JP2790822B2 - Recombinant lymphotoxin derivative - Google Patents

Description

The present invention relates to a novel recombinant lymphotoxin derivative, a gene recombination technique relating to its production, and a pharmaceutical composition containing the derivative. Therefore, the present invention can be used in the field of prescription drugs.

[Conventional technology]

There are various humoral factors involved in the body's defense mechanisms in living organisms, and these are trace bioactive substances mainly produced by leukocytes, lymphocytes, macrophages, etc. in response to stimuli, such as interferon (IFN). −α,
β, γ), interleukin (IL1,2,3,4), lymphotoxin (LT), cancer necrosis factor (TNF), and colony stimulating factor (G-, GM-CSF). Of these, lymphotoxin and TNF have been identified as polypeptides that exhibit direct anticellular activity against neoplastic cell lines. Lymphotoxin is produced from lymphocytes and TNF is produced from macrophages upon stimulation with mitogens.

It has already been reported that lymphotoxin and TNF are produced by cloning the gene for lymphotoxin and INF and culturing Escherichia coli transformed with a vector incorporating the gene (Nature, 312 , 721-). 724,1984 (Nature, 312,721-724,198
Four). And lymphotoxin and T produced in E. coli
NF has also been shown to have a necrotic effect on transplanted cancer in in vivo experiments, such as Meth transplanted into mice.
A Sarcoma-induced cancer has been found to be significantly cured by intravenous or local administration of lymphotoxin or TNF (J. Immunology, MAPALLADINO et al., 13).
8 4023 (1987)).

However, it has been revealed that these lymphotoxins and TNF do not specifically damage all cancer cells, and that many cancer cells have no effect at all (Sugarman et al, Science, 230 , 943 ( 1985), Watanab
e et al, J. Cancer Res. (Gann) 76 1115 (1985)). For example, human bladder cancer T-24, human malignant melanoma G-361, colon adenocarcinoma LS174-T, human laryngeal carcinoma HEp2, human fibroma HT1080, etc.
It is less susceptible to damaging effects in in vitro experiments.

By the way, it has been revealed that lymphotoxin and TNF show a wide range of actions other than antitumor action, and lipoprotein lipase inhibitory action, IL1 inducing action, prostaglandin inducing action, blood coagulation promoting action, etc. have been reported so far. . When using lymphotoxin or TNF clinically, it is expected that these effects will be exposed as side effects.In fact, in a phase I clinical trial of TNF, fever, chills,
Horror, blood pressure lowering, GOT / GPT rise, general malaise, ALP increase has been observed (cancer and chemotherapy, 13, 3491,1986).

Above all, it has been reported that hypotension is a dose limiting factor (Immunobiobiology, 175 , 8,198).
7), it is an obstacle for high dose TNF. In addition, it has been reported that the blood pressure lowering effect of TNF can be reduced by pre-administration of a cyclooxygenase inhibitor such as indomethacin or ketoprofen (Proc. Natl. Acad. Scad.
ci. USA, 84 , 4273, 1987).
Suggesting the possibility that based on prostaglandin E ₂ production induced effect by.

By the way, lymphotoxin has α (molecular weight of 70,000 to 90,000) and β (molecular weight of 25,000 to 500)
00), γ (molecular weight 10,000-20,000), and complex (molecular weight 200,000 <) (Paul F. Torrance, Biological Response)
Modifiers 293,1985 (Paul F. Torrence, Biolog
ical Response Modifiers, 293, 1985), and the α and β classes can be further classified into subclasses by charge. That is, lymphotoxin is used as a general term for various heterogeneous substances.

Aggarwal et al. Reported the amino acid structure of lymphotoxin (JBC, 260 2334 (1985) (JBC260,
2334 (1985)), and the lymphotoxin gene structure is also reported by Gray et al., And it is thought that a part of the cause of the lymphotoxin heterogeneity can be explained by the difference in the number of amino acid residues and the association formation of the lymphotoxin.

On the other hand, Granger et al. Have reported that there is a lymphotoxin that does not show an immunological cross with this lymphotoxin (J. Immunol 137 , 1878, 1986 (J. Immunol).
ogy 137 , 1878, 1986), which allegedly has a different spectrum of anticellular activity from the previously elucidated lymphotoxin. In addition, human tumor necrosis factor (TNF), which has been reported as a factor having antitumor cell activity, shows 28% similarity in amino acid structure to human lymphotoxin, and is reported to have a common receptor on the tumor cell surface. Nevertheless, the spectrum of antitumor cell activity is believed to be somewhat different.

Based on the above findings, the present inventor has made various polypeptides from the lymphotoxin to exhibit an antitumor effect.
Finding the amino acid sequence of gion and enhancing the antitumor effect and reducing clinical side effects were examined as problems.

[Means for solving the problem]

As a result of various studies, it was found that the amino acid sequence consisting of 143 amino acid residues has an antitumor effect,
Continuing any amino acid sequence on the NH ₂ side, or having a stronger antitumor effect by converting a specific amino acid in the sequence, or has a damaging effect on lymphotoxin-resistant cancer cells, and We know that the novel polypeptide can be obtained and the attenuation of prostaglandin E ₂ production induced ability, and have completed the present invention.

That is, the present invention is an amino acid sequence composed of 143 amino acid residues for the purpose of enhancing the antitumor effect, expanding the spectrum of the antitumor effect, and reducing clinical side effects. Novel lymphotoxin derivatives containing amino acids with several variations are disclosed. However, since the derivative can be effectively achieved by using a gene recombination technique, the gist of the present invention is to provide a recombinant lymphotoxin derivative containing the above amino acid sequence.

Hereinafter, the present invention will be described in detail by dividing into sections of configuration and method.

Structure The recombinant lymphotoxin derivative of the present invention has an amino acid sequence represented by the following primary structural formula (hereinafter, referred to as an amino acid sequence according to the present invention).

(Where A ₁ is Lys or Ser Thr Leu Lys, A ₂ is Pro or Va
l, A ₃ is Lys or Gly, A ₄ represents a Lys or Asn. Preferred as such recombinant lymphotoxin derivatives are those shown below.

Recombinant lymphotoxin derivatives A ₁ is Lys, A ₂ is Pro, A ₃ is Lys, A ₄ is Lys.

Recombinant lymphotoxin derivatives A ₁ is Lys, A ₂ is Pro, A ₃ is Gly, A ₄ is Asn.

Recombinant lymphotoxin derivatives A ₁ is Lys, A ₂ is Pro, A ₃ is Lys, A ₄ is Asn.

Recombinant lymphotoxin derivatives A ₁ is Lys, A ₂ is Val, A ₃ is Lys, A ₄ is Lys.

A ₁ is Ser Thr Leu Lys, A ₂ is Val, A ₃ is Lys, A ₄ is Lys
A recombinant lymphotoxin derivative which is

These recombinant lymphotoxin derivatives have excellent antitumor activity.

The final target substance of the present invention can be produced by gene recombination technology. Accordingly, the cDNA encoding the recombinant lymphotoxin derivative of the present invention, and the expression plasmid containing the cDNA as a foreign gene and continuously obtained so as to be capable of regulation and expression in a selected host are all of the present invention. It is an intermediate substance necessary for the production of the final target substance, and can be integrated as an invention in the sense of solving the same problem. As specific examples of the expression plasmid, pMLT5001A, pEH5118, pEH5123, pBRD5037, pBRD703
There may be mentioned the plasmids identified by 7, all of which are shown in the examples below. These expression plasmids each contain a cDNA encoding the recombinant lymphotoxin derivative shown above as a foreign gene, and are obtained by ligation such that control and expression are possible in a selected host. . Also, the host transformed with the expression plasmid can be integrated as the invention together with the cDNA and the expression plasmid, and the host may be Escherichia coli or animal cells, particularly
BHK cells are used. Examples of transformed E. coli include RB791-pMLT5001A, JM83-pEH5118, JM83-pEH5123, R
Escherichia coli identified and indicated by R1-pBRD5037 and RR1-pBRD7037 can be mentioned, each of which is shown in the Examples below and deposited with the Microtechnical Laboratory with each indication. Each accession number corresponds to FERM P-9560, P-9562, P-9563, P-1025 corresponding to the above order.
1, P-10284.

Although the recombinant lymphotoxin derivative of the present invention, which is the final target substance, is a mutant of natural lymphotoxin, the activity of natural lymphotoxin, that is, the antitumor activity, is as high as that of natural lymphotoxin, as shown in the experimental examples described below. It has above. Thus, just as natural lymphotoxin is an essential active ingredient of a pharmaceutical composition and provided for medical purposes utilizing its activity, the recombinant lymphotoxin derivative of the present invention is also 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 activity of the recombinant lymphotoxin derivative of the present invention is at least four times greater than that of the natural lymphotoxin.

In this case, the pharmaceutical composition is mainly an injection and is administered intravenously. In order to produce it as an injection, it may be carried out according to a conventional method when a trace amount of a physiologically active substance is used as an injection. Therefore, for example, the recombinant lymphotoxin derivative of the present invention may be used alone or in an aqueous solution together with a suitable excipient and dissolving agent, aseptically filtered and filled, lyophilized, and attached with an aqueous solution for dissolution to prepare a ready-to-use injection. I just need.

Method The production method and the measurement method of the substance of the present invention will be described.

First, a lymphokine cDNA library required for producing the substance of the present invention is obtained by extracting mRNA from a suitable cell, screening for a substance showing lymphotoxin activity in an expression system, performing reverse transcription, and transforming E. coli as a suitable plasmid. (Nature, 312 , 721-72
4,1984 (Nature, 312,721-724,1984)). For example, as shown in Examples below, mRNA is fractionated from Daudi cells, injected into Xenopus oocytes, lymphotoxin activity is measured on the culture supernatant, and the target mRNA is screened, and the obtained mRNA is obtained. Is reverse-transcribed by a standard method,
A plasmid may be prepared from the obtained DNA by a conventional method and transformed into Escherichia coli X1776.

In addition, Daudi cells are human Burkitt lymphoma, ATC
It is generally available as C CCL 213 (Cancer Lisa. 28 , 1300-1310, 1968 (Cance Res. 28 , 1300).
-1310, 1968)).

Next, a plasmid containing a cDNA encoding lymphotoxin is selected from the cDNA library by colony hybridization. Colony hybridization was performed by the method of Wallace (Nucleic Acids Res., 9 , 879, 198).
Performed according to 1), and probed C
The following DNA corresponding to the eight amino acid sequences at the ends is synthesized,
Purify and use.

A plasmid may be prepared for a positive colony, the nucleotide sequence may be determined, and a plasmid containing a lymphotoxin cDNA may be selected.

Next, in order to obtain DNA encoding the recombinant lymphotoxin derivative of the present invention from a plasmid containing the cDNA of lymphotoxin, known gene recombination operations may be appropriately combined. In the present invention, the amino acid sequence at a specific position is exclusively removed and the amino acid at a specific position is substituted,
Zoller and Smith site-directed mutagenesis (site-di-re
cted mutagenesis) can be used. That is, the lymphotoxin cDNA was incorporated into a phage vector such as M13, and a single-stranded DNA was prepared from a culture solution of E. coli transformed with the resulting double-stranded M13 DNA, and a predetermined synthetic oligonucleotide was used as a primer thereto. Anneal and induce mutation. After performing site-directed mutagenesis, Escherichia coli is transformed with the obtained reaction solution, and plaques obtained by culturing the resultant are hybridized with radiolabeled synthetic oligonucleotides and screened to encode the target substance. A double-stranded M13 phage DNA containing the desired cDNA as a foreign gene can be obtained. If necessary, a predetermined cDNA may be cut out from the phage DNA and ligated to an appropriate expression vector used depending on the host, according to a conventional method.

As the expression vector, a commercially available expression vector, for example, pKK233-2, pUC12, pUC18 (Pharmacia), PUG1
30, PUG 131, (M13tg130,131
Plasmids replaced with 2,13) may be used, but the necessary promoter and terminator are selected and ligated as shown below, and a special expression vector is prepared.
This may be used. That is, generally, in order to express a gene derived from a mammal in Escherichia coli, yeast, or animal cells, a promoter is arranged upstream of ATG (Met) which is a protein synthesis initiation signal, and a protein synthesis stop codon (TAA, TGA, TA
A terminator must be connected downstream of G). As a promoter, lac, trp, tac, trc,
sac, PL, etc., α-factor, TPI, suc, GAL, PGI, etc. in yeast, and metallothionein, SV 40 earl in animal cells.
y, LTR, etc. are usually used. In particular, in order to express a gene efficiently in E. coli, the promoter, Shine-Dalgarno sequence (SD sequence) and ATG must be accurately ligated.
It is necessary to optimally select the number of bases from the D sequence to ATG. In consideration of these points, in Examples described later, trc promoter, SD sequence and ATG were used as E. coli expression vectors.
In this order, and adjust so that ATG is located 11 bases downstream from the SD sequence.
l Prepare the one constructed to insert the II site,
I'm using this.

Most of the individual genetic recombination operations used in the intermediate stages of the above manufacturing process are known, and will be described below in the literature or a brief description.

Cleavage of the plasmid with restriction enzymes and separation, recovery and binding of the DNA fragments obtained by agarose gel electrophoresis or polyacrylamide gel electrophoresis are described in Maniatis, T., et al., Molecular Cloning, Laboratory Manual, Cold Manual. Spring Harbor Laboratory 1982 (Maniati
s, T, et al., Molecular Cloning, A Laboratory Manual, C
old Spring Harbor Laboratory 1982).

For example, the present invention is applied to a case where a plasmid containing a cDNA encoding a natural lymphokine and an M13 phage vector are each cleaved, and fragments are ligated to obtain a double-stranded M13 DNA into which the cDNA is integrated.

Transformation of the plasmid into Escherichia coli is described in DNA Cloning Volume 1, Chapter 6, De. M. Glover, (IR Press 1985 (DNA clonig Vol.1, Chap.6, ed.D.
M. Glover IRL press 1985).

For example, it is applied when transforming Escherichia coli with a double-stranded M13 DNA containing a cDNA encoding a natural lymphokine or an expression plasmid containing a cDNA encoding a mutant lymphokine.

Synthesis of oligonucleotides for mutagenesis and screening by the phosphoamidite method and labeling at the 5 'end are described in S.D. El. View cage, ethanol.,: Tetrahedron let., 22 1859, (1981) (SLB
eaucage, etal., Tetrahedron Lett., 22 1859 (1981))
And A. Maxam and W. Gilbird, Methods in Enchimology, 65, 499-560
Page, Academic Press 1980 (A. Maxam and W. Gilbert,
Methods in Enzymology, Vol. 65 p499-560 Academic Pr
ess 1980).

The determination of the DNA base sequence is performed by F.S. Sanger Ethal.,
P. N. A. S. 74 5463, 1977 (F. Sanger et
al., PNAS 74 5463, 1977).

For example, mutagenized and screened single-stranded M13 DN
It is applied when determining the nucleotide sequence of A and confirming whether or not the desired mutation has been induced.

Double-stranded and single-stranded M13 phage DNA are prepared as follows.

That is, the transformed E. coli was transformed into Meshing, Methods in Enchimology 101, 20-78 19
83 (J. Messing, Method in Enzymology 101 , 20-78 198
Culture by the method described in 3), culture with shaking, and centrifuge. From the Escherichia coli in the precipitate, Maniatis, Tea, Etal., Molecular Cloning, Laboratory Manual. Cold Spring Harbor Laboratory 1982 (Maniatis, T, et al., Molecular Clonin
g, A Laboratory Manual, Cold Spring Harbor Laborator
y 1982) to obtain double-stranded M13 DNA. Also, from the supernatant, add 1.3µL of 2.5M NaCl ^- 20%
PEG6000 mixed solution was added, and the mixture was centrifuged in an Eppendorf microtube.
Was suspended in 10 mM Tris-HCl buffer (pH 7.9, 0.1 mM EDTA), extracted with phenol, precipitated with ethanol, washed with 70% ethanol, dried, and dried, and 30 μl of 10 mM Tris-HCl buffer (pH 7.9, 0.1 resuspended in single-stranded M13 DNA
Get.

The site-directed mutagenesis reaction is described in Zoller and Smith, Methods In Entology 100 468-500,198.
3 (Zoller and Smith, Methods in Enzymology, 100 468
-500, 1983).

That is, a single-stranded M was added to 20 pmol of a synthetic oligonucleotide (primer) for mutagenesis end-labeled with non-radioactive phosphate.
13 μg of DNA, ₂ μ of buffer for annealing (50 mM Tris-HCl, pH 8.0, 0.25 mM MgCl ₂ ), 8 μ of B buffer (0.2 M Tris-HCl, pH 7.5, 0.1 M MgCl ₂ , 0.1 M dithiothreitol), dATP, dGTP, dCTP, mixture 4μ of each dTTP each 2.5 mM, 5 mM rATP 2.mu., distilled water 1 [mu], T ₄ DNA ligase 5 units of DNA polymerase I Klenow fragment 5 units added, 14 ° C. 3 the final total liquid volume as 20μ Let react for hours.

E. coli in then the reaction mixture was subjected to mutagenesis using the DNA to be transformed, Kramer Ettal., Cell 38 879-887,1984 (Kramer et al., Cell 38 879-8
87, 1984), followed by Grunstein and Hogness, P. N. A. S. USA
72 3961, 1975 (Grunstein and Hogness, PNASUSA 72
E. coli JM1 transformed according to E. coli JM1
Spread on agar medium by mixing with 03, incubate overnight at 37 ° C, and transfer the formed plaque onto a nitrocellulose filter.

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

Filter nitrocellulose filter 6 times SSC (1 times SSC
Is 150 mM NaCl, 15 mM sodium citrate), 10 times Denhar
dt's (1x Denhard's is 0.02% polyvinylpyrrolidone, 0.02% ficoll, 0.02% bovine serum albumin), 5
0 μg of sonicated salmon sperm DNA is added and treated at 65 ° C. for 3 hours to form a preliminary hybrid. The filter is then mixed with the radiolabeled synthetic oligonucleotide for mutagenesis under the same buffer conditions and left at 65 ° C. overnight to form a hybrid. Wash the filter with 6x SSC,
After draining the water, an intensifying screen is overlaid and exposed to an X-ray film to select a target plaque.

CDNA encoding mutant lymphokine is converted to double-stranded M13 DNA
The expression plasmid obtained by ligating this to an expression vector may be used for transformation depending on the host.For example, for transformation of BHK cells,
Reuters Ethal. N. A. S. USA, 79 422, 1982 (Loyter et al., PNASUSA, 79
422, 1982) according to the method of Loyter et al.
That is, the obtained expression plasmid is, for example, pSV2-dhfr
With BHK ⁵⁷⁰ cell line (ts, tk ^-) and transformed into. Note that pS
For V2-dhfr, see Sabramani, etal: mol. cell. Violo. 1 854-864, (1981) (Sabramani, et a
l: Mol. Cell. Biol. 1 854-864 (1981)).

Cloning is the limiting dilution method
Can be performed as follows using

From several days after the transformation, Dulbecco's modified Eagle's medium containing 200 nM Amethopterin (Dulbeco's modified Eagle's medium, glucose 3.5%, sodium bicarbonate 7.5%, fetal calf serum (FC
S) 5%, tranexamic acid 3 mM, glutamine 2 mM, (+)-A
(methopterin 200 nM) and perform limiting dilution with medium exchange every 2-3 days for 1-2 weeks to obtain a production strain. Next, the produced strain was inoculated into 300 ml of 200 nM Amethopterin-containing double-beco modified Eagle medium containing 200 nM Amethopterin, cultured at 37 ° C., and 3 to 4 days after the start of the culture, the medium was replaced with the same amount of medium for 3 to 4 hours every day. The culture supernatant may be collected.

In Examples described later, E. coli JW83, E. coli RRI or RB791
After shaking culture, the cells were disrupted, and the supernatant fraction was collected.

Purification may be carried out by appropriately combining gel filtration, dye adsorption chromatography, high performance liquid chromatography and the like, fractionating and obtaining a required fraction by lymphotoxin activity.

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

Indicator cells such as mouse fibroblast cell lines
LP3 cells, which are a sub-strain of L cell and highly sensitive to lymphotoxin, were used,
/ ml of actinomycin D at 37 ° C under a CO ₂ stream.
Incubate for 18 hours. The medium used here is RPMI 1640 containing 10% inactivated fetal serum. After completion of the culture, the dead cells are washed away, and the remaining living cells are fixed and stained with 0.5% crystal violet (methanol: water = 1: 4) solution.
A dye is extracted from the stained cells, and the absorbance is measured to determine the number of living cells. Titer is 50% lys of LP3 cells
Indicate by the sample dilution when giving is. In addition,
Since this value changes depending on the growth phase of LP3 cells when the sample is added, it is necessary to always use a lymphotoxin standard titer sample as a control and correct it.

When the above assay is performed using logarithmic growth phase LP3 cells passaged in a serum-free medium (DM-153 medium), the active concentration of LT that causes 50% lysis after 24 hours is defined as 1 U / ml.

The cytotoxicity and growth inhibition test of LT against human malignant tumor cell lines is performed as follows.

Various cell lines are added to a 96-well microtiter plate at a density of 0.5 to 1 × 10 ³ wells, and after overnight culture, a medium containing an LT sample is added. Thereafter, the cells are cultured for about one week in the presence of CO _{2 in a} wet state. Viable cells are determined by extraction of Crystal wiolet in the same manner as described above.

Alternatively, Assay can be performed in the presence of actinomycin D at 0.1 μg / ml. In that case, the cell line is added at a density of 2-5 × 10 ⁴ / well, the LT sample and actinomycin D are added, and the number of viable cells is determined in 12 to 15 hours.

Estimation of specific activity by enzyme immunoassay (EIA) is performed as follows. Using a rabbit anti-human LT antibody obtained by immunization with LT, or a monoclonal or anti-human LT antibody produced by a hybridoma obtained by cell fusion from spleen cells of an immunized mouse, the amount of LT in a sample by a sandwich method Is estimated. That is, plastic we
After adsorbing an anti-human LT antibody (monoclonal or polyclonal) to the II, LT is added and allowed to bind. Further, a monoclonal antibody or a polyclonal antibody having a recognition site different from that of the primary antibody is added. As this secondary antibody, one to which bition is previously bound is used. Thereby, if avidin bound to horseradish peroxidase is subsequently added, peroxidase is fixed to the well in proportion to the amount of the secondary antibody bound to LT by a specific reaction between avidin and biotin of the secondary antibody. Here, for example, if a substrate such as orthophenylenediamine is added,
Color development is quantitatively observed with peroxidase, and the LT concentration in the sample can be estimated from the absorbance. That is, a sample containing an unknown amount of LT may be compared with an EIA value of LT having a known content concentration, which is previously purified and determined by amino acid analysis. When the former antitumor activity is examined using L-P3 cells as an indicator as described above, its specific activity can be determined.

〔Example〕

 Hereinafter, the present invention will be described in more detail with reference to examples.

Reference Example 1 Preparation of mRNA 2 × 10 ⁸ Daudi cells were prepared using 4M guanidium thiocyanate, 0.5% sodium N-lauroyl sarcosine, 25 mM sodium citrate pH 7.0, 0.1 M mercaptoethanol, and 0.1% mercaptoethanol.
The cells were suspended in 10 ml of a 1% antiform (Sigma) solution and homogenized with a polytron. Add this homogenate to 0.
Overlaid on a 5.7M cesium chloride aqueous solution containing 1M EDTA, and used an ultracentrifuge (SRP28A rotor, Hitachi) for 2600
The mixture was centrifuged at 0 rpm at 20 ° C for 23 hours. The precipitated RNA was dissolved in 10 ml of 10 mM Tris-HCl, pH 7.4, added with 2.5 volumes of ethanol, and left at -70 ° C for 1 hour. Next, the RNA precipitated by centrifugation was dissolved in 14 ml of 10 mM Tris-HCl pH 7.4, and 4M
An additional 2 ml of potassium chloride was added. This RNA solution was applied to an oligo (T) cellulose column (φ2 × 15 cm), washed thoroughly with 0.5 M potassium chloride, 10 mM Tris-HCl, pH 7.4, and then washed with 10 mM
The mRNA was eluted with M Tris-HCl pH 7.4 aqueous solution.

Purification of mRNA 500 μg of Daudi cell-derived mRNA was added to 50 mM Tris HCl pH 7.4,
The solution was layered on 15 ml of a 10% to 28% sucrose concentration gradient solution containing 0.2M NaCl and 1mM EDTA, and was subjected to ultracentrifugation (SRP28A
The mixture was centrifuged at 26000 rpm at 20 ° C. for 16 hours using a rotor (Hitachi). Next, fractionate 500μ each, add ethanol to each fraction, m
RNA was precipitated. Each fraction mRNA is dissolved in sterile water, and the mRNA
The concentration was adjusted to 1 μg / μ.

mRNA Screening Xenopus females (one year old or older) were injected with 200 U of serum gonadotropin (Pimex: Sankyo). After one night, remove the oocytes and add modified b
It was stored in arth saline medium (MBS). Next mRN
Inject 50-100 ng / egg of each fraction of A into eggs and add 1 mM phenyl met
25 ℃, 36h in MBS containing hyl sulfonyl fluoride (PMSF)
r was cultured. After removing the supernatant, the oocytes were homogenized, extracted with MBS containing 1 mM PMSF, and used as a sample for each fraction of mRNA. Lymphotoxin activity was measured for each sample, and the fraction of mRNA showing lymphotoxin activity was screened.

Preparation of cDNA library 5 μg of the mRNA fraction showing lymphotoxin activity in a Xenopus oocyte mRNA expression system
M Tris HCl pH3.8 Dissolve in 7μ and incubate at 65 ℃ for 5 minutes
After that, 1.4 μg of pcDVI DNA (Pharmacia) was added, and the reaction was performed under the following conditions. 50mM Tris HCl pH8.3,8mM MgCl
_{2, 30mM KCl, 0.3mM DTT,} 2mM dNTP, reversetranscripteas
e 5U, reaction solution volume 20μ.

After performing the reaction at 37 ° C. for 60 minutes, 2 μm of 0.25 M EDTA was added to stop the reaction. After phenol-chloroform treatment, 4 M NH ₄ OAc 20 μ and EtOH 80 μ were added, the mixture was allowed to stand at −70 ° C., then centrifuged to remove the supernatant, and the same operation was repeated. The precipitate was washed with 75% EtOH water and dried, and then dried at 10 mM s.
odium cacodylate, 2.5mM HEPES pH7.6,0.1mM dCTP, poly
(A) The reaction solution was adjusted to 0.2 μg / μ with a terminal deoxyn
Add ucleotidyl transferase (TdT) 10U and finally 20
μ. After reacting at 37 ° C for 10 minutes, 0.25M EDTA
1 μm was added to stop the reaction, and post-treatment was performed according to a conventional method.

A precipitate of the obtained DNA was dissolved in 10 mM Tris HCl pH 7.4 containing 8 mM MgCl ₂ , 6 mM mercaptoethanol, and Hind III 10 U.
It was dissolved in 11 µ and reacted at 37 ° C for 2 hours. 0.25M EDTA 1
After stopping the reaction, post-treatment was performed according to a conventional method, and 10 mM Tris HCl pH 7.4, 1 mM EDTA solution 10 μ and Et were added.
OH was dissolved in a mixed solution of 3μ.

Next, 77 ng of pL-1 DNA (Pharmacia) was added to 11 µ of the DNA-containing solution, and 10 mM Tris HCl pH 7.4, 1 mM EDTA, 0.1 M Na
The Cl solution was 120 μm. After standing at 65 ° C. for 5 minutes and at 42 ° C. for 30 minutes, the following components were added under ice cooling to a volume of 1.2 ml. 20mM Tris HCl pH7.5,4mM MgCl ₂ , 10mM (NH ₄ ) ₂ SO ₄ ,
0.1MKCl, 0.1mM β-NAD, 50μg / ml Bovine Serum Albumi
n, E. coli ligase 1 μg After overnight reaction at 12 ° C., 4 mM dNTP 10 μ, 20 mM
β-NAD 10μ, E. coli ligase 1μg, E. coli DNA polym
Add eraseI 17.5U, RNase H 120U, 12 ℃ for 1 hour, 25 ℃
The reaction was further performed for 1 hr.
776 strains were transformed.

That is, E. coli x1776 strain was transformed into x1776 medium (Maniatis
et al, Molecular cloning) at 37 ° C (550n
After culturing until m) reached 0.2, the cells were collected and the competent cells were prepared by the method of Maniatis et al. This compitent ce
After the above DNA was added to II, the E. coli x1776 strain was transformed to prepare a cDNA library.

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

The structure of the synthetic probe is GTC TTC TTT GGA GCC TTC GCT
CTG, which was synthesized by an automatic solid phase synthesizer (Applied Biosystems) and then purified by HPLC. Hybridization is performed by the method of Wallace et al. (Nucleic Acids
Res, 9 879, 1981). Out of about 100,000 cDNA libraries, 60 were positive colonies. Among them, plasmids of three colonies were prepared and their nucleotide sequences were determined. One of them was a plasmid containing lymphotoxin cDNA. FIG. 1 shows the nucleotide sequence of the amino acid coding region of the obtained lymphotoxin cDNA.

Construction of Expression Vector The plasmid containing the lymphotoxin cDNA obtained in the previous section was
After digestion with mHI, an approximately 1.1 kbp fragment containing the lymphotoxin cDNA was ligated to the poly li of pUG131 (pUC13 (Pharmacia)).
After inserting the nker portion into the BamHI site of a plasmid in which the polylinker portion of M13tg131 (Amersham) was replaced (plasmid number pMLT2000), a DNA fragment of about 1.1 Kbp digested with HincII and BalI was obtained. Next, pKK23
3-2 (Pharmacia) was digested with NcoI and then the 1.1 Kbp fragment was inserted into a vector treated with Klenow enzyme (plasmid number pMLT2100). pKK233-2 has a trc promoter (an improved version of the tca promoter),
Inducible expression is possible with G. Next, pMLT2100 is digested with EcoRl and HindIII, containing approximately 1.
5 kbp fragment to M13mp10 double-stranded DNA (Pharmacia)
Inserted. Next, after transfection into E. coli JM103, single-stranded DNA was prepared according to a conventional method, and in vitro mutagenesis was performed using the following synthetic oligonucleotides.

Synthetic DNA: GCT TGC TGG GAT CCA TGA GAT CTG TTT CCT The site-specific mutagenesis reaction was basically performed according to the method of Zoller et al. (Method in Enzymology 100 468, 1983). That is, 1 μg of template DNA (single-stranded D) was added to 20 pmol of the synthetic oligonucleotide labeled with a non-radioactive phosphate.
NA), 2μ of 5x annealing buffer (50mM Tris HCl, pH
8.0,25mM MgCl ₂ ), 8μB buffer (0.2M Tris HCl
pH 7.5, 0.1 M MgCl ₂ , 0.1 M DTT), 4 μm of 2.5 mM dNTP,
2μ of 5mM ATP, 1μ of distilled water, 5U of T4 DNA ligas
e, 5 U of DNA polymerase I Klenow fragment was added to make the total amount 20 μm. After performing the reaction at 14 ° C for 3 hours, E. coli
Transformed JM103 (Kramer et al., Cell 38 879,198
Four).

The transformed bacteria were mixed with E. coli JM103, spread on an agar medium, cultured at 37 ° C. overnight, and the formed plaques were transferred onto nitrocellulose (Grunstein et al PNAS 72 39).
61, 1975). Next, a hybridization test was performed with a synthetic oligonucleotide labeled with ³² P to select phage plaques in which the mutation was induced. Double-stranded DNA was prepared from the phage plaques selected as described above, and EcoRl
And the DNA fragment obtained by digestion with Hind III
3-2 was inserted into a plasmid prepared by digestion with EcoRl and HindIII (plasmid number pMLT3020).

pMLT3020 starts protein synthesis from ATG 11 nucleotides downstream of the Shine-Dargaro sequence of the trc promoter, and can insert any gene downstream of the promoter by Bgl II digestion, and is an initiation codon when digested with BamHI. An expression vector into which a gene can be inserted downstream of ATG. pMLT3020 encodes a polypeptide in which seven amino acid residues have been deleted from the NH ₂ terminus of the polypeptide of the present invention. The DNA sequence around the NH ₂ terminus is as follows.

FIG. 2 shows a construction process diagram of pMLT3020.

Example 1 pMLT3020 was digested with BamHI and HindIII and was
NA fragments were recovered by agarose gel electrophoresis.
In addition, after synthesizing the oligonucleotide shown below and phosphorylating the 5 'end with polynucleotide kinase (NEB) and ATP, pUG13 was added together with the above 500 bp DNA fragment.
0 was enzymatically ligated with the vector obtained by digestion with EcoRl and HindIII (plasmid number pUG5001).

After stopping the reaction, E. coli JM83 was transformed according to a standard method,
White colonies were selected on an agar plate containing X-Gal.
Next, after digesting plasmid pUG5001 with Bgl II and Hind III, an approximately 500 bp DNA fragment was recovered from agarose electrophoresis, and combined with about 4..kbp DNA obtained by digesting pMLT3020 with Bgl II and Hind III. (Plasmid number pMLT5001A). The construction process diagram is shown in FIG.

Reference Example 2 About 50 obtained by digesting pMLT3020 with BamHI and HindIII
The following synthetic oligonucleotide obtained by phosphorylating the DNA fragment of 0 bp at the 5 'end and pUG130 were used as EcoRl and Hind
It was ligated enzymatically with the vector obtained by digestion III (plasmid number pUG5004). The construction process diagram is shown in FIG.

Reference Example 3 An approximately 540 bp DNA fragment obtained by digesting plasmid pUG5004 with Sac II and Hind III, the following synthetic oligonucleotide phosphorylated at the 5 ′ end, plasmid pUG130,
An approximately 2.8 kbp DNA fragment obtained by digestion with EcoRl and HindIII was ligated enzymatically (plasmid number pUG501
1).

Next, an approximately 570 bp DNA fragment obtained by digesting plasmid pUG5011 with Bgl II and Hind III and plasmid pM
About 4.6 kbp obtained by digesting LT3020 with Bgl II and Hind III
Was enzymatically ligated (plasmid number pMLT5011).

 The construction process diagram is shown in FIG.

Reference Example 4 An approximately 800 bp DNA fragment obtained by digesting plasmid pMLT2000 with Hinc II and Bal I was used to convert plasmid pUG131 to Hi
It was enzymatically ligated with an approximately 2.8 kbp DNA fragment obtained by digestion with nc II. At this time, two types of plasmids are produced. When digested with BamH1, a plasmid that produces a DNA fragment of about 800 bp and 2.8 kbp is converted to a plasmid of pMLT2010 and about 3.5 kbp.
The plasmid from which the DNA fragment was generated was named pMLT2011. Next, plasmid pMLT2010 was transformed with Pvu II and Hind
The approximately 600 bp DNA fragment obtained by digestion with III and the plasmid pUG130 were digested with Sma I and Hind III to obtain approximately 2.8
The kbp DNA fragment was ligated enzymatically (plasmid number pUCLT3011). The construction process diagram is shown in FIG.

Reference Example 5 A plasmid fragment of about 600 bp obtained by digesting plasmid pML2010 with Pvu II and Hind III, the following synthetic oligonucleotide phosphorylated at the 5 ′ end, and plasmid pUG131 was converted to Bg
A DNA fragment of about 2.8 kbp obtained by digestion with lII and HindIII was ligated enzymatically (plasmid number pUG300
0).

Next, pUG3000 was digested with Bgl II and Hind III to about 6
A 00 bp DNA fragment was obtained. This DNA fragment and a plasmid pMLT3020 were digested with EcoRl and Bgl II, a DNA fragment of about 300 bp, and plasmid pUG130 was converted into EcoRl.
and a DNA fragment of about 2.8 kbp obtained by digestion with HindIII were ligated enzymatically (plasmid number pEH300
0). The construction process diagram is shown in FIG.

Reference Example 6 A DNA fragment of about 600 bp obtained by digesting plasmid pMLT2010 with Pvu II and Hind III, the following synthetic oligonucleotide phosphorylated at the 5 'end, M13tg130 double-stranded DNA
(Pharmacia) was digested with EcoRl and HindIII, and the resulting DNA fragment of about 7.3 kbp was ligated enzymatically.

Synthetic oligonucleotides When E. coli JM103 was transformed with the obtained double-stranded DNA, M
13 phage plaques were generated. Single-stranded DNA was prepared from this phage plaque, and in vitro mutagenesis was performed using the following synthetic oligonucleotides as primers.

Synthetic oligonucleotides After preparing double-stranded DNA from the obtained mutant M13 phage
Digestion with Acc I and Hind III yielded a DNA fragment of approximately 300 bp. Further, a DNA fragment of about 430 bp obtained by digesting plasmid pMLT 5001A with EcoRl and AccI,
The approximately 2.8 kbp DNA fragment obtained by digesting plasmid pUG130 with EcoRl and Hind III and the above-mentioned approximately 300 bp DNA fragment were ligated enzymatically (plasmid number pE
H5153).

 The construction process diagram is shown in FIG.

Reference Example 7 Approximately 5 kbp DNA obtained by digesting plasmid pMLT5001A with Bgl II and Sac II and pFGKI DNA were
Approximately 2 kbp DNA obtained by digestion with ac II was ligated enzymatically (plasmid number pLRM2000).

Next, about 5 kbp DNA obtained by digesting pLRM2000 with BglII and SacII was ligated enzymatically with the following synthetic DNA, and then transformed into Escherichia coli RRI to obtain a plasmid library. This library is pMLT50
It includes LT in which all kinds of amino acids have been substituted at the site of the second amino acid Lys from the NH ₂ terminus of 01A.

A plasmid having high activity was selected from this library using the strength of the damaging effect on mouse LP3 cells as an index.
The selected plasmid was digested with EcoR I and Hind III to recover a DNA of about 800 bp, and the M13mp10 double-stranded DNA was enzymatically ligated with the DNA digested with EcoR I and Hind III and transfected into JM103. Thereafter, the DNA base sequence was determined according to a conventional method. The plasmids produced by this method and their amino acid structures are shown below.

FIG. 9 shows a diagram of the process for constructing puasmid).

Reference Example 8 An approximately 300 bp DNA fragment obtained by digesting pMLT5001A with EcoR I and Sac II, an approximately 600 bp DNA fragment obtained by digesting pMLT5001 with BamH I and Hind III, and pFGKl as BomH I S
A 1.6 kb DNA fragment obtained by digestion with coII was digested with pUC12 using EcoR.
Enzymatic ligation was performed with l, Hind III digestion to obtain pLRM300.

Approximately 3.5 kb obtained by digesting pLRM300 with Bam HI and Sac II
DNA is purified and a synthetic oligonucleotide mixture (N represents A, G, C, T) and transformed into Escherichia coli RRI to obtain a plasmid library.

By screening this library using the strength of the inhibitory effect on LP3 as an index, a plasmid in which the lymphotoxin amino acids His ⁴ , Ile ⁶ and Gly ⁷ were converted as follows was isolated.

The construction process diagram is shown in FIG.

Reference Example 9 After digestion of pMLT5001A with restriction enzymes EcoRl and HindIII, a fragment of about 800 bp was recovered by agarose gel electrophoresis, and M13mp10 RF DNA was digested with EcoRl and HindIII and digested with about 7.2 bp DNA and enzymatically. Was connected. This DNA
Was used to prepare single-stranded DNA from white plaques formed by transforming E. coli JM103. Next, using synthetic DNA as primer
In vitro mutagenesis was performed.

As a result, a double-stranded DNA was prepared from the obtained phage, digested with EcoRl and HindIII to obtain a DNA fragment of about 800 bp, and pUG130 was digested with EcoRl and HindIII to obtain an about 2.8 kbp fragment and an enzyme. (Plasmid pEH5073).

 The construction process diagram is shown in FIG.

Reference Example 10 Using the same single-stranded DNA as in Reference Example 9, in vitro mutagenesis was performed using the following synthetic DNA as a primer.

Double-stranded DNA was prepared from the phage obtained by Mutagenesis, and after digestion with EcoRl and HindIII, a DNA fragment of 800 bp was obtained. Next, pUG130 was added to EcoRl and Hind III.
The fragment was enzymatically ligated with the approximately 2.8 kbp fragment obtained by digestion with (Plasmid pEH5177).

Example 2 Plasmid pUC12 was digested with BamH1 and PstI and
Synthesis of bp DNA fragment obtained, phosphorylated at 5 'end
The DNA was enzymatically ligated to obtain plasmid pUC7692.

After digestion of plasmid pUC7692 with AccI and AvaII, 7
% Polyacrylamide gel electrophoresis to recover a DNA fragment of about 100 bp, plasmid pMLT5001 'was digested with EcoRl and Acc
About 450b each obtained by digestion with I, Ava II and Hind III
p, about 300 bp DNA fragment and plasmid pUG130
Enzymatically ligated with the approximately 2.8 kbp fragment obtained by digestion with l and Hind III (plasmid pEH5123).

 The construction process diagram is shown in FIG.

Example 3 After digestion of plasmid pEH5123 with AccIII and XhoI,
5 'terminal dephosphorylation was performed by bacterial alkaline phosphorylase treatment. An approximately 3.5 kbp fragment was recovered by agarose electrophoresis and ligated enzymatically with synthetic DNA (plasmid pEH5118).

The construction process diagram is shown in FIG.

Reference Example 11 Approximately 500 bp DNA obtained by digesting plasmid pEH5073 with EcoRl and SalI, and approximately 3.1 kbp DNA similarly obtained by digestion with BamH1 and SalI, and the following synthetic DNA were enzymatically ligated. Then, it was transformed into Escherichia coli strain JM83 to obtain a plasmid library.

A plasmid having an antitumor activity was selected from this library, and the structure of the plasmid was determined to obtain a plasmid having the following structure.

The construction process diagram is shown in FIG.

Reference Example 12 After digesting pMLT5001A with restriction enzymes EcoRl and HindIII, a fragment containing approximately 800 bp of lymphotoxin was recovered. This fragment was inserted into EcoRl and HindIII site of M13mp10RF DNA, and then single-stranded DNA was isolated. next,
In vitro mutagenesis using the following synthetic DNA as primer
Was done.

After preparing double-stranded DNA from plaques selected by plaque hybridization, EcoRl and HindIII
Digestion with を 得 800 bp DNA fragment to obtain pUG130 with EcoRl
And about 2.8 kbp fragment obtained by digestion with Hind III (plasmid number pEH5096).

 The construction process diagram is shown in FIG.

Reference Example 13 Plasmid pBR322 was cut with Bal I and Pvu II to
p DNA was obtained. Next, a plasmid pBR322 d-rop consisting of 3741 bp was obtained by enzymatic ligation. The plasmid pBR322 d-rop thus obtained controls the replication of plasmid DNA
This plasmid lacks the function of the repressor of primer gene, and is expected to have a higher plasmid copy number than prB322.

Next, plasmids pMLT5011 and pBR322 d-rop were
And PvuI to obtain DNA fragments of about 1800 bp and 3100 bp, respectively, and both were ligated enzymatically (plasmid number pBRD
5011). The construction process diagram is shown in FIG.

Furthermore, pBRD5011 was digested with EcoRl and ScaII to obtain about 400 bp.
While obtaining a DNA fragment, pEH5071-24 was converted to SacII and Hind.
About 450 bp DNA obtained by digestion with III is enzymatically
It was inserted into the EcoRl and HindIII sites of G130 (plasmid number pEH8071). The construction process diagram is shown in FIG.

Plasmid pEH8071 contains a stop codon TAG derived from the natural lymphotoxin cDNA and an untranslated region of about 100 bp, aiming to improve mRNA stability in Escherichia coli and prevent skipping of the stop codon by amber mutant Escherichia coli. The plasmid was remodeled.

That is, about 2400 bp D obtained by first cutting pUG130 with Pvu II.
About 900 bp DN obtained by cutting NA and pEH300 with EcoRl and BamHl
A was enzymatically ligated, and a plasmid in which the lymphotoxin gene was inserted in the same direction as the ampicillin gene was selected (plasmid number pTRC3000). The construction process diagram is shown in FIG.

Next, pTRC3000 was cut with EcoRl and ScaI to obtain 1549
An 823 bp DNA fragment obtained by cutting the bp DNA fragment and pKK233-2 (Pharmacia) with EcoRl and ScaI was enzymatically ligated. An approximately 2000 bp DNA fragment obtained by digesting this plasmid with EcoRl and HindIII, an approximately 800 bp DNA fragment obtained by digesting pMLT5001A with EcoRl and ScaI, and the following synthetic DNA were ligated enzymatically (plasmid number pTT5001). . The construction process diagram is shown in FIG.

Next, pEH8071 was cut with EcoR1 and PstI to obtain about 600
A bp DNA fragment, an about 1700 bp DNA fragment obtained by cutting pTT5001 with PstI and PvuI, and an about 3100 bp DNA fragment obtained by cutting pBR322 d-rop with EcoRl and PvuI were enzymatically ligated (plasmid number pBRD8071). The construction process diagram is shown in FIG.

Example 4 An approximately 3000 bp DNA fragment obtained by cutting plasmid pEH5073 with BamHl and SalI, an approximately 500 bp DNA obtained by cutting pEH5073 with EcoRl and SalI, and the following synthetic DNA were enzymatically ligated.

The plasmid was recovered again from E. coli transformed with the plasmid prepared as described above, and its DNA structure was examined.
(Plasmid number pEH503
7).

Next, pEH5037 was cut with EcoRl and PstI to obtain about 600
Approximately 1700 bp DNA fragment obtained by cutting the bp DNA fragment and pTT5001 with Pat I and Pvu I and pBR322 d-rop were EcoRl and Pvu I
An approximately 3100 bp DNA fragment obtained by digestion with the above was ligated enzymatically (plasmid number pBRD5037).

 The construction process diagram is shown in FIG.

Reference Example 14 An approximately 450 bp DNA fragment obtained by digesting plasmid pEH5037 with Sac II and Hind III and an approximately 400 bp DNA fragment obtained by digesting pBRD5011 with EcoRl and Sac II were used to obtain EcoRl and Hind I of pUG130.
Enzymatic insertion into site II (plasmid number pEH803
7).

Subsequently, about 6 obtained by cutting pEH8037 with EcoRl and Pat I.
An approximately 1700 bp DNA fragment obtained by cleaving a 00 bp DNA fragment and pTT5001 with Pst I and Pvu I, pBR322 d-rop was EcoRl and Pvu I
Approximately 3100 bp DNA fragment obtained by digestion was ligated enzymatically (plasmid number pBRD8037).

 The construction process diagram is shown in FIG.

Example 5 Approximately 350 bp DNA obtained by cutting plasmid pEH7084 with EcoRl and SacII, approximately 1.3 kbp DNA obtained by cutting pBRD5037 with ScaII and PvuI, pBR322 d-rop was cut with EcoRl and PvuI. The three DNA fragments of about 3.1 kbp DNA thus obtained were ligated enzymatically (plasmid number pBRD7037).

 The construction process diagram is shown in FIG.

Reference Example 15 Approximately 350 bp DNA obtained by cutting plasmid pEH7084 with EcoRl and SacII, approximately 1.3 kbp DNA obtained by cutting pBRD8071 with SacII and PvuI, pBR322 d-rop was cut with EcoRl and PvuI. The three DNA fragments of about 3.1 kbp DNA thus obtained were ligated enzymatically (plasmid number pBRD7071).

 The construction process diagram is shown in FIG.

Example 6 Escherichia coli was transformed with the plasmids of the above Examples and Reference Examples by a conventional method, cultured, and the target substance was purified. pUCL
The case of T3011 is shown below as an example.

Recombinant E. coli JM83 / pUCLT3011 with ampicillin 100 μg / m
l of LB medium 2 (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl) at 37 ° C. for 14 hours with shaking. Next, after centrifugation, 10 mM PBS (pH 10
7.4), crush the cells using a sonicator under cooling, and centrifuge at 20,000 rpm for 30 minutes to remove the supernatant fraction (crude extract).
28 ml were obtained. LT activity of this crude extract is 7.4 × 10 ⁶ units / mg
It was a protein. Next, 200 mM Phosph
It was applied to Sephacryl S-200, a gel filtration carrier sufficiently equilibrated with ate Buffer (pH 6.8). Then Sephacryl S-2
100 ml of the fraction having LT activity obtained through 200 mM Pho
Apply to Procion red agarose, a dye-adsorbing affinity carrier that has been sufficiently equilibrated with sphate Buffer (pH 6.8), wash well with equilibrated Buffer, then 100 mM Tris Buffer (pH 9.4)
To which arginine having a linear concentration gradient of 0 mM to 400 mM was added. As a result, the fraction having LT activity was eluted at an arginine concentration of 180 mM to 200 mM. Then LT
12.5 ml of the active fraction was added to 100 mM Tris Buffer (pH 9.4)
/ 2 M Na ₂ SO ₄ , diluted with 0.2 M Arg to a total volume of 25 ml.

Subsequently, high performance liquid chromatography was performed using TSK gel Ether-5PW (7.5 φ × 75 mm). 2.0 ml of a fraction showing LT activity equilibrated to 1 M Na ₂ SO ₄ was previously prepared in 100 mM Tris Buff
er (pH 9.4) / 1 TSK gel Ether fully equilibrated with Na ₂ SO ₄
Elution was carried out by applying 5 PW and applying a linear concentration gradient from 100 mM Tris Buffer (pH 9.4) / 1 M Na ₂ SO ₄ to 100 mM Tris Buffer (pH 9.4) /0.2 M Arg. As a result, the fraction showing LT activity was Na
It was eluted at a concentration of ₂ SO ₄ between 220 mM and 180 mM. This was used as the LT final sample. The specific activity of LT of this sample is 2.7 × 10 ⁸
Unit / mg protein, and could be purified about 36 times from the crude extract.

Example 7 Recombinant E. coli JM83 / pMLT5001A was used under the same conditions as in Example 6
After culturing, 21 ml of a crude extract was obtained. Thereafter, purification was performed in the same manner as in Example 6, and the purity was 95% or more (purity test by HPLC).
And got a single item. NH ₂ -terminal sequence and amino acid composition analysis of pMLT5001A shown in Table 1. The specific activity calculated from the amino acid composition analysis value was 3.4 × 10 ⁸ U / mg.

Here, the amount by which pMLT5001A kills 50% of mouse LP3 cells was defined as 1 U.

Example 8 Escherichia coli recombinant (RRl / pBRD5037) was ligated to ampicillin (10
0 μg / ml) LB medium (Bacto tryptone 10 g /, NaCl
5g /, Yeast extract 10g /) Using 400ml, 37 ℃, 15
Cultured for hours. Next, an M9CA medium (1% casamino acid, 1% lactose, Na ₂ HPO ₄ 6 g /, KH ₂ PO ₄ 3 g /, NaCl 0.5 g /, NCl
H ₄ Cl 1g /, FeSO ₄・ 7H ₂ O 0.5mg /, 2mM MgSO ₄ , 0.1mM Ca
Cl ₂ ) 10 and shaking culture was performed at 37 ° C. After 15 hours, 500 ml of 20% casamino acid was added, and the cells were further cultured for 25 hours. The cells were collected by centrifugation and suspended in 100 ml of PBS (Dulbecco). Next, this suspension was crushed by sonication under ice cooling, and then centrifuged at 20,000 rpm for 30 minutes to obtain 132 ml of a supernatant.
Using this crude extract, purification was performed by the method described below.

First, 132 ml of the crude extract (2.8 × 10 ¹⁰ U / ml) was sufficiently equilibrated with 0.2 M phosphate buffer pH 6.8 to Sephadex S-200 (φ13
3 x 900 mm). Next, the fractions in which LT activity was observed were collected, applied to a Sepharose column (φ55 × 210 mm) conjugated with an anti-LT monoclonal antibody, and the column was sufficiently washed with 0.2 M phosphate buffer pH 6.8, and then containing 3 M KSCN. Elution was performed with 0.2 M phosphate buffer pH 6.8. 232 ml of the obtained LT fraction was concentrated to 82 ml with an ultrafiltration apparatus, and then desalted using Sephadex G-25 (φ55 × 210 mm) sufficiently equilibrated with 20 mM phosphate buffer pH 6.8. . The LT derivative thus obtained is SD
Analysis by S-PAGE showed a purity of 98% or more.

Example 9 Escherichia coli recombinants (RRl / pBRD7037) were cultured for 10 times in the same manner as in Example 8, and after collecting and disrupting cells, 150 ml of a crude extract (1.1 × 10 ¹⁰ U / ml) was obtained. This crude extract was subjected to Sephadex S-200 and anti-LT monoclonal antibody-bound S
epharose and Sephadex G-25 columns were sequentially performed, and SDS-P
Analysis by AGE gave a purified protein with a purity of 98% or more.

Reference Example 16 A recombinant Escherichia coli (RRl / pBRD8037) was cultured for 20 times in the same manner as in Example 8, and after collecting and disrupting cells, a crude extract of 160 ml (4.8 × 10 ¹⁰ U / ml) was obtained. This crude extract was subjected to Sephadex S-200 and anti-LT monoclonal antibody-bound S
epharose and Sephadex G-25 columns were sequentially performed, and SDS-P
Analysis by AGE gave a purified protein with a purity of 98% or more.

〔The invention's effect〕

 The effects of the present invention will be described using experimental examples.

Experimental Example 1 Each plasmid obtained in the above example was expressed and purified as described in Example 6, and the final target substance was obtained.
Using these as samples, the antitumor activity was measured. Table 2 shows the results
Shown in In the table, the plasmid column shows the plasmid used to obtain each final target substance, and the antitumor activity column shows the antitumor activity (specific activity to pMLT5001A) of each final target substance.

From Table 2, it is known that the 143 amino acid sequence shown in the present invention has antitumor activity of lymphotoxin.

Experimental Example 2 <Samples> The plasmids pEH5123, pEH5118, pMLT5001A, and pBRD7037 were respectively expressed and purified, and the final target substances A, B, C, and D were obtained.
This was used as a specimen sample. Separately, natural lymphotoxin was prepared as a control sample.

<Method> Human lung cancer cells (PC-10) and human colon adenocarcinoma cells (WiDr) were used as indicator cells. Each cell line is 2-5
The medium containing each sample was added to a 96-well microtiter plate at a density of × 10 ⁴ / Well and actinomycin D (PC-
0.1 μg / ml for 10 and 0.5 μg / ml for WiDr). Incubated for 20 hours in a wet CO ₂ presence, the viable cells C
The cells were fixed and stained with rystal violet, a dye was extracted from the stained cells, and the absorbance was measured. Assuming that the survival value before the sample addition was 100%, the survival rate due to the sample addition was determined.

<Results> FIG. 25 shows the anticancer activity of pEH5118, pEH5123, pMLT5001A and natural lymphotoxin against human lung cancer-derived PC-10 cells (0.
The evaluation results of 1 μg / ml Actinomycin D) are shown.

As is clear from FIG. 25, pEH5118 and pEH5123 attract attention in terms of their effectiveness on cancer cells. These suggest that substitutions and removals at amino acid positions 56-61 will provide more useful compounds.

FIG. 26 shows the anticancer activity of pBRD7037, pMLT5001A and natural lymphotoxin against human colon adenocarcinoma cells (WiDr) (0.5 μg / m 2).
l The results of the evaluation of Actinomycin D) are shown.

As is clear from FIG. 26, pBRD7037 is noted for its efficacy against cancer cells.

Experimental Example 3 (PGE ₃ production inducibility test) <Samples> Plasmids pMLT5001A, pBRD7037, and pBRD5037 were respectively expressed and purified to obtain final target substances C, D, and E, which were used as specimen samples. Separately, natural lymphotoxin and recombinant TNF were prepared as control samples.

<Method> Rat synovial cells (cultured for 2 or 3 passages after collection) were added to a 96-well microculture plate at 1 × 10 ⁴ cells / well, and added.
After culturing and attaching for ~ 5 hours, the medium is aspirated off and replaced with fresh medium (10% FCS-RPMI-1640 medium) (50 μl).
/ Well). Furthermore, each sample previously diluted to various concentrations was added at 50 μ / Well, and cultured at 37 ° C. under 5% CO ₂ for 12 hours, and the supernatant was recovered.

The measurement of the amount of prostaglandin E ₂ (PGE ₂ ) in the supernatant
Performed by RIA KIT (Dupont / NEN research product).

<Results> The results are shown in FIG. Prostaglandin E ₂ production inducing ability than 27 strongest in recombinant TNF, natural lymphotoxin and recombinant lymphotoxin (substance C) is weakened subsequently. However, substances D and E of the present invention are further attenuated, and thus the recombinant lymphotoxin derivative of the present invention is expected to reduce clinical side effects.

[Brief description of the drawings]

FIG. 1 is a diagram of the nucleotide sequence of the amino acid coding region of lymphotoxin cDNA. FIG. 2 is a construction process diagram of pMLT3020. FIG. 3 is a diagram showing the construction steps of pUG5001 and pMLT5001A. FIG. 4 is a construction step diagram of pUG5004. FIG. 5 is a construction process diagram of pMLT50111. FIG. 6 is a construction step diagram of pUCLT3011. FIG. 7 is a construction process diagram of pEH3000. FIG. 8 is a construction process diagram of PEH5153. FIG. 9 is a diagram showing the construction process of a plasmid. FIG. 10 is a diagram showing the construction process of a plasmid. FIG. 11 is a construction step diagram of pEH5073. FIG. 12 is a construction step diagram of pEH5123. FIG. 13 is a construction step diagram of PEH5118. FIG. 14 is a construction step diagram of pEH5071. FIG. 15 is a construction step diagram of pEH5096. FIG. 16 is a construction step diagram of pBRD5011. FIG. 17 is a construction step diagram of pEH8071. FIG. 18 is a construction step diagram of pTRC3000. FIG. 19 is a construction step diagram of pTT5001. FIG. 20 is a construction step diagram of pBRD8071. FIG. 21 is a construction step diagram of pBRD5037. FIG. 22 is a construction step diagram of pBRD8037. FIG. 23 is a construction step diagram of pBRD7037. FIG. 24 is a construction step diagram of pBRD7071. Figure 25 is a graph showing the evaluation results of the anti-cancer activity against human lung cancer-derived PC-10 cells, Figure 26 is a graph showing the evaluation results of the anti-cancer activity against WiDr, Figure 27 is the production of PGE ₂ induced potential in Rat Synovial Cells It is a graph which shows an evaluation result.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) Accession number of microorganism FERM P-10254 Accession number of microorganism FERM P-10283 Accession number of microorganisms FERM P-10251 Accession number of microorganisms FERM P-10284 Accession number of microorganisms FERM P-10252 Accession number of microorganisms FERM P-10160 19-13 Eisai Shizan Ryo 311 (72) Inventor Manabu Kuwata 3010-118 Ushiku-cho, Ushiku City, Ibaraki Prefecture (56) References JP-A-62-181298 (JP, A) JP-A-62-151182 (JP, A) JP-A-63-8398 (JP, A) JP-A-63-8399 (JP, A) (58) Survey The field (Int.Cl. ^6, DB name) C12N 15/00 - 15/90 C07K 14/52 A61K 37/02 GenBank / EMBL / DDBJ (G ENETYX) WPI (DIALOG) BIOSIS (DIALOG)

Claims (10)

(57) [Claims] 1. A recombinant lymphotoxin derivative having an amino acid sequence represented by the following primary structural formula. (Where A ₁ is Lys or Ser Thr Leu Lys, A ₂ is Pro or Va
l, A ₃ is Lys or Gly, A ₄ represents a Lys or Asn. ) 2. A recombinant lymphotoxin derivative according to claim 1, wherein A ₁ is Lys, A ₂ is Pro, A ₃ is Lys, A ₄ is Lys. 3. A recombinant lymphotoxin derivative according to claim 1, wherein A ₁ is Lys, A ₂ is Pro, A ₃ is Gly, A ₄ is Asn. 4. A recombinant lymphotoxin derivative according to claim 1, wherein A ₁ is Lys, A ₂ is Pro, A ₃ is Lys, A ₄ is Asn. Wherein A ₁ is Lys, A ₂ is Val, recombinant lymphotoxin derivatives according to claim 1, wherein A ₃ is Lys, A ₄ is Lys. Wherein A ₁ is Ser Thr Leu Lys, A ₂ is Val, A ₃ is Ly
s, recombinant lymphotoxin derivatives according to claim 1, wherein A ₄ is Lys. 7. A cDNA encoding the recombinant lymphotoxin derivative according to any one of claims 1 to 6. 8. An expression plasmid containing the cDNA of claim 7 as a foreign gene and ligated so as to enable control and expression in a selected host. 9. A host transformed with the expression plasmid according to claim 8. 10. An antitumor agent comprising the recombinant lymphotoxin derivative according to any one of claims 1 to 6 as an active ingredient.