JP2843048B2 - Novel recombinant lymphotoxin derivatives - 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 mitogen.

It has already been reported that lymphotoxin and TNF are produced by cloning the gene for lymphotoxin and TNF and culturing Escherichia coli transformed with a vector having the gene incorporated therein (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, 1).
38 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)). , Watanabe
et al, J. Cancer Res. (Gann) 76 1115 (1985)). For example, human bladder cancer T-24, human melanoma G-361, colon adenocarcinoma L
S174-T, human pharyngeal carcinoma HEp2, human fibroma HT1080, etc.
It is less susceptible to damaging effects in in vitro experiments.

By the way, it has been clarified that lymphotoxin and TNF show a wide range of actions other than antitumor action, and lipoprotein lipase inhibitory action, ILI 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).

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, Biolo
gical Response Modifiers, 293, 1985), and α, β
Classes can be divided 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 gene structure of lymphotoxin is also reported by Gary et al., And it is thought that a part of the cause of the heterogeneity of lymphotoxin can be explained by differences 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.
The purpose of this study was to find the amino acid sequence of gion and to enhance its antitumor effect.

[Means for solving the problem]

As a result of various studies, it was found that the amino acid sequence consisting of 137 amino acid residues has an antitumor effect,
Either continuous any amino acid sequence NH ₂ side, or a stronger anti-tumor effect by converting the specific amino acid in the sequence, or a new poly with cytotoxic action against lymphotoxin resistant cancer cells Knowing that a peptide could be obtained, the present invention was completed.

That is, the present invention is an amino acid sequence composed of 137 amino acid residues for the purpose of enhancing the antitumor effect and expanding the spectrum of the antitumor effect, and a specific amino acid in the sequence has several kinds of variations. Novel lymphotoxin derivatives containing certain 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 contains, in its molecule, an amino acid sequence represented by the following primary structural formula (hereinafter referred to as the amino acid sequence according to the present invention), which exhibits excellent antitumor activity.

(In the formula, A ₁ represents Pro or Val) Other amino acid sequences linked to the NH ₂ side and the COOH side of the amino acid sequence according to the present invention can be freely selected and are not particularly limited. Examples of amino acids linked to the NH ₂ side include the following (1) to (3).

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. Specific examples of expression plasmids include plasmids identified by pEH6084, pEH7084, pEH8084, pBRD8050, and pEH5050, all of which are shown in the Examples below. Also, the host transformed with the expression plasmid can be integrated as the invention like the cDNA and the expression plasmid, and the host may be Escherichia coli or animal cells, especially BHK.
Cells are used. As an example of transformed E. coli, JM
83-pEH6084, JM83-pEH7084, JM83-pEH8084, RR1-pBRD8
Escherichia coli identified by 050 can be mentioned, all of which are shown in the Examples below and deposited with the respective companies with the respective designations. The accession numbers are FERM P-10041, P-10040, P-10042, and P-10253 corresponding to the above order.

Despite the fact that 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, that is, the antitumor activity is comparable to or higher than that of natural lymphotoxin as shown in the experimental examples described below Have. Therefore, natural lymphotoxin is an essential active ingredient of the pharmaceutical composition,
Just as provided for medical purposes that take advantage of its activity,
The recombinant lymphotoxin derivative of the present invention can also be an essential active ingredient of a pharmaceutical composition for therapeutic purposes utilizing its activity. As shown by the experimental examples described later, the activity of the recombinant lymphotoxin derivative of the present invention on, for example, cells derived from human lung cancer (PC-10) is 50 to 80 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 , 130).
0-1310, 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.

Since the individual recombination operations used in the intermediate stages of the above-mentioned production process are almost known, they will be described below in the literature or a brief description.

Cleavage of plasmid with restriction enzymes and separation, recovery and binding of DNA fragments obtained by agarose gel electrophoresis or polyacrylamide gel electrophoresis are described in Maniatis, Tee, Etal, Molecular Cloning, Laboratory Manual, Cold 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 the case where a plasmid containing a cDNA encoding a natural lymphokine and an M13 phage vector are each cut, 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. Clover, IR L Press 1985 (DNA cloning Vol.1, Chap.6, ed.DM
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, Vol. 65, pp. 499-560, Academic Press 1980 (A. Maxam and W. Gilbert, Me.
thods in Enzymology, Vol. 65 p499−560 Academic Pres
s 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 1983
(J. Messing, Method in Enzymology 101, 20-78 1983)
And culturing with shaking, followed by centrifugation. From the Escherichia coli in the precipitate, Maniatis, Tea, Etal., Molecular Cloning, Laboratory Manual. Cold Spring Harbor Laboratory 1982 (Maniatis. T, et al., Molecular Cloning,
A Laboratory Manual, Cold Spring Harbor Laboratory
1982) to obtain double-stranded M13 DNA. Also, from the supernatant, 1.3 μl of 260 μM of 2.5 M NaCl-20% P
Add the EG 6000 mixed solution, centrifuge the mixture in an Eppendorf microtube, and pellet the resulting
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 resuspend in single-stranded M13 DNA
Get.

Site-directed mutagenesis reactions are described in Zoller and Smith, Methods In Entology 100 468-500,19.
83 (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 μm of annealing buffer (50 mM Tris-HCl, pH 8.0, 0.25 mM MgCl ₂ ), 8 μm of B buffer (0.2 M Tris-HCl, pH 7.5, 0.1 M MgCl ₂ , 0.1 M dithiothreitol) A mixture of 2.5 μm each of dATP, dGTP, dCTP and dTTP was added at 4 μm, 2 μm of 5 mM rATP, 1 μm of distilled water, 5 units of T ₄ DNA ligase, and 5 units of DNA polymerase I Klenow fragment. Let react for hours.

E. coli in then the reaction mixture was subjected to mutagenesis using the DNA to be transformed, Kramer Ettal., Cell <br/> Le 38 879-887,1984 (Kramer et al., Cell 38 879- 88
7, 1984), followed by Grunstein and Hogness, P. N. A. S. US 72
3961, 1975 (Grunstein and Hogness, PNASUSA 72 3
961, 1975) and E. coli JM103.
Mix, spread on an agar medium, 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
d's (1x Denhardt's is 0.02% polyvinylpyrrolidone,
0.02% ficoll, 0.02% bovine serum albumin), 50μg
The 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. After the filter is washed with 6 times SSC to remove moisture, 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
At the same time, transform into a BHK ⁵⁷⁰ cell line (ts, tk ⁻ ). Note that pS
For V2-dhfr, see Sabramani, etal: mol. cell. Violo. 1 854-864, (1981) (Sabramani, et.
al: Mol. Cell. Biol. 1 854-864, (1981)).

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

Several days after the transformation, modified Dulbecco's 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 a double becco modified Eagle medium containing 200 nM Amethopterin prepared in a roller bolt, 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 every day for 3 to 4 weeks. The culture supernatant may be collected.

In Examples described later, Escherichia coli JW83 was used, and the cells were disrupted after shaking culture, and the supernatant 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 are sensitive to lymphotoxin, were used, containing the sample and a final concentration of 1 μg.
/ 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 a 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 of LP3 cells
It is indicated by the sample dilution when giving% lysis.
Since this value changes depending on the growth phase of LP3 cells when a sample is added, it is necessary to always correct using a lymphotoxin standard titer sample as a control.

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.

90 wells of various human cancer cell lines at a density of 0.5-1 × 10 ³ wells
After adding to a microtiter plate and culturing overnight, a medium containing the LT sample is added. Thereafter, the cells are cultured for about one week in the presence of CO _{2 in a} humid environment. The surviving cells were treated with Crystal vio as described above.
Judge by extracting let.

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 different confirmation site from the primary antibody is added. As this secondary antibody, one to which bition is previously bound is used. Thereby, after that, if adipine to which horseradish peroxidase is bound is added, peroxidase is fixed to the well in proportion to the amount of the secondary antibody bound to LT by a specific reaction between adipine and the secondary antibody bition. Here, if a substrate such as orthophenylenediamine and hydrogen peroxide is added, color development is quantitatively observed by 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.

Example 1 Preparation of mRNA 2 × 10 ⁸ Daudi cells were treated with 4 M guanidium thiocyanate, 0.5% sodium N-lauroyl sarcosine, 25 mM sodium citrate pH 7.0, 0.1 M mercaptoethanol, 0.1 M 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 28 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 is 50 mM TrisHCl pH 7.4, 0.2
The solution was layered on 15 ml of a solution containing MNaCl and 1 mM EDTA and having a concentration gradient of 10% to 28% sucrose, and centrifuged at 26,000 rpm at 20 ° C. for 16 hours using an ultracentrifuge (SRP28A rotor, Hitachi).
Next, fractionate 500μ each, add ethanol to each fraction, and mRNA
Was precipitated. Each fractionated mRNA was dissolved in sterile water and adjusted to an mRNA concentration of 1 μg / u.

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
After dissolving in M TrisHCl pH 8.37μ and incubating at 65 ° C for 5 minutes, 14μg of pcDVI DNA (Pharmacia) was added, and the reaction was performed under the following conditions. 50mM TrisHCl pH8.3mM MgCl ₂ , 30m
MKCl, 0.3mM DTT, 2mM dNTP, reversetranscriptease 5U,
Reaction liquid 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) Adjust to 0.2 μg / u reaction solution, and add terminal deoxynu
Add 10 U of cleotidyl transferase (TdT) and finally add 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 TrisHCl pH 7.4, 1 mM EDTA, 0.1 M NaC was added.
The 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. Two
_{0mM Tris HCl pH7.5,4mH MgCl 2, 10mM} (NH 4) 2 SO 4, 0.1MK
Cl, 0.1mM β-NAD, 50μg / ml Bovine Serum Albumin, Ec
oli ligase 1μg After overnight reaction at 12 ° C, 4mM dNTP 10μ, 20mM
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 at
76 strains were transformed.

That is, E.colix1776 strain was transformed into x1776 medium (Maniatis
et al, Molecular cloning) at 37 ° C (550nm)
After culturing until the cell concentration became 0.2, the cells were collected and a competent cell was prepared by the method of Maniatis et al. This competent cell
After adding the above DNA, transform E.colix1776 strain to c
A DNA library was created.

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 resulting lymphotoxin cDNA.

Construction of Expression Vector The plasmid containing the lymphotoxin cDNA obtained in the previous section was
After digestion with mH1, an approximately 1.1 kbp fragment containing the lymphotoxin cDNA was ligated with the poly li of pUG131 (pUC13 (Pharmacia)).
After inserting the nker portion into a BamHI site (plasmid in which the polylinker portion of M13tg131 (Amersham) was replaced) (plasmid number pMLT2000), it was digested with Hinc II and Bal I to obtain a DNA fragment of about 1.1 kbp. Then, 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 tac promoter),
Inducible expression is possible with G. Next, pMLT2100 is digested with EcoR1 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.

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 TrisHCl, pH
8.0,25mM MgCl ₂ ), 8μB buffer (0.2M TrisHCl p
H7.5, 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 cultured overnight at 37 ° C. spread on an agar medium mixed with E. coli JM103, the formed plaques were O'in duplicate onto nitrocellulose (Grunstein et al PNAS 72 3
961, 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,
The DNA fragment obtained by digestion with R1 and Hind III was
233-2 was inserted into a plasmid prepared by digestion with EcoR1 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 Bg1 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.

pMLT3020 is digested with BamH1 and HindIII to obtain a DNA of about 500 bp.
The A fragment was 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 agar plates containing X-Gal. Next, plasmid pUG5001 was replaced with Bal II and Hind III
After digestion, a DNA fragment of about 500 bp was recovered from agarose gel electrophoresis, and pMLT3020 was digested with Bgl II and Hind III.
Was combined with about 4.6 kbp DNA obtained by digestion with, and ligated enzymatically (plasmid number pMLT5001A).

 The construction process diagram is shown in FIG.

Plasmid pUC12 was digested with BamHl and PstI to about 2.8 kb
A pDNA fragment was obtained, and the following synthetic DNA phosphorylated at the 5 ′ end was enzymatically ligated to obtain a plasmid pUC7692.

After digestion of plasmid pUC7692 with AccI and AvaII, 7
% Polyacrylamide electrophoresis to recover a DNA fragment of about 100 bp, and plasmid pMLT5001A was transferred to EcoR1 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.

Plasmid pEH5123 was digested with restriction enzymes AccIII and XhoI, and then digested with Bacterial Alkalin Phosphatase.
The terminal dephosphorylation group was removed. Next, about 3.5 kbp fragment was recovered by 0.8% agarose gel electrophoresis,
It was enzymatically ligated to the following synthetic DNA phosphorylated at the 5 'end (plasmid number pEH6084).

The construction process diagram is shown in FIG.

As a result of the nucleotide sequence analysis, this pEH6084 plasmid was found to have 1-amino acid sequence of the lymphotoxin amino acid sequence shown in FIG.
62-position and lack the 118-123 amino acid position, Met the NH ₂ -terminal
It was confirmed that -Lys encodes the added lymphotoxin derivative.

Example 2 An approximately 300 bp fragment obtained by digesting plasmid pEH6084 with restriction enzymes EcoR1 and BglII, and SacII and HindIII
Approximately 500 bp fragment obtained by digestion with Escherichia coli and the following synthetic DNA phosphorylated at the 5 'end were ligated with EcoR1 of plasmid pUG130.
And into the Hind III site enzymatically (plasmid number pEH7084).

The construction process diagram is shown in FIG.

As a result of the nucleotide sequence analysis, this pEH7084 plasmid was found to have 1-amino acid sequence of the lymphotoxin amino acid sequence shown in FIG.
62-position and lack the 118-123 amino acid position, -S to NH ₂ -terminal
It was confirmed that er-Thr-Leu-Lys encodes the added lymphotoxin derivative.

Example 3 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 ligated with EcoR1 and Hind.
It was ligated enzymatically with the vector obtained by digestion III (plasmid number pUG5004).

 The construction process diagram is shown in FIG.

Approximately 540 bp DNA fragment obtained by digesting plasmid pUG5004 with Sac II and Hind III
A DNA fragment of about 2.8 kbp obtained by digestion with EcoR1 and Hind III 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 pL
About 4.6 kb obtained by digesting MLT3020 with Bgl II and Hind III
The DNA fragment of p was ligated enzymatically (plasmid number pMLT5011).

 The construction process diagram is shown in FIG.

After cutting plasmid pEH7084 with restriction enzymes Acc I and Hind III, about 450 bp was obtained by 1% agarose gel electrophoresis.
Was collected. Similarly, plasmid pMLT5
11450 bp D obtained by digesting 011 with restriction enzymes EcoR1 and AccI
The NA fragment and the fragment were converted to plasmid pUG130.
Was enzymatically inserted into the EcoR1 and HindIII sites (plasmid number pEH8084).

 The construction process diagram is shown in FIG.

As a result of the nucleotide sequence analysis, this pEH8084 plasmid contained 1-amino acid sequence of the lymphotoxin aminic acid sequence shown in FIG.
The amino acids at positions 62 and 118-123 were confirmed to encode a lymphotoxin derivative deleted.

Example 4 Approximately 580 bp DNA fragment obtained by cutting plasmid pEH6084 with EcoR1 and PstI, approximately 1700 bp DNA fragment obtained by cutting pTT5001 with PstI and PvuI, pBR322d-rop with EcoR1 and PvuI
Approximately 3,100 bp DNA fragments obtained by digestion were ligated enzymatically (plasmid number pBRD6084).

Next, about 1100 bp DNA fragment obtained by cutting pBRD6084 with EcoR1, AccI and PvuI and about 450 bp DNA fragment obtained by cutting pEH8037 with EcoR1 and AccI, pBR322 d-rop was used for EcoR1 and P
Three of the approximately 3100 bp DNA fragments obtained by digestion with vu I were ligated enzymatically (plasmid number pBRD8050).

 The construction process diagram is shown in FIG.

As a result of the nucleotide sequence analysis, this pBRD8005 plasmid was found to have the amino acid sequence 1- of the lymphotoxin amino acid sequence shown in FIG.
It was confirmed that the amino acids at positions 62 and 118-123 were deleted, and that proline at position 71 encoded a lymphotoxin derivative converted to valine.

Example 5 Plasmid pEH7084 was digested with restriction enzymes AccI and HindIII to obtain a DNA fragment of about 300 bp. Plasmid pEH503
7 was cleaved with EcoR1 and AccI to obtain a DNA fragment of about 450 bp.
dIII site (plasmid number pEH5050).

 The construction process diagram is shown in FIG.

As a result of the nucleotide sequence analysis, this pEH5050 plasmid was found to have 1-amino acid sequence of the lymphotoxin amino acid sequence shown in FIG.
It was confirmed that the amino acids at positions 62 and 118-123 were deleted, the proline at position 71 was converted to valine, and the gene encoded a lymphotoxin derivative with Met-Lys added to the NH ₂ terminus.

Example 6 Escherichia coli W3110 transformed with plasmid pEH6084 was inoculated into LB medium 20 containing 100 mg / ampicillin, and incubated at 37 ° C. for 20 minutes.
Time stirring culture was performed. Next, the cells were collected by centrifugation, washed with PBS, and suspended in 100 ml of PBS. After disrupting the cell fluid with an ultrasonic generator, centrifugation (Tomy Seiko: R
B20BH, 15,000 rotations, 30 minutes) to obtain 125 ml of the bacterial cell extract. The lymphotoxin activity of this crude extract is 4.4 × 10 ⁸ U /
ml. Next, it was applied to a gel filtration carrier Sephacryl S-200 (Pharmacia) φ113 × 900 mm, which was sufficiently equilibrated with a 0.2 M phosphate buffer in advance.

After collecting fractions showing lymphotoxin activity, a portion was applied to a 30 ml anti-lymphotoxin mouse monoclonal antibody column. The antibody column is a monoclonal antibody prepared by immunizing mice with recombinant lymphotoxin, Sepharose 4B
(Pharmacia). 0.2M
After sufficient equilibration with a phosphate buffer, the previously partially purified lymphotoxin derivative is adsorbed, and then washed with the same buffer.
Elution was performed with 0.2 M phosphate buffer containing 3 M KSCM. Then Sep
Desalting was performed with hadex G-25 (Pharmacia), and purity was determined by SDS-polyacrylamide electrophoresis. As a result, a single band having a molecular weight of about 16,000 was obtained. When the NH ₂ terminal sequence was examined, the structure was found to be Met Lys Pro Ala Ala Hi
s Leu IIe Gly Asp.

Example 7 Escherichia coli JM83 transformed with the plasmid pEH7084 was inoculated into 200 ml of LB medium containing 100 mg / ampicillin.
Shaking culture was performed for 8 hours. Next, the cells were collected by centrifugation, washed with PBS (Dalbecco), and suspended in 2 ml of PBS. After crushing the cell fluid with an ultrasonic generator, a cell extract was obtained by centrifugation. After the crude extract was subjected to aseptic filtration (0.22 μm), the filtrate was applied to a 2 ml anti-lymphotoxin mouse monoclonal antibody column. The column is 50 mM
After sufficient equilibration with Tris HCl pH 8.0 buffer, the above lymphotoxin derivative was adsorbed, and then washed with the same buffer. Next, elution was carried out with 50 mM Tris HCl pH 8.0 buffer containing 3 M KSCN. The eluate was dialyzed immediately against PBS (Dulbecco) buffer.

Example 8 Escherichia coli JM83 transformed with plasmid pEH8084 was inoculated into 200 ml of LB medium containing 100 mg / ampicillin.
Shaking culture was performed for 8 hours. Next, the cells were collected by centrifugation, washed with PBS (Dulbecco), and suspended in 2 ml of PBS. After crushing the cell fluid with an ultrasonic generator, a cell extract was obtained by centrifugation. The lymphotoxin activity of the crude extract was 6.6 × 10 ⁷ / ml. Next, the extract was subjected to aseptic filtration (0.22 μm), and then applied to a 2 ml anti-lymphotoxin mouse monoclonal antibody column. The mixture was eluted in the same manner as described above, and dialyzed against PBS to obtain a 2 ml solution containing 2.9 × 10 ⁷ U / ml activity. SDS- gave a single band with a molecular weight of about 16,000 in polyacrylamide electrophoresis, NH ₂ terminal sequence P
ro Ala Ala His Leu IIe Gly Asp.

〔The invention's effect〕

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

Experimental Example 1 <Sample> The plasmid pEH6084 was expressed and purified to obtain the final target substance A, which was used as a sample. Separately, natural lymphotoxin was prepared as a control sample.

<Method> As indicator cells, human lung cancer cells (PC-10), human colon adenocarcinoma cells (WiDr) and human fibrosarcoma cells (HT108)
0) was used. Each cell line is grown at a density of 2-5 × 10 ⁴ / Well 9
The medium containing each sample and actinomycin D (0.1 μg in the case of PC-10) were added to a 6-well microtiter plate.
/ ml, 0.5 μg / ml for WiDr and HT1080). And 12 to 15 hours at a wet CO ₂ presence, viable cells Cry
The cells were fixed and stained with stal violet, a dye was extracted from the stained cells, and the absorbance was measured. Survival values before sample addition
The survival rate by adding the sample was determined as 100%.

<Results> The results are shown in FIGS. FIGS. 12 to 14 show the results when PC-10, WiDr, and HT1080 were used as indicator cells, respectively.
The circles indicate the relationship between the lymphotoxin activity and the survival rate when the target substance A was used as a sample.

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 <Samples> Plasmids pHE6084, pH7084, and pEH8084 were respectively expressed and purified to obtain final target substances B, C, and D, which were used as specimen samples. Separately, natural lymphotoxin was prepared as a control sample.

<Method> As indicator cells, human lung cancer cells (PC-10), human colon adenocarcinoma cells (WiDr) and human fibrosarcoma cells (HT108)
0) was used. Each cell line is grown at a density of 2-5 × 10 ⁴ / Well 9
The medium containing each sample and actinomycin D (0.1 μg in the case of PC-10) were added to a 6-well microtiter plate.
/ ml, 0.5 μg / ml for WiDr and HT1080). Incubated for 20 hours in a wet CO ₂ presence, the viable cells Crystal
The cells were fixed and stained with violet, a dye was extracted from the stained cells, and the absorbance was measured. Survival value before sample addition is 100
%, The survival rate due to the addition of the sample was determined.

<Results> The results are shown in FIG. (A), (b) and (c) of FIG. 15 show PC-10, WiDr and HT1080 as indicator cells, respectively.
Are the results when is used.
The □ and ▽ marks are graphs showing the relationship between the lymphotoxin activity and the survival rate when natural lymphotoxin and target substances B, C, and D were used as samples, respectively.

FIGS. 15A, 15B and 15C show the present invention.
It is known that the 137 amino acid sequence has antitumor activity of lymphotoxin.

Experimental Example 3 For the samples B, C, and D of Experimental Example 2, the activity on mouse LP3 cells was determined by enzyme immunoassay,
The biological activity of each sample was calculated using the activity of natural lymphotoxin (0.5-1 × 10 ⁸ U / mg protein) as a standard.

index Natural lymphotoxin 1.0 B 7.3 C 5.6 D 6.2 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 the natural lymphotoxin.

[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 pEH5123. FIG. 5 is a construction process diagram of pPU5004. FIG. 6 is a construction step diagram of pMLT5011. FIG. 7 is a construction step diagram of pEH6084. FIG. 8 is a construction step diagram of pEH7084. FIG. 9 is a construction step diagram of pEH8084. FIG. 10 is a construction step diagram of pBRD8050. FIG. 11 is a construction step diagram of pEH5050. 12, 13, and 14 are graphs showing the results of Experimental Example 1, respectively.
FIG. 15 is a graph showing the results of Experimental Example 2.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) (72) Inventor Yasushi Shikata Tsukuba, Ibaraki 4-19-13, Kasuga-shi, Eisai Mt. Shiyama Ryo 311 (72) Inventor Manabu Kuwata 3010-118, Ushiku-cho, Ushiku-shi, Ibaraki (58) Field surveyed (Int.Cl. ⁶ , DB name) C12N 15/12- 15/28 C12N 1/21 C07K 14/51-14/57 A61K 37/02 C12P 21/00-21/02 GenBank / EMBL / DDBJ, BIOSIOS (DIALOG) WPI (DIALOG)

Claims (10)

(57) [Claims] 1. A novel recombinant lymphotoxin derivative having an amino acid sequence represented by the following primary structural formula in the molecule. (Where A ₁ represents Pro or Val) 2. The recombinant lymphotoxin derivative according to claim 1, wherein 1 to 4 amino acids are linked to the NH ₂ terminus of the amino acid sequence, or no amino acids are linked. 3. The recombinant lymphotoxin derivative according to claim 2, wherein the amino acid linked to the NH ₂ terminus of the amino acid sequence is Met-Lys. 4. The recombinant lymphotoxin derivative according to claim 2, wherein the amino acid linked to the NH ₂ terminus of the amino acid sequence is Ser-Thr-Leu-Lys. A cDNA encoding the recombinant lymphotoxin derivative according to any one of claims 1 to 4. 6. An expression plasmid comprising the cDNA according to claim 5 as a foreign gene and ligated so as to enable control and expression in a selected host. 7. The expression plasmid according to claim 6, wherein the expression plasmid is identified by the following. pEH6084, pEH7084, pEH8084, pBRD8050, pEH5050 (8) A host transformed with the expression plasmid according to (6) or (7). 9. An antitumor agent comprising the recombinant lymphotoxin derivative according to claim 1 as an active ingredient. 10. A method for producing a recombinant lymphotoxin derivative according to claim 1, comprising culturing the host according to claim 8, and recovering the host from the culture solution.