg-Ly is added to the N-terminal of ⁹ Lys.
¹⁵⁴ Ala converted to ¹⁵⁴ Trp by adding s-Arg. (6) Described in JP-A-63-270697: ¹⁰⁰
Gln to ¹⁰⁷ Ala deleted. ¹ Ser to 2
One to eight amino acid residues deleted. (7) Description in Japanese Patent Application No. 2-193935: ⁶⁸ P
Ro converted into ⁶⁸ Asp, ⁶⁸ Met, or ⁶⁸ Tyr, respectively. (8) Description in Japanese Patent Application No. 2-311129: ¹⁰⁶
Gly deleted or replaced with another amino acid. (9) Described in Japanese Patent Application No. 2-311130: ²⁹ A
Substitution of rg with another amino acid.

Throughout the specification, amino acids, polypeptides, bases and their sequences are represented by the following list.

[0010]

The abbreviations used in this specification mean the following. dATP: Deoxyadenosine triphosphate (Deoxy
adenosine triphosphate) dGTP: Deoxyguanosine triphosphate (Deoxy)
Guanosine triphosphate) dCTP: Deoxycytidine triphosphate (Deoxy c)
ytidine triphosphate TTP: thymidine triphosphate (Thymidine tr)
iphosphate) ATP: adenosine triphosphate (Adenosine t)
Riphosphate) SDS: Sodium Dodecyl Sulfate (Sodium do)
decyl sulphate BPB: Bromophenol blue (Bromophen
ol Blue) DTT: Dithiothreit
ol) BSA: Bovine serum albumin
albumin) PMSF: phenylmethylsulfonyl fluoride (P
phenylmethylsulfone Fluor
ide) EDTA: ethylenediaminetetraacetic acid (Ethylene
diaminetracetic Acid) CPG resin: Controlled-Pore Gla
ss resin

The amino acid sequence having the laminin cell-adhesive peptide sequence may be composed of only the cell-adhesive peptide sequence, but may be composed of the cell-adhesive peptide sequence and one or more amino acids. And may have one or more cell-adhesive peptide sequences of the laminin in question. The number of constituent amino acids of the amino acid sequence having the laminin cell-adhesive peptide sequence is 5 to 30, preferably 5 to 20, and more preferably 5 to 17. Specific examples thereof include the amino acid sequences shown in SEQ ID NOS: 2 to 6 of the sequence listing, or the like. Among them, the amino acid sequences shown in SEQ ID NOS: 2, 3 or 4 are preferable.

The laminin cell-adhesive peptide sequence is 3 in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
The one having the amino acid sequence represented by the Tyr at the 9th position to the Arg at the 7th position is preferable.

Furthermore, the amino acid sequence other than the amino acid sequence having the cell-adhesive peptide sequence of the laminin of the polypeptide of the present invention, that is, the amino acid corresponding to the amino acid sequence of human TNF from Lys at position 9 to Leu at position 155 of SEQ ID NO: 1. Regarding the sequence, 0 to 15 amino acids may be modified by addition, deletion or substitution, or a combination thereof, and among them, the 9th Ly of SEQ ID NO: 1
Amino acid sequence of human TNF from s to 155th Leu or its 19th Gln, 29th Ar
g, the 68th Pro, the 97th Ser, the 106th Gly or the 126th Lys is deleted or is substituted with another amino acid, or 10
An amino acid sequence in which at most 8 amino acids before and after the Gly at the 6th position is modified with the modification of amino acids at other positions is preferable. The 106th G
104 for the modified amino acid sequence centered on ly
In the amino acid sequence of Proth to 108th Glu, it is preferable that the amino acid is simply substituted with another amino acid, that at most 8 amino acids are added, or that these substitutions and additions are combined.

On the other hand, when the 29th Arg is substituted, for example, Met, Trp, Pro, Ala, Val,
Neutral amino acid such as Leu, Gln, Tyr or Ser, preferably Val, Leu, Pro, Gln or S
er, more preferably those substituted with Val, Leu or Gln, respectively, and Lys or Hi
Examples thereof include basic amino acids such as s, which are preferably substituted with Lys, and acidic amino acids such as Asp or Glu, which are preferably substituted with Asp.

When the 68th Pro is replaced,
Examples thereof include those substituted with the same neutral amino acid or acidic amino acid as in the case of Arg at position 29 above, preferably those substituted with Met, Tyr or Asp, more preferably those substituted with Met or Asp. There are some.

When the 106th Gly is further modified, for example, it is deleted or the above-mentioned 29th Arg.
Examples thereof include those substituted with a neutral amino acid, a basic amino acid or an acidic amino acid, which are preferably deleted or are Trp, Pro, Ala,
Substituted with Arg, Lys, Asp or Glu,
More preferably deleted or Trp, Pro, A
Examples thereof include those substituted with la, Arg or Asp.

In the polypeptide of the present invention, a laminin cell-adhesive peptide sequence is contained at a certain position, and
The amino acid sequence containing 5 to 30 amino acids is considered in consideration of the type of amino acid sequence called so-called laminin cell-adhesive peptide, antitumor activity, degree of cancer metastasis, side effects, applicability of gene recombination technology, etc. And decided accordingly.

Thus, the polypeptide of the present invention is composed of 137 to 192 amino acids.

D having a nucleotide sequence represented by the first T to the 465th G shown in SEQ ID NO: 7 in the sequence listing
NA is DN that encodes the amino acid sequence of human TNF.
It is one form of A.

D encoding the amino acid sequence of human TNF
The mutation-introduced DNA of NA is one obtained by appropriately modifying one or two or more pairs of codons in the nucleotide sequence shown in SEQ ID NO: 7 in the sequence listing, individually or in combination, and modifying the nucleotide sequence as described above. Is a DNA encoding the amino acid sequence of the human TNF conversion product of. Therefore, the modification is SEQ ID NO: 7.
Since it covers the entire region of the base sequence represented by the 1st T to the 465th G shown in Fig. 2, not only the base sequence from the 1st T to the 24th C, but also the 25th A to 46
It extends to the base sequence up to the fifth G.

Next, the embodiments of the present invention will be described in detail. Regarding the gene manipulation technique of the present invention, the methods and means described in many documents are appropriately adjusted and applied. The documents are listed below for reference.

T. Maniatis et al, (1
982): Molecular Cloning, A
Laboratory Manual (hereinafter, Mole
abbreviated as "Clar Cloning") Cold S
pring Harbor Laboratory, R.P.
Wu et al, (1983): Methodsin.
Enzymology, 100 and 101 , R.S. Wu
et al, (1987): Methods in E.
nzymology, 153 , 154 and 155

The polypeptide of the present invention can be produced by various methods, means and machines. Typical production methods are described below.

(1) Acquisition of human TNF gene The nucleotide sequence and amino acid sequence of the human TNF gene have been clarified by Pennica et al.
The human TNF gene sequence is designed by appropriately changing the base sequence. The sequence is designed by designing a DNA double strand in which the DNA strand of SEQ ID NO: 7 is the upper strand (coding strand) and the complementary sequence strand corresponding thereto is the lower strand. At that time, it is desirable to select a codon suitable for the host cell (such as Escherichia coli), and to select an appropriate position so as to facilitate the cloning by the ligation of DNA fragments described below and the gene modification for producing the transformant. It is desirable to place an appropriate restriction enzyme cleavage site. Of course, a translation initiation codon (ATG) is provided upstream of the human TNF gene,
Downstream translation stop codon (TAA, TGA or TAG)
Must be placed so that they match the open reading frame. Appropriate restriction enzyme cleavage sites should be placed upstream of the translation start codon and downstream of the translation stop codon, respectively, and they are applicable to vectors and easy to clone. Preferably.

Here, it is prepared on the basis of the above-mentioned DNA double strand, and its upper strand connects 5'-ATG-3 'to the 1st T in the upstream direction and 5'-TAA to the 465th G.
474, which connects TGA-3 'in the downstream direction
Bases (hereinafter coding 474 U chain). Therefore, the lower chain contains 474 bases starting from T and ending at T if the lower chain is described as being complementary to the coding 474 U chain (hereinafter, complementary sequence chain 474 L chain).

The human TNF gene can be produced by a method of chemically synthesizing several oligonucleotides for each of the upper chain and the lower chain, and appropriately ligating each block sequentially. For example, in the DNA duplex prepared based on the DNA duplex consisting of the coding 474 U chain and the complementary sequence chain 474 L chain, each strand of the human TNF gene is divided into 10 oligonucleotides each having about 50 bases, A total of 20 chemicals are chemically synthesized.

The synthetic method thereof is the diester method [H.
G. Khorana, "SomeRecent Dev"
elements in Chemistry of
Phosphate Esters of Biolo
physical Interest ", John Wile
y and Sons, Inc. , New York
(1961)], triester method [R. L. Letsi
nger et al. Am. Chem. So
c. , 89 , 4801 (1967)] and the phosphite method [M. D. Matteucci et al, Tet
rahedron Lett. , 21 , 719 (198
0)], but the phosphite method using a fully automatic DNA synthesizer is preferably used from the viewpoint of operability and the like.

The synthesized oligonucleotide is purified by a conventional purification method such as high performance liquid chromatography using a reverse phase chromatography column and electrophoresis using a polyacrylamide gel. The oligonucleotides are then phosphorylated, eg using T4 polynucleotide kinase, annealed and then ligated using T4 DNA ligase. Here, the oligonucleotides are divided into several blocks, which are sequentially ligated to obtain the desired human TNF gene sequence, which is cleaved with a restriction enzyme or T4 DNA.
After blunting with a polymerase, it is purified by electrophoresis or the like. Regarding the obtained DNA fragment, for example, pUC8, ibid 9, ibid 18, ibid 19 [J. Messing et al,
Gene, 19 , 259 (1982)], and a competent cell is transformed by a conventional method and cloned. Plasmid DNA is extracted and purified from the obtained clone according to a known method, and it is checked whether or not the nucleotide sequence of the DNA fragment inserted into the vector has achieved the desired gene sequence. Human TN achieved
Each part of the F gene was excised from a plasmid vector containing the same by using a restriction enzyme, ligated again into the vector, and then incorporated into the desired full-length human TN.
A plasmid vector having the F gene is obtained. The desired human TNF gene can be obtained by digesting the thus obtained plasmid vector with a restriction enzyme and then separating and purifying by gel electrophoresis.

On the other hand, in the above-mentioned method, cDNA encoding human TNF may be prepared from mRNA derived from human cells expressing TNF, and the methods using the cDNA may be appropriately combined.

(2) Construction of human TNF expression vector The human TNF expression vector is constructed by appropriately inserting the human TNF gene obtained in (1) above into the expression vector. The expression vector has a transcription promoter region and a translation signal SD upstream of the translation initiation codon (ATG).
It must have a (Shine-Dalgarno) sequence and a transcription terminator region downstream of the translation stop codon (TAA, TGA or TAG). The transcription promoters include trp promoter, lac promoter, t
ac promoter, P _L promoter, β-lactamase promoter, α-amylase promoter, P
H05 promoter, ADCI promoter and the like can be used, and as the transcription terminator, trp terminator, rrnB terminator, ADCI terminator and the like can be used. Such an expression vector is, for example,
pKK223-3 _(Pharmacia), pP L _-lamb
It can be easily obtained from commercially available products such as da (same as left) and pDR720 (same as left), but it is also possible to use those with improved expression or handleability by improving these.

(3) Construction of human TNF converter or its transformant expression vector As a method for preparing a DNA encoding the human TNF converter or its transformant polypeptide, for example, the following method can be mentioned.

(A) It is prepared by appropriately ligating chemically synthesized oligonucleotides according to the method described in (1) Acquisition of human TNF gene. According to this method, substitutions, additions, deletions or modifications of combinations thereof of amino acids, polypeptides and the like are possible.

(B) The human TNF gene prepared in (1) above is cleaved with an appropriate restriction enzyme to remove a specific region in the gene, and then a synthetic oligonucleotide having a mutated base sequence (eg, upper and lower regions) The strand is annealed to form a ligated double-stranded DNA fragment) or an appropriate other gene. By this method, as in the case of (A), the modification can be freely performed.

(C) Mutagenesis is carried out by extending a DNA chain using a synthetic oligonucleotide having a mutation-introduced nucleotide sequence as a primer [Part-specific mutagenesis (TA Kunkel et al, Metho).
ds in Enzymology, 154, 367
(1987))]. This method is difficult to apply to addition and insertion of a relatively long DNA chain exceeding 10 base pairs, but other modifications can be performed freely as in the case of (A) above. It is particularly suitable for substituting any amino acid.

In the present invention, generally, the site-directed mutagenesis method of (C) and the method of (B) utilizing a restriction enzyme cleavage site are appropriately combined and used, and the method will be described below.

After the DNA encoding the transformant or the transformant polypeptide is prepared by the site-directed mutagenesis method, it is appropriately inserted into an expression vector.

First, in performing the site-specific mutation method, a template DNA is prepared. The human TNF gene obtained in (1) above was prepared according to the method of Messing et al. [Methods in
Enzymology, 153 , 3 (1987)] and ligated to a plasmid vector for preparing single-stranded plasmid DNA (pUC118, pUC119, etc.) and introduced into an E. coli strain. A clone having the desired plasmid is selected from the obtained transformants.

The dut This plasmid ^- and ung ^- mutant E. coli were introduced into the strain (such as CJ236 strain), was incorporated uracil in a gene, by infecting mutant helper phage such as M13K07 to E. coli strain single strand of interest Obtain plasmid DNA.

On the other hand, an oligonucleotide primer of about 15 to 50 bases having a mutation introduction site and base sequences before and after it according to the present invention is chemically synthesized. The primer and the uracil-introduced single-stranded plasmid DNA obtained in the above step are annealed and then double-stranded using, for example, T4 DNA polymerase and T4 DNA ligase. In order to inactivate the DNA chain containing the template uracil and increase the frequency of mutation introduction, the double-stranded plasmid is introduced into the ung ⁺ E. coli strain.

Colonies are hybridized using the above-mentioned primers as a probe, and a clone having a plasmid containing a DNA encoding the desired transformant or transformant polypeptide is selected from the obtained transformants.

From the plasmid obtained above, a DNA fragment encoding the human TNF converter or the same polypeptide is excised by restriction enzyme digestion treatment, and inserted into an expression vector in the same manner as in the above (2), Human TN
An F-converted or transformant expression vector can be constructed.

A portion into which a mutation is to be introduced is cut out by cutting with an appropriate restriction enzyme, and is replaced with a DNA fragment prepared by chemical synthesis or the like according to the conversion design to construct an expression vector of the converter or the converter polypeptide.

In order to modify the amino acid sequence near the N-terminal of human TNF, it is preferable that an appropriate restriction enzyme cleavage site be present near the N-terminal. Therefore, first, an appropriate restriction enzyme cleavage site is introduced near the N-terminus of human TNF by the site-directed mutagenesis method. It is more preferable to use two types of cleavage sites, the restriction enzyme cleavage site and the restriction enzyme cleavage sites placed before and after the translation initiation codon located upstream thereof.

On the other hand, according to the conversion design of the present invention, oligonucleotides corresponding to the upper and lower chains are chemically synthesized in the same manner as in the above (1). Of course, it is necessary to install the above restriction enzyme cleavage sites at both ends in consideration of integration into an expression vector. This oligonucleotide (upper and lower chains) is phosphorylated using, for example, T4 polynucleotide kinase and then annealed to form a double-stranded DNA fragment. This double-stranded DNA fragment is inserted and ligated using, for example, T4 DNA ligase into an expression plasmid vector containing most of human TNF cleaved with the above two types of restriction enzymes (deleted at the N-terminal portion),
Human T having a modified amino acid sequence near the N-terminal of interest
An expression vector for the NF convert can be constructed.

In addition, recombination between respective expression vectors is carried out by utilizing an appropriate restriction enzyme cleavage site in the gene encoding the transformant or transformant polypeptide,
Furthermore, a novel transformant or transformant polypeptide expression vector can be constructed.

Regarding the introduction of the expression vector into a host cell such as E. coli strain, for example, a known method using competent cells of Escherichia coli strain prepared by the calcium chloride method [Molecular Cloning, T. et al. M
aniatis et al, (1982)]. Although microbial cells such as Escherichia coli, Bacillus subtilis, and yeast can be used as the host cells, JM is used as the E. coli.
83, JM103, HB101, etc. coli K
-12 mutants are mentioned.

(4) Obtaining Human TNF Transformant Polypeptide In the present invention, the transformed microbial cell described in (3) above is cultured to produce the desired human TNF transformant polypeptide in the culture. Accumulate, extract and separate. As a method for culturing microbial cells, in particular Escherichia coli, for example, a culture solution containing nutrients required by Escherichia coli is inoculated with E. coli, and usually about 12 to about 32 to 37 ° C.
A method of culturing a large amount in a short time by shaking or stirring for 24 hours can be used. The medium is, for example, L medium,
M9 medium, M9CA medium, etc. [said Molecular
Cloning.
When using the ac promoter and tac promoter, a drug such as isopropyl-β-D-thiogalactopyranoside can be added, and when using the trp promoter, a drug such as 3-β-indoleacrylic acid can be added.

After culturing, the human TNF transformant polypeptide of the present invention is generally subjected to a disruption treatment by sonicating a microbial cell suspension suspended in a Tris buffer, followed by a centrifugation operation to remove bacteria. Obtained by removing body residues. Further, the thus obtained product can be further purified by a combination of treatment with a nucleic acid / endotoxin removing agent, filtration through a filter, anion exchange chromatography, and other conventional methods for separating and purifying proteins. The human TNF transformant gene of the present invention can be directly produced by once applying the above-mentioned method, once produced after producing the human TNF gene, or produced after producing the human TNF gene and then its transformant mutant gene. You can

The human TNF transformant polypeptide of the present invention has the same antitumor activity as that of human TNF or its transformant, and has the same antitumor activity as that of human TNF or its transformant. On the other hand, the antitumor agent, which shows almost no cancer metastasis-promoting effect observed in them,
It is effective as an active ingredient of medicines. Human TNF of the present invention
Specific examples of the transformant polypeptide include F4 described below.
236, F4146, F4419, F4422, F44
23, F4603, F4604, F4616, F461
7, F4630, F4631, F4636, F463
7, F4618, F4619, F4632, F463
3, F4640, F4641, F4620, F462
1, F4622, F4623, F4624, F464
7, F4648, F4649, F4650, F465
1, F4653, F4655, F4657 and the like, among which F4236, F4146, F4419, F
4603, F4616, F4617, F4621, F4
649, F4653 are desirable, F4236, F414
6, F4419 is more desirable. In the formulation of the pharmaceutical composition, the pharmaceutical composition can be formulated with a pharmacologically acceptable carrier or diluent. Examples of the dosage form of the pharmaceutical composition of the present invention include external preparations, oral administration agents, injections and the like, and the administration method suitable for each dosage form is used. As the administration method, administration by injection is preferable. Administration by injection includes systemic administration and local administration. Systemic administration includes intravenous injection, subcutaneous injection, intramuscular injection, intradermal injection and the like, and local administration includes intratumoral injection and the like. The pharmaceutical composition of the present invention can be used in any method, but systemic administration is preferable in consideration of applicability to various cancer types, and intravenous injection is particularly preferable from the nature of the drug. Examples of cancer types include solid cancers such as colon cancer, lung cancer, gastric cancer, pancreatic cancer, malignant melanoma, and hematological cancers. The pharmaceutical composition of the present invention can be used for any cancer type, but application to solid cancer is particularly desirable.

[0051]

【Example】

Example 1 (Design of human TNF gene) Human T reported previously [Pennica et al., Supra].
Based on the amino acid sequence of the NF structural gene, the DNA sequence of SEQ ID NO: 7 in the sequence listing is coded 4 for the nucleotide sequence of the human TNF gene for convenience of gene construction and conversion.
D of 74 U chain and complementary sequence chain 474 L chain
The base sequence of the NA double strand and the DNA double strand prepared by slightly adjusting it was designed. Here, a restriction enzyme cleavage site is incorporated at appropriate intervals, and a cleavage site by the restriction enzyme EcoRI is added upstream of the translation initiation codon (ATG) so that it can be easily ligated to the plasmid vector. Downstream, the restriction enzyme Hind I
A cleavage site according to II was provided.

Example 2 (Chemical Synthesis of Oligonucleotide) The DNA designed in Example 1 was obtained by using an automatic DNA synthesizer (Applied Biosystems, Model 381).
Using A), it was chemically synthesized by the phosphite method. The synthesis includes U-1 to 10 and L-1 to 1 having the above-mentioned base sequence.
It was performed by dividing it into 10 20 oligonucleotides.

These oligonucleotides will be described below based on the DNA strand of SEQ ID NO: 7 in the sequence listing.

The lower strand (complementary sequence strand) corresponding to the DNA strand of SEQ ID NO: 7 will be described based on SEQ ID NO: 7, but the explanation of the flow direction of the lower strand, that is, upstream, downstream, 5 '
-And 3'- are exactly the opposite of that of the upper chain.

Cleavage of the synthesized oligonucleotide from CPG resin (sold by Funakoshi Co., Ltd.) and removal of the protecting groups were in accordance with the manual of Applied Biosystems. Separation and purification of each oligonucleotide was performed by HPLC (high performance liquid chromatography) using a reverse phase chromatography column or polyacrylamide gel electrophoresis containing 7 M urea (gel concentration 10 to 20%).

For the HPLC method, nucleosil 5C
Separation and purification was carried out by reverse phase chromatography using 18 columns (φ4.6 × 150 mm: sold by Chemco), eluting with triethylaminoacetic acid (100 mM) buffer (pH 7.0) containing acetonitrile.
The above elution was performed using a linear concentration gradient of acetonitrile of 5 to 35
% (30 minutes) and the peak at about 15 minutes was collected.

Regarding the polyacrylamide gel electrophoresis method, each synthetic oligonucleotide sample is separated by electrophoresis, the band portion of the desired size is cut out from the result of observation of the migration pattern by the ultraviolet shadowing method, and the polyacrylamide gel fragment Minced to a size of about 1 to 2 mm ³ , and about 2 ml of elution buffer (0.5MN
H ₄ OAc and 1 mM EDTA) were added and shaken at 37 ° C. overnight. The elution buffer containing each oligonucleotide was collected and extracted with phenol (50% phenol / 5
A 0% chloroform solution was used), isobutanol extraction was performed, and ethanol precipitation was performed to obtain a purified sample of each oligonucleotide.

For some of the synthesized and purified oligonucleotides, the Maxam-Gilbird method [A. M. Maxa
m et al, Methods in Enzymo
, 65 499 (1980)], it was confirmed that it has the target nucleotide sequence.

The following (Examples 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, and 15), the reaction conditions of the restriction enzyme and other related enzymes are mainly Molecular Clon.
According to the method described in ing (supra). The enzymes and the like were mainly obtained from Takara Shuzo, and the Takara Shuzo manual was also referred to.

Example 3 (Construction of human TNF gene by ligation of synthetic oligonucleotides) (1) First, the construction of human TNF gene was tried according to FIG. The synthetic oligonucleotides obtained in Example 2 were divided into three groups (U and L-1 to 4, U and L-5 to 7).
And U and L-8-10) were separately cloned. That is, U-2, 3, 4, 6, 7, 9 and 10
And 5'end of each of L-1, 2, 3, 5, 6, 8 and 9 oligonucleotides (1 to 2 μg) using 2 to 5 units of T4 polynucleotide kinase (Takara Shuzo),
Each was phosphorylated separately. Phosphorylation reaction is 10 μl
(50 mM Tris-HCl pH 7.6, 10
mM MgCl ₂ , 0.1 mM spermidine, 0.1 m
MEDTA, 10 mM DTT and 1 mM ATP),
The reaction was carried out at 37 ° C for 1 hour, and after completion of the reaction, T4 polynucleotide kinase was inactivated by treating at 70 ° C for 10 minutes. Newly prepare 10 μl of an aqueous solution having the same composition as described above and separately containing 1 to 2 μg of each of U-1, 5 and 8 and L-4, 7 and 10 oligonucleotides. At this time, each oligonucleotide (U-1 to 10 and L-1 to 10) aqueous solution is mixed (2
0 μl), boiled at 100 ° C. for 5 minutes, and then annealed by slow cooling. Next, the obtained 10 annealing bodies (double-stranded DNA fragments) were treated with an aqueous solution (66 mM Tris-HCl PH7.
6, 6.6 mM MgCl ₂ , 10 mM DTT, 1 m
MATP and 100 μg / ml BSA) (total liquid volume 120 to 160 μl), after heating to 40 ° C. and annealing by slow cooling, 700 units of T4 DNA ligase (Takara Shuzo) was added and the mixture was heated at 16 ° C. for 15 hours. The ligation reaction was performed.

After completion of the reaction, the respective reaction solutions were separated by polyacrylamide gel electrophoresis (gel concentration 6%), and the target size (176 bp, 150 bp and 153 b) was obtained from the result of observing the migration pattern by the ethidium bromide staining method.
The band portion of p) was cut out, and the three target DNA fragments were recovered by the electro-elution method.
Further, phenol extraction (50%
Phenol / 50% chloroform solution is used), isobutanol extraction is performed, and the target D
NA was purified. According to the above method, the three purified double-stranded DNA fragments were individually phosphorylated at their 5'ends with T4 polynucleotide kinase, mixed in an aqueous solution for ligation reaction, and then 40 Annealing was performed by heating at 0 ° C, and T4 DNA ligase was added for ligation. The ligated DNA was recovered by an ethanol precipitation operation, and 50 μl of high salt buffer (50 mM Tris-containing 1 mM DTT and 100 μg / ml BSA was collected.
HCl pH 7.5, 100 mM NaCl, 10 mM
MgCl ₂ ) and dissolved in 15 units of restriction enzyme E
coRI (Takara Shuzo) and 15 units of restriction enzyme H
Ind III (Takara Shuzo) was added, and a cleavage reaction was performed at 37 ° C. for 2 hours. After completion of the reaction, the target DNA fragment (about 480 bp) was separated and purified by polyacrylamide gel electrophoresis (gel concentration 4%) according to the above method.

On the other hand, 5 μg of the plasmid vector pUC9 (distributed by the Institute for Genetic Information, Kyushu University) was treated with restriction enzymes EcoRI and Hind III according to the method described above.
The DNA fragment of about 2.7 Kbp was separated and purified by agarose gel electrophoresis (gel concentration 1%). According to the method described above, the previously purified DNA fragment of about 480 bp (including the human TNF gene) and this pUC9 fragment were
After mixing in 20 μl of the ligation reaction solution, 350 units of T4 DNA ligase was added, and the ligation reaction was performed at 16 ° C. for 3 hours. Calcium chloride method [Molecular C
E.L. coli K-
12 Competent cells of JM83 strain (distributed by Kyushu University Genetic Information Laboratory) were transformed with the above ligation reaction solution according to a conventional method [Molecular Clon].
ing]].

From the obtained ampicillin resistant clone,
A plasmid was prepared using a known method, and the restriction enzymes (EcoR I and Hind II were prepared according to the method described above.
I) After treatment, pU of human TNF gene was analyzed by analyzing its migration pattern by agarose gel electrophoresis.
The insertion into the C9 vector was investigated. As a result, about 250b
The insertion of the gene of p can be confirmed, and the nucleotide sequence of the inserted gene of the clone was determined by the dideoxy method [F. San
ger, Science, 214 , 1205 (198).
1)], it was confirmed to be a gene fragment having a target human TNF gene nucleotide sequence of about 130 bp downstream from the EcoR I site and about 90 bp upstream from the Hind III site. This clone and plasmid were designated pUA41 / JM83 and p
It was named UA41.

(2) Subsequently, according to FIG. 2, human TNF
We tried to construct the gene. Around the region that has not been achieved in the nucleotide sequence of the human TNF gene by the cloning in the step (1) is divided into two groups (U and L-3 to 6 and U and L-6 to 9), and the step (1) is performed. In the same manner as above, after phosphorylating each oligonucleotide and annealing,
Ligation was performed with T4 DNA ligase. Polyacrylamide gel electrophoresis (gel concentration 6%) according to the above method
The two DNA fragments (201
bp and 200 bp) each separately in 100 μl of 6
7 mM Tris-HCl (pH 8.8), 6.7 mM M
gCl ₂ , 16.6 mM (NH ₄ ) ₂ SO ₄ , 6.7 μ
M EDTA, 1 mM DTT, 200 μg / ml BS
A and each 330 μM deoxyribonucleotide triphosphate etc. (dATP, dGTP, dCTP and TTP) dissolved in an aqueous solution, 2 to 5 units of T4 DNA polymerase (Takara Shuzo) was added, and the DNA fragment was reacted at 37 ° C. for 30 minutes. Both ends were blunted. After the reaction,
The T4 DNA polymerase was inactivated by treatment at 68 ° C. for 10 minutes, and two target DNA fragments were recovered by ethanol precipitation.

On the other hand, 5 μg of pUC9 vector was added to 1 mM.
50 μl containing DTT and 100 μg / ml BSA
Medium salt buffer (10 mM Tris-H
Cl, pH 7.5, 50 mM NaCl and 10 mM
MgCl ₂ ) and dissolved in 15 units of restriction enzyme Hi
ncII (Takara Shuzo) was added, and the mixture was reacted at 37 ° C. for 2 hours, and then recovered by ethanol precipitation. To the obtained cleaved / opened pUC9 vector, the two DNA fragments previously blunted and recovered were separately separated according to the above-mentioned method.
Integration with 4 DNA ligase and E. coli K-1
2 JM83 strain was transformed. For each of the obtained clones, the nucleotide sequence of the inserted DNA was examined in the same manner as in the above-mentioned step (1), and it was confirmed that it was the target nucleotide sequence. These clones were designated pUA4
2 / JM83 and pUA43 / JM83 and the plasmids were named pUA42 and pUA43.

The pUA41 obtained in the above step (1) was treated with restriction enzymes EcoR I (high salt buffer) and Sac I (Takara Shuzo: low salt buffer), restriction enzymes Hae II (Takara Shuzo) and Hind III.
(Medium salt buffer), pUA42 obtained in the above step (2) with restriction enzymes SacI (low salt buffer) and Hpa I (Takara Shuzo: KCl buffer), and pUA43 with restriction enzymes Hpa I and Hae II (KCl). Buffer), and each was cut according to the above method. Raw salt buffer (1
0 mM Tris-HCl pH 7.5 and 10 mM Mg
Cl ₂ ) and high salt buffer or KCl buffer (20 mM Tris-HCl pH 8.5, 100 mM
The combination of KCl and 10 mM MgCl ₂ ) was handled by dividing the cleavage reaction into two and performing an ethanol precipitation operation during the reaction. Ec from pUA41
oRI-SacI DNA fragment (127 bp) and Hae II-HindIII DNA fragment (80b
p), Sac I-Hpa IDN from pUA42
A fragment (147 bp) as well as Hpa from pUA43
The I-Hae II DNA fragment (126 bp) was separated and purified by polyacrylamide gel electrophoresis (gel concentration 6%) according to the above method.

On the other hand, 5 μg of pUC19 plasmid vector (distributed from Kyushu University Genetic Information Laboratory) was treated with the restriction enzymes EcoRI and Hind according to the above method.
After cutting with III, a DNA fragment of about 2.7 kbp was separated and purified by agarose gel electrophoresis (gel concentration 1%). The previously purified four DNA fragments were sequentially added as shown in the figure (FIG. 2), ligated using T4 DNA ligase according to the method described above, and finally the purified pUC1
9 vector (about 2.7 Kbp fragment) and integrated into JM83
The strain was transformed. Regarding the plasmid contained in this transformant, the nucleotide sequence of the inserted gene was examined according to the method described above, and the desired full length (about 480 b
It was confirmed that the clone had a plasmid vector (about 3.2 Kbp) containing the human TNF gene of p). This clone was named pUA44 / JM83 and the plasmid was named pUA44.

Example 4 (Construction of human TNF expression vector) (1) The following improvements were attempted in order to make the expression vector pKK223-3 (obtained from Pharmacia) having the E. coli tac promoter easier to handle. i) Reduce the molecular weight of the expression vector. ii) The restriction enzyme BamHI has a unique cleavage site. iii) The orientation of the tac promoter is reversed from that of the ampicillin resistance gene.

FIG. 3 illustrates the method. According to the method of Example 3, 5 μg of the plasmid vector pBR322 (distributed by Kyushu University Genetic Information Laboratory) was used for restriction enzyme E.
After cutting with coR I and Hind III, both ends were blunted using T4 DNA polymerase. According to the method of Example 3, a DNA fragment of about 4.4 Kbp was separated and purified by agarose gel electrophoresis (gel concentration 1%), and 100 ng of nonphosphorylated Bgl II linker (restriction enzyme was added to the ring-opening site. 10 bp double stranded DNA fragment containing the Bgl II cleavage site: Catalog N
o. 4721A Takara Biotechnology Catalog 1991 Vol. 1. Obtained from Takara Shuzo)
Insertion was ligated using ligase. From the transformants obtained according to the method of Example 3 above, the plasmid was examined for the ability to be cleaved with a restriction enzyme. A clone having a plasmid pBR9333 (about 4.4 Kbp) having a newly generated cleavage site was obtained.

5 of the plasmid pBR9333 obtained above
According to the method of Example 3, μg was dissolved in high salt buffer, and the restriction enzymes Bgl II (Takara Shuzo) and P were added.
About 2.3 including the origin of replication after cutting with vuII (Takara Shuzo)
The Kbp DNA fragment was separated and purified by agarose gel electrophoresis (gel concentration 1%). On the other hand, the expression vector pK
As in the above case, 5 μg of K223-3 (about 4.6 Kbp) was dissolved in high salt buffer, and the restriction enzyme B was added.
It was cut with amHI (Takara Shuzo) and Sca I (Takara Shuzo). The cleavage reaction with the restriction enzyme BamH I
The amount of added enzyme is about 1/2 of the usual amount, and the reaction time is 5 to 30.
It was performed by partial cutting for a minute. After cutting, tac
A DNA fragment of about 1.1 Kbp containing a promoter, rrnBT ₁ T ₂ terminator, etc. was prepared as in the above case.
It was separated and purified by agarose gel electrophoresis. The previously purified DNA fragment of about 2.3 Kbp containing the origin of replication was added to the above-mentioned DNA fragment of about 1.1 Kbp in the same manner as described above using T4DN.
E. coli produced by the calcium chloride method according to the method of Example 3 was inserted and ligated using A ligase. coli K
-12 It was introduced into competent cells of JM103 strain (Kyushu University Genetic Information Laboratory). A clone having an expression vector (about 3.4 Kbp) containing the target tac promoter and the like was selected from the obtained transformants, and this expression vector was named pKK101.

(2) The next step will be described with reference to FIG. 5 μg of the expression vector pKK101 obtained in the above step (1) was added to the restriction enzyme EcoR I according to the above method.
And Hind III, and a DNA fragment of about 3.4 Kbp containing an origin of replication and a transcriptional regulatory region was separated and purified by agarose gel electrophoresis (gel concentration 1%). Further, the plasmid pUA44 (about 3.2 Kbp) containing the human TNF gene obtained in Example 3 was similarly cleaved with restriction enzymes EcoR I and Hind III to obtain about 480 bp DN containing the entire human TNF gene.
The A fragment was separated and purified by agarose gel electrophoresis.
This DNA fragment containing the entire region of the human TNF gene was inserted and ligated into a DNA fragment of about 3.4 Kbp purified from the expression vector pKK101, using T4 DNA ligase according to the above method, and E was inserted according to the above method. ． coli
It was introduced into the K-12 JM103 strain. From the resulting transformants, the desired human TNF expression vector (about 3.9
A clone having Kbp) was selected, and this expression vector was named pKF4102.

Example 5 (plasmid pUC119-F4
Construction of 104) (1) A description will be given with reference to FIG. The plasmid pUA44 obtained in Example 3 was treated with the restriction enzyme E according to the method described above.
cleaved with coR I and Hind III, human TN
A DNA fragment of the F gene (about 480 bp including the entire region) was separated and purified by agarose gel electrophoresis. On the other hand, Me
ssing et al. [Methods in Enzymol
pg., 153, 3 (1987)] for the preparation of single-stranded plasmid DNA.
C119 (obtained from Takara Shuzo) was similarly digested with restriction enzymes EcoRI and HindIII, and a DNA fragment of about 3.2 Kbp containing the IG region was separated and purified by agarose gel electrophoresis. Due to the presence of this IG region (intergenic region of M13 phage DNA), the plasmid pUC119 is preferentially converted to single-stranded DNA after infection with the helper phage M13K07, and is encapsulated in the phage particles and released outside the cells. DN of about 480 bp including the entire human TNF gene purified above
About 3.2 Kbp of pUC119 containing A fragment and IG region
Fragments were ligated using T4 DNA ligase as described above and E. coli as described in Example 3 above. coli K-1
2 was introduced into JM83 strain. From the resulting transformants, a clone having the desired plasmid (about 3.7 Kbp) was selected, and this clone was designated as pUC119-hTNF.
/ JM83 and the plasmid was designated as pUC119-hT.
It was named NF.

Plasmid pUC119-hT obtained above
Escherichia coli CJ236 strain (dut ⁻ , un) prepared by the calcium chloride method according to the method of Example 3 in order to retain uracil by incorporating uracil into its DNA.
g ^-) were introduced into competent cells of. The CJ236 strain has a mutation (du) in the deoxyuridine triphosphate degrading gene.
t ^-) competitive reaction occurs due to its, can make the accepted DNA part uracil instead of thymine, further ung ^- uracil N- glycosylase are deficient by mutation, retained the uracil in DNA You can keep it. This CJ236 strain was obtained from Bio-Rad. The clone obtained by the above introduction (pUC119-
hTNF / CJ236) to helper phage M13KO
7 (obtained from Takara Shuzo), 100 μg / ml ampicillin, 70 μg / ml kanamycin and 30 μg / ml
2 × YT broth containing 1.6 ml chloramphenicol (1.6% tryptone, 1% yeast extract and 0.5% N)
The target uracil-introduced single-stranded plasmid DNA (about 3.7
Kbases) was released outside the cells in the form of being wrapped in phage particles. The released phage particles were recovered from the culture supernatant, and the target single-stranded plasmid DNA was prepared according to the method for preparing single-stranded phage DNA.

(2) Next, a description will be given with reference to FIG. Primer 4104 was designed to mutate the human TNF gene using the coding strand oligonucleotides. Primer 4104 (base number 16) It has a base sequence from C at the 5th position to G at the 20th position in SEQ ID NO: 7 in the sequence listing, and A at the 13th position is replaced with G. The chemical synthesis and purification of this oligonucleotide were carried out according to the method of Example 2 above.

The site-directed mutagenesis of the human TNF gene is carried out by the Bio-Rad system (Muta-Gene ^™).
in vitro mutagenesis ki
It carried out according to t). That is, about 0.
The 5'end of 5 μg of the primer was converted to T4 according to the method described above.
Part phosphorylated by polynucleotide kinase,
The uracil-introduced single-stranded plasmid DNA (p
UC 119-hTNF) with about 200 ng of 1
0 μl of annealing buffer (20 mM Tris-
HCl pH 7.4, ₂ mM MgCl ₂ and 50 mM
Annealing (NaCl) (heating to about 70 ° C. followed by slow cooling) was performed. After completion of annealing, 10 times synthesis buffer (5 mM each deoxyribonucleotide triphosphate etc. (dATP, dGTP, dCTP and TT
P), 10 mM ATP, 100 mM Tris-HCl
pH 7.4, 50 mM MgCl ₂ and 20 mM DT
T) was added in 1/10 volume, and a double-strand formation reaction (37 ° C., 90 minutes) was performed using 1 unit of T4 DNA polymerase and 2 to 4 units of T4 DNA ligase. TE
The reaction was stopped by adding about 8 volumes of buffer (10 mM Tris-HCl pH 7.5 and 1 mM EDTA) and freezing. E. coli produced by the calcium chloride method according to the method of Example 3 above. coli K-12
Double-stranded DNA was introduced into a competent cell of TG1 strain (ung ⁺ : Amersham) by treating the above reaction solution.

When the hetero double-stranded DNA is introduced into the ung ⁺ strain, the DNA chain containing the template uracil is inactivated and is not a target for replication. (Therefore, the frequency of mutation introduction is as high as 50% or more.) Among the obtained transformants, the colony hybridization method using the primer used for the introduction of mutation as a probe was used. A clone having a plasmid (about 3.7 Kbp) containing the transformant DNA of interest was selected. Regarding the selected clone, the nucleotide sequence around the mutation introduction site of the plasmid was determined by the dideoxy method [F. Sanger:
As described above, it was confirmed that the transformant DNA was mutated as designed. This plasmid is called pUC11
It was named 9-F4104. pUC119-F4104
Is a conversion of the 13th A in the sequence of SEQ ID NO: 7 into G, which results in restriction enzyme Xho.
An I 2 cleavage site was generated.

Example 6 (Construction of human TNF transformant expression vector pKF4146) Using the plasmid pUC119-F4104 having the restriction enzyme XhoI cleavage site obtained in Example 5, the N-terminal side of the restriction enzyme XhoI cleavage site was used. An expression vector (named as pKF4146) of an N-terminal addition type human TNF convertible polypeptide (named as F4146) was constructed by ligating a chemically synthesized oligonucleotide (double-stranded by annealing) with. The construction method will be described with reference to FIG.

5 μg of the plasmid pUC119-F4104 (about 3.7 Kbp) was dissolved in a high salt buffer according to the method of Example 3, and the restriction enzyme Xho was used.
Approximately 460 bp D containing the F4104 gene which was cleaved with I (Takara Shuzo) and Hind III and deleted the N-terminal part
The NA fragment was separated and purified by agarose electrophoresis (gel concentration 1%). Further, the upper chain (U-4146) and the lower chain (L-4) of an oligonucleotide encoding an oligopeptide designed to be added to the N-terminal portion of F4104
146) was chemically synthesized and purified according to the method of Example 2 above. The upper chain (U-4146) has the sequence shown in SEQ ID NO: 8 in the sequence listing. The lower chain (L-4146) is complementary to the upper chain, but it is the first to 4th of SEQ ID NO: 8.
There is no base sequence complementary to the 5th-AATT-3 'in the 5th position, while a base sequence connecting 5'-TCGA-3' in the 5'direction to G complementary to the 43rd C in SEQ ID NO: 8. Have. The two oligonucleotides (1 μg each) obtained after purification were phosphorylated at their 5'ends using T4 polynucleotide kinase according to the method of Example 3 above, and then double-stranded by annealing. It was a fragment (43 bp). On the other hand, according to the method of Example 4, the plasmid vector pKK101 was digested with restriction enzymes EcoRI and Hin.
It was cleaved with dIII, and a DNA fragment of about 3.4 Kbp containing an origin of replication, a transcriptional regulatory region and the like was separated and purified.

The three DNA fragments obtained according to the above method were ligated with T4 DNA ligase according to the method of Example 3 described above, and E. coli K-12 JM10
It was introduced into 3 competent cells. Among the obtained transformants, the desired F4146 expression vector pKF41
The clone having 46 (about 3.9 Kbp) was confirmed for its cleavage pattern by restriction enzymes EcoR I, Xho I and Hind III, and N of the transforming polypeptide.
It was selected by examining the base sequence around the terminal addition site.

The transformant expression vector constructed according to the above method produces a novel bioactive polypeptide having the following conversion in E. coli by expression induction. Vector pKF4146: polypeptide F4 in which the 1st to 8th amino acid sequence shown in SEQ ID NO: 1 of the sequence listing is replaced with the amino acid sequence shown in SEQ ID NO: 3 of the sequence listing
Code 146.

Example 7 (Construction of human TNF convertible expression vector pKF4422 and pKF4423) 5 μg of the human TNF convertible expression vector pKF4146 (about 3.9 Kbp) obtained in Example 6 was used in the method of Example 3 above. According to the above, about 47 containing the F4146 gene, which was dissolved in a high salt buffer, cleaved with restriction enzymes Bgl II and Hind III, and deleted at the N-terminal portion.
The 0 bp DNA fragment was separated and purified by agarose gel electrophoresis (gel concentration 1%). In addition, an upper chain (U-4422 or U-4423) and a lower chain (L-4422 or L-442) of an oligonucleotide encoding an oligopeptide designed to be added to the N-terminal portion of F4146.
3) was designed.

U-4422 (SEQ ID NO: 9 in the sequence listing, 21 bases) L-4422 (21 bases) Complementary to U4422, the first to fourth 5'-AATT- in SEQ ID NO: 9 It has no base sequence complementary to 3 ', while T complementary to the 21st A of SEQ ID NO: 9.
, Which has a base sequence connecting 5'-GATC-3 'in the 5'direction.

U-4423 (SEQ ID NO: 10 in the sequence listing, number of bases 36) L-4423 (number of bases 36) Complementary to U-4423, the first to fourth 5'- of SEQ ID NO: 10 It has no base sequence complementary to AATT-3 ', while having a base sequence connecting 5'-GATC-3' in the 5'direction to T complementary to the 36th A of SEQ ID NO: 10.

These oligonucleotides were chemically synthesized and purified according to the method of Example 2 above. The two upper and lower oligonucleotides obtained after purification (1 μ each)
g) is phosphorylated at its 5'end with T4 polynucleotide kinase according to the method of Example 3 above,
Double-stranded DNA fragments (21 bp or 36 bp by annealing)
bp).

On the other hand, according to the method of Example 4, the plasmid vector pKK101 was transformed with the restriction enzyme EcoRI.
And Hind III, and a DNA fragment of about 3.4 Kbp containing an origin of replication and a transcriptional regulatory region was separated and purified.

Three types of DNA obtained according to the above method
Each of the fragments was subjected to T4D according to the method of Example 3 above.
Ligation was performed using NA ligase and E. coli K-12
It was introduced into a competent cell of JM103 strain. Among the obtained transformants, clones having the desired F4422 expression vector pKF4422 (about 3.9 Kbp) or F4423 expression vector pKF4423 (about 3.9 Kbp) were digested with restriction enzymes EcoR I, Bgl II and H.
It was selected by confirming the cleavage pattern by ind III and examining the nucleotide sequence around the N-terminal addition site of the transformant polypeptide.

The transformant expression vector constructed according to the above method produces a novel physiologically active polypeptide having the following conversion in E. coli by inducing expression. Vector pKF4422: Polypeptide F442 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 5 of the sequence listing
Code 2. Vector pKF4423: polypeptide F442 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 6 of the sequence listing
Code 3

Example 8 (Construction of human TNF convertible expression vector pKF4236) (1) A method for constructing an expression vector having a trp promoter will be described with reference to FIGS. 8 to 11. A plasmid vector (named pSK407) having a trp promoter as a transcription promoter, two translation initiation signals (SD sequences) arranged in tandem, and having a rrnBT ₁ T ₂ terminator as a transcription terminator is shown in FIGS. 8 to 10.
It was constructed according to the method shown in. Plasmid vector p
5 μg of GF101 (about 4.3 Kbp, distributed by Osaka University Faculty of Pharmaceutical Sciences) was dissolved in medium salt buffer according to the above method to prepare 10 units of restriction enzyme Cla.
It was cut by adding I (Takara Shuzo). This cleaved DNA fragment was prepared according to the method of Example 3 above.
After smoothing with T4 DNA polymerase and ethanol precipitation, NaCl was added to adjust the NaCl concentration to 175 m.
It was dissolved in a high salt buffer (M) and cleaved by adding 15 units of a restriction enzyme Sal I (Takara Shuzo). According to the method of Example 3, a DNA fragment of about 4.0 Kbp was separated and purified by agarose gel electrophoresis (gel concentration 1%).

On the other hand, Casadaban et al. [J. Bac
teriol. , 143 , 971 (1980)], and plasmid pMC1403 (about 9.9 Kb).
p., 5 μg from the Institute for Virus Research, Kyoto University) was digested with the restriction enzyme EcoRI according to the above method, followed by blunting and SalI digestion in the same manner as in the above method, followed by agarose gel electrophoresis. 6.2 Kbp D
The NA fragment was separated and purified. According to the method of Example 3, a ligation reaction was performed with the previously purified DNA fragment using T4 DNA ligase, and E. coli produced by the calcium chloride method was used. Escherichia coli K-12 HB101 strain (distributed by Kyoto University Virus Research Institute) was introduced into competent cells. Among the obtained transformants, the desired trp promoter and the restriction enzymes EcoR I and BamHI are selected.
A clone having a plasmid vector of about 10.2 Kbp (named pSK101) containing a cleavage site was selected.

Plasmid vector pSK obtained above
5 μg of 101 was separately dissolved in each of the two tubes in a high salt buffer according to the method described above, and digested by adding the restriction enzymes EcoR I and Sal I and the restriction enzymes BamHI and SalI, and then agarose. DNA fragments of about 4.0 Kbp and about 6.2 Kbp were separated and purified by gel electrophoresis. On the other hand, a portable translation initiation site oligonucleotide (abbreviated as SD-ATG linker, translation initiation signal SD
Oligonucleotide having 21 bases including sequence and translation initiation codon ATG: Catalog No. 27-4878-01
And 27-4898-01 Pharmacia Molecular Biologicals 1985 May, obtained from Pharmacia, Inc., upper and lower chains (100 ng each) were subjected to phosphorylation and annealing of the 5'end according to the method of Example 3 above. It was used as a double-stranded DNA fragment (21 bp). The three DNA fragments obtained above were treated with T4 according to the above method.
Ligation using DNA ligase, E. coli K-1
2HB101 strain was introduced into competent cells. Among the obtained transformants, the SD-ATG linker of interest was located downstream of the trp promoter and the restriction enzyme BamH.
A clone having a plasmid vector of about 10.2 KbP (designated pSK211) inserted immediately before the I cleavage site was selected. This plasmid vector will have two SD sequences arranged in tandem downstream of the trp promoter.

Restriction enzyme Sal of this plasmid vector
The I-cleavage site disappears, and the restriction enzyme Hind
The following operations were performed for the purpose of creating a III cleavage site. According to the above method, 5 μg of the plasmid vector pSK211 was cleaved with the restriction enzyme SalI, and then T4DN.
It was blunted using A polymerase. The DNA fragment recovered by ethanol precipitation was treated with the restriction enzyme BamHI
Cleaved with agarose gel electrophoresis and about 4.0 Kb
The p DNA fragment was separated and purified. On the other hand, a plasmid vector pGFK503-1 (about 5.7 Kbp) constructed by recombination using the plasmid vector pKK223-3 (Pharmacia) having the tac promoter and SD-ATG linker according to the method for constructing pSK211 described above. Was cleaved with the restriction enzyme Hind III according to the above method, and then blunted with T4 DNA polymerase. The DNA fragment recovered by the ethanol precipitation operation was cleaved with the restriction enzyme BamHI and the DNA fragment of about 1.1 Kbp was separated and purified by agarose gel electrophoresis. The two DNA fragments purified above were
Ligation was performed using T4 DNA ligase according to the method described above, and E. E. coli K-12 HB101 strain was introduced into competent cells. Among the resulting transformants, Hind III lacking the desired restriction enzyme SalI cleavage site
Approximately 5.1 containing the trp promoter with a new cleavage site
A clone having the Kbp plasmid vector (named pSK301) was selected.

5 of this plasmid vector pSK301
According to the method described above, μg was dissolved in a wax / salt buffer, and 5 units of a restriction enzyme Bal I (Takara Shuzo) was added to perform a cleavage reaction. After completion of the reaction, Na of the reaction solution
Cleavage was performed with the restriction enzyme Hind III at a Cl concentration of 50 mM, and a DNA fragment of about 4.3 Kbp was separated and purified by agarose gel electrophoresis. On the other hand, 5 μg of the plasmid vector pKK223-3 was dissolved in a high salt buffer according to the method described above, and the restriction enzyme Hi was added.
cleaved with nd III and Sca I, about 0.8 Kb
The DNA fragment of p was separated and purified by agarose gel electrophoresis. The two DNA fragments purified above were ligated using T4 DNA ligase according to the method described above, and E. c
It was introduced into a competent cell of oliK-12HB101 strain. The target rrn is selected from among the obtained transformants.
The plasmid vector pSK407 (about 5. BT ₁ T ₂ terminator is placed downstream of the trp promoter).
A clone having 1 Kbp) was selected.

Next, a TNF converter expression vector under the control of transcription and translation signals of the plasmid vector pSK407 constructed by the above method was designed.
Encoded converter polypeptide (designated F4113)
Is an oligopeptide Asp-Pr at the N-terminus of human TNF.
o-Gly-Arg-Gly-Asp-Ser-Pro
Is added to convert the fifth Thr to Ala. Two oligonucleotides (U-4113 and L-4) are added according to the amino acid sequence of the oligopeptide to be added.
-4113) was designed.

U-4113 (SEQ ID NO: 11 in the sequence listing, base number 32) L-4113 (base number 32) Complementary to U-4113, the first to fourth 5'- in SEQ ID NO: 11 It has no base sequence complementary to GATC-3 ', whereas it has a base sequence connecting 5'-TCGA-3' in the 5'direction to G complementary to the 32nd C of SEQ ID NO: 11.

Chemically synthesized according to the method of Example 2,
The oligonucleotides (1 μg each) produced by purification were phosphorylated at their 5 ′ ends with T4 polynucleotide kinase according to the method of Example 3 above, and then double-stranded by annealing. DNA fragment (32b
p). Also, the trp promoter and rrnBT
Plasmid vector p having ₁ T ₂ terminator and the like
According to the method of Example 3, 5 μg of SK407 (about 5.1 Kbp) was dissolved in high salt buffer and cleaved with restriction enzymes BamHI and HindIII,
Approximately 4.0 Kbp of D including origin of replication and transcriptional regulatory region
The NA fragment was separated and purified by agarose gel electrophoresis (gel concentration 1%). On the other hand, 5 μg of the plasmid pUC119-F4104 (about 3.7 Kbp) obtained in Example 5 was treated with the restriction enzyme Xho in the same manner as in Example 6.
A DNA fragment of about 460 bp containing the F4104 gene, which had been cleaved with I 2 and Hind III and had the N-terminal portion deleted, was separated and purified (FIG. 11).

The three DNA fragments obtained according to the above method were ligated using T4 DNA ligase according to the method of the above Example 3, and E. coli K-12 HB10
It was introduced into one competent cell. From the obtained transformants, a clone having the desired F4113 expression vector of about 4.5 Kbp (designated pKF4113) was transformed into
Restriction enzymes BamH I, Xho I and Hind
It was selected by confirming the cleavage pattern with III and examining the base sequence around the N-terminal addition site of the converter polypeptide.

(2) The expression vector pKF4 obtained by constructing the transcription promoter (tac promoter) of the expression vector pKF4146 obtained in Example 6 in the above item (1).
It is replaced with the trp promoter contained in 113. This substitution also involves conversion of the N-terminal amino acid sequence. Figure 1
This replacement method will be described according to 2.

Plasmid pkF4146 (about 3.9 Kb
p) 5 μg with 1 mM DTT and 100 μg / ml B
50 μl of SmaI buffer containing SA (10 mM Tris-HCl pH 8.0, 20 mM KCl and 10
mM MgCl ₂ ) and 15 units of restriction enzyme Sma I (Takara Shuzo) was added, and a cleavage reaction was carried out at 37 ° C. for 2 hours. After completion of the reaction, the DNA fragment was recovered by ethanol precipitation and redissolved in 50 μl of medium salt buffer. 15 units of restriction enzyme H
After cleavage reaction with ind III at 37 ° C. for 2 hours,
A DNA fragment of about 490 bp was separated and purified by agarose gel electrophoresis (gel concentration 1%). On the other hand, 5 of plasmid pKF4113 (about 4.5 Kbp) having the trp promoter obtained by the construction of the above item (1)
μg was added to the restriction enzyme Sma I in the same manner as described above.
And Hind III, and a DNA fragment of about 4.0 Kbp containing the trp promoter was separated and purified by agarose gel electrophoresis (gel concentration 1%). The two DNA fragments thus obtained were ligated using T4 DNA ligase according to the method of Example 3,
E. E. coli K-12 HB101 strain was introduced into competent cells. From the obtained transformants, a clone having the target transformant polypeptide expression vector (about 4.5 Kbp) by the trp promoter was confirmed for the restriction enzyme cleavage pattern and the N-terminal addition site of the transformant polypeptide. It was selected by examining the surrounding nucleotide sequence. By this selection, the restriction enzyme S that should be regenerated
A non-regenerating clone of ma I was obtained by deleting the 3 base pair 5′-CCC-3 ′ 3′-GGG-5 ′ encoding Pro. This expression vector was named pKF4236.

The transformant expression vector constructed according to the above method produces a novel bioactive polypeptide having the following conversion in E. coli by expression induction. Vector pKF4236: polypeptide F423 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 of the sequence listing
Code 6

Example 9 (Construction of human TNF transformant expression vector pKF4419) Using the primer 4419 shown in SEQ ID NO: 12 of the sequence listing, the desired mutation was performed according to the method of Example 5 (1) and (2) above. Plasmid containing introduced DNA (about 3.7 Kbp)
Got Converted DN as designed by the dideoxy method
Confirm that it is mutated to A,
It was named C119-F4419.

Human TNF of interest obtained by mutagenesis
The transformant gene was incorporated into the expression vector pKK101 having the tac promoter according to the method for constructing the human TNF expression vector of Example 4 to construct a human TNF transformant expression vector. Human TNF transformant gene (about 480b
p) is the plasmid p of about 3.7 Kbp obtained above.
From UC119-F4419, according to the method described above, it was separated and purified after digestion with restriction enzymes EcoRI and HindIII. The target human TNF transformant expression vector (pKF4419) is a human TNF expression vector (pKF4419).
KF4102) and E. coli as a host. coli K-
12 JM103 strain was used.

The above transformant expression vector produces a novel bioactive polypeptide having the following conversion in E. coli by inducing expression. Vector pKF4419: A polypeptide F441 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 4 of the sequence listing.
Code 9.

Example 10 (Construction of human TNF transformant expression vectors pKF4603 and pKF4604) (1) Primers 4291 and 4292 were designed. These primers are C to 213 at the 193rd position of SEQ ID NO: 7.
The 5'-CCA-3 'from the 202nd to the 204th nucleotide having a base sequence up to T is 5'-GAT-3' in the case of the primer 4291 and 5'-ATG-3 'in the case of the primer 4292. It is an oligonucleotide consisting of 21 bases each substituted by.

Chemical synthesis and purification of the oligonucleotide were carried out according to the method of Example 2 above. Using these primers 4291 or 4292, the above-mentioned Example 5 (1) was used.
According to the method of (2) and (2), a plasmid (about 3.7 Kbp) containing the target mutation-introduced DNA was obtained. It was confirmed by the dideoxy method that the transformant DNA was mutated as designed, and this plasmid was designated as pUC119-F4291 or pUC119-F4292. According to the method for constructing the human TNF expression vector of Example 4, the target human TNF converter gene obtained by the mutation introduction was transformed into ta.
A human TNF converter expression vector was constructed by incorporating it into the expression vector pKK101 having the c promoter. Human T
The NF converter gene (about 480 bp) is obtained by the above-mentioned plasmid of about 3.7 Kbp, pUC119-F4291.
Alternatively, from pUC119-F4292, the restriction enzymes EcoR I and Hind III were prepared according to the method described above.
After cutting with, the product was separated and purified. Target human TNF converter expression vector pKF4291 or pKF4922
(About 3.9 Kbp) is a human TNF expression vector (pK
F4102), as a host, E. coli K-1
2 Obtained using the JM103 strain.

(2) Human TNF obtained in Example 8 above
Transformant expression vector pKF4236 (about 4.5 Kbp)
Was dissolved in medium salt buffer and cleaved with restriction enzymes Sac I and Hind III according to the method of Example 3 to obtain a DNA fragment of about 4.1 Kbp containing an origin of replication, a transcriptional regulatory region and the like, and agarose. It was separated and purified by gel electrophoresis (gel concentration 1%). on the other hand,
Human TNF converter expression vector p obtained in (1) above
5 μg of KF 4291 or pKF 4292 was treated with the restriction enzymes Sac I and Hind III in the same manner as above.
A DNA fragment of about 360 bp containing the human TNF conversion gene in which the N-terminal portion was deleted was isolated and purified.

The two DNA fragments obtained above were ligated using T4 DNA ligase according to the method of Example 3 described above, and E. E. coli K-12 HB101 strain was introduced into competent cells. From among the resulting transformants,
F4603 or F4604 expression vector of interest pKF4
A clone having 603 or pKF4604 (about 4.5 Kbp) was cloned into restriction enzymes Sac I and Hind II.
It was selected by confirming the cleavage pattern by I and examining the base sequence around the conversion site (derived from the converter polypeptide).

The transformant expression vector constructed according to the above method produces a novel physiologically active polypeptide having the following conversion in E. coli by expression induction. Vector pKF4603: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 of the sequence listing, and the 68th Pro is A
Encodes the polypeptide F4603 converted to sp. Vector pKF4604: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 of the sequence listing, and Pro at the 68th is M
Encodes the polypeptide F4604 converted to et.

It was confirmed that each host Escherichia coli having the above expression vector expressed and produced a polypeptide having antitumor activity by centrifugation of the E. coli suspension subjected to ultrasonic disruption (supernatant fraction after centrifugation). ) Was confirmed by performing a test according to the method of Example 17 described later.

Example 11 (Human TNF transformant expression vector pKF4616, pKF4617, pKF4630,
pKF4631, pKF4636, pKF4637, p
KF4618, pKF4619, pKF4632, pK
Construction of F4633, pKF4640 and pKF4641) (1) Primers 4268, 4150, 4222,
Designed 4267, 4123, and 4223. The primer 4268 is the 301st A of SEQ ID NO: 7.
It is an oligonucleotide consisting of 30 bases having a base sequence from C to 333rd C and having 316th to 318th 5'-GGC-3 'deleted. On the other hand, the other primers have a nucleotide sequence from A at the 307th position to C at the 327th position in SEQ ID NO: 7, and 316th to 318th 5'-GGC-3 'are 5'in the case of primer 4150.
-TGG-3 ', 5'for primer 4222
-CCC-3 ', 5'for primer 4267
-GCT-3 ', 5'for primer 4123
-GAC-3 ', 5'for primer 4223
-An oligonucleotide consisting of 21 bases each substituted with CGC-3 '.

The chemical synthesis and purification of the oligonucleotide were carried out according to the method of Example 2 above. Using these primers, a plasmid containing the target mutation-introduced DNA (about 3.7K) was prepared according to the method of Example 5 (1) and (2) above.
bp) was obtained. It was confirmed by the dideoxy method that the transformant DNA was mutated as designed, and this plasmid was used for pUC119-F4268 and pUC119-F415.
0, pUC119-F4222, pUC119-F42
67, pUC119-F4123 or pUC119
-F4223 was named. According to the method for constructing the human TNF expression vector of Example 4, the desired human TNF transformant gene obtained by the mutation introduction was incorporated into the expression vector pKK101 having the tac promoter to construct the human TNF transformant expression vector. The human TNF conversion gene (about 480 bp) was obtained from the above-obtained plasmid of about 3.7 Kbp according to the above-mentioned method according to the restriction enzyme Eco.
After cleavage with RI and Hind III, separation and purification were performed. Targeted human TNF converter expression vector pKF4
268, pKF4150, pKF4222, pKF42
67, pKF4123 or pKF4223 (about 3.
9 Kbp) is a human TNF expression vector (pKF410).
As in 2), E. coli K-12 JM
It was acquired using 103 strains.

(2) According to the method of Example 10 (2), 5 μg of the human TNF transformant expression vector pKF4236 (about 4.5 Kbp) obtained in Example 8 was treated with restriction enzymes Sac I and Hind. Cleavage with III, DN of about 4.1 Kbp including origin of replication and transcriptional regulatory region
The A fragment was separated and purified. On the other hand, the human TNF converter expression vectors pKF4268 and pKF4 obtained in (1) above
150, pKF4222, pKF4267, pKF41
In the same manner as described above, 5 μg of 23 or pKF4223 was cleaved with restriction enzymes Sac I and Hind III, and a DNA fragment of about 360 bp containing the human TNF converter gene lacking the N-terminal portion was separated and purified.

The two DNA fragments obtained above were ligated using T4 DNA ligase according to the method of Example 3 described above, and E. E. coli K-12 HB101 strain was introduced into competent cells. From among the resulting transformants,
Target F4616, F4617, F4630, F463
Expression vector pKF4 of 1, F4636 or F4637
616, pKF4617, pKF4630, pKF46
31, pKF4636 or pKF4637 (about 4.5K
bp) was cloned into the restriction enzymes SacI and H
It was selected by confirming the cleavage pattern by ind III and examining the base sequence around the conversion site derived from the conversion polypeptide.

The transformant expression vector constructed according to the above method produces a novel physiologically active polypeptide having the following conversion in E. coli by expression induction. Vector pKF4616: encodes a polypeptide F4616 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 of the sequence listing, and 106th Gly is deleted. To do. Vector pKF4617: A polypeptide F4617 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 of the sequence listing, and 106th Gly is converted to TrP. To code. Vector pKF4630: A polypeptide F4630 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 in the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 in the sequence listing, and 106th Gly is converted to Pro. To code. Vector pKF4631: A polypeptide F4631 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 in the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 in the sequence listing, and 106th Gly is converted to Ala. To code. Vector pKF4636: A polypeptide F4636 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 in the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 in the sequence listing, and 106th Gly is converted to Asp. To code. Vector pKF4637: A polypeptide F4637 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 in the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 in the sequence listing, and 106th Gly is converted to Arg. To code.

It was confirmed that each host Escherichia coli having the above-mentioned expression vector expresses and produces a polypeptide showing antitumor activity, and is centrifuged supernatant of the E. coli suspension subjected to ultrasonic disruption (supernatant fraction after centrifugation). ) Was confirmed by performing a test according to the method of Example 17 described later.

(3) Human TNF obtained in Example 9 above
Transformant expression vector pKF4419 (about 3.9 Kbp)
Was dissolved in medium salt buffer and cleaved with restriction enzymes Sac I and Hind III in accordance with the method of Example 3 to obtain a DNA fragment of about 3.5 Kbp containing an origin of replication, a transcriptional regulatory region and the like, and agarose. It was separated and purified by gel electrophoresis (gel concentration 1%). on the other hand,
Human TNF converter expression vector p obtained in (1) above
KF4268, pKF4150, pKF4222, pK
5μ of F4267, pKF4123 or pKF4223
g in the same manner as above with the restriction enzymes Sac I and H
Human TN cleaved with ind III and lacking the N-terminal part
A DNA fragment of about 360 bp containing the F-converter gene was separated and purified. The two DNA fragments obtained above were ligated using T4 DNA ligase according to the method of Example 3, and E. E. coli K-12 JM103 strain was introduced into competent cells. From the obtained transformants, the desired F4618, F4619, F4632, F463
3, F4640 or F4641 expression vector pKF
4618, pKF4619, pKF4632, pKF4
633, pKF4640 or pKF4641 (about 3.9
A clone having Kbp) was selected by confirming the cleavage pattern with restriction enzymes SacI and HindII1 and examining the nucleotide sequence around the conversion site derived from the conversion polypeptide.

The transformant expression vector constructed according to the above method produces a novel physiologically active polypeptide having the following conversion in E. coli by expression induction. Vector pKF4618: encodes a polypeptide F4618 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 4 of the sequence listing, and 106th Gly is deleted. To do. Vector pKF4619: A polypeptide F4619 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 in the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 4 in the sequence listing, and 106th Gly is converted to Trp. To code. Vector pKF4632: A polypeptide F4632 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the Sequence Listing are replaced with the amino acid sequences shown in SEQ ID NO: 4 of the Sequence Listing, and 106th Gly is converted to Pro. To code. Vector pKF4633: A polypeptide F4633 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 in the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 4 in the sequence listing, and 106th Gly is converted to Ala. To code. Vector pKF4640: A polypeptide F4640 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 in the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 4 in the sequence listing, and 106th Gly is converted to Asp. To code. Vector-pKF4641: A polypeptide F4641 in which the 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 4 of the sequence listing, and 106th Gly is converted to Arg. Code

It was confirmed that each host Escherichia coli having the above-mentioned expression vector expresses and produces a polypeptide exhibiting antitumor activity by centrifugation of the E. coli suspension subjected to ultrasonic disruption (the supernatant fraction after centrifugation). ) Was confirmed by performing a test according to the method of Example 17 described later.

Example 12 (plasmid pUC119
(Construction of (B) -F4236) The construction method of the plasmid (named pUC119 (B) -F4236) in which the human TNF transformant F4236 gene was inserted into the plasmid vector pUC119 for preparing single-stranded plasmid DNA is shown in FIG.

1 μg of plasmid vector pUC119
Was dissolved in Sma I buffer and cleaved with the restriction enzyme Sma I according to the method of Example 8 (2). The obtained ring-opened plasmid was recovered by an ethanol precipitation operation, and the non-phosphorylated Bgl was inserted into the ring-opened site.
2 μg of II linker (Takara Shuzo) was used in Example 4 (1).
Insertion and ligation were performed using T4 DNA ligase according to the method described in 1. Among the transformants obtained according to the method of Example 3, the restriction enzyme Bgl I was selected for the plasmid.
By examining whether or not it can be cleaved by I, the plasmid pUC1 in which the desired restriction enzyme Bgl II cleavage site was newly generated
A clone with 19 (B) (about 3.2 Kbp) was obtained.

The plasmid pUC119 obtained above.
According to the method of Example 3, 5 μg of (B) was dissolved in high salt buffer, and the restriction enzymes Bgl II and H were dissolved.
About 3.2 Kbp including the origin of replication, cleaved with indIII
Agarose gel electrophoresis (DNA concentration 1
%) And separated and purified. On the other hand, 5 μg of the human TNF transformant expression vector pKF4236 (about 4.5 Kbp) obtained in Example 8 was cleaved with restriction enzymes Bgl II and Hind III in the same manner as described above to delete the N-terminal portion. About 470 bp D containing the F4236 gene
The NA fragment was separated and purified. The above-mentioned approximately 470 bp D fragment was added to the approximately 3.2 Kbp DNA fragment containing the origin of replication.
The NA fragment was insert ligated using T4 DNA ligase as described above. Of the transformants obtained according to the method of Example 3, the restriction enzyme B was used for the plasmid.
By confirming the cleavage pattern by gl II and Hind III, the desired plasmid pUC119
(B) A clone having 4236 (about 3.7 Kbp) was selected.

Example 13 (Human TNF transformant expression vector pKF4620, pKF4621, pKF4622,
pKF4623, pKF4624, pKF4647, p
KF4648, pKF4649, pKF4650 and p
Construction of KF4651) (1) Primers 4134, 4391, 4392,
Designed 4409 and 4410. These primers are the 76th T to the 96th T of SEQ ID NO: 7.
Having a base sequence of up to 85th to 87th 5'-C
5'-CAA when GC-3 'is primer 4134
-3 ', 5'-AAG in the case of primer 4391
-3 ', 5'-GAC in case of primer 4392
-3 ', 5'-GTC in case of primer 4409
-3 ', 5'-CTC for primer 4410
An oligonucleotide consisting of 21 bases each substituted with -3 '. The chemical synthesis and purification of the oligonucleotide were carried out according to the method of Example 2 above.

(2) In the above-mentioned Example 5 (1), pU
PUC obtained in Example 9 instead of C119-hTNF
Single-stranded plasmid D of interest using 119-F4419
NA was prepared. Next, this pUC119-F4419
Using the single-stranded plasmid DNA containing uracil and the primers 4134, 4391, 4392, 4409, or 4410 purified in (1) above, site-specification was carried out according to the method of Example 5 (2) above. Mutagenesis was performed to obtain a plasmid (about 3.7 Kbp) containing the DNA in which the desired mutation was introduced. It was confirmed by the dideoxy method that the mutations as designed were introduced, and these plasmids were cloned into pUC119-F4620 and pUC1.
19-F4621, pUC119-F4622, pUC
It was named 119-F4623 or pUC119-F4624.

Human TNF of interest obtained by mutagenesis
The transformant gene was incorporated into the expression vector pKK101 having the tac promoter according to the method for constructing the human TNF expression vector of Example 4 to construct a human TNF transformant expression vector. Human TNF transformant gene (about 480b
p) is the plasmid p of about 3.7 Kbp obtained above.
UC119-F4620, pUC119-F4621,
pUC119-F4622, pUC119-F4623
Alternatively, it was isolated and purified from pUC119-F4624 according to the above-mentioned method after being cleaved with restriction enzymes EcoRI and HindIII. Target human TNF transformant expression vector pKF4620, pKF4621, pKF4
622, pKF4623 or pKF4624 (about 3.9
Kbp is a human TNF expression vector (pKF410).
As in 2), E. coli K-12 JM1
It was acquired using 03 strains.

The above transformant expression vector produces a novel physiologically active polypeptide having the following conversion in E. coli by inducing expression. Vector pKF4620: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 in the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 4 in the sequence listing, and the 29th Arg is G.
Encodes the polypeptide F4620 converted to In. Vector pKF4621: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 in the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 4 in the sequence listing, and the 29th Arg is L.
Encodes the polypeptide F4621 converted to ys. Vector pKF4622: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 in the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 4 in the sequence listing, and the 29th Arg is A.
Encodes the polypeptide F4622 converted to sp. Vector pKF4623: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 4 of the sequence listing, and the 29th Arg is V.
Encodes the polypeptide F4623 converted to al. Vector pKF4624: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 4 of the sequence listing, and the 29th Arg is L.
Encodes the polypeptide F4624 converted to eu.

It was confirmed that each host Escherichia coli having the above-mentioned expression vector expresses and produces a polypeptide showing antitumor activity, and is centrifuged supernatant of the E. coli suspension subjected to ultrasonic disruption (supernatant fraction after centrifugation). ) Was confirmed by performing a test according to the method of Example 17 described later.

(3) pUC in Example 5 (1)
PUC obtained in Example 12 instead of 119-hTNF
The desired single-stranded plasmid DNA was prepared using 119 (B) -F4236. Next, this pUC119 (B)
-Single-stranded plasmid DNA containing uracil of F4236
And the primers 4134 and 43 purified in the above (1)
91, 4392, 4409 or 4410, site-directed mutagenesis was carried out according to the method of Example 5 (2), and a plasmid containing the DNA having the desired mutation introduced therein (about 3. 7 Kbp) was acquired. It was confirmed by the dideoxy method that the mutation as designed was introduced, and these plasmids were cloned into pUC119-F4647,
pUC119-F4648, pUC119-F464
9, pUC119-F4650 or pUC119-F4
It was named 651.

The novel human TNF transformant gene obtained above was transformed into the expression vector pKF having the trp promoter.
4236 was incorporated to construct a novel human TNF transformant expression vector of interest.

5 μ of the human TNF transformant expression vector pKF4236 (about 4.5 Kbp) obtained in Example 8 was obtained.
g in accordance with the method of Example 12 with the restriction enzyme Bgl I
It was cleaved with I and Hind III, and a DNA fragment of about 4.0 Kbp containing an origin of replication, a transcriptional regulatory region and the like was separated and purified by agarose gel electrophoresis (gel concentration 1%).
On the other hand, the plasmid pUC119-F46 obtained above was used.
47, pUC119-F4648, pUC119-F4
649, pUC119-F4650 or pUC119-
5 μg of F4651 was treated with the restriction enzyme B in the same manner as above.
A DNA fragment of about 470 bp containing a human TNF transformant gene in which the N-terminal portion was deleted was cleaved with glII and HindIII, and purified.

The two DNA fragments obtained above were ligated using T4 DNA ligase according to the method of Example 3 above, and E. E. coli K-12 HB101 strain was introduced into competent cells. From among the obtained transformants, the desired F4647, F4648, F4649, F4
650 or F4651 expression vector pKF464
7, pKF4648, pKF4649, pKF4650
Alternatively, a clone having pKF4651 (about 4.5 Kbp) was selected by confirming the cleavage pattern by restriction enzymes BglII and HindIII.

The transformant expression vector constructed according to the above method produces a novel physiologically active polypeptide having the following conversion in E. coli by expression induction. Vector pKF4647: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 of the sequence listing, and the 29th Arg is G.
Encodes the polypeptide F4647 converted to In. Vector pKF4648: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 of the sequence listing, and the 29th Arg is L.
Encodes the polypeptide F4648 converted to ys. Vector pKF4649: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 of the sequence listing, and the 29th Arg is A.
Encodes the polypeptide F4649 converted to sp. Vector pKF4650: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 of the sequence listing, and the 29th Arg is V.
Encodes the polypeptide F4650 converted to al. Vector pKF4651: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 of the sequence listing, and the 29th Arg is L.
Encodes the polypeptide F4651 converted to eu. Each host Escherichia coli having the above expression vector expresses and produces a polypeptide exhibiting antitumor activity, with respect to the centrifugal supernatant of the E. coli suspension ultrasonically disrupted (supernatant fraction after centrifugation), It was confirmed by testing according to the method of Example 17 described later.

Example 14 (Construction of human TNF transformant expression vectors pKF4653 and pKF4655) (1) Primers 4427 and 4429 were designed. These primers have a nucleotide sequence from A at the 301st position to C at the 333rd position in SEQ ID NO: 7, and are 310 'to 312'5'-CCC-3', 313 to 3'.
The 15th 5'-GAG-3 ', the 319th to 321st 5'-GCA-3' and the 322nd to 324th 5'-GAG-3 'are 5'-GGT-in the case of the primer 4427, respectively. 3 ', 5'-AGA-3', 5'-GA
Primer 4 with T-3 'and 5'-AGC-3'
429 is 5'-TAC-3 ', 5'-ATT-, respectively.
It is an oligonucleotide consisting of 33 bases respectively substituted with 3 ', 5'-AGC-3' and 5'-AGA-3 '.

The chemical synthesis and purification of the oligonucleotide were carried out according to the method of Example 2 above. Using these primers, a plasmid containing the target mutation-introduced DNA (about 3.7K) was prepared according to the method of Example 5 (1) and (2) above.
bp) was obtained. It was confirmed by the dideoxy method that the transformant DNA was mutated as designed, and this plasmid was designated as pUC119-F4427 or pUC119.
-F4429 was named.

Human TNF of interest obtained by mutagenesis
The transformant gene was incorporated into the expression vector pKK101 having the tac promoter according to the method for constructing the human TNF expression vector of Example 4 to construct a human TNF transformant expression vector. Human TNF converter gene (about 480b
p) is the restriction enzymes EcoRI and Hi from the above-obtained plasmid of about 3.7 Kbp according to the method described above.
After cleavage with nd III, separation and purification were performed. Targeted human TNF converter expression vector pKF4427 or pKF
4429 (about 3.9 Kbp) was used as a host in E. coli, similar to the human TNF expression vector (pKF4102). coli
It was acquired using K-12 JM103 strain.

(2) According to the method of Example 10 (2), 5 μg of the human TNF transformant expression vector pKF4236 (about 4.5 Kbp) obtained in Example 8 was treated with restriction enzymes Sac I and Hind. Cleavage with III, DN of about 4.1 Kbp including origin of replication and transcriptional regulatory region
The A fragment was separated and purified.

On the other hand, 5 μg of the human TNF converter expression vector pKF4427 or pKF4429 obtained in (1) above was cleaved with the restriction enzymes Sac I and Hind III in the same manner as described above to delete the N non-portion. A DNA fragment of about 360 bp containing the human TNF converter gene was separated and purified.

The two DNA fragments obtained above were ligated using T4 DNA ligase according to the method of Example 3 described above, and E. E. coli K-12 HB101 strain was introduced into competent cells. From the resulting transformants, the respective expression vector pKF4653 or pKF4655 of F4653 or F4655 of interest (about 4.5 Kbp)
Clones containing the restriction enzymes Sac I and Hind
It was selected by confirming the cleavage pattern by III and examining the base sequence around the conversion site derived from the conversion polypeptide.

The transformant expression vector constructed according to the above method produces a novel bioactive polypeptide having the following conversion in E. coli by inducing expression.

Vector pKF4653: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 in the sequence listing were replaced with the amino acid sequences shown in SEQ ID NO: 2 in the sequence listing, and the 104th Pro was Gly and the 105th Glu is Arg
, 107th Ala is Asp, 108th Gl
Polypeptide F465 in which u is converted to Ser
Code 3

Vector pKF4655: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing were replaced with the amino acid sequences shown in SEQ ID NO: 2 of the sequence listing, and the 104th Pro was Tyr and the 105th Glu is Ile
, 107th Ala is Ser, 108th Gl
Polypeptide F465 in which u is converted to Arg, respectively
Code 5

[0140] Each host Escherichia coli having the above-mentioned expression vector expresses and produces a polypeptide having antitumor activity. Min) was confirmed by a test according to the method of Example 17 described later.

Example 15 (Construction of human TNF convertible expression vector pKF4657) (1) Substitution of 314th A for G in the nucleotide sequence from Gth 304th to Gth 324th shown in SEQ ID NO: 7 The designed oligonucleotide consisting of 21 bases was designed and named as primer 4255.

The chemical synthesis and purification of this oligonucleotide were carried out according to the method of Example 2 above. By using this primer 4255 in place of the primer 4104 in the above Example 5 (2), the target transformant DNA
Was obtained (about 3.7 Kbp). The nucleotide sequence around the mutation-introduced site was examined by the dideoxy method, and it was confirmed that the transformant DNA was mutated as designed, and this plasmid was named pUC119-F4255.

(2) In the above-mentioned Example 5 (1), pU
PU obtained in (1) above instead of C119-hTNF
The desired single-stranded plasmid DNA was prepared using C119-F4255. In addition, according to the method of Example 2, the primers 4424 and 4425 were chemically synthesized and purified according to the following design.

Primer 4424: An oligonucleotide consisting of 21 bases in which the 289th A in the base sequence from the 280th G to the 300th G shown in SEQ ID NO: 7 is replaced with G. Primer 4425: An oligonucleotide consisting of 21 bases in which the 333rd C is replaced by A in the base sequence from the 322nd G to the 342nd G shown in SEQ ID NO: 7.

Next, pUC119-F4 prepared above was prepared.
255 single-stranded plasmid DNA and primer 442
4 or 4425 was used to introduce site-directed mutagenesis into the human TNF converter gene according to the method of Example 5 (2) above, and a plasmid containing the target converter DNA (about 3.
7 Kbp) was acquired. It was confirmed by the dideoxy method that the transformant DNA was mutated as designed, and these plasmids were named pUC119-F4424 or pUC119-F4255 (N), respectively.

Plasmid pUC119-obtained above
F4424 and pUC119-F4255 (N) were digested with the restriction enzyme Sma produced by introducing the mutation in (1) above.
The plasmid pUC1I9-F4 having both mutations introduced above by recombining using the I cleavage site
424 (N) (about 3.7 Kbp) was constructed. The plasmids pUC119-F4424 and pUC1
5 μg of 19-F4255 (N) was added to 50 μl of Sma I buffer (10 mM trimer HCl pH 8.0, 20 containing 1 mM DTT and 100 μg / ml BSA).
Each of them was dissolved in mM KCl and 10 mM MgCl ₂ ), 15 units of restriction enzyme SmaI (Takara Shuzo) was added, and a cleavage reaction was carried out at 37 ° C. for 2 hours.

After completion of the reaction, D
The NA fragment was recovered and redissolved in 50 μl of high salt buffer. After cleavage reaction with 15 units of restriction enzyme EcoRI for 2 hours at 37 ° C., plasmid pUC
A small DNA fragment (about 320 bp) was obtained from the reaction solution of 119-F4424 as plasmid pUC119-F42.
A large DNA fragment (about 3.
4 Kbp) was separated and purified. The two DNA fragments thus obtained were ligated using T4 DNA ligase according to the method of Example 3 above, and E. coli
It was introduced into competent cells of K-12 strain. From the resulting transformants, by confirming the restriction enzyme cleavage pattern of the plasmid and examining the base sequence around the restriction enzyme Sma I cleavage site, the desired plasmid pUC119-F4424 (N) (about 3.7 Kb
The clone with p) was selected.

(3) The plasmid p obtained in (2) above
UC119-F4424 (N) is SEQ ID NO: 1 in the sequence listing.
It has restriction enzyme Apa I, Sma I and Nco I cleavage sites around the 106th Gly-encoding nucleotide sequence shown in. Oligonucleotides U-4431 and L-4431 were designed for the purpose of introducing a mutation around the 106th Gly shown in SEQ ID NO: 1 using these restriction enzyme cleavage sites.

U-4431 (SEQ ID NO: 13 in the sequence listing, number of bases 43) L-4431 (number of bases 47) Complementary to U-4431, but to G complementary to C at position 43 of SEQ ID NO: 13 On the other hand, 5'-CA in the 5'direction
It has a base sequence in which TG-3 'is connected.

These oligonucleotides were chemically synthesized and purified according to the method of Example 2 above. The upper and lower two oligonucleotides (1 μg each) obtained after purification were phosphorylated at their 5 ′ ends with T4 polynucleotide kinase according to the method of Example 3 above, and then annealed to each other. This was used as a single-stranded DNA fragment.

On the other hand, the plasmid p obtained in (2) above
5 μg of UC119-F4424 (N) was added to the above (2).
According to the method described in 1. above, it was cleaved with restriction enzyme Sma I, the DNA fragment was recovered by ethanol precipitation, and then dissolved in 50 μl of medium salt buffer. After cleavage reaction with 15 units of restriction enzyme Nco I (Takara Shuzo) at 37 ° C. for 2 hours, a DNA fragment of about 3.7 Kbp was separated and purified by agarose gel electrophoresis.

The two DNA fragments obtained according to the above method were ligated using T4 DNA ligase according to the method of Example 3, and E. E. coli K-12 strain was introduced into competent cells. From among the resulting transformants,
A clone having a plasmid (about 3.7 Kbp) containing the transformant DNA into which the desired mutation was introduced was selected by examining the nucleotide sequence around the mutation introduction site in the plasmid. This plasmid was designated as pUC119-F44.
It was named 31.

The desired human TNF transformant gene obtained above was used as an expression vector pK having a tac promoter according to the method for constructing the human TNF expression vector of Example 4.
It was integrated into K101 to construct a human TNF converter expression vector. The human TNF converter gene (about 500 bp) is
About 3.7 Kbp plasmid pUC11 obtained above
From 9-F4431, according to the method described above, the restriction enzyme E
After cleavage with coRI and HindIII, separation and purification were performed. Targeted human TNF converter expression vector pKF
Similar to the human TNF expression vector (pKF4102), E. 4431 (about 3.9 Kbp) was used as a host in E. coli. coli
It was acquired using K-12 JM103 strain.

(4) According to the method of Example 10 (2), 5 μg of the human TNF transformant expression vector pKF4236 (about 4.5 Kbp) obtained in Example 8 was treated with restriction enzymes Sac I and Hind. A DNA of about 4.1 Kbp that is cleaved with III and contains an origin of replication, a transcriptional regulatory region, and the like
The fragment was separated and purified.

On the other hand, 5 μg of the human TNF converter expression vector pKF4431 obtained in the above (3) was cleaved with restriction enzymes Sac I and Hind III in the same manner as described above, and human TNF lacking the N portion was deleted. A DNA fragment of about 380 bp containing the transformant gene was separated and purified.

The two DNA fragments obtained above were ligated using T4 DNA ligase according to the method of Example 3 described above, and E. E. coli K-12 HB101 strain was introduced into competent cells. Of the obtained transformants, the desired F4657 expression vector pKF4657 is selected.
A clone having (about 4.5 Kbp) was selected by confirming the cleavage pattern with a restriction enzyme and examining the base sequence around the conversion site derived from the converter polypeptide.

The transformant expression vector constructed according to the above method produces a novel bioactive polypeptide having the following conversion in E. coli by expression induction. Vector pKF4657: The 1st to 8th amino acid sequences shown in SEQ ID NO: 1 of the sequence listing are replaced with the amino acid sequences shown in SEQ ID NO: 2 of the sequence listing, and the 97th Ser is G
In ly, the 105th Glu had the amino acid sequence shown by SEQ ID NO: 14 (Gly-Met-Asp-Gly-Tyr).
-Ile-Gly-Ser-Arg) encoded polypeptide F4657. Regarding the centrifugal supernatant of the Escherichia coli suspension sonicated (the supernatant fraction after centrifugation) that the host Escherichia coli having the expression vector expresses and produces the polypeptide showing antitumor activity, It was confirmed by testing according to the method of Example 17 described later.

Example 16 (Expression and purification of human TNF and its transformant polypeptide in E. coli) The human TNF expression vector (pKF4) obtained in Example 4
102) and the human TNF transformant expression vectors (pKF4419, pKF) obtained in Examples 6, 7, 9 and 13.
4146, pKF4422, pKF4423 and pKF
E. 4621). coli K-12 JM10
The three strains were treated with 25 to 50 μg / ml ampicillin and 0.0
M9 medium containing 0.6% Vitamin B1 (0.6% Na ₂
HPO ₄ , 0.3% KH ₂ PO ₄ , 0.05% Na
_{Cl, 0.1% NH 4 Cl,} 2mM MgSO 4, 0.
20 ml of 2% glucose and 0.1 mM CaCl ₂ )
And cultured at 37 ° C. for 18 hours with shaking. 20 ml of this culture solution was added to 1 liter of the above medium,
Shaking culture was performed at ℃ for 2-3 hours. Next, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the mixture was further incubated at 37 ° C. for 18 hours.
The shaking culture was continued for an hour.

Human TNF transformant expression vector (pKF423) obtained in Examples 8, 10, 11, 13 and 14.
6, pKF4603, pKF4616, pKF461
7, pKF4649 and pKF4653).
For the K. coli K-12 HB101 strain, 25-
50 μg / ml ampicillin, 0.001% vitamin B
M9CA medium containing 1 and 5 μg / ml tryptophan (0.6% Na ₂ HPO ₄ , 0.3% KH ₂ P
O ₄ , 0.05% NaCl, 0.1% NH ₄ Cl, 2
mM MgSO ₄ , 0.2% glucose and 0.1 mM
20 ml of CaCl ₂ and 0.2% casamino acid) was inoculated and shake culture was performed at 37 ° C. for 18 hours. 5 μg / m
20 ml of this culture solution was added to 1 liter of the above-mentioned medium not containing 1-tryptophan, and shake culture was carried out at 37 ° C. for 18 hours.

After collecting the E. coli cells by centrifugation,
TP buffer (10 mM Tris-HCl pH 8.0
And the cells were washed with 100 μM PMSF). After washing, the cells are suspended in 10 volumes (ml) of TP buffer per wet weight (gram) of the cells, and the cells are sonicated using an ultrasonic generator (Heat Systems; Model W-225). It was crushed. The resulting suspension was centrifuged to remove the bacterial cell residue and collect the supernatant fraction. The purification process after this ultrasonic crushing process was mainly performed under low temperature (0 degreeC-4 degreeC).

After filtering this supernatant through a 0.45 μm filter, anion exchange chromatography using SepaBeads FP-DA13 (Mitsubishi Kasei) (column size:
φ2.5 × 1.5 cm and flow rate: 0.5 ml / min), and the presence or absence of activity is assayed according to the method of Example 17 described below using SDS-polyacrylamide gel electrophoresis described below and L929 cells. Thus, an active fraction was obtained. Use TP buffer containing NaCl for elution
The concentration was gradually increased from 0.05M to 0.1M, 0.2M, 0.5M, and human TNF (1) was 0.1M NaC.
l, human TNF (2), human TNF (3), human TN
F-converter F4419, F4422, F4603, F4616, F4617, F4621, F464
9 and F4653 were 0.2M NaCl, and human TNF transformants F4146 and F4236 were 0.5M.
Elute with NaCl respectively. Furthermore, in order to remove endotoxin and the like derived from Escherichia coli cells, treatment with a nucleic acid / endotoxin removing agent C-9 (manufactured by Kurita Water Industries Ltd., Dainippon Pharmaceutical Co., Ltd.) was carried out according to the attached manual. Thus, human TN
A one-step purified sample of F and its transformant polypeptides was obtained.
This sample was used to evaluate the antitumor activity and the effect on experimental lung metastasis of Examples 17, 18 and 19 described below. The transformant polypeptide F4423 was used for the host E. coli E. coli K-12 JM
Due to instability in 103 strains, the active fraction could not be obtained. However, the active fraction may be obtained using other hosts.

After filtering the above sample with a 0.20 μm filter, a MonoQ (registered trademark) (HR10 / 10 and HR5 / 5) prepack column (manufactured by Pharmacia LKB Biotechnology) under the control of the FPLC system.
By anion exchange chromatography using
Containing TPQ buffer (20 mM Tris-HCl pH
Elution by 8.0 and 10 μM PMSF), and fractionation by SDS-polyacrylamide gel electrophoresis and L
An active fraction was obtained by assaying the presence or absence of activity according to the method of Example 17 described below using 929 cells. The elution method was performed according to the following program under the control of the FPLC system. The third step was repeated several times until a single band was obtained by SDS-polyacrylamide gel electrophoresis in order to improve the purification purity.

First step: Mono Q (registered trademark) (HR
10/10) Column use (flow rate: 4 ml / min) 0-5 min; 0-0.2M NaCl linear concentration gradient 5-16 min; 0.2M NaCl 16-20 min; 0.2-0.5M NaCl linear Concentration gradient 20 to 24 minutes; 0.5 M NaCl The fractionation results (NaCl concentration and retention time) of the active fractions according to the above method were 0.2 for human TNF (1), respectively.
M, 6.6 min, human TNF (2) 0.2 M, 5.6
Min, human TNF (3) 0.2M, 5.0 min, human TN
F conversion body F4146 is 0.2M, 6.5 minutes, same F423
6 is 0.2M, 6.4 minutes, the same F4419 is 0.2M,
5.4 minutes, same F4603 0.2M, 7.6 minutes, same F4
616 is 0.2M, 5.0 minutes, F4617 is 0.2
M, 6.7 minutes, the same F4621 was 0.2M and 5.0 minutes, the same F4649 was 0.18M, 4.0 minutes and the same F4653 was a non-adsorbed fraction.

Second step: Mono Q (registered trademark) (HR
5/5) Column use (flow rate: 1 ml / min) 0-2.5 minutes; 0-0.2M NaCl linear concentration gradient 2.5-8 minutes; 0.2M NaCl 8-10 minutes; 0.2-0 .5M NaCl linear concentration gradient 10 to 12 minutes; 0.5M NaCl The fractionation results (NaCl concentration and retention time) of the active fractions according to the above method were 0.2 for human TNF (1), respectively.
M, 3.7 min, human TNF (2) 0.2 M, 4.1
Min, human TNF (3) 0.2M, 3.8 min, human TN
F converter F4146 is 0.2M, 4.1 minutes, same F423
6 is 0.2M, 3.8 minutes, F4419 is 0.2M,
3.8 minutes, same F4603 0.2M, 5.7 minutes, same F4
616 is 0.2M, 3.4 minutes, F4617 is 0.2
M, 4.4 minutes, the same F4621 was 0.2M, 3.7 minutes, the same F4649 was 0.2M, 3.4 minutes, and the same F4653 was a non-adsorbed fraction.

Third step: Mono Q (registered trademark) (HR
5/5) Column use (flow rate: 1 ml / min) 0 to 6 minutes; 0-0.15M NaCl linear concentration gradient 6 to 11 minutes; 0.15-0.2M NaCl linear concentration gradient 11 to 13 minutes; 2-0.5M NaCl linear concentration gradient 13 to 15 minutes; 0.5M NaCl The fractionation results (NaCl concentration and retention time) of the active fractions according to the above method were 0.16 for human TNF (1).
M, 6.5 minutes, human TNF (2) 0.16M, 6.5
Min, human TNF (3) 0.16M, 6.4 min, human TNF (3)
NF converter F4146 was 0.17M, 7.1 minutes, same F4
236 is 0.17M, 6.7 minutes, F4419 is 0.1
6M, 6.3 minutes, same F4603 0.23M, 10.8
Minute, same F4616 0.15M, 5.9 minutes, same F461
7 is 0.18M, 8.7 minutes, F4621 is 0.15
M was 5.9 minutes, the same F4649 was 0.16M, 6.3 minutes and the same F4653 was a non-adsorbed fraction.

Thus, purified samples of human TNF and its transformant polypeptide were obtained. This sample was used to evaluate the antitumor activity and the effect on experimental lung metastasis of Examples 17, 18 and 19 described below. The transformant polypeptide F4422 was found to have a deletion at the N-terminal portion in the purification process, and therefore the purification was stopped halfway. However, there is a possibility that the final purified sample can be obtained by improving the purification method.

SD of the above purification process and purified sample
S-polyacrylamide gel electrophoresis is performed to detect human TN
The expression and purification of F and its transformant polypeptides were confirmed. Laemml containing each sample with 10 mM DTT
i's sample buffer (62.5 mM Tris-H
Cl pH 6.8, 2% SDS, 0.01% BPB and 10% glycerol), and Laemmli [Nat
ure, 227 , 680 (1970)],
Electrophoresis was performed using a 15% preparative gel. After the electrophoresis was completed, the protein in the separation gel was confirmed by staining with Coomassie Brilliant Blue. 14,
Some of the results are shown in 15 and 16. The expression levels of the human TNF and the transformant polypeptide which were able to obtain the active fraction were almost the same, and the obtained stained gel was analyzed by a chromatographic scanner (Shimadzu,
When the expression efficiency was calculated by applying CS-920 type), it was about 20% of the total bacterial cell protein of E. coli. The final purified sample was a single band in the obtained stained gel. From the migration position, the molecular weights of human TNF and its transformant polypeptide were calculated and shown in Table 1 and Table 2.

As one of the physical properties of protein, human T
The isoelectric points of NF and its transformant polypeptide were measured by isoelectric focusing gel electrophoresis using Amforin (manufactured by LKB), and the obtained results are shown in Tables 1 and 2.

[0169]

[Table 1]

[0170]

[Table 2]

Example 17 (Evaluation of In Vitro Antitumor Activity) Mouse-derived connective tissue cells L929 (ATCC CCL1) were prepared for the one-step purified sample and the purified sample of human TNF and its transformant polypeptide obtained in Example 16. The cytotoxic activity against A. galgarwal et al [J. Bio
l. Chem. , 260, 2345 (1985)]. That is, L929 cells were seeded at 3 × 10 ⁴ cells / 0.1 ml / well in a 96-well tissue culture microplate (manufactured by Corning) and cultured overnight at 37 ° C. in the presence of 5% carbon dioxide. The medium is 1
Eagle's minimum essential medium modified by Dulbecco with 0% fetal bovine serum (D
ME medium, manufactured by Sigma) was used. Next day, final concentration 1μ
The medium was replaced with the above medium supplemented with g / ml of actinomycin D, and a sample serially diluted with this medium was treated in each well (total medium amount 0.1 ml). After culturing for another 20 hours, 0.5% crystal violet solution (0.5%
Viable cells attached to the plate were stained with (% crystal violet / 20% methanol) (room temperature, 15 minutes). Phosphate buffer PBS containing 1 mM CaCl ₂ and 1 mM MgCl ₂ (10 mM Na · K phosphate pH 7.4, 0.8% NaCl and 0.02% KCl)
After sufficiently washing with 0.01N containing 0.01% ethanol
The crystal violet remaining on the plate was extracted with 0.1 ml of hydrochloric acid solution, and its absorbance (492 nm)
EIA leader (Bio-Rad, model 255
It was measured in 0). This absorbance is proportional to the number of viable cells.
Therefore, the final dilution rate of the sample in the well showing the absorbance corresponding to 50% of the absorbance of the sample-untreated well was obtained, and the reciprocal of this sample dilution rate was defined as the number of units per unit of the sample [unit / ml]. Define.

In order to calculate the specific activity (unit / mg protein) of each sample from the L929 cytotoxic activity (unit / ml) determined according to the above method, protein quantification of each sample was performed. The quantification was carried out by the Bradford method [Anal. Bio
chem. , 72 , 248 (1976)], using bovine serum albumin as a standard sample, the protein concentration (mg
/ Ml) was determined. Based on these results, the specific activities calculated for human TNF and the same transformant polypeptide sample are shown in Tables 3 and 4.

[0173]

[Table 3]

[0174]

[Table 4]

From Tables 3 and 4 above, the human TN of the present invention
It can be seen that the F-converting polypeptide has a cytotoxic activity against mouse-derived connective tissue cell L929, similar to human TNF.

Example 18 (Evaluation of In Vivo Antitumor Activity) The single-step purified sample or the purified sample of human TNF and its transformant polypeptide obtained in Example 16 was tested against mouse transplantable fibrosarcoma MethA tumor. Tumor therapeutic activity was determined. The test was 1 × 10 ⁶ suspended in physiological saline.
Cells / 0.2 ml of MethA tumor cells (distributed by Sasaki Research Institute) were subcutaneously transplanted to the back of BALB / c mice (male, 5 weeks old, Charles River), and the tumor diameter became 6 to 10 mm after 8 days. After confirming the arrival, a sample (0.2 ml / mouse) serially diluted with physiological saline was administered through the tail vein. Each dose sample was prepared by making the lethal dose the highest dose and diluting it in several stages.

Observation of tumor growth was continued for about 2 weeks after the administration. For tumor growth, tumor volume (the long diameter × short diameter ^2/2 tumor mass) was measured to obtain the tumor volume ratio with respect to tumor volume of the sample dosing day (Day 0), a sample administration this value becomes 2 or 5 Calculate the number of days from the day (D2 or D5). Then, the ratio to the control group (administered with physiological saline) was calculated, and the D2% control value and the D5% control value were obtained. The larger the value, the lower the tumor growth ability and the higher antitumor activity. The results obtained according to the above are shown in Table 5, Table 6 and Table 7.

[0178]

[Table 5]

[0179]

[Table 6]

[0180]

[Table 7]

From Table 5, Table 6 and Table 7, the human TNF transformant polypeptide is similar to human TNF in that the mouse transplanted Met.
It can be seen that it has an antitumor therapeutic activity against hA tumor.

Example 19 (Effect of Tumor on Lung Metastasis) A clone showing high metastasis to lung by cloning B16F10 mouse malignant melanoma cells (distributed by Chiba University School of Medicine) by the following method. Was established. That is, Eagle's minimum essential medium (MEM
2 × 10 ⁴ cells / 0.
Add 2 ml of B16F10 cells to C57BL / 6 NCr
j mouse (female, 6 weeks old, Charles River) was injected into the tail vein, and the lung was extracted 14 days later. After washing with DME medium, one of the metastatic nodules with a diameter of about 1 mm formed on the lung surface was aspirated using a syringe with a 26G injection needle (Terumo), suspended in 1 ml of DME medium, and then 10%. Colonies were formed by culturing at 37 ° C. in the presence of 5% carbon dioxide on soft agar containing DME medium containing fetal bovine serum. This colony formation method is called "tissue culture technology"
(Pages 35 to 36, 1984, edited by the Japanese Society for Tissue Culture, Asakura Shoten). 10-12 days later,
When the diameter of the colony reached 1-2 mm, the colony was aspirated using a Pasteur pipette and cultured in DME medium containing 10% fetal bovine serum. After culturing, the proliferated cells were used again in MEM medium to obtain 2 × 10 ⁴ cells / 0.
The cells were prepared to a cell concentration of 2 ml and injected into the tail vein of the same mouse as in the above case. After 14 days, the lung was removed, and one of the metastatic nodules was isolated and cultured in the same manner as the previous time. This operation was repeated 5 times to obtain a clone (designated as B16F10 / L5) showing a high metastatic property in the lung.

B16F10 / L5 cells in the logarithmic growth phase, which had been cultured in a DME medium containing 10% fetal bovine serum, using a tissue culture dish (Corning (registered trademark), manufactured by Iwaki Glass Co., Ltd.) having a diameter of 10 cm were prepared. In the adhered state, washed once with phosphate buffer PBS and MEM
A cell suspension was prepared by adding 5 ml of medium and pipetting. Collect cells by centrifugation and 2 ml
Of MEM medium. On the other hand, the one-step purified sample or purified sample of human TNF and its transformant polypeptide obtained in Example 16 was treated at a predetermined concentration (in the antitumor treatment test of Example 18, a dose showing a therapeutic effect was used). 2 × 10 ⁴ cells / 0.2 by adding the B16F10 / L5 cell suspension prepared above to the solution.
A cell suspension containing ml of each one-step purified sample or purified sample was prepared. This cell suspension was added to C57BL / 6NCrj.
It was administered through the tail vein of a mouse (female, 6 weeks old) (0.2 m
1 / mouse). The control group was administered with B16F10 / L5 cells only. Fourteen days after the administration, the lung was removed and the number of metastatic nodules on the lung surface was measured. The results obtained according to the above are shown in Table 8, Table 9 and Table 10.

[0184]

[Table 8]

[0185]

[Table 9]

[0186]

[Table 10]

From Tables 8, 9 and 10, human TNF and its transformant polypeptide have the effect of promoting experimental lung metastasis, whereas the cell-adhesive peptide sequence of laminin was introduced near its N-terminal. It can be seen that the human TNF transformant polypeptide of the present invention does not promote experimental lung metastasis.

[0188]

INDUSTRIAL APPLICABILITY According to the present invention, in human TNF or its transformant, the amino acid sequence from Ser at position 1 to Asp at position 8 in SEQ ID NO: 1 of the Sequence Listing or the amino acid sequence of the corresponding human TNF converter Contains at least one laminin cell-adhesive peptide sequence, and
By having an amino acid sequence containing 30 to 30 amino acids, it has an antitumor activity similar to that of human TNF or its transformant, while not exhibiting the cancer metastasis-enhancing effect observed in them. Human TNF transformants are provided.

[Sequence list]

[Brief description of drawings]

FIG. 1 Human TN by ligation of synthetic oligonucleotides
It is a process drawing which shows the construction process of F gene.

FIG. 2 Human TN by ligation of synthetic oligonucleotides
It is a process drawing which shows the construction process of F gene.

FIG. 3 is a process diagram showing a construction process of an expression type plasmid vector.

FIG. 4 is a process diagram showing a process of constructing a human TNF expression vector.

FIG. 5 is a process drawing showing a process for producing a human TNF converter or a gene for the same by a site-directed mutagenesis method.

FIG. 6 is a process diagram showing a process for producing a human TNF converter or a transformant gene by the site-directed mutagenesis method.

FIG. 7 is a process drawing showing the process of constructing a human TNF transformant expression vector by gene recombination utilizing a restriction enzyme cleavage site.

FIG. 8 is a process chart showing the process of constructing an expression type plasmid vector.

FIG. 9 is a process drawing showing the process of constructing an expression type plasmid vector.

FIG. 10 is a process drawing showing the process of constructing an expression type plasmid vector.

FIG. 11 is a process drawing showing the process of constructing a human TNF converter expression vector by gene recombination utilizing a restriction enzyme cleavage site.

FIG. 12 is a process drawing showing the process of constructing a human TNF transformant expression vector by gene recombination utilizing a restriction enzyme cleavage site.

FIG. 13 is a process drawing showing the construction process of the human TNF transformant F4236 gene-containing plasmid pUC119 (B) -F4236.

FIG. 14 shows the results of electrophoresis of purified samples of human TNF and its transformant polypeptide expressed by Escherichia coli.

FIG. 15 shows the results of electrophoresis of purified samples of human TNF convertor polypeptide expressed by E. coli.

FIG. 16 shows the results of electrophoresis of purified samples of human TNF convertible polypeptide expressed by E. coli.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12N 15/70 C12P 21/02 L 8214-4B // (C12N 1/21 C12R 1:19) ( C72P 21/02 C12R 1:19) (72) Inventor Yoshiyuki Aoyama 2-3-1, Nishi-Shibukawa, Kusatsu City, Shiga Ishihara Sangyo Co., Ltd. Central Research Institute (72) Inventor Hiroshi Shiogama 2-chome, Nishi-Shibukawa, Kusatsu City, Shiga Prefecture No. 3 Ishihara Sangyo Co., Ltd. Central Research Laboratory

Claims (29)

[Claims] 1. The first S shown in SEQ ID NO: 1 in the sequence listing
in the tumor necrosis factor polypeptide having an amino acid sequence represented by er to 155th Leu or a transformant thereof, the amino acid sequence from 1st Ser to 8th Asp of SEQ ID NO: 1 or the transformant corresponding thereto A polypeptide comprising at least one laminin cell-adhesive peptide sequence and is substituted with an amino acid sequence comprising 5 to 30 amino acids. 2. The laminin cell-adhesive peptide sequence is 3 in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
The polypeptide according to claim 1, which is an amino acid sequence represented by the Tyr of the second position to the Arg of the seventh position. 3. An amino acid sequence containing at least one laminin cell-adhesive peptide sequence and containing 5 to 30 amino acids is shown in SEQ ID NO: 2, 3, 4, 5 or 6, respectively. The polypeptide according to claim 1, which has an amino acid sequence. 4. An amino acid sequence containing at least one laminin cell-adhesive peptide sequence and containing 5 to 30 amino acids is the amino acid sequence shown in SEQ ID NO: 2, 3 or 4, respectively. The polypeptide according to claim 1. 5. The tumor necrosis factor polypeptide or its transformant comprises the amino acid sequence represented by the 9th Lys to the 155th Leu shown in SEQ ID NO: 1 or the 29th Arg, 68th of the amino acid sequence. 4. The polypeptide according to claim 1 or 3, which has an amino acid sequence in which the Pro or Gly at position 106 has been deleted or replaced with another amino acid. 6. The 29th Arg is Met, Tr
p, Pro, Ala, Val, Leu, Gln, Ty
The polypeptide according to claim 5, which is substituted with r, Ser, Lys, His, Asp or Glu. 7. The 29th Arg is Gln, Ly
6. The polypeptide of claim 5 substituted with s, Asp, Val or Leu. 8. The 68th Pro is Met, Tr
p, Ala, Val, Leu, Gln, Tyr, Se
The polypeptide according to claim 5, which is substituted with r, Asp or Glu. 9. The 68th Pro is Asp or Me
The polypeptide of claim 5 substituted with t. 10. The 106th Gly is deleted or Met, Trp, Pro, Ala, Val, L
eu, Gln, Tyr, Ser, Arg, Lys, Hi
The polypeptide according to claim 5, which is substituted with S, Asp or Glu. 11. The polypeptide according to claim 5, wherein the 106th Gly is deleted or replaced by Trp, Pro, Ala, Asp or Arg. 12. A tumor necrosis factor polypeptide having an amino acid sequence represented by the first Ser to the 155th Leu shown in SEQ ID NO: 1 of the Sequence Listing, or a transformant thereof, wherein the first SEQ ID NO: 1 The amino acid sequence from Ser to the 8th Asp or the amino acid sequence corresponding to the above-mentioned transformant is replaced by an amino acid sequence containing at least one cell adhesive peptide sequence of laminin and containing 5 to 30 amino acids. A recombinant plasmid comprising a DNA encoding the said polypeptide. 13. The recombinant plasmid is pKF414.
6, pKF4236, pKF4419, pKF442
2, pKF4423, pKF4603, pKF460
4, pKF4616, pKF4617, pKF463
0, pKF4631, pKF4636, pKF463
7, pKF4618, pKF4619, pKF463
2, pKF4633, pKF4640, pKF464
1, pKF4620, pKF4621, pKF462
2, pKF4623, pKF4624, pKF464
7, pKF4648, pKF4649, pKF465
0, pKF4651, pKF4653, pKF4655
The recombinant plasmid according to claim 12, which is pKF4657. 14. A tumor necrosis factor polypeptide having an amino acid sequence represented by the first Ser to the 155th Leu shown in SEQ ID NO: 1 of the Sequence Listing or a transformant thereof, wherein the first SEQ ID NO: 1 The amino acid sequence from Ser to the 8th Asp or the amino acid sequence corresponding to the above-mentioned transformant is replaced by an amino acid sequence containing at least one cell adhesive peptide sequence of laminin and containing 5 to 30 amino acids. Recombinant microbial cell transformed with a recombinant plasmid containing DNA encoding the said polypeptide. 15. The recombinant microbial cell according to claim 14, wherein the recombinant microbial cell is Escherichia coli. 16. A tumor necrosis factor polypeptide having an amino acid sequence represented by the first Ser to the 155th Leu shown in SEQ ID NO: 1 of the sequence listing, or a transformant thereof, wherein the first SEQ ID NO: 1 The amino acid sequence from Ser to the 8th Asp or the corresponding amino acid sequence of the above-mentioned transformant is replaced with an amino acid sequence containing at least one laminin cell-adhesive peptide sequence and containing 5 to 30 amino acids. Of a recombinant microbial cell transformed with a recombinant plasmid containing a DNA encoding the present polypeptide, the polypeptide having the amino acid sequence of interest is produced and isolated. Method. 17. A tumor necrosis factor polypeptide having an amino acid sequence represented by the first Ser to the 155th Leu shown in SEQ ID NO: 1 of the sequence listing, or a transformant thereof, wherein The amino acid sequence from Ser to the 8th Asp or the amino acid sequence corresponding to the above-mentioned transformant is replaced by an amino acid sequence containing at least one cell adhesive peptide sequence of laminin and containing 5 to 30 amino acids. A pharmaceutical composition containing the polypeptide as an active ingredient. 18. The laminin cell-adhesive peptide sequence contains a polypeptide which is the amino acid sequence represented by Tyr at the 3rd position to Arg at the 7th position in the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing. 17. The pharmaceutical composition according to 17. 19. An amino acid sequence containing at least one laminin cell-adhesive peptide sequence and containing 5 to 30 amino acids is shown in SEQ ID NO: 2, 3, 4, 5 or 6 of the sequence listing, respectively. 18. The pharmaceutical composition according to claim 17, which comprises a polypeptide that is an amino acid sequence. 20. An amino acid sequence containing at least one laminin cell adhesive peptide sequence and containing 5 to 30 amino acids is the amino acid sequence shown in SEQ ID NO: 2, 3 or 4, respectively. The pharmaceutical composition according to claim 17, which comprises a polypeptide. 21. The amino acid sequence of the tumor necrosis factor polypeptide or its transformant represented by the 9th Lys to the 155th Leu shown in SEQ ID NO: 1 or the 29th Arg, 68th amino acid sequence of the amino acid sequence. 20. The pharmaceutical composition according to claim 17 or 19, containing a polypeptide having an amino acid sequence in which Pro or Gly at position 106 has been deleted or substituted with another amino acid. 22. The 29th Arg is Met, Tr
p, Pro, Ala, Val, Leu, Gln, Ty
22. The pharmaceutical composition according to claim 21, containing a polypeptide substituted by r, Ser, Lys, His, Asp or Glu. 23. The 29th Arg is Gln or Ly.
22. The pharmaceutical composition according to claim 21, containing a polypeptide substituted by s, Asp, Val or Leu. 24. The 68th Pro is Met, Tr
p, Ala, Val, Leu, Gln, Tyr, Se
22. The pharmaceutical composition according to claim 21, which contains a polypeptide substituted with r, Asp or Glu. 25. The 68th Pro is Asp or M
A polypeptide containing a polypeptide replaced by et.
The pharmaceutical composition according to 1. 26. The 106th Gly is deleted or Met, Trp, Pro, Ala, Val, L
eu, Gln, Tyr, Ser, Arg, Lys, Hi
22. The pharmaceutical composition according to claim 21, which contains a polypeptide substituted by s, Asp or Glu. 27. The pharmaceutical composition according to claim 21, which contains a polypeptide in which the 106th Gly is deleted or substituted by Trp, Pro, Ala, Asp or Arg. 28. A tumor necrosis factor polypeptide having an amino acid sequence represented by the first Ser to the 155th Leu shown in SEQ ID NO: 1 of the sequence listing, or a transformant thereof, wherein the first SEQ ID NO: 1 The amino acid sequence from Ser to the 8th Asp or the amino acid sequence corresponding to the above-mentioned transformant contains at least one amino acid sequence having a laminin cell-adhesive peptide sequence,
A polypeptide which is substituted with an amino acid sequence containing 5 to 30 amino acids and further has Met at its N-terminus. 29. A tumor necrosis factor polypeptide DNA having a nucleotide sequence represented by the first T to the 465th G shown in SEQ ID NO: 7 of the Sequence Listing or its mutated DNA, wherein 1-3 TCA
To the 22 to 24th GAC or the corresponding nucleotide sequence of the mutation-introduced DNA containing at least one laminin cell-adhesive peptide sequence and 5 to 30 amino acid sequences A DNA characterized by being replaced by a nucleotide sequence encoding