JPH02227080A - New recombinant lymphotoxin derivative - Google Patents

Abstract

PURPOSE:To increase anti-tumor action and enlarge anti-tumor action spectrum by synthesizing recombinant lymphotoxin derivative containing polypeptide connecting to NH2 end of amino acid sequence and comprising 1-28 amino acid residues. CONSTITUTION:Host such as colibacillus is transformed by using plasmid containing DNA sequence coding recombinant lymphotoxin derivative containing polypeptide connected to NH2 end of amino acid sequence and composed of 1-28 amino acid residues. Next, said transformed host is cultured in a medium and the above-mentioned recombinant lymphotoxin derivative is recovered. Resultant recombinant lymphotoxin derivative has, e.g. activity of 50-80 times of natural lymphotoxin to cell derived from human lung cancer (PC-10) and thus able to be essential active ingredient of drug composition for curing object.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel recombinant lymphotoxin derivative, genetic recombination technology for its production, and a pharmaceutical composition containing the derivative. Therefore, the present invention can be utilized in the field of medical drugs.

[Conventional technology]

There are various humoral factors involved in biological defense mechanisms in living organisms, and these are mainly trace amounts of physiologically active substances produced by white blood cells, lymphocytes, macrophages, etc. in response to stimuli, such as interferon (IFN). −α, β
, T), interleukin (ILL, 2, 3.4),
Mention may be made of lymphotoxin (LT), tumor necrosis factor (TNF), colony stimulating factor (G-, GM-C3F).

Among these, lymphotoxin and TNF have been confirmed as polypeptides that exhibit direct anti-cell activity against neoplastic cell lines.
P is produced by macrophages upon stimulation with mitogenes, respectively.

It has already been reported that lymphotoxin and TNF can be produced by cloning the lymphotoxin and TNF genes and culturing E. coli transformed with a vector incorporating the genes (Nature, Dango 721- 724.1984 (Natur
re, 312.721-724.1984).

and lymphotoxin and TNF produced in E. coli.
It has been proven that Meth A Sarcoma has a necrotic effect on transplanted cancers in in vivo experiments, and for example, cancer caused by Meth A Sarcoma transplanted into mice was found to be significantly cured by intravenous or local administration of lymphotoxin or TNF. (J, Immun
ology, M, A, PALLADINO etal,
1384023 (1987)).

However, it has become clear that these lymphotoxin and TNF do not specifically damage all cancer cells, and that there are many cancer cells that are not affected at all (Sugarman et al.
230.943 (1985), Watanabe
et al., J.

Cancer Res, (Gann) 761115
(1985)). For example, human bladder cancer T-24, human malignant melanoma G-361. Colon adenocarcinoma 5174-T, human laryngeal carcinoma HEp2, human fibroinoma HT1080, etc.
It is not susceptible to damaging effects in vitro experiments.

By the way, it has become clear that lymphotoxin and TNF have a wide range of effects other than antitumor effects, and so far, riboprotein lipase inhibitory effects, ILI-inducing effects, prostaglandin-inducing effects, and blood coagulation-promoting effects have been reported. . When lymphotoxin or TNF is used clinically, it is expected that these effects will be exposed as side effects, and in fact, in phase clinical trials of TNF, fever, chills, shivering, decreased blood pressure, increased GOT/GPT, and general malaise were observed. increased ALP has been observed (cancer and chemotherapy,
13.3491.1986).

Now, due to differences in molecular weight, charge, and heat resistance, lymphotoxin has α (molecular weight 70,000 to 90,000), β (molecular weight 25,000 to 50,000), and -r (molecular weight 1).
It is divided into four classes: molecular weight (molecular weight < 200,000) and complex (molecular weight < 200,000).

Torrence, Biological Response Mof,r
77 I'r -x 293.1985 (Paul
F. Torrence.

Biological Re5ponse Modif
Iers, 293.1985), and the α and β classes can be further classified into subclasses according to charge. That is, lymphotoxin is used as a general term for various heterogeneous substances.

Aggarwal et al. reported the amino acid structure of lymphotoxin. B, C, Plate 2334 (1985
) (J, B, C, 260, 2334 (1985)), and the gene structure of lymphotoxin was also reported by Gray et al., and it is believed that part of the cause of lymphotoxin heterogeneity is due to the number of amino acid residues and association formation of the lymphotoxin. It is thought that this difference can be explained.

On the other hand, Granger et al. reported that there is a lymphotoxin that does not show immunological crosstalk with this lymphotoxin (J. Immunology 137, 1878.
1986 (J, Immunology 137.1
878° 1986), it is said that this has a different spectrum of anti-cellular activity from lymphotoxin, whose structure has already been elucidated. Furthermore, human tumor necrosis factor (TNF), which has been reported to have anti-tumor cell activity, shows 28% similarity to human lymphotoxin in its amino acid structure, and it has been reported that they share a receptor on the surface of tumor cells. Despite this, the spectrum of antitumor cell activity is thought to be somewhat different.

Based on the above findings, the present inventors created various polypeptides from the lymphotoxin and developed reagents that exhibit antitumor effects.
The aim of this study was to discover the amino acid sequence of Gion and to enhance its antitumor effect.

[Means to solve the problem]

As a result of various studies, it was discovered that an amino acid sequence consisting of 137 amino acid residues has an antitumor effect. The present invention was completed based on the knowledge that a novel polypeptide having stronger antitumor effects or even damaging effects on lymphotoxin-resistant cancer cells can be obtained by conversion.

That is, the present invention provides an amino acid sequence consisting of 137 amino acid residues, with several variations of specific amino acids in the sequence, for the purpose of enhancing antitumor action and expanding the spectrum of antitumor action. Disclosed are novel lymphotoxin derivatives containing: However, since such derivatives can be effectively achieved through the use of genetic recombination techniques, the gist of the present invention is to provide recombinant lymphotoxin derivatives containing the above amino acid sequences.

The present invention will be explained in detail below, divided into sections of configuration and method.

Composition The recombinant lymphotoxin derivative of the present invention has the following in its molecule:
It contains an amino acid sequence represented by the structural formula (hereinafter referred to as the amino acid sequence according to the present invention).

Pro^la Ala^, Leu A2A, Asp
A, ^, ^, GlnAsn Ser Leu A?
^a As Ago Asn Thr Act A+a
^1a Phe Leu Gln Asp Gly P
he Ser Leu Ser^,, Asn Ser
Leu Leu Val Pro Thr Ser
Glylle Tyr Phe Val Tyr Se
r Gin Vat Val PheSer A, 4^
2. Thr Ser Ser Pro A,, Ty
r LeuAla His Glu Val Gin
Leu Phe Ser Ser Ser GinTyr Pr
o Phe His Val Pro Leu Leu
Ser 5erGin A23 Met Val T
yr Pro Gly Leu Gin GluPro
Trp Leu His Ser Met Tyr
)lis Gly Ala^1a Phe Gln L
eu Thr A24 Ass A211^17Leu
Ser Thr )lis Thr Asp Gly
lie Pro His LeuVal Leu A2
s Ass A30 A31 Val A32 Phe
GlyAla Phe Ala Leu (In the formula, ^l''141 A21432 represents any amino acid) In particular, A1 is His, Asn, Leu, A2 is l
ie, Met, Gly.

A3 is Leu, Gin, His, Cys,
Ile, Pro, Val, Ser.

Gty SA, Pro, Arg, Leu,
Gin, Val, ^, Ser, Phe,
Cys, A6 is Lys, Asn, Gin
, Glu.

^Sps: ^, Leu, Ala, Gin SA,
are Trp, Phe.

Leu SA, is Arg, Leu, A,, is ^^
la, Arg, Glu.

A mouth is ^sp, Asn, Ala, Arg
, Al1 is ^rg, Asn.

Gly 5A13 is ^sn, Arg, Asp,
Ala, Ser, A14 is Gly, A
rg, Lys, Pro, Ala, Set,
Thr 5A21 is Ala, Lys, Ser
, Glu, A22 is Leu, Val,
A23 is Lys, Asn, ^2. is Gln,
Lys, Arg, GlySA2s is Gly,
Asp, Ser, A2@ is Asp,
Glu, Gin.

A21 is Gin, Arg, Pro, 5er
SAas is Ser, Thr.

Glu, Phe, Trp 5A21 is Pro,
Thr, Trp, Tyr.

^3o is Ser, Thr, Tyr, A
31 is Thr, Val, Phe.

When A32 represents either Phe or Tyr, excellent antitumor effects are exhibited.

The amino acid sequence that is the same as that contained in the so-called lymphotoxin precursor, and which exhibits the antitumor effect of lymphotoxin, is A, former sSA, and ■
1eSA3 is ety SA4 is Pr.

Or Val , ^, is Ser SAs is Lys , ^,
is leu, As is Trp, A, is Arg s
Ago is Ala and Act is Asp.

A, 2 is Arg, A+s is ^SnS A+s is G
ty 5A21 is Ala 5A22 is Leu 5A23
is Lys, ^3. is Gln.

A, S are Gty, A26 is Asp, 1A2t is Gln, 5A211 is Ser, ^2. is Pro,
A311 is Ser, ^, 1 is Thr.

^5. is Phe.

The NO3 side and C0O side of the amino acid sequence according to the present invention
Other amino acid sequences linked to the H side can be freely selected,
There are no particular restrictions. As an amino acid linked to the NO3 side,
For example, the following ■~0 can be mentioned.

■ Met ■ Met Lys ■ Met Thr Met lie Thr
Asn Ser AspLeu Met
Arg Arg Arg Lys Met
Thr Met Ile Thr Asn
Ser Pr.

Ala Gin Thr Ala Arg
Gin His Pr.

Lys Met His Leu Ala
His Ser ThrLeu Lys ■ Met Leu Pro Gly Val
Gly Leu TbrPro Ser
Ala Ala Gin Thr Ala
ArgGln His Pro Lys Me
t His Leu AlaHis Ser
Thr Leu LysNet Arg Net Thr Net 5et Net Pr.

Met Gln Met Ser Thr Leu Lys No Ser 'Thr Leu Lys The final target substance of the present invention can be produced by genetic recombination technology. Therefore, any cDN^ that encodes the recombinant lymphotoxin derivative of the present invention, and an expression plasmid that contains the cDN^ as a foreign gene and that has been continuously obtained so as to be able to control and express it in a selected host, are the present invention. They are intermediate substances necessary for the production of the final target substance of the invention, and can be integrated together as an invention in the sense that they solve the same problem. As a specific example of an expression plasmid, pEH
6084, p8H7084゜pt! H8084, pBR
Examples include plasmids identified by D8G5G and pBH5050, both of which are shown in the Examples below. In addition, a host transformed by an expression plasmid can also be integrated into the invention as well as a cDN^ and an expression plasmid, and E. coli or animal cells, especially cells for BH, are used as the host. As an example of transformed E. coli, JM83-pB
H6084, JM83-pH) 17084. JM83
-pBH8084 and RRI-pBRD8050 can be mentioned, and these are shown in the examples below, and have been deposited with the respective designations at FIKEN. Each accession number is FERM P-10041, FERM P-10041, corresponding to the above order.
-10040, P-10042, and P-10253.

Although the recombinant lymphotoxin derivative of the present invention, which is the final target substance, is a mutated version of natural lymphotoxin, it has the same level of activity as natural lymphotoxin, that is, antitumor activity, as shown by the experimental examples described later, and is superior to that of natural lymphotoxin. has. Therefore, just as natural lymphotoxin becomes an essential active ingredient of a pharmaceutical composition and is provided for medical purposes utilizing its activity, the recombinant lymphotoxin derivative of the present invention is also provided as a pharmaceutical for therapeutic purposes utilizing its activity. It can be an essential active ingredient of the composition.

As shown in the experimental examples below, the recombinant lymphotoxin derivative of the present invention was used, for example, in human lung cancer-derived cells (PC-10
) activity against natural lymphotoxin is 5% higher than that of natural lymphotoxin.
0 to 80 times larger.

In this case, the pharmaceutical composition is mainly an injection and is administered intravenously. In order to manufacture it as an injection, it may be carried out according to the conventional method for making a trace amount of physiologically active substance into an injection. Therefore, for example, the recombinant lymphotoxin derivative of the present invention may be made into an aqueous solution alone or together with appropriate excipients and solubilizers, sterile-filtered, filled, and lyophilized, and then an aqueous solution for dissolution may be added to prepare an injectable preparation for dissolution at the time of use. Bye.

Method The method for producing and measuring the substance of the present invention will be explained.

First, the lymphokine cDNA library necessary for producing the substance of the present invention is obtained by extracting mRNA from appropriate cells, screening for those that exhibit lymphotoxin activity in an expression system, reverse transcribing, and transforming Escherichia coli as an appropriate plasmid. Obtained (Nature, J Mue 7
21-724°1984 (Nature, 312.
721-724.1984)), for example, as shown in the Examples below (fractionating mRNA from Daudi cells,
The target mRNA was injected into African frog oocytes and the lymphotoxin active plate was measured in the culture supernatant.
A, the obtained mRN^ is reverse transcribed by a conventional method, a plasmid is prepared from the obtained DNA by a conventional method, and E. coli strain X1776 is transformed.

Furthermore, Daud i cells are human 8urkitt lym.
phoma and is commonly available as ATCCCCL 213 (Cancer Rear.
300-1310゜1968 (Cance Res,
28, 1300-1310.1968)).

Next, a plasmid containing cDNA encoding lymphotoxin is selected from the cDNA library by colony hybridization. Colony hybridization was performed using the method of WallaCe (Nucleic A
c1ds Res, 9.879.1981), the following DNA corresponding to the C-terminal 8 amino acid sequence of lymphotoxin is synthesized, purified, and used as a probe.

GTCTTCTTT GGA GCCTTCG
Plasmids may be prepared from CTG-positive colonies, their base sequences determined, and plasmids containing lymphotoxin cDNA selected.

Next, in order to obtain DNA encoding the recombinant lymphotoxin derivative of the present invention from the plasmid containing lymphotoxin cDNA, known genetic recombination operations may be carried out in an appropriate combination. In the present invention, site-di-rected mutagenesis of Zollerand Sm1th is particularly used since amino acid sequences at specific positions are removed and amino acids are substituted at specific positions.
sis) can be used.

That is, lymphotoxin cDNA is inserted into a phage vector such as M13, and the resulting double-stranded M
Single-stranded DNA may be prepared from a culture of Escherichia coli transformed with 13 DNA, and may be annealed with a predetermined synthetic oligonucleotide as a primer to induce mutations. After site-directed mutagenesis, E. coli is transformed with the resulting reaction solution, and the resulting plaques are screened by hybridization with radiolabeled synthetic oligonucleotides, which encode the target substance. A double-stranded M containing a predetermined cDNA as a foreign gene
13 phage DNA can be obtained. If necessary, a predetermined cDNA may be excised from the phage DNA and ligated to an appropriate expression vector used depending on the host, as appropriate, according to a conventional method.

The expression vector may be a commercially available expression vector, such as I) KK 233-2. pUc12. pUc
1g (7y Rumashi 7), PUG 130. P.I.G.
131. (Plasmid in which one polylinker site of M13tg130.131 is replaced with puc 12.13)
Alternatively, a special expression vector may be prepared and used by selecting and ligating the required promoter and terminator as shown below. That is, in order to express mammalian-derived genes in E. coli, yeast, or animal cells, a promoter is generally placed upstream of ATG (Met), which is a protein synthesis initiation signal.
Protein synthesis stop codon (TAA).

A terminator must be connected downstream of TG^, TAG). In E. coli, l is used as a promoter.
ac, trp, tac, trc, sac, P
L etc., but in yeast a-factor, TPl, s
uc, GAL, PGI, etc., and in animal cells, metallothionein, SV 40early 5LTR, etc. are commonly used. In particular, in order to express genes efficiently in E. coli, promoters, Shine-Dalgarno sequences (
SO sequence) and ATG must be connected accurately, and the number of bases from the SO sequence to ATG must be optimally selected. Taking these points into consideration, in the example described later, the trc promoter, SD sequence, and ATG were linked in this order as an E. coli expression vector, and the ATG was adjusted so that it was located 11 bases downstream from the SO sequence. I have prepared a structure in which Bgl ll5ite is inserted immediately before ATG, and am using this.

Since most of the individual genetic recombination operations used in the intermediate stages of the above manufacturing process are known, they will be explained below using literature or a brief description.

Cleavage of plasmids with restriction enzymes and separation, recovery, and ligation of DNA fragments obtained by cleavage by agarose gel electrophoresis or polyacrylamide gel electrophoresis are performed by experts.

Tee, etal 0. molekinira cloning, a
Laboratory Manual, Cold Spring Barber Laboratory 1982 (Maniatis).

T. et al., Molecular Clone
tng, A Laboratory Manual,
Co1d Spring Harbor Labora
(1982).

For example, cutting a plasmid and an M13 phage vector containing cDNA encoding a natural lymphokine,
Combine the fragments to create a double-stranded M13D cDNA.
It is applied when obtaining NA.

The transformation of plasmids into Escherichia coli is described in DNA Cloning Vol.
at, l, Chap, 5. ed, DlM, G.
lover IRL press 1985).

For example, it is applied when E. coli is transformed with a double-stranded M13 DNA into which a cDNA encoding a natural lymphokine has been integrated, or an expression plasmid into which a cDNA encoding a mutant lymphokine has been integrated.

Oligonucleotides for mutagenesis and screening were synthesized by the phosphoamidite method and labeled at the 5'' end using Nis, L, Beaucage, ethanol (0.0: Tetrahedron), etc.
22 1859. (1981) (S, L, Be
aucage, etal,.

Tetrahedron Lett,, 22185
9 (1981)) and Nie Maxam and Tahriya Kilbird, Methods in Enzymology,
Vol. 65, pp. 499-560 Academic Press 1980
(A.

Max and W, G11bert, Meth
ods in Bnzymology.

Vol, 65 p499-560 Academic
Press 1980).

Determination of DNA base sequence was performed by F., Sanger Etal 0
．． P, N, Knee, Varnish, 745463°197
7 (F, Sanger et al., P.N.
A, S 745463゜1977) dideox
This can be done using the y method.

For example, mutagenized and screened single chain M13
It is applied to determine the base sequence of DNA and confirm whether or not the desired mutation has been induced.

Double-stranded and single-stranded M13 phage []NA are prepared as follows.

That is, the transformed E. coli was subjected to
Methods in Enzymology 101, 20-781
983 (JoMessing, Method rnE
zymology 101.20-781983), followed by shaking culture and centrifugation.

Escherichia coli in the sedimentation area contained 0. Molecular Kroninck, Araporaatory Manual, Cold Spring Harbor Laboratory-198
2 (Maniatis, T. et al.

Molecular Cloning, A Labo
ratory Manual, ColdSpring
Double-stranded M13 DNA is obtained by the method described in (Harbor Laboratory, 1982). Also, from the supernatant liquid, that 1.3mj! 2.5 of 260 UE
Add M NaCl-20% PBG6000 mixed solution,
The mixture was centrifuged in an Etzpendorf microtube and
The resulting pellet was suspended in 120771 (10mM) Lis-HCl buffer (pH 7, 9, 0, 1mM BDTA), phenol extracted, ethanol precipitated, and 70%
After washing with ethanol and drying, 1QmM of 30iie
Tris-HCl buffer (pH 7, 9, 0, 1mM BDTA
) to obtain single-stranded M13 DNA.

Site-directed mutagenesis reactions are performed by Schiller and Smith, Mentz's In Enzymeology-100468-500,1
983 (Zoller and Sm1th, Met
hodsin Bnzymology, 10t)
468-500.1983).

That is, 2Qp mol of synthetic oligonucleotide for mutagenesis (brimer) terminal-labeled with non-radioactive phosphoric acid, 1 μg of original M13 DNA, annealing buffer (5QmM), lithium-hydrochloric acid, p) 18.0.0.25m
MMgC1,) 2μe, B buffer (0,2M) Lis-HCl, pH 7,5,0, IM MgCl□, 0.1M
dithiothreitol) 8/jf, dATP, dGT
P, dCTP, dTTP 2.5mM each mixture 4pe, 5mM rATP 2ttl, distilled water 1μ
g, 5 units of T* DNA ligase, and 5 units of DNA polymerase I Klenow fragment were added, and the final total volume was 20 μe, and the reaction was carried out at 14° C. for 3 hours.

Next, in order to transform E. coli with the mutagenized DNA using the reaction mixture, Kramer nital,...
Cell 38879-887.1984 (rameret
al,, Ce1l 38879-887.1984
) and further according to Grunstein and Hognes.

B, N, Knee, Nice, Knee Nishnee 723961
, 1975 (Grunstein and Hogn
ess, P, N, ^, S.

E. coli transformed according to USA 723961.1975) is mixed with E. coli JM 103 and spread on an agar medium, cultured overnight at 37° C., and the plaques formed are transferred onto a nitrocellulose filter.

Screening for plaques transferred onto nitrocellulose filters is performed as follows.

6x (7) SSC (1
1x SSC (150mM NaCl, 15mM sodium citrate), 10x Denhardt's (1x Denhardt's 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin), 50 μg It is added to sonicated salmon sperm DNA and treated at 65°C for 3 hours to form a preliminary hybrid. The filters were then mixed with radiolabeled synthetic oligonucleotides for mutagenesis under the same buffer conditions.
Leave to form a hybrid overnight at °C.

After washing the filter with 6x SSC and draining the water,
Overlay an intensifying screen and expose to X-ray film to select the desired plaque.

The cDNA encoding the mutant lymphokine was converted into a double-stranded M13
The expression plasmid obtained by cutting out 0NA and ligating it to an expression vector may be used for transformation depending on the host. For example, for the transformation of BHK cells, Reuter etal. 79422.1982 (Loyt
er et al., P.N.A.S.

USA, 79422.1982)
This may be done by the method of R et al. That is, the obtained expression plasmid is used together with, for example, pSV2-dhfr in auKs.
? Transform into G cell line (ts, tk-). In addition, p
For SV2-dhfr, Subramani, Etal:
Mol, Cell, Bio O, 1854-864. (
1981) (Sabramani.

et al: Mol, Ce11. Biol, 18
54-864. (1981)).

Cloning is done using the limiting dilution method.
utlon method) as follows.

Several days after transformation, 200 nM Amethopte
Dulbecco's modified Eagle's medium containing rin (Dulbeco's modified Eagle's medium, glucose 3.5%, sodium bicarbonate 7
．． 5%, fetal calf serum (FCS) 5%, tranexamic acid 3mM, glutamine 2mM, (+) -Ametho
A production strain is obtained by limiting dilution with pterin (200 nM) while replacing the medium every 2 to 3 days for 1 to 2 weeks. Next, the production strain was prepared in a roller bottle with 200 nM Amethop.
It is sufficient to inoculate terin-containing Double Beco modified Eagle medium 300d, culture at 37°C, replace the medium with the same amount of medium every day for 3 to 4 weeks from 3 to 4 days after the start of culture, and collect the culture supernatant.

In the Examples described later, Escherichia coli JW83 was used, and after culturing with shaking, the bacterial cells were crushed and the supernatant was collected.

Purification can be carried out by appropriately combining gel filtration, dye adsorption chromatography, high performance liquid chromatography, etc., and fractionating to obtain the required two components depending on lymphotoxin activity.

The lymphotoxin activity, that is, the antitumor activity of lymphotoxin, is determined as follows.

As an indicator cell, for example, mouse fibroblast cell line L
LP3P2O3, which is a sub-strain of cell and highly sensitive to lymphotoxin, was used, and the sample sample and actinomycin D at a final concentration of 1 μg/wdl were added to C(b).
Incubate for 12-18 hours at 37°C under air flow. Here, the medium used is RPMI 1640 containing 10% inactivated fetal serum. After culturing, dead cells are washed and removed, and living cells remaining attached are fixed and stained with 0.5% crystal violet (methanol:water=1=4) solution. The dye is extracted from the stained cells, and its absorbance value is measured to determine the number of living cells. The titer is expressed by the sample dilution when giving LP3P2O30% 1ysis. Note that this value changes depending on the LP3P2O3 growth phase when the sample is added, so it is necessary to always use a lymphotoxin standard titer sample as a control and make corrections.

When performing the above assay using logarithmically growing LP3 cells passaged in serum-free medium (DM-153 medium),
The active concentration of LT that causes 50% lysis after 4 hours is defined as 10/ml.

Tests for cytotoxicity and growth inhibition of LT against human malignant tumor cell lines are conducted as follows.

Various human cancer cell lines were added to a 95-well microtiter plate at a density of 0.5 to 1 x 1 Q' well.
After late incubation, add the medium containing the LT sample. Then about -
Culture in humidified, CO□ presence for weeks. Viable cells are determined by extraction of Crystal Violet in the same manner as described above.

Alternatively, 0.1 μg/ml actinomycin D
It is also possible to coexist and say ^5.

In that case, add the cell line at a density of 2-5 x 10'/well, add the LT sample and actinomycin D, and determine the number of viable cells in 12-15 hours.

Specific activity is estimated by enzyme immunoassay (BIA) as follows. Using a rabbit anti-human LT antibody obtained by immunizing LT, or a monoclonal anti-human LT antibody produced from a hybridoma obtained by cell fusion method from the tile cells of an immunized mouse, the amount of LTO in the sample was determined by the Sand-Deutsch method. Estimate. That is, after adsorbing an anti-human LT antibody (monoclonal or polyclonal) to a plastic well, LT is added and bound. Furthermore, a monoclonal or polyclonal antibody having a different recognition site from the primary antibody is added.

This secondary antibody is used to which biotin has been bound in advance. Thereby, when avidin bound to horseradish peroxidase is subsequently added, peroxidase is immobilized on the well in proportion to the amount of secondary antibody bound to LT due to the specific reaction between avidin and biotin of the secondary antibody. If a substrate such as orthophenylenediamine and hydrogen peroxide is added here, peroxidase will quantitatively develop a color, and the LT concentration in the sample can be estimated from the absorbance. That is, it is sufficient to compare the BIA value of a sample containing an unknown amount of LT with that of LT whose content concentration is known and which has been purified in advance and determined by amino acid analysis.

Further, by examining the antitumor activity of the former as described above using L-P3 cells as an indicator, its specific activity can be determined.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Preparation of mRNA Daudi cells 2 XIO' cells were treated with 4M guanidinium thiocyanate), 0.5% N-lauroylsarcosine sodium, 25mM sodium citrate pH 7.0.0
, 1M mercaptoethanol, 0.1% antiform (
Sigma) solution was suspended in 10 ml and homogenized using a polytron. This homogenate is 0, IM BD
It was layered on a 5.7 M cesium chloride aqueous solution containing TA, and centrifuged at 2600 Orpm at 20° C. for 23 hours using an ultracentrifuge (SRP28A rotor Hitachi). The precipitated RNA was dissolved in 10mM) lithium-hydrochloric acid pH 7,410-
After dissolving in the solution, add 2.5 times the volume of ethanol and heat at -70°C.
It was left for 1 hour. Next, the RNA precipitated by centrifugation
(10mM) was dissolved in 4141nl of lithium-hydrochloric acid (pH 7), and 21nl of 4M potassium chloride was further added. This RN
Solution A was transferred to an oligo(T) cellulose column (φ2x15c
After washing thoroughly with 0.5M potassium chloride and 10mM lithium-hydrochloric acid p)17.4, the mRNA was eluted with a 10mM lithium-hydrochloric acid pH 7.4 aqueous solution.

Purification of mRNA 500 μg of mRNA from Audi I cells was prepared at 50 mM.
Tris) IcI pH 7,4, 0,2M NaCl, 1
10% to 28% 5ucros with mM IEDTA
e layered on 15 ml of the solution in which a concentration gradient was formed, and centrifuged at 2600 m using an ultracentrifuge (SRP28A rotor, Hitachi).
Centrifuged at 20°C for 3 hours at Orpm. Then, centrifuged at 500μ
The mixture was fractionated by ε and ethanol was added to each fraction to precipitate mRNA. Each fractionated mRNA was dissolved in sterile water and adjusted to an mRNA concentration of 1 μg/μ2.

Screening for mRNA: Female African frog frogs (over 1 year old) were given 200 u of serum gonadotropin (Beamex: Sankyo).
Injected. - After a few hours, remove the oocytes and add modi
fied barth 5aline medium (
MBS). Next, each fraction of the previous mRNA 50
~1100n/egg was injected into eggs and 1mM phen
yl meth-yl 5ulfonyl fluor
ide (PMSF) in MBS at 25 °C, 36
Cultured for hr. After removing the supernatant, oocytes were homogenized and extracted with MBS containing 1mM PMSF to extract mRNA.
Samples were taken for each portion of the sample. Lymphotoxin activity was measured for each sample, and mRNA showing lymphotoxin activity was determined.
Both parts were screened.

Creation of cDNA library 5μ of the mRNA fraction that showed lymphotoxin activity in an mRNA expression system using African frog oocytes
pcD was dissolved in 88.37μe of 5mM TrisHCl
Add 1.4 μg of VI DNA (Pharmacia),
The reaction was carried out under the following conditions. 50mM TrisHCl
pH8, 3, 8mM MgCl□, 30mM KCl,
Q, 3mM DTT, 2mM dNTP, rever
setranscrlptease51, reaction liquid volume 20
μe0 After 60 minutes of reaction at 37°C, 0.25M EDT
The reaction was stopped by adding 2 μi of A. After phenol chloroform treatment, 4M NH, OAc 20μm,
80μ2 of 1EtOH was added, left to stand at -70°C, and then centrifuged to remove the supernatant, and the same operation was repeated. 75
%EtO) After washing the precipitate with 1 water and drying, 1QmM
sodium cacodylate, 2.5mM H
3PB5 pH 7.6, 0.1mM dCTP.

poly(A) 0.2 pg/p (l) reaction solution, terminal deoxynucleotide
100 l transferase (TdT) was added to make the final volume to 20 μl. After reacting at 37° C. for 10 minutes, 1 μe of 0.25 M BDTA was added to stop the reaction, and post-treatment was performed according to a conventional method.

The resulting DNA precipitate was mixed with 8mM MgClz, 6mM
Mme Jl/captoethanol, )lindIIllo
It was dissolved in 11μ2 of a 10mM Tris HCI pH 7.4 solution containing U, and reacted at 37°C for 2 hours. 0.
After adding 1 μl of 25M BDTA to stop the reaction, post-treatment was performed according to a conventional method, and 10mM TrisHCI p
) 17.4.1mM EDTA solution 1ipe and Bt
O) It was dissolved in a mixed solution of 13/Jf.

Next, 77 ng of pL-IDNA (Pharmacia) was added to 11 μ2 of the DNA-containing solution and diluted with 10 mM TrisHCl.
pH7, 4, 1mM EDTA, 0.1M NaC
One solution was 120 pe. After leaving it at 65℃ for 5 minutes and at 42℃ for 30 minutes, add the following ingredients under water cooling and reduce the liquid volume to 1.
．． It was set to 2mM. 20mM TrisHCI p)! 7
．． 5, 4mM MgClg, 10mM (NHa)
2sO*, 0.1M KCl, 0.1mM β-NA0
．． 5OA1g/m1Bovine SerumAlbu
min, Il! , coli ligase 1g
g After overnight reaction at 12°C, 4mM dNTP 1
0. c+L20mM β-NAD 10pe, B, co
li Iigase l pg, B.

coli DNA polymerase I 1
After adding 7.5[1, RNase H1200 and carrying out lhr reaction at 12°C and further lhr reaction at 25°C, this solution was used to transform ε, coli χ1776 strain.

That is, ε, coli z 1776 strain z1776
medium (Maniatis et al, M
After culturing at 37°C during ocular cloning until the absorbance (550 nm) reached 0.2, the bacteria were collected and made competent using the method of Maniatis et al.
Adjusted the cell. This competent cell
After adding the previous DNA to l, E, col i Z 17
A cDNA library was created by transforming 76 strains.

Screening of cDNA In order to select a plasmid containing cDNA encoding lymphotoxin from the cDNA library obtained in the previous section, DNA corresponding to the C-terminal 8 amino acids of lymphotoxin was synthesized and colony hybridization was performed using this as a probe. went.

The structure of the synthetic probe is GTCTTCTTT GGA G
CCTTCGCT CTG, which was synthesized using an automatic solid phase synthesizer (Applied Biosystems),
Purified by LC. Hybridization is Wal
The method of Nucleic Ac1ds Re
s, 9879, 1981). cDN
Of the approximately 10,000 A library, 60 were positive colonies, and the plasmids of 3 of them were adjusted.
When the base sequence was determined, one of the bases was found to be lymphotoxin cD.
It was a plasmid containing N^. The base sequence of the amino acid coding region of the obtained lymphotoxin cDNA is shown in FIG.

Construction of expression vector The plasmid containing the lymphotoxin cDNA obtained in the previous section was digested with BamHl, and the approximately 1.1 kbp fragment containing the lymphotoxin C-NA was transformed into p[lG131 (pt
The poly llnker part of lc13 (77 Luasia) is the poly ll of M13tg131 (amajam)
BamHI of the plasmid that replaced the nker part)
After inserting into 5ite (plasmid number pMLT200
0), digested with Hincff and BaII to approximately 11
A DNA fragment of Kbp was obtained. Next, pKK23
3-2 (Pharmacia) Nc.

After 1.n digestion, the vector was treated with Klenow enzyme. IKbp fragment was inserted (plasmid number pMLT2100), pKK233-2 was tr
c promoter (improved version of tac promoter)
, and its expression can be induced by IPTG. Then p
HLT2100 was digested with BcoRl and old ndrfl, and the approximately 1.5 kbp fragment containing lymphotoxin cDNA was inserted into A113mp10 double-stranded DNA (Pharmacia).

Next, transfect ε, coli JM103.
After tion I, the double-stranded DNA was prepared according to a conventional method, and inoculated using the synthetic oligonucleotide shown below.
vitr.

mutagenes Is was performed.

Synthesis ON＾: GCT TGCTGG GAT Cl
1l'A TGA GAT ["TGTTT CCT Site-specific mutagenesis reaction is basically performed using the method of Zoller et al. (Method in Bnzymology).
100468.1983). That is,
20 pmol of the synthetic oligonucleotide end-labeled with non-radioactive phosphoric acid, 1 pg of template DNA (-wood-like DNA), 5x annealing of 2/j/
buffer (50mM TrisHCl, p)1
8.0.

25mM MgClg), 8pl B buffer
(0,2M TrisflC1 pH7,5,0,1M
MgCl2. 0.1M DTT), 2 of 4pe
．． 5mM dNTP, 5mM ATPll at 2 pH
pe distilled water, 5υ T4 DNA ligase,
51 mouth NA polymerase IKle
Now fragment was added to bring the total amount to 20 pg.

After a 3-hr reaction at 14°C, B. coli JM
103 was transformed (in Ramer etc.,
Ce1l 38879.1984>.

The transformed bacteria were spread on an agar medium mixed with E. coli JM103 and cultured overnight at 37°C, and the formed plaques were transferred onto nitrocellulose (Grunste
in et al PNAS 723961.1975
). Next, a hybridization test was performed with a synthetic oligonucleotide labeled with 32p to select phage plaques in which mutations had been induced. Double-stranded DNA was prepared from the phage plaques selected as described above, and Bco
The DNA fragment obtained by digestion with Rl and old ndIII was digested with fEcoRl and Hind of pKK233-2.
Plasmid number pMLT3020) is inserted into the plasmid prepared by Ill digestion.

pMLT3020 is 5hine of trc promoter
-A 11 nucleotides downstream from the Dargaro sequence
Protein synthesis starts from TG, and any gene can be inserted downstream of the promoter by BgI n digestion, and by BamHI digestion, the start codon ATG
This is an expression vector that allows genes to be inserted downstream of the vector.

pMLT3020 encodes a polypeptide in which 7 amino acid residues are deleted from the N1 (2nd terminus) of the polypeptide of the present invention, and the ON^ sequence around the NH2 terminus is as follows.

A diagram of the construction process of pMLT3020 is shown in FIG. 2.

pMLT3020 was digested with Bam) 11 and old ndnI, and a DNA fragment of about 500 bp was recovered by agarose gel electrophoresis. In addition, the oligonucleotides shown below were synthesized and polynucleotide kinase (NEB
), after phosphorylating the 5” end with ATP, the previous 50
Bc pUG130 with 0bp DNA fragment
It was enzymatically ligated with the vector obtained by digestion with oRl and old ndI[n (plasmid number ptlG5001).

5° ^^TTCAGATCTCATGAAA
3'NLT-53° GTCTAGAGTACT
TTGGGCGC5°NLT-65° CCCGCGG
CTCATCTGAT[:GGG 3°NLT-1
3° CGAGTAGACTAGCCCCTAG
After stopping the 5' NLT-2 reaction, E, col was added according to a conventional method.
i JM83 was transformed and white colonies were selected on an agar plate containing X-Ga1. Next, plasmid pUG
After digesting 5001 with Bgl■ and old ndIII,
Approximately 5oobp DNA fragment was recovered by agarose electrophoresis, and approximately 4.13kbp DNA was obtained by digesting pMLT3020 with Bgl II and HindIII.
A and enzymatically linked together (plasmid number pML
T5001^).

The construction process diagram is shown in Figure 3.

Plasmid pUc12 was digested with BamHl and Pst I to obtain an approximately 2.8 kbp DNA fragment, which was 5′
Plasmid pUc7692 was obtained by enzymatically ligating the following synthetic NA with phosphorylated ends.

BamHI Acc m
5' GATCCGTCTACTCCCCAGGTGGT
TTTCT 3゜3' GCAGATG
AGGGTCCCACCAAAAGAGGGCC5 “Acc
m xho15°C
CGGAAAAGCTTACTCTCCGAACGCT
ACC3'3° TTTTCGAATGAGAGGC
TTGr:GATGGAGCT 5'xhol
Pst I5' TC
GAGCCCGCTGTACCTGGCTCACGAG
GTCCTGCA 3”3”CGGGCGACA
TGGACCGAGTGCTCCAGG 5゜plasmid pUc7692 with A″ccI and Ava ■
After digestion with 7% polyacrylamide electrophoresis, approximately 100
bp DNA fragment was recovered and plasmid pML
T5001^ was digested with BcoRl and Acc I, Av
a If and HindI [[about 45 ml each obtained by digestion]
0bp, approx. 300bp low NA75 component and plug
Mi) 'pLIG130, BcoR1 and Hindln
It was enzymatically ligated with the approximately 2.3 kbp fragment obtained by digestion (plasmid pε85123).

The construction process diagram is shown in Figure 4.

After cutting plasmid pEH5123 with restriction enzymes Acc ffl and Xho 1, Bacterial A'
The 5° terminal dephosphorylation group was removed using 1kalin Phosphatase. Next, an approximately 3.5 kbp fragment was recovered by 0.8% agarose gel electrophoresis and enzymatically ligated with the following synthetic NA phosphorylated at the 5° end (plasmid number pE86084).

5° CCGGAGCTACC3' 3° TCG^TGGAGCT 5'' construction process diagram is shown in FIG.

Plasmid pEH6084 was digested with restriction enzymes EcoR1 and B.
An approximately 300 bp fragment obtained by cutting with gl II and an approximately 500 bp fragment obtained by cutting with 5acll and old ndIII.
The bp fragment and the following synthetic NA phosphorylated at the 5° end were enzymatically inserted into the EcoRl and HindI sites of plasmid pUG130 (plasmid number pBH7084).

5” GATCTCATGAGCACCCTCAAAC
CCGC3゜3“ ＾GTACTCGTGGGAG
A diagram of the construction process of TTTGGG 5° is shown in FIG.

Example 3 An approximately 500 bp DNA fragment obtained by digesting pMLT3020 with [3amHI and old ndI[[] was digested with 5°
The following synthetic oligonucleotide with a phosphorylated end and a vector obtained by digesting pUG130 with BcoRl and old ndlII were enzymatically ligated (plasmid number p
HG5004).

The construction process diagram is shown in Figure 5.

5° AATTCAGATCTCCATGCGTCGTC
GTAAA 3°NLT-3 3° GTCTAGAGTACGCAGCAGCA
TTTGGGCGf 5゜NLT-4 5” CCCGCGGCTCATCTGATCGGG
3'NLT-13" CGAGTAGA
CTAGCCCCTAG 5' Approximately 540 bp DNA fragment obtained by digesting NLT-2 plasmid puGsoo4 with Sac II and former ndIII Te, 5"
The following synthetic oligonucleotides with phosphorylated ends, plasmid p[JG130, BcoRl and former ndII
The approximately 2.3 kbp DNA fragment obtained by digestion with I was enzymatically ligated (plasmid number pUG5011
).

Next, plasmid pUG5011 was transformed into BglII and H
A DNA fragment of about 570 bp obtained by digestion with ind■ and a DNA of about 4.6 khp obtained by digesting plasmid pMLT3020 with BglII and )lindII
To enzymatically ligate the A fragment, use plasmid number pMLT5011).

The construction process diagram is shown in Figure 6.

5° AATTCAGATCTCCATGCCCGC3'
NLT-153° GTCTAGAGTACGGG
5'NLT-16 plasmid pE! 87084
After cutting with restriction enzymes AccI and Hind■, 1
Approximately 450 bp of DNA was obtained by % agarose gel electrophoresis.
The fragments were separated.

Similarly, plasmid pMLT5011 was added with restriction enzyme BcoR.
The approximately 450 bp DNA fragment obtained by cutting with 1 and Accl and the fragment were combined into plasmid pUG13.
0 (plasmid number pBH8084).

The construction process diagram is shown in Figure 9.

Example 4 Plasmid pH) 16084 with BcoRl and PstI
pTT50, an approximately 580 bp DNA fragment obtained by cutting with
Approximately 1700 obtained by cutting 01 with Pstl and Pvul
bp DNA fragment, pBR322d-rop. co
Approximately 3100hp DN obtained by cutting with R1 and pvul
The three A fragments were enzymatically ligated (plasmid number pB
R06084).

Next, an approximately 1100 bp DNA fragment obtained by cutting pBRD6084 with BcoRl, Accl, and pvul, and an approximately 450 bp DNA fragment obtained by cutting PBH8037 with BCORI and ACCI, pBR322d-rop.
About 3100b obtained by cutting with 8coR1 and pvul
The three p DNA fragments were enzymatically ligated (plasmid number pBRD8050).

The construction process diagram is shown in Figure 10.

Plasmid pB87084 was digested with restriction enzymes ^ccl and old n
A DNA fragment of about 300 bp was obtained by cutting with dl[[.

In addition, plasmid pBH5037 was used as BcoRl and Acc.
A DNA fragment of about 450 bp was obtained by cutting with
E! of plasmid pUG130 enzymatically together with the A fragment. c.
oR1,)IindII [plasmid number pB) 15050].

The construction process diagram is shown in Figure 11.

Example 6 E. coli wa transformed with plasmid pE)16084
Iio was inoculated into LB medium 201 containing 100 mg/l ampicillin, and cultured with stirring at 37°C for 20 hours.

Next, the bacterial cells were collected by centrifugation, washed with PBS, and then
ld in PBS. After crushing this bacterial body fluid with an ultrasonic generator, centrifugation (Tomy Seiko: RB20BH,
15,000 revolutions for 30 minutes) to make bacterial cell extract 125
m1! I got it. The lymphotoxin activity of this crude extract was 4
．． 4X10"U/-. Next, 0.2 MIJ
5e, a gel filtration carrier equilibrated in acid buffered wave buffer.
phacryl S-200 (Pharmacia) φ113
It was attached to X 900mm.

After collecting both fractions showing lymphotoxin activity, a portion was applied to anti-lymphotoxin mouse monoclonal antibody column 30-. The antibody column is a monoclonal antibody created by immunizing mice with recombinant lymphotoxin.
se 4B (Pharmacia). The same column was sufficiently equilibrated with 0.2M phosphate buffer, and after adsorbing the previously partially purified lymphotoxin derivative, it was washed with the same buffer and added with 0.2MIJ phosphate buffer containing 3M KSCN. . Next 5ephadex G-25
(Pharmacia) and purity assay by 5O8-polyacrylamide electrophoresis, it was found that the molecular weight was 1.
There were 6,000 single bands. In addition, when the NH terminal sequence was examined, the structure was Met Lys Pr.

Ala Ala)is Leu lie Gly
It was Asp.

Example 7 E. coli JM8 transformed with plasmid pB87084
LB medium 2 containing ampicillin 100 mg/12
00ml and cultured with shaking at 37°C for 18 hours.

Next, the bacterial cells were collected by centrifugation and dissolved in PBS (Dalbecc
After washing with o), 2ml! of PBS. This bacterial cell fluid was disrupted using an ultrasonic generator, and then centrifuged to obtain a bacterial cell extract. This crude extract was sterile filtered (0.22μm
), the filtrate was applied to an anti-lymphotoxin mouse monoclonal antibody column 2Wdl.

The column was sufficiently equilibrated with 5QmM Tris HCI pH 8.0 buffer, and after the lymphotoxin derivative was adsorbed, it was washed with the same buffer. Next sin KSCN
Elution was performed with 50mM Tris HCI pH 8,0 buffer containing. The eluate was immediately diluted with PBS (Dulbecc
o) Dialyzed into buffer.

Example 8 E. coli JM8 transformed with plasmid pEH8084
3 in LB medium containing 100 mg/1 ampicillin 20
The cells were inoculated into 0ml and cultured with shaking at 37°C for 18 hours.

Next, the bacterial cells were collected by centrifugation and dissolved in PBS (Dulbecc
After washing with o), the suspension was suspended in two volumes of PBS. This bacterial cell fluid was disrupted using an ultrasonic generator, and then centrifuged to obtain a bacterial cell extract. The lymphotoxin activity of this crude extract is 6.6
It was 10'U/ml. Next, the same extract was sterile filtered (0.22 μm), and then anti-lymphotoxin mouse monoclonal antibody A2m7! It was attached to. It was eluted in the same manner as above and dialyzed against PBS to give 2.9 x 1
A 2Wdl solution containing 0'U/1nl of activity was obtained. 5O
8-Polyacrylamide electrophoresis shows a molecular weight of 16.0.
gave a single band of 00 and the NH2-terminal sequence was Pro A
la Ala His Leu Ile Gly As
It was p.

〔Effect of the invention〕

The present invention will be explained in detail using experimental examples.

Experimental Example 1 Sample Plasmid pB86084 was expressed and purified to obtain the final target substance A, which was used as a test sample.

Natural lymphotoxin was separately prepared as a control sample.

Method> Human lung cancer cells (PC-10) were used as indicator cells,
Human colon adenocarcinoma cells (WiDr) and human fibrosarcoma cells (
HT1080) was used. 2-5 x 10 each cell line
'/Wall in a 96-Wall microtiter plate at a density of 96-Wall microtiter plates containing each sample and actinomycin D (0.1 μg/m for PC-10 and 0.5 pg/m for H71080). The cells were cultured for 12 to 15 hours in the presence of humid CO□.
The living cells were fixed and stained with crystal violet, the dye was extracted from the stained cells, and its absorbance value was measured. The survival rate by sample addition was determined by setting the survival value before sample addition as 100%.

Results> The results are shown in FIGS. 12 to 14. Figures 12 to 14 show PC-IQ, WiDr, and PC-IQ as indicator cells, respectively.
These are the results when using HT1080, and in the figure, Δ
A graph showing the relationship between lymphotoxin activity and survival rate when the marked line is natural lymphotoxin and the O marked line is target substance A, respectively.

From FIGS. 12 to 14, it is known that the 137 amino acid sequence shown in the present invention has antitumor activity of lymphotoxin.

Experimental Example 2 Sample> Plasmid pE) 16084. pEH7084, pH
1H8084 was expressed and purified, and the final target substance B.

C and D were obtained and used as specimen samples. Natural lymphotoxin was separately prepared as a control sample.

Method> Human lung cancer cells (PC-10) as indicator cells,
Human colon adenocarcinoma cells (WiDr) and human fibrosarcoma cells (
HT1080) was used. 2-5 x 10 each cell line
'/well of the culture medium containing each sample and actinomycin D (0.1 μg/rn11wi in the case of PC-10).
In the case of leaf and HT1080, 01.5 μg/−) was added. The cells were cultured for 20 hours in the presence of humid CO2, and the surviving cells were fixed and stained with crystal violet, and the dye was extracted from the stained cells and its absorbance value was measured. The survival rate by sample addition was determined by setting the survival value before sample addition as 100%.

Results> The results are shown in Figure 15. (a), (b), (
C) shows the results when PC-10°WiDr and HT1080 were used as indicator cells, respectively. In the figure, the lines marked with *, the line marked with Δ, the landmark line, and the marked line represent natural lymphotoxin, target substances B and C, respectively.

It is a graph showing the relationship between lymphotoxin activity and survival rate when D is used as a sample.

From FIGS. 15(a), (6), and (C), it is known that the 137 amino acid sequence shown in the present invention has antitumor activity of lymphotoxin.

Experimental Example 3 The activity of samples B, C, and D of Experimental Example 2 against mouse L-P3 cells was determined by the enzyme immunoassay method, and the activity of natural lymphotoxin (0.5 to I
The biological activity of each sample was calculated using "U/mg protein" as a standard.

1ndex Natural Lymphotoxin 1.0 B7.3 05.6 D6.2 From the above results, it is known that the activity of the lymphotoxin derivative of the present invention is 5 to 8 times greater than that of natural lymphotoxin.

[Brief explanation of the drawing]

FIG. 1 is a diagram of the base sequence of the amino acid coding region of lymphotoxin cDNA. FIG. 2 is a diagram of the construction process of pMLT3020. FIG. 3 is a diagram showing the construction steps of p[lG5001 and pMLT5001A. FIG. 4 is a diagram of the construction process of pEH5123. FIG. 5 is a diagram of the construction process of pUG5004. FIG. 6 is a diagram of the construction process of pMLT5011. FIG. 7 is a diagram of the construction process of J) BH6084. FIG. 8 is a diagram showing the construction process of plEH7084. FIG. 9 is a diagram of the construction process of pBH8084. FIG. 10 is a diagram of the construction process of pBRD8050. FIG. 11 is a diagram of the construction process of pEH5050. 12, 13, and 14 are graphs showing the results of Experimental Example 1, and FIG. 15 is a graph showing the results of Experimental Example 2.

Claims (1)

[Scope of Claims] A novel recombinant lymphotoxin derivative containing an amino acid sequence represented by the following primary structural formula in one molecule. [There is a gene sequence] (In the formula, A_1_～_1_4, A_2_1_～_3_2
represents any amino acid) 2 A_1 is His, Asn, Leu, A_2 is Ile
, Met, Gly, A_3 is Leu, Gln, His,
Cys, Ile, Pro, Val, Ser, G1y, A
_4 is Pro, Arg, Leu, Gln, Val, A_
5 is Ser, Phe, Cys, A_6 is Lys, Asn
, Gln, Glu, Asp, A_7 is Leu, Ala,
Gln, A_8 is Trp, Phe, Leu, A_9 is A
rg, Leu, A_1_0 are Ala, Arg, Glu,
A_1_1 is Asp, Asn, Ala, Arg, A_1
_2 is Arg, Asn, Gly, A_1_3 is Asn,
Arg, Asp, Ala, Ser, A_1_4 is Gly
, Arg, Lys, Pro, Ala, Ser, Thr,
A_2_1 is Ala, Lys, Ser, Glu, A_2
_2 is Leu, Val, A_2_3 is Lys, Asn,
A_2_4 is Gln, Lys, Arg, Gly, A_2
_5 is Gly, Asp, Ser, A_2_6 is Asp,
Glu, Gln, A_2_7 is Gln, Arg, Pro
, Ser, A_2_8 is Ser, Thr, Glu, Ph
e, Trp, A_2_9 is Pro, Thr, Trp, T
yr, A_3_0 is Ser, Thr, Tyr, A_3_
1 is Thr, Val, Phe, A_3_2 is Phe, T
2. The recombinant lymphotoxin derivative according to claim 1, which represents each of yr. 3 A_1 is His, A_2 is Ile, A_3 is Gly
, A_4 is Pro or Val, A_5 is Ser, A_6
is Lys, A_7 is Leu, A_8 is Trp, A_9 is Arg, A_1_0 is Ala, A_1_1 is Asp, A
_1_2 is Arg, A_1_3 is Asn, A_1_4 is Gly, A_2_1 is Ala, A_2_2 is Leu, A
_2_3 is Lys, A_2_4 is Gln, A_2_5 is Gly, A_2_6 is Asp, A_2_7 is Gln, A
The recombinant lymphotoxin derivative according to claim 1 or 2, wherein _2_8 is Ser, A_2_9 is Pro, A_3_0 is Ser, A_3_1 is Thr, and A_3_2 is Phe. 4. The recombinant lymphotoxin derivative according to any one of claims 1 to 3, wherein the polypeptide linked to the NH_2 terminus of the amino acid sequence consists of 1 to 28 amino acid residues. 5 cDN encoding a recombinant lymphotoxin derivative containing the amino acid sequence shown by the following primary structural formula in its molecule
A. [There is a gene sequence] (In the formula, A_1_～_1_4, A_2_1_～_3_2
represents any amino acid) 6 A_1 is His, Asn, Leu, A_2 is Ile
, Met, Gly, A_3 is Leu, Gln, His,
Cys, Ile, Pro, Val, Ser, Gly, A
_4 is Pro, Arg, Leu, Gln, Val, A_
5 is Ser, Phe, Cys, A_6 is Lys, Asn
, Gln, Glu, Asp, A_7 is Leu, Ala,
Gln, A_8 is Trp, Phe, Leu, A_9 is A
rg, Leu, A_1_0 are Ala, Arg, Glu,
A_1_1 is Asp, Asn, Ala, Arg, A_1
_2 is Arg, Asn, Gly, A_1_3 is Asn,
Arg, Asp, Ala, Ser, A_1_4 is Gly
, Arg, Lys, Pro, Ala, Ser, Thr,
A_2_1 is Ala, Lys, Ser, Glu, A_2
_2 is Leu, Val, A_2_3 is Lys, Asn,
A_2_4 is Gln, Lys, Arg, Gly, A_2
_5 is Gly, Asp, Ser, A_2_6 is Asp,
Glu, Gln, A_2_7 is Gln, Arg, Pro
, Ser, A_2_8 is Ser, Thr, Glu, Ph
e, Trp, A_2_9 is Pro, Thr, Trp, T
yr, A_3_0 is Ser, Thr, Tyr, A_3_
1 is Thr, Val, Phe, A_3_2 is Phe, T
cDNA according to claim 5, each representing any one of yr.
. 7 A_1 is His, A_2 is Ile, A3 is Gly,
A_4 is Pro or Val, A_5 is Ser, A_6 is Lys, A_7 is Leu, A_8 is Trp, A_9 is A
rg, A_1_0 is Ala, A_1_1 is Asp, A_
1_2 is Arg, A_1_3 is Asn, A_1_4 is G
ly, A_2_1 is Ala, A_2_2 is Leu, A_
2_3 is Lys, A_2_4 is Gln, A_2_5 is G
ly, A_2_6 is Asp, A_2_7 is Gln, A_
2_8 is Ser, A_2_9 is Pro, A_3_0 is S
The cDNA according to claim 5 or 6, wherein er, A_3_1 is Thr, and A_3_2 is Phe. 8. The cDNA according to any one of claims 5 to 7, wherein the polypeptide linked to the NH_2 terminus of the amino acid sequence consists of 1 to 28 amino acid residues. 9 cDN encoding a recombinant lymphotoxin derivative containing the amino acid sequence shown by the following primary structural formula in its molecule
An expression plasmid obtained by containing A as a foreign gene and ligating it so that it can be controlled and expressed in a selected host. [There is a gene sequence] (In the formula, A_1_～_1_4, A_2_1_～_3_2
represents any amino acid) 10 A_1 is His, Asn, Leu, A_2 is Il
e, Met, Gly, A_3 is Leu, Gln, His
,Cys,Ile,Pro,Val,Ser,Gly,
A_4 is Pro, Arg, Leu, Gln, Val, A
_5 is Ser, Phe, Cys, A_6 is Lys, As
n, Gln, Glu, Asp, A_7 is Leu, Ala
, Gln, A_8 is Trp, Phe, Leu, A_9 is Arg, Leu, A_1_0 is Ala, Arg, Glu
, A_1_1 is Asp, Asn, Ala, Arg, A_
1_2 is Arg, Asn, Gly, A_1_3 is Asn
, Arg, Asp, Ala, Ser, A_1_4 is Gl
y, Arg, Lys, Pro, Ala, Ser, Thr
, A_2_1 is Ala, Lys, Ser, Glu, A_
2_2 is Leu, Val, A_2_3 is Lys, Asn
, A_2_4 is Gln, Lys, Arg, Gly, A_
2_5 is Gly, Asp, Ser, A_2_6 is Asp
, Glu, Gln, A_2_7 is Gln, Arg, Pr
o, Ser, A_2_8 is Ser, Thr, Glu, P
he, Trp, A_2_9 is Pro, Thr, Trp,
Tyr, A_3_0 is Ser, Thr, Tyr, A_3
_1 is Thr, Val, Phe, A_3_2 is Phe,
10. An expression plasmid according to claim 9, each of which represents Tyr. 11 A_1 is His, A_2 is Ile, A_3 is Gl
y, A_4 is Pro or Val, A_5 is Ser, A_
6 is Lys, A_7 is Leu, A_8 is Trp, A_9
is Arg, A_1_0 is Ala, A_1_1 is Asp,
A_1_2 is Arg, A_1_3 is Asn, A_1_4
is Gly, A_2_1 is Ala, A_2_2 is Leu,
A_2_3 is Lys, A_2_4 is Gln, A_2_5
is Gly, A_2_6 is Asp, A_2_7 is Gln,
A_2_8 is Ser, A_2_9 is Pro, A_3_0
The expression plasmid according to claim 9 or 10, wherein is Ser, A_3_1 is Thr, and A_3_2 is Phe. 12. The expression plasmid according to any one of claims 9 to 11, wherein the polypeptide linked to the NH_2 terminus of the amino acid sequence consists of 1 to 28 amino acid residues. 13. The expression plasmid of claim 9, wherein the expression plasmid is identified by: pEH6084, pEH7084, pEH8084, p
BRD8050, pEH5050 14 cD encoding a recombinant lymphotoxin derivative whose molecule contains the amino acid sequence shown by the primary structural formula below.
A host that is transformed with an expression plasmid containing NA as a foreign gene and ligated to enable control and expression within the selected host. [There is a gene sequence] (In the formula, A_1_～_1_4, A_2_1_～_3_2
represents any amino acid) 15 A_1 is His, Asn, Leu, A_2 is Il
e, Met, Gly, A_2 is Leu, Gln, His
,Cys,Ile,Pro,Val,Ser,Gly,
A_4 is Pro, Arg, Leu, Gln, Val, A
_5 is Ser, Phe, Cys, A_6 is Lys, As
n, Gln, Glu, Asp, A_7 is Leu, Ala
, Gln, A_8 is Trp, Phe, Leu, A_9 is Arg, Leu, A_1_0 is Ala, Arg, Glu
, A_1_1 is Asp, Asn, Ala, Arg, A_
1_2 is Arg, Asn, Gly, A_1_3 is Asn
, Arg, Asp, Ala, Ser, A_1_4 is Gl
y, Arg, Lys, Pro, Ala, Ser, Thr
, A_2_1 is Ala, Lys, Ser, G1u, A_
2_2 is Leu, Val, A_2_3 is Lys, Asn
, A_2_4 is Gln, Lys, Arg, Gly, A_
2_5 is Gly, Asp, Ser, A_2_6 is Asp
, Glu, Gln, A_2_7 is Gln, Arg, pr
o, Ser, A_2_8 is Ser, Thr, Glu, P
he, Trp, A_2_9 is Pro, Thr, Trp,
Tyr, A_3_0 is Ser, Thr, Tyr, A_3
_1 is Thr, Val, Phe, A_3_2 is Phe,
15. A host according to claim 14, each of which represents Tyr. 16 A_1 is His, A_2 is Ile, A_3 is Gl
y, A_4 is Pro or Val, A_5 is Ser, A_
6 is Lys, A_7 is Leu, A_8 is Trp, A_9
is Arg, A_1_0 is Ala, A_1_1 is Asp,
A_1_2 is Arg, A_1_3 is Asn, A_1_4
is Gly, A_2_1 is Ala, A_2_2 is Leu,
A_2_3 is Lys, A_2_4 is Gln, A_2_5
is Gly, A_2_6 is Asp, A_2_7 is Gln,
A_2_8 is Ser, A_2_9 is Pro, A_3_0
The host according to claim 14 or 15, wherein is Ser, A_3_1 is Thr, and A_3_2 is Phe. 17. The host according to any one of claims 14 to 16, wherein the polypeptide linked to the NH_2 terminus of the amino acid sequence consists of 1 to 28 amino acid residues. 18. The host of claim 14, wherein the expression plasmid is identified by: pEH6084, pEH7084, pEH8084, p
BRD8050, pEH5050 19 A pharmaceutical composition containing as an active ingredient a recombinant lymphotoxin derivative whose molecule contains an amino acid sequence represented by the following primary structural formula. [There is a gene sequence] (In the formula, A_1_～_1_4, A_2_1_～_3_2
represents any amino acid) 20 A_1 is His, Asn, Leu, A_2 is Il
e, Met, Gly, A_3 is Leu, Gln, His
,Cys,Ile,Pro,Val,Ser,Gly,
A_4 is Pro, Arg, Leu, Gln, Val, A
_5 is Ser, Phe, Cys, A_6 is Lys, As
n, Gln, Glu, Asp, A_7 is Leu, Ala
, Gln, A_8 is Trp, Phe, Leu, A_9 is Arg, Leu, A_1_0 is Ala, Arg, Glu
, A_1_1 is Asp, Asn, Ala, Arg, A_
1_2 is Arg, Asn, Gly, A_1_3 is Asn
, Arg, Asp, Ala, Ser, A_1_4 is Gl
y, Arg, Lys, Pro, Ala, Ser, Thr
, A_2_1 is Ala, Lys, Ser, Glu, A_
2_2 is Leu, Val, A_2_3 is Lys, Asn
, A_2_4 is Gln, Lys, Arg, Gly, A_
2_5 is Gly, Asp, Ser, A_2_6 is Asp
, Glu, Gln, A_2_7 is Gln, Arg, Pr
o, Ser, A_2_8 is Ser, Thr, Glu, P
he, Trp, A_2_9 is Pro, Thr, Trp,
Tyr, A_3_0 is Ser, Thr, Tyr, A_3
_1 is Thr, Val, Phe, A_3_2 is Phe,
20. A pharmaceutical composition according to claim 19, each of which represents Tyr. 21 A_1 is His, A_2 is Ile, A_3 is Gl
y, A_4 is Pro or Val, A_5 is Ser, A_
6 is Lys, A_7 is Leu, A_8 is Trp, A_9
is Arg, A_1_0 is Ala, A_1_1 is Asp,
A_1_2 is Arg, A_1_3 is Asn, A_1_4
is Gly, A_2_1 is Ala, A_2_2 is Leu,
A_2_3 is Lys, A_2_4 is Gln, A_2_5
is Gly, A_2_6 is Asp, A_2_7 is Gln,
A_2_8 is Ser, A_2_9 is Pro, A_3_0
The pharmaceutical composition according to claim 19 or 20, wherein is Ser, A_3_1 is Thr, and A_3_2 is Phe. 22. The pharmaceutical composition according to any one of claims 19 to 21, wherein the polypeptide linked to the NH_2 terminus of the amino acid sequence consists of 1 to 28 amino acid residues. 23. Claims 19 to 2, wherein the pharmaceutical composition is an antitumor agent.
2. The pharmaceutical composition according to any one of 2.