JPH0265793A - Novel recombinant type lymphotoxin - Google Patents

Abstract

PURPOSE:To obtain a polypeptide that is an LT mucin in which an amino acid or peptide having antibody-linking functional group linked to the N-terminal part of an N-terminal deficient lymphotoxin (LT) is bound to a lysozyme enzyme-sensitive linker peptide with hardly any side effects and useful as an anticancer agent. CONSTITUTION:A protein having an amino acid sequence expressed by the formula (R1 is Cys, Lys, Ser, Cys-Cer or Lys-Ser; R2 is Ala-Leu-Ala, Leu-Ala- Leu, Leu-Ala-Leu-Thr, Ala-Leu-Ala-Leu, Ala-Leu-Ala-Leu-Ser-Asp-Lys-Pro, etc.; R3 is Ala-Ala-Gln-Thr-Ala-Arg-Gln-His-Pro-Lys-Met-His-Leu-A-la-His-Ser- Thr-Leu or part thereof; m and n each are 0 or 1) or part of an active part thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel physiologically active polypeptide useful as an anticancer agent and polydeoxyribonucleic acid (hereinafter abbreviated as DNA) having genetic information thereof.

The present invention also provides a replicable recombinant DNA comprising the DNA.
The present invention also relates to microorganisms or cells transformed with the replicable recombinant DNA, and also to a novel physiologically active polypeptide obtained by expressing the genetic information contained in the DNA, and a method for producing the same.

Amino acids and peptides herein are referred to as IUPAC
-It18 is designated by the abbreviations adopted by the Biochemical Nomenclature Committee (CB!II); for example, the following abbreviations are used. In addition, when an optical isomer is possible for an amino acid, etc., the L-isomer is indicated unless otherwise specified.

Gln: Glutamine residue A sp: Aspartic acid residue Pro Nibroline residue Tyr: Tyrosine residue Val: Valine residue Lys: Lysine residue Glu: Glutamic acid residue Ala: Alanine residue Asn: Asparagine residue Leu : Leucine residue Phe: Phenylalanine residue Gly Nigericine residue His: Histidine residue Ser: Serine residue Thr: Threonine residue 11e: Isoleucine residue Trp: Tryptophan residue Arg: Arginine residue M et: Methionine residue Cysnidistine Residues In this specification, DNA polymers or oligomers are designated by the following abbreviations.

A: 2'-deoxyadenylic acid residue C: 2'-deoxycytidylic acid residue G: 22-deoxyguanylic acid residue T: Thymidylic acid residue Unless otherwise specified, direction from left to right of the sequence is 5'
3'.

[Conventional technology]

Lymphotoxin (hereinafter abbreviated as LT) is a protein derived from lymphocytes and has antitumor activity, and there are expectations for its clinical application along with tumor necrosis factor (hereinafter abbreviated as TNF) derived from macrophages. These LT and TNF were obtained by adding endotoxin or phorbol ester to lymphocytes and macrophages and activating them, respectively.
With recent advances in genetic engineering technology, their structures have been clarified, and by using the genes encoding these proteins, it has become possible to produce these proteins in microorganisms or cell culture. Gray et al. [Nature,
Volume 312 (1984) Page 721] and Penn1ca
[Nature, Volume 312 (19
(1984) page 724] successfully cloned each gene by making full use of this genetic manipulation technology.

[Problem to be solved by the invention]

With the progress of these gene technologies, LT and TN are now available.
Mass production of F has become possible, and it has come to be used clinically. However, the results of many clinical trials obtained to date, especially clinical data on TNF, are not necessarily good.
Tetsuo Taro: Cancer and Chemotherapy, 13.3491 (198
6) Oncologia by Akira Urushisaki
a), 20.105 (1987), A, Kl.
ansne: Biotechnology (Biotech)
+ 5 + 335 (1987)), the biggest problem is the occurrence of serious side effects, and administration of TNF can cause fever, chills,
It is accompanied by shivering, a drop in blood pressure, etc., and therefore has the disadvantage that administration cannot be stopped to prevent tumor growth.

The reason for this is that the tumor selectivity of TNF is not as good as originally thought, and in fact many normal cells and
TNF receptors have also been found in fibroblasts [Yoji Niitsu, Nitera Boitei Soku Research (Therap
eu, Res, ), 7, 275 (1987)
A substance that reacts with LTNF has been found.

Clinical results regarding LT are still limited.Although not as many as regarding TNF have been obtained, it is important to note that LT has a very similar chemical structure to TNF, and also has the property of binding to common receptors on cells.

Rubin, et al.: Journal of
Experimental Medicine (J, Exp, )
Led, ), 162.1099 (1985)) also exhibits nonspecific cytotoxicity to non-tumor organs, tissues, and cells, and is thought to cause side effects similar to those of TNF.

In this way, regarding TNF and LT themselves.

It is difficult to obtain the clinical effect that was originally expected.

Application to new formulations including new modifications and combination therapy is awaited.

On the other hand, anti-tumor immune complexes, which combine anti-cancer antibodies with chemotherapeutic agents or biological toxins, are used as drugs that selectively kill tumor cells.
So-called ``missile therapeutic agents'' have been developed.These drugs recognize and bind to tumor-specific antigens or tumor-associated antigens on tumor cells, and stop DNA or protein synthesis in these tumor cells, leading to their death. It has the characteristics of At present, the results are as follows.

Based on this technical background, the present inventors developed a novel immunotoxin by binding it to an anticancer antibody in order to make LT, a human-derived toxin with a strong cytotoxic effect, more tumor-specific. This research was carried out with the aim of developing. Two major problems have been considered in the production of such immunotoxins. One is that with conventional LT or LT mutin, there is a high possibility that antibodies bind to the active site on the molecule, and the biological activity of the LT itself is impaired in the binding process.Second, even if the biological activity of LT is Even if the immunotoxin is synthesized in a somewhat preserved form, the toxin moiety, namely L
It is predicted that the T molecule will not be released from the antibody, therefore it will be difficult to migrate to the target site within the cell, and the expression of its activity will be extremely small.

[Means to solve the problem]

In order to solve the above problems, we developed a new recombinant L that binds to an antibody at the inactive site of the LT molecule and that allows the LT molecule to be easily released from the antibody after being taken up into tumor cells.
As a result of research on T mutins, we found that the N-terminus of N-terminally deleted LT contains an amino acid or peptide with an antibody-binding functional group, and a linker that is sensitive to lysozyme enzymes.
Create a new LT mutin bound to a peptide,
This completes the present invention.

That is, the present invention relates to a protein having the following amino acid sequence or a part of its active portion.

H-(Met)n-R-R,-(Rt)n+-Lys
-Pro-^1a-Ala-His-Leu-11e-
Gly-Asp-Pro-5er-Lys-Gln-A
sn-5er-Leu-Leu-Trp-Arg-Al
a-Asn-Thr-Asp-Arg-Ala-Phe
-Lau-Gln-Asp-Gly-Phe-5er-
Leu-5er-Asn-Asn-5er-Leu-L
eu-Val-Pro-Thr-5er-G1. y-1
1e-Tyr-Phe-Val-Tyr-5er-Gl
n-Val-Val-Phe-5er-Gly-Lys
-Ala-Tyr-5er-Pro-Lys-Ala-
Thr-5er-Ser-Pro-Leu-Tyr-L
eu-Ala-His-Glu-Va 1-Gln-seu-Phe-5er-3er-Gln-Tyr-Pr
o-Phe-His-Val-Pro-Leu-Leu
-5er-5er-Gln-Lys-Net-Val-
Tyr-Pro-Gly-Leu-Gln-Glu-P
ro-Trp-Leu-His-5er-Met-Ty
r-t(is-Gly-Ala-Ala-Phe-GL
n-Leu-Thr-Gln-Gly-Asp-Gin
-Leu-5er-Dinghr-His-Thr-Asp-
Gly-11e-Pro-11is-Leu-Val-
Leu-3er-Pro-3er-Thr-Val-P
ha-Phe-Gly-Ala-Phe-Ala-Le
u-OH [wherein R1 is Cys, Lys, Ser+
Cys-Ser or Lys-5er, R is A
la -Leu -Ala, Leu-Ala-Leu
, Leu-Ala-Leu-Thr, Ala-Leu-
Ala-Leu, Ala-Leu-Ala-Leu-3
er-Asp-Lys-Pro, Ala-
Leu-Ala-Leu-5er-Asp,
Gly-Phe-Leu-GLy-Ser.

Gly-Phe-Leu-Gly-Ser
-Leu -Lys -Pro or Gly-Phe-L
eu-G], y, R, is Ala-Ala-Gin-T
hr-Ala-Arg-Gln-His-Pr.

-Lys-Met-His-Leu-Ala-His-
1m represents 0 or 1. n represents O or 1. ] In the above formula, R1 is an amino acid residue or a peptide chain having a functional group for antibody binding, R2 is a linker peptide chain sensitive to lysosome, R1 is the N-terminal part of N-terminally deleted LT, and Ala -Ala-Gln-Thr-Ala
-Arg-G l n-Hl s-P r o-Ly
s-Me t-Hi s-Leu-Ala-His-
LT No. 1 expressed as 5er-Thr-Leu
0-No, all or part of the 27 amino acids.

The present invention also includes polydeoxyribonucleic acids containing a base sequence encoding the above polypeptide and polydeoxyribonucleic acids containing a sequence complementary thereto.

The present invention relates to a replicable recombinant DNA capable of expressing a polypeptide comprising the above-described amino acid sequence in transformed microorganisms or cells. Examples of such recombinant DNAs include p"r B 773.ρTB775, pT
B 85g, pT B 86 (1, PTB864, ρ
TBI004, pT B 1005, pT B 100
6, pT B 1007, etc.

Furthermore, the present invention relates to a microorganism or cell transformed with a replicable recombinant DNA capable of expressing a polypeptide having the amino acid sequence of LT. Such microorganisms or cells include Escherichia coli, Bacillus subtilis, mesenchyme, and higher animal cells.

Furthermore, the present invention provides L
This relates to a method for manufacturing T. For more information, see Replicatible Recombinant D
The present invention relates to a method for producing the polypeptide, which comprises growing a microorganism or cell transformed with NA and efficiently recovering the peptide.

DNA encoding the novel LT mutin of the present invention can be prepared, for example, by the following method.

1, 12-0-tetradecanoylphorbol-acetate (TPA) and concanavalin A (ConA)
m-RNA can be collected by a known method from human peripheral lymphocytes in which LT synthesis has been induced, and a cDNA library of approximately 5×10 5 cDNAs can be constructed from the m-RNA.

2. Normally 10- which encodes the partial peptide chain of LT
A 50-mar oligonucleotide was synthesized from mar and used as a probe. Perform LT cDNA screening. For example, the C-terminal 18-mer (T
Approximately 50 clones can be obtained when using the synthetic nucleotide CAAAGAAGACAGTACT).

3. Isolate a plasmid from the obtained LT cDNA clone and determine its nucleotide sequence. A plasmid encoding the previously reported amino acid sequence of LT was selected.

This can be cut with an appropriate restriction enzyme and introduced into an appropriate expression vector to produce a recombinant DNA containing the DNA.

4, 3. Transform various hosts, such as E. coli, using the vector prepared in
It is possible to obtain a strain that retains the .

5, 4. After culturing the transformant and isolating the plasmid, DNA encoding the novel LT mutin can be prepared as follows.

■ In the case of the human LT gene, 20-2 from the N-terminal side
A restriction enzyme Nsil recognition site exists in the region encoding the first methionine-histidine. Therefore, by cutting the LT gene with NsiI, a DNA fragment encoding the N-terminal LT can be obtained. In this fragment. A DNA encoding the novel LT mutin is prepared by linking the adapter sequence described below with a DNA encoding an amino acid sequence containing an antibody-binding amino acid or peptide and a linker peptide, and inserting it into an appropriate vector.

3'5' (D Similarly, in the case of the human LT gene, there is a restriction enzyme pvulI recognition site in the region encoding serine-alanine at position 9-10 from the N-terminus. By cleavage, a DNA fragment encoding N-terminally deleted LT can be obtained. To this fragment, the adapter sequence of n) and a DNA encoding an amino acid sequence containing an antibody-binding amino acid or peptide and a linker peptide are ligated. A DNA encoding the new LT mutin is prepared, and an appropriate vector is inserted.

■ The DNA fragment encoding the N-terminally deleted LT after cleavage with P vu U or Nsil is further removed with an exonuclease, such as nuclease BAL31, to remove the region encoding 1-18 amino acids, and then the adapter sequence of ■ and the antibody are added. DNA encoding an amino acid sequence containing a binding amino acid or peptide and a linker peptide
was combined. By selecting among them those whose gene reading frame is correctly maintained, LT (x-1
71) It is possible to prepare a DNA encoding a novel LT mutin in which an amino acid or peptide for antibody binding and a linker peptide are bound to the N-terminus of x=10 to 28.

■ Also, site-directed mutations (site-directed mutations)
ed mutagenesis) (SLlith, M
．． and Gillam, S. r.
g) J.

3, l (1981)) can also be applied. That is, N-terminally deleted LT after P vu If cleavage
A DNA fragment encoding
-L Biochemicals). After growth, M13 phage released into broth can be precipitated with polyethylene glycol and then treated with phenol to obtain M13 phage single-stranded DNA.

Next, some amino acids or peptides in the peptide chain from the 10th alanine to the 27th leucine on the N-terminal side of LT are deleted, and an amino acid or peptide for antibody binding and a linker peptide are added to the N-terminus. A DNA encoding a polypeptide having the following can be produced by chemical synthesis and used as a primer. This primer and the previously ml M13 phage DNA can be mixed, made into double strands by the action of DNA polymerase I large fragment, and then circularized by the action of T4 DNA ligase. This circular DNA was transferred to E. coli JM10.
After transferring the released M13 phage DNA to a filter, plaque hybridization (
Maniatis, T., et al., “Molecular Cloning, Laboratory Manual”.
r Cloning, A Laboratory Ma
nual)”, Co1d Spring Harbo
r Laboratory, P., 312 (1982
))I do. D from the phages for which a strong signal was detected.
Prepare DNA, cut it out with an appropriate restriction enzyme, and cut out this DNA.
By incorporating the fragment into a plasmid, a DNA encoding a new modified LT mutin is obtained.

Plasmid DN containing all or part of the human LT gene
A, cosmid DNA, phage DNA, etc. can be used as template DNA for site-directed mutagenesis. M13 phage and φX174 phage are single-stranded DNA
Because A can be easily prepared.

More desirable as template DNA. For example, when using M13 phage or φx174 phage into which all or part of the human LT gene has been integrated, phage particles present in the broth are precipitated with polyethylene glycol, protein is removed by phenol treatment, and then ethanol is used. By performing precipitation, the single-stranded DNA present within the phage particles can be obtained. Also, double-stranded plasmid DNA into which all or part of the human LT gene has been incorporated as template DNA.
When using cosmid DNA or cosmid DNA, double-stranded DNA is
After heat treatment at ℃ for 1 minute to 10 minutes, preferably 3 minutes to 5 minutes, it can be quenched with ice water and used after being denatured into one MDNA.

Primers for specific site-directed mutations may have any NA sequence as long as they have the DNA sequence to be converted and can hybridize with template DNA and function as primers during DNA synthesis. . Also, how to make primers.

Any method may be used, but one of the appropriate sequences can be synthesized by chemical synthesis.
It is desirable to make double-stranded DNA, first with such a primer! l! ! Mix the prepared single-stranded DNA and
After being repaired to double-stranded DNA by the action of A polymerase ■ large fragment, it can be circularized by the action of T4 DNA ligase. After introducing this circular DNA into E. m. , plaque hybridization (Maniatis, T., et al. [Molecular Cloning, Laboratory Manual (Mo.
Regular Cloning, A Laborat
ory Manual) J, Co1d Sprin
g Harbor Laboratory, P, 31
2 (1982)) and colony hybridization [Ibid. P, 326].

The desired mutant can be selected. Phage DNA and plasmid DNA are prepared from the plaques and colonies thus obtained, and the DNA is used to create a new LT.
Mutin genes can be gl.

The DNA encoding the novel LT mutin thus obtained was transferred to various hosts (eg, Escherichia coli, Bacillus subtilis).

By inserting the mutin into the 3' end of a promoter region that functions in yeast, animal cells), a recombinant DNA capable of expressing a DNA encoding a novel LT mutin can be constructed.

The promoter region may be any region as long as it contains a site necessary for initiating mRNA synthesis upon binding of RNA polymerase.

For example, when E. coli is used as a host, a recombinant DNA capable of expressing the DNA encoding the novel LT mutin can be constructed by inserting the DNA encoding the novel LT mutin into the 3' end of the promoter region that functions in E. coli. In addition, pBR322, pBR325, ptrp 7 are used as vectors when using large*i as a host.
81. pUC8, pUC9, pUC19, pJ I3
8 is used, and DNA encoding a new LT mutin is inserted into this by the action of T 4 DNA ligase. using this reaction solution.

Escherichia coli (e.g. C600 strain, 41M 294 strain, DHI
strain, W3110 strain, RRI strain, PH10 strain, etc.) using known methods (Cohen, S. N., et al. [Procedures of the National Academy of Sciences (Proc.
Natl, Acad, sci, U, s, A) J 69,
2110 (1972)) or a similar method.

The promoter used is the trp promoter (trp
-p), and for example, recA promoter [JP-A-59-65099], lac promoter, λPL promoter, etc. may be used.

A transformant carrying the novel recombinant plasmid DNA containing the DNA encoding the novel LT mutin obtained as described above can be selected as having, for example, ampicillin resistance, tetracycline resistance, or resistance to both of these drugs as a phenotype.

The above transformant is cultured in a medium known per se. As a medium, for example, L broth, Penass
ay) M- containing broth and glucose, casamino acids
9 medium (Miller, J., “Experiment In Molecular Genetics (IE Experiment)
s in Molecular Genetics)J
, 431-433 (Cold Spring Harbo
r Laboratory, New York, 197
2)). If necessary, a drug such as 3β-indolyl acrylic acid can be added here to make the promoter work efficiently.

The transformant is usually cultured at 15-43°C, preferably at 2°C.
It is carried out at 8 to 40°C for 2 to 24 hours, preferably 4 to 16 hours, and aeration and stirring can be added if necessary.

For example, when using animal cells as a host, promoters that can function in animal cells (e.g., SV40
A DNA encoding a novel LT mutin was inserted into the 3' end of the region of the promoter), and by a method known per se,
A novel LT mutin can be produced by transforming a host with the recombinant DNA and culturing the transformant.

When using Bacillus subtilis or yeast as a host, for example, a DNA encoding a novel LT mutin is placed at the 3' end of a promoter region that can function in Bacillus subtilis or yeast.
A novel LT mutin can be produced by inserting the recombinant DNA, transforming a host with the recombinant DNA by a method known per se, and culturing the transformant.

Among the above hosts, E. coli is more preferred.

After culturing, the bacterial cells are collected by a known method, and in the case of an E. coli transformant, the bacterial cells are suspended in an appropriate buffer such as Tris-HCl buffer (PH 7,5), and treated with sonication, lysozyme and /or after destroying the bacterial cells by freezing and thawing,
A method of obtaining a supernatant containing the new LT mutin by centrifugation can be used as appropriate. Preferably, the bacterial cells are collected, suspended in a buffer solution, lysozyme is added, and the mixture is incubated at 0 to 10°C for 10 minutes to 30 minutes.
A method is used in which the mixture is incubated for a period of time, subjected to ultrasonic treatment at 0 to 10°C for 30 seconds to 5 minutes, and then centrifuged to obtain L clear.

Separation and purification of the new LT mutin from the extract can be performed, for example, by gel filtration, hydroxyapatite column chromatography, ion exchange column chromatography, ultracentrifugation,
It can be performed by affinity chromatography using human LT antibodies.

As the amino acid or peptide having an antibody-binding functional group contained in the N-terminus of the novel LT mutin of the above invention, (:, ys, Lys, Ser, C70-5er, Lys-5er, etc. are used.Cys and C
In the case of ys-8er, it is attached to the antibody via the sulfhydryl group it contains. Especially in the case of human LT,
Since it does not contain Cys in its molecule, it can be bound to antibodies very specifically through the N-terminal part, which is the non-active site of LT. In the case of Lys and Lys-5ep, they can be linked to antibodies via the alpha- and E-amino groups they contain. Also Ser, Cys-5er and Lys-
In the case of 5er, it can be attached to the antibody through the hydroxyl group it contains.

Various known methods are used to bind these antibodies to human LT (V, P, Butler: Pharmacological Review (Pharmacol,
Rev, ), Kawa, +03 (1978), Tsunehiro Kitayo:
Organic Synthetic Chemistry, Dan.

283 (1984)L For example,
After modification with N-(γ-maleimido butyryloxy)-sufushiimide (abbreviated as PDP), the anticancer antibody is added to the LT mutin containing Cys at the N-terminus of the present invention and a complex is created by a thiol exchange reaction. After maleimidation with GMBS), the L containing Cys at the N-terminus of the present invention
LT mutin containing Lys at the N-terminus is modified with 5PDP and then reduced, and added to maleimidized anticancer antibody to create a complex with thioether bond. ■ 5PDP of LT mutin that also contains Lys at the N-terminus
It is conceivable to modify and reduce the antibody with 5PDP, add it to an anti-cancer antibody modified with 5PDP, and create a complex through a thiol exchange reaction. In these methods, it is also possible to use antibody fragment F(ab')z, Fab' or Fab instead of the anti-cancer antibody IgG. Also, anti-cancer antibodies and LT
It is also possible to create complexes by completely reversing the mutin treatments.

Next, the lysozyme enzyme-sensitive linker peptide contained in the N-terminus of the novel LT mutin of the present invention includes Ala
-Leu-Ala, Leu-Ala-Leu, Leu-
Ala-Leu-Thr.

Ala-Leu-Ala-Leu, Ala-Leu-A
la-Leu-3er-Asp-Lys-Pro and Gly-Phe-Leu-Gly are used, but
Any known peptide chain sensitive to lysosomal enzymes may be used. Especially the above-mentioned Ala-Leu-Ala,
Leu-Ala-Leu, Ala-Leu-Ala-
A linker containing Leu or Gly-Phe-Leu-Gly is effective for release into a drug after being taken into cells and is preferably used (B, Rihova a
tal,: Clinical Immunology and Immunotherapy (C1in, Immunol, Io+
+aunopath1. ), 46, 100 (19
88), A. Trouat at al.: Bulletin of the National Academy of Sciences (Proc. Natl. Acod.
Sci, U, S, A).

79, 626 (1982)).

As described above, the present inventor obtained a novel LT mutin gene and demonstrated a method for producing a novel LT mutin using this gene; however, the present invention is not limited to the above.

In the present invention, part or all of the base sequence is replaced with artificial DNA synthesized organically without changing the amino acid sequence due to reasons such as differences in the frequency of use of codons (genetic code) corresponding to each amino acid. is also possible.

The novel LT mutin of the present invention has an amino acid or peptide that can be used for binding to an antibody at the N-terminus that is not involved in the activity, and further contains a lysosomal enzyme-sensitive linker peptide following the sequence.

Therefore, upon binding with anti-cancer antibodies, it is possible to form an immune complex without impairing LT's original biological activity, and furthermore, after it is taken up into tumor cells, LT molecules are activated by the influence of intracellular lysozyme enzymes. is expected to be released from the antibody and effectively exert its cytotoxicity. These properties are extremely advantageous for creating tumor-selective anticancer drugs, and L
It is effective in enhancing the action of T and reducing side effects.

The present invention will be specifically explained below using reference examples and examples, but it goes without saying that these do not limit the scope of the present invention.

In carrying out the present invention, the preparation of recombinant DNA and the introduction of the recombinant into microorganisms were carried out according to the following experimental zone unless otherwise specified.

(1) T, Maniatis, E, F, Fr1tsc
h, J., Sambrook.

[Molecular Cloning (Molecular C)
1゜ning) J, Co1d Spring Har
Published by Bor Laboratory (USA) (2) Edited by Yasutaka Takagi, "Gene Manipulation Experimental Method", Published by Kodansha Reference Example 1. L929m 1-wound j ('!'M-
The cytotoxicity of LT was evaluated using L929 cells [Journal of Immunology (J, Immunol), 1
26 (1981) p. 235] or [Journal of Immunological Methodology (J, lm1uno
1. Methods), Volume 70 (1984) 25
7]. That is, RPMI 1 containing 10% fetal calf serum (FC5) was prepared using a 96-sized tissue culture microplate (Flow Laboratory).
4X suspended in the above medium containing 4 μg/ml of mitomycin C was added to 50 μQ of a 2-fold serially diluted sample in 640 medium.
10' cells/m Q (7) J degree L929 cells 50
μfl was added and cultured for 148 hours at 37°C in 5% carbon dioxide gas. After culturing, live cells were stained with dimethylthiazoyl diphenyltetrazolium bromide salt (NTT) at 10% 5050. OIN HCQ'? ’fl after solving 5
The absorbance at 90 nm was measured using Titertic Multiscan (Flow Laboratory) 6 The obtained absorbance is proportional to the number of living cells. The amount of biological activity required to kill 50% of L929 cells was defined as 1 unit/IIQ, and the biological activity of the sample was expressed as unit/mQ.

Reference example 2. Form change of large intestine strain ■Escherichia coli DHI
Colonies of the strain were placed in SOB medium [Experiment i (1) page 69]
was used to culture until the absorbance at 550 nm reached 0.5. Collect 30mQ of the culture and add 0.1M of 12mQ
RbCQ-10a+M CaCR, -50mM MnC
Q2-0.2M acetic acid buffer containing 15% glycerol (
Suspended in pH 5.8) and centrifuged on a water-cooled plate for 5 minutes, then centrifuged for 10 minutes.
mM RbCQ - 75mM CaCQ2 - 10mM Mops buffer containing 15% glycerol (PH6,5
), cooled in water for 615 minutes, quenched with dry ice-ethanol, and stored at -70°C.

Reference example 3. Form change Escherichia coli ■DH1, C6
00, MM 294 colonies of various E. coli strains were SOB
The cells were cultured using medium 10++ until the absorbance at 550 nm reached 0.3. After cooling with water and centrifuging, the resulting bacterial cells were
Washed with 10rrl of nQ NaCQ. The bacterial cells were resuspended in 5nQ of 50+++M CaCQ and allowed to stand for 15 minutes under water cooling. After centrifugation, 0.5nQ of 50mM Ca
CQ, and used immediately.

Example 1 Lymphocytes prepared from human peripheral blood were treated with TPA (15 g/
1 (1) and ConA (40 μg/mQ) at 37°C in RPMI 1640 medium (containing 10% FO5), 524 hours after LT was induced.
The induced 1 x 1010 human lymphocytes were treated with 5M guanidine thiocyanate and 11 ci of 5% mercaptoethanol! pH 7.6, lomME DTA
N-Lauroylsarcosine was disrupted and denatured in a Teflon homogenizer in a solution containing sodium chloride, then sodium was added to the solution to give a concentration of 4%, and the homogenized mixture was mixed. 11, 7 Fv
iCesium chloride solution (5.7M cesium chloride, 0.1
M EDTA ) layered on SmQ, Beckman SW
24,000 rp11 at 15°C using 28 rotors
Centrifugation was performed for 148 hours to obtain RNA precipitate. This R
After dissolving the NA precipitate in 0.25% N-lauroyl sodium sarcosinate solution, it was precipitated with ethanol, and 10
I got the RNA of ■, this RN! '． in a high salt solution (
0.5M NaCQ.

]OmM Tris ・HCQp H 7,6, l
oligos (in mM EDTA, 0.3% SDS)
dT) adsorbed on a cellulose column, v R :”i A containing poly(A) was added to a low salt solution (10mM Tris
-HCQ・pH 7.6. 1 rnM EDTA
Poly(O, 3% SOS) was eluted with
300 μg of mRNA containing A) was collected.

This mRNA was further precipitated with ethanol.

0.2m solution of Q (10mM Trisi(CQpH
7.6. 2mM EDTA, 0.3%SOS)
The mixture was dissolved in water, treated at 65°C for 2 minutes, and fractionated by 10-35% sucrose density gradient centrifugation (centrifugation at 20°C and 25.00 rpm for 21 hours using a Beckman SW28 rotor).

A portion of the RNA from each fraction was injected into Xenopus oocytes and the LT activity in the synthesized protein was measured.
activity was detected. LT mRNA in this fraction is approximately 25μ
It was g.

Using this poly(A) RNA as a template, a cDNA library was prepared using the method of Okayama and Berg [Molecular and Cellular Biology (Mo1.Ce11.Bi
ol. ), vol. 2 (1982), p. 161; same magazine, vol. 3 (
1983) p. 280], the pcDV1 vector,
It was produced using a PLI linker. Circularized cDNA
The vector plasmid containing the vector was infected with E. coli DHI.
Starting from 5 μl of poly(A) RNA, approximately 5×1
c using E. coli DHI consisting of 0s clones as a host.
We were able to obtain a DNA library.

Example 2 The human cDNA library using E. coli DH1 was filtered using a nitrocellulose filter (Millipore, IIATF).
A total of 20 replica filters were prepared, with each set of 2 replica filters using this filter as a master filter. E. coli on this replica filter was dissolved with 0.5N, 5N NaOH solution, and the denatured plasmid DNA was dried and fixed on the filter (Grunstein, M., & Hognes et al.
Proceeding of the National Academy of Sciences (Proc, Nat.
l, Acad, sci, USA), vol. 72 (1975
) 3961 pages].

On the other hand, the base sequence of the LT gene (Gr
ayv et al., Nature J, 31
2 (1984), p. 721] (amino acid N0.
Gene portion corresponding to 162-167) was synthesized to synthesize an oligonucleotide corresponding to the human LTC DNA.
It was used as a screening probe.

The 5' ends of these oligonucleotide probes were labeled with 32p using T4 polynucleotide kinase, [γ-32P]ATP.

Labeled probes were separately associated with DNA-immobilized replica filters. The association reaction was performed using 5x SSC (0,15M NaCQ,
0.015M Sodium cimrate)
, 5 X Denhardt's.

0.1% SO5, 100μg/IIIQ denatured salmon sperm D
The reaction was carried out at 40°C for 16 hours in a 10 mQ solution of NA, and after the reaction,
The filter was washed with 6XSSC, 0.1% SDS solution three times for 30 minutes each at room temperature and twice for 60 minutes each at 43°C.

(Actual Di(1), page 3o9). Radioautograms were taken from the washed filters, and bacterial strains that reacted to the probe were searched for by superimposing the radioautograms of two replica filters in a set. By this method, From about 3 x 10' colonies, 50 E, colj D H1 strains that reacted to the probe were obtained.

Plasmid DNA was extracted using the alkaline method [Bi
Nucleic Acids Research (Nucleic Acids Research)
Res, ), Vol. 11 (1979), p. 1513]. DNA with restriction enzyme BamHI
(manufactured by Zenshuzo Co., Ltd.) and fractionated by agarose gel electrophoresis, the DNA fragments were filtered from a 7-galose gel using a nitrocellulose filter (Nis & Nis Co., Ltd., BA85).
Moved above [Southern blotting method, experimental book (1) 3
82 pages]. When this filter was associated with the oligonucleotide probe described above, the plasmid DNA fragments reacted with the probe.

Therefore, the largest BamHI DNA fragment (c
One strain E, c that has a plasmid that produced a DNA portion)
oli K l 2 DH1/pTB618 was selected. The base sequence of the cDNA portion of this plasmid DNA was extracted using the dideoxynucleotide synthetic chain termination method (J, Mess.
ing et al.
Cleic Ac1ds Res, ), 9 volumes (198
1 year) page 309] 6 As a result, the L contained in plasmid pT B 618
It was found that the T gene was not complete, but included upstream of the untranslated portion at the 3' end up to the third C of the Pro codon CCC, which is No. 1 and 18 amines.

Example 3 Human L T l -171 vector TB 694 conjugation step 1 (Preparation of pTB693 plasmid DNA)
As shown in Figure 1, 2μ of plasmid pTB618
using 0.6 units of BaQI (manufactured by Takara Shuzo).
Digested at ℃ for 6 hours.

On the other hand, B[p indicated by 8-a+er of CAGATCTG
After phosphorylating 2 μg of n linker with 0.5 mM ATP and 2.5 units of T4 polynucleotide kinase, 0.2 μg of the phosphorylated linker was added to 1.6 μg of the above Ba
Add 35 units of T to I-digested pT B 618.
4. Reaction was carried out at 14° C. overnight in the presence of DNA ligase.

After inactivation at 65° C. for 5 minutes, the cells were trimmed using 30 units of BgQIT and subjected to 1.2% agarose gel electrophoresis.

The main band corresponding to 4.3 Kbp was cut out and
After extraction with salt a Fjia solution, it was purified using an RDP mini column (Biorat). The above linear DNA 110
0n was added with 10 units of T4 DNA ligase to obtain pTB693 plasmid-containing DNA, which was then transformed into E. coli strain DHI according to a conventional method. For more information,
D for transformation prepared in Reference Example 3 and stored frozen at -70°C
Gradually thaw the H1 bacterial cells under water cooling, and add 100μ of the suspension.
30 ng of pTB693-containing DNA was added to Q. After reacting for 30 minutes under water cooling, heat shock was applied at 42° C. for 90 seconds, followed by water cooling for 1 to 2 minutes. After adding SOB medium containing 0.2 mQ of 2On+M glucose and culturing at 37°C for 1 hour, the suspension was plated on an LB agar plate containing 35 μg/va Q of ampicillin and cultured at 37°C overnight.

As a result, ampicillin-resistant transformed colonies could be obtained from plasmid PTB693.

The above large 111iWD containing plasmid pTB693
H1 strain was transformed into L containing 35 μg/mQ of ampicillin.
After culturing in B medium [Experimental force (1) p. 68) 25OmQ,
Use the plasmid according to the method on page 88 of Experiment J (1)! ! '
-About 300 μg of p”r B 693 was obtained.

Step 2 (Creation of human LT-cDNA) pTB6
93 plasmid 50μ& was incubated at 37°C with 100 units of N5iI and 120 units of BglII (manufactured by Takara Shuzo).
The cells were digested for 1 hour and subjected to 2% agarose gel electrophoresis. N5il-BgQ containing L T -cDNA
A 0.56 Kbp band corresponding to the II fragment was excised and purified using the RDP minicolumn described in step 1.

On the other hand, the following six oligonucleotide chains encoding the N-terminal peptide (1-20) of LT were applied.
Model 380 from Biosystems (USA)
Chemically synthesized using A-DMA 5ynthesizer [[
Tetrahedron Letters
Lett,) J, Vol. 21 (1980), p. 3243]
, 12.5 units of T4 polynucleotide kinase and 1mM ATP were added to the mixture of 1 μm each, and the mixture was incubated at 37°C.
, phosphorylated in 1 hour.

5' end of LT geneLeu Pro Gly Val Gly Leu T
hr Pro Ser Ala-AATTCT ATG
CTCCCT GGT GTT GGCCTCACA
CCT TCA GCT-GA DingACGAG
GGA CCA CAA CCG GAG
TGT GGA AGT CGA-(EcoRI
) (Pvu H) Ala Gln Thr Ala ArgGin 1l
is Pro l, ys MetGCCCAG ACT
GCCCGT CAG CACCCCAAG ATG
CACGG GTCTGA CGG GCA GTC
GTG GGG TTCT (Nsi + ) Synthetic oligonucleotide After inactivation at 70°C for 5 minutes, an additional 350 units of T4
React with DNA ligase for 1 il °C for -night, then incubate at 65 °C.
After inactivation for 5 min at °C, the reaction solution (approximately 3.5μ & DN
45 units of EcoRI (manufactured by Takara Shuzo Co., Ltd.) and 35 units of N5il were added to the mixture (containing A), decomposed at 37°C for 2 hours, and subjected to 10% polyacrylamide gel electrophoresis.

A band corresponding to approximately 70 bp was cut out and purified using an RDP mini column.

The above approximately 70 bp EcoRI-Nsi r fragment 30n
0.56 Kbp Nsil-BgQII fragment 110 in g
ng was added and reacted for 2 hours at 14°C in the presence of 35 units of T4 DNA ligase. The reaction solution was mixed with 6 units of BgQH and 9 units of EcoRI at 37°C for 1 hour.
Time-resolved trimmed 0.63Kbp human LT (L-
We were able to construct a cDNA encoding the entire amino acid sequence of [17]).

Step 3 (made by !l!l of ρTB692 plasmid DNA)
Plasmid ptrp781 of 2μ& 32 units of P
It was decomposed with st■ (manufactured by Takara Shuzo) at 37°C for 1 hour. After the reaction was completed, TNE buffer [Experiment 1 (1) p. 448] and SDS at a final concentration of 0.2% were added, followed by extraction and purification with phenol-chloroform.

The above Pst [0.1mM in digested ptrp7811 μg
Add XTP and 4 units of T4 DNA polymerase, react at 37°C for 5 minutes, then add THE buffer and SD
S was added, and the mixture was extracted and purified with phenol-chloroform.

Next, the phosphorylated BgQ II linker described in step 1
0.2 μg and 35 units of T4 DNA ligase were added to 0.8 fig of the above ptrp781 DNA and reacted overnight at 14°C. After inactivation at 65°C for 5 minutes, the residue was trimmed with 30 units of BgQ II, extracted with phenol-chloroform, and further purified on a Sepharose 4B column. Then 10 units of T4
DNA ligase was added to obtain ρT B 692-containing DNA, which was then transformed into E. coli DHI strain according to the conventional method described in step 1. However, L containing IOμg/mQ of tetracycline instead of 35μg/mQ of ampicillin
Colonies of tetracycline-resistant transformed bacteria were obtained using B agar plates. 10 μg/m
Culture in LB medium containing Q tetracycline, step 1
pTB692 plasmid was obtained according to the method of .

Step 4 (Preparation of pT8694 plasmid D, NA) pTB 692 plasmid DNA described in step 3 10μ
54 units of EcoRI and 30 units of JIII were added to the mixture, and after reacting overnight at 5°C, the mixture was purified by 1% agarose gel electrophoresis and a 3.3 Kbp DNA band was excised. Add 15 ng of the 0.63 Kbp DNA described in Step 82 to this 3.3 Kbp DNA band g, use 10 units of T4 DNA ligase to obtain pTB694-containing DNA, and create a tetracycline-mogenic transformant according to the method described in Step 3. , a plasmid pTB694 for expressing human LT (1-171) could be obtained.

Example 4 As shown in FIG. 2, the plasmid pT described in Example 3
B694 was digested with restriction enzymes EcoRr and N5il to remove the DNA fragment encoding the N-terminal portion of LT.

The large fragment was then given Lys-Lau-Al
Code a-Leu-Thr and Ec with A T G
.

Oligonucleotides containing RI linkers were ligated using T4 DNA ligase. After inactivation at 65°C for 5 min, E
It was trimmed with coRr and subjected to 0.8% agarose gel electrophoresis.

After cutting out the main band corresponding to 3.8 Kbp and purifying it using an RDP mini column, T4 DNA ligase was added and p
The DNA containing T B 773 was then transformed into Escherichia coli strain DH1 according to the conventional method described in Example 3, Step 3, and the PTB773 plasmid could be obtained from the transformant.

Example 5 Human LT (Lys-5er-Ala-Leu-Al
a-Leu-3er-Asp-1 and as shown in FIG.
An oligonucleotide encoding eu-5er-Asp-Lys-Pro and containing an EcoRI linker with ATG was ligated with T4DNA ligase.

Hereinafter, pTB858 plasmid could be obtained using the same method as in Example 4.

Example 7 In the same manner as in Example 4, pT B 694 was treated with the restriction enzyme Ec.
.

Cut between R1 and N5il and encode 5er-Leu-Ala-Leu in the large fragment.
An oligonucleotide containing an EcoRr linker with TG was ligated using T4 DNA ligase. Thereafter, the pT B 775 plasmid could be obtained using the same method as in Example 4.

Example 6゜1 As shown in Figure 4, pT B 694 was digested with restriction enzyme B.
After digestion with gfl1, T4 DNA polymerase was reacted to prepare a DNA fragment encoding the jrp-promoter and the entire amino acid sequence of 1.2 Kbp human LT (1-171,). Then plasmid pUC19 (
(manufactured by Zenshuzo) was cut with Pvull, and the above 1.2Kb
P T B 867 was obtained by adding the p DNA fragment and T 4 D NΔ ligase.

On the other hand, p T B 858 prepared in Example 6 was
Decomposed with RI and ngQrr, 0.6Kbp DNA
I picked up the fragments. Same<pTB867 with EcoRI and B
The above 0.6K b p DNA fragment and T4D N were decomposed into this large fragment 1.
By adding A ligase, pTB864 plasmid could be obtained.

Example 8゜Similarly to Example 4, p T B 694 was limited butyric N5i
After cutting with l, T4 DNA polymerase was reacted.

Next, after digestion with EcoRI, an oligonucleotide encoding Cys-5er-Ala-Leu-Ala and containing an EcoRI linker with ATG was ligated to the large fragment using T 4 DNA ligase. Hereinafter, in the same manner as in Example 4, p T B 8
We were able to obtain 60 plasmids.

Construction of Example 9゜ pT B 864 obtained in Example 7 was digested with EcoRI and Eco 47 III, and the large fragment was coated with Cys-5er-Ala-Leu-Ala and EcoRI linker with ATG. An oligonucleotide containing - was ligated using T4 DNA ligase. Hereinafter, in the same manner as in Example 4, p T B 865
We were able to obtain a plasmid.

Example 1o.

After pTB864 was digested with N5il, it was reacted with T4 DNA polymerase. Next, after decomposing with Eco 4711T to remove small fragments, T4D
The ring was closed by the action of NA ligase. Thereafter, the pT B 866 plasmid was obtained in the same manner as in Example 4.

Example 11. Example 1 of human LT expression in E. coli strains
and plasmid pTB694 created in steps 4-10.
pTB773. pTB775. pTB85g.

pTB864. pTB860. pTB865. p
Large Il described in Reference Example 3 using TB 866! Bacterial DHI
The strain was transformed.

The obtained transformant was cultured in M9-CA medium [Experimental Report (1) 6
(Page 9) The cells were cultured for 4 hours at 37°C in 4mQ, 25 μg/mQ of indole acrylic acid was added, and the culture was continued for an additional 4 hours. After bacterial collection, 0.01% lysozyme and 10
Tris-HCl buffer (pH 7.5) containing % sucrose 0.
The suspension was suspended in 3mQ solution and reacted at 5° C. for 1 hour, followed by ultrasonication for 45 seconds while cooling with water.

The obtained bacterial cell extract was subjected to the L929 cytotoxic activity test described in Reference Example 1, and the LT activity was measured, and the results shown in Table 1 were obtained.

Table 1 L929 1.2〃 1.2X] by DHI Plasmi F p
E. coli strain DHI for transformation described in Reference Example 3 was transformed using TB865. The obtained transformant was cultured by the method described in Example 11, and then disrupted by lysozyme and ultrasound treatment.

A bacterial cell extract containing human LT was obtained.

Next, the extract was diluted with 20mM phosphate buffer (pH 8,0
) was added to a column of DEAE-Sepharose CL-6B (Pharmacia) equilibrated with
A crude purified solution was obtained by elution with the same buffer containing 0.1M NaCQ.

After adjusting the pH of the crude purified liquid to 6.0 using hydrochloric acid.

20mM phosphate buffer containing 0.1M NaCQ (pH 6)
, 0) to a Blue Sepharose CL-6B column, and after thorough washing, add 0.5M NaC (l
Elution was performed with 20mM phosphate buffer (pHLO) containing

Furthermore, the eluate was diluted with 20mM phosphate saline buffer (p +-+
Gel filtration was performed using a Sephacryl S-200 column equilibrated with 7.3), and human L with a specific activity of 5.7xlO'U/mg was
A T specimen was obtained.

Example 13 Human LT created using a peptide synthesizer (Applied System, model 43OA type) using a known solid phase synthesis method
A 10 x 15 mQ aqueous solution of the -C-terminal peptide (152-171) was added to BTG40 x 15 ΦQ water, and after being lightly sonicated, a 2% GLA solution was cooled at 1 mA with water.

It was gently added dropwise and allowed to react for 5 hours. After dialysis with physiological saline three times (3QX3), it was cryopreserved and used as an immunogen.

Peptide-BTG complex 4 wy, / i, 5m Q
Complete adjuvant was added to an equal volume of Freund 1 to a physiological saline solution, and rabbits (male, n=3: 1.3■/1mQ/
Immunization was started subcutaneously on the back and palms of the hind limbs of rabbits.

Booster immunizations were carried out by adding an equal amount of Freund's incomplete adjuvant to the immunogen and inoculating the mice 5 times every 4 weeks.

Blood was collected from the ear vein 7 to 10 days after the final immunization and centrifuged to obtain antiserum.

For production of specific antibodies, the above serum was subjected to salting out and column chromatography according to known methods, and the resulting antibody IgG fraction was purified by affinity chromatography using an insolubilized human LT-cellulofine column. That is, rabbit anti-human LT-1gG was added to a human LT binding column equilibrated with 0.02M borate buffer (p) (8,0) containing 0.15 to 1 NacQ, and washed for +/min.
-'P, 0,02 M glycine-HCl buffer (pH
2, 3) Neutralizing specific antibody LT-Rl which has high affinity for human LT by releasing l8.

LT-R2 and LT-R3 were obtained.

(2) Construction of pTB953 As shown in FIG. 5, pTB864 obtained in Example 7 was cleaved with N5il, and Ba131 (manufactured by Takara Shuzo) was added to degrade and remove the terminal oligonucleotide.

Next, the ends were blunted by reacting with T4 DNA polymerase, and then cut with Eco RI. Phe-5er-Thr-L was added to the obtained DNA large fragment.
An oligonucleotide encoding eu-Lys-Pro and containing an EcoRI linker with TATG was ligated with T4 DNA ligase.

After transforming the colon 1DH1 strain using the obtained plasmid, a bacterial cell extract was prepared according to Example 11. Manufactured by ljl.

Then rabbit anti-human LT-CE peptide antibody LT-
LT-expressing strains were selected by immunoblotting using R2, and the plasmid?
) By preparing NA:A and confirming the nucleotide sequence, we were able to obtain a plasmid pTB953 expressing human hLT [Phe-(25-171)).

Example 1・1 pT B 953 obtained in Example 13 was converted to EcoR[
Lys-5er-A was added to the resulting I]NA large fragment.
la-Leu-Ala-1, eu-5er-Asp-L
An oligonucleotide encoding ys-Pro and containing an ATG-plated EcoRI linker was ligated with T4 DNA ligase. below.

A plasmid was prepared in the same manner as in Example 4, and as shown in FIG. 6, Nhe!
By confirming the presence of the Eco47111 cleavage site, the target pTB1004 plasmid could be obtained.

Example 15 EcoR p T B to04 obtained in Example 14
Cys-5er-A]
, a-16U and containing an EcoRI linker with ATG were ligated using T4 DNA ligase. Hereinafter, a plasmid was prepared in the same manner as in Example 4 to obtain the target pT B 1005 plasmid that does not contain the Nhel cleavage site (see Figure 6) but contains the Eco47■ cleavage site, as shown in Figure 7. I was able to do that.

Example 16 pT B 953 obtained in Example 13 was converted to EcoRI
and Pvull, and then the obtained DNA
In the large fragment, Cys-5er-Gly-P
he-Leu-Gly-5er-Leu-Lys-Pr
An oligonucleotide encoding EcoRI o and containing an EcoRI linker with ATG was ligated using T4 DNA ligase. Hereinafter, plasmid 3A was prepared in the same manner as in Example 4.
As shown in FIG. 8, the target pT B 1006 plasmid containing the Acc■ cleavage site could be obtained.

Example 17 The residue p T B 867 obtained in Example 7 was converted to Eco
Cys-5er-
Gly-Phe-Leu-Gly-5er-Leu-L
EcoRI with ys-Pro code and ATG
Oligonucleotides containing linkers were ligated using T4 DNA ligase. A plasmid was then prepared in the same manner as in Example 4, and the desired PT B 1007 plasmid containing the Acc■ cleavage site as shown in FIG. 9 was obtained.

Example 18 C+ of human LT in E. coli strain Plasmid pTBIO0 created in Examples 14-17
4, pTBlo05. pTBlo06. pTB
An extract of an E. coli transformant was prepared by the method described in Example 11 using 1007. Next, an L929 cytotoxic activity test was conducted according to the method described in Reference Example 1, and the results shown in Table 2 were obtained.

Example 19 Novel Human LT Mutin Essence (1) Human LT (21-171) Expression Vector pT
Construction of B 622 Plasmid pTB 618 prepared above was digested with restriction enzyme N
Cut with 5iT (manufactured by Takara Shuzo) and Bam HI, LT
1.1 kilobase pairs (hereinafter abbreviated as Kbp) containing the gene
DNA fragments were isolated. T4 to this DNA
After reacting with DNA polymerase (PL Inc.) to blunt the ends, add the 16-mer so that they fit in frame.
EcoRI linker with ATG (AACATGAAT
TCATGTT) were ligated by T4 DNA ligase. After T4 DNA ligase was inactivated by heat treatment at 65°C for 10 minutes, it was further cleaved with the restriction enzyme EcoRI, and a 0.6 Kbp DNA fragment containing the linker-bound LT gene was separated by agarose electrophoresis.

On the other hand (Kurokatsu, T. et al., “Nucl, Ac1cls Re.
5) Cut the plasmid ρtrp781 described in Vol. 411 (1983) p. 3077 with the restriction element EcoR,
The 5' terminal phosphate was removed by alkaline phosphatase treatment. This DNA was mixed with a 0.6 Kbp) LT DNA fragment linked with an EcoRI linker with ATG, and treated with T4 DNA ligase to generate human LT expression in E. coli in which the LT gene was inserted downstream of the tryptophan promoter. Vector pTB622 was constructed.

Escherichia coli strain DHI was transformed using plasmid PTB622 prepared as described above.

The obtained transformant was cultured in M9-CA medium [Experimental Report (1) 6
(page 9) Incubate in 2.5Q at 37°C for 4 hours, add 25 μg/m Q f of indole acrylic acid, and further incubate 4 hours.
After culturing for 8 hours, the bacteria were collected using a Tris-HCl buffer (pH 7,
5) Suspended in room Q, reacted at 5°C for 1 hour, and treated with ultrasonic waves for S minutes under water cooling.
DEAE-Sepharose CL equilibrated with ) (8,0)
-6B (Pharmacia) column. After washing with the same buffer, it was eluted with the same buffer containing 0.1M NaCQ to obtain a crude 1-Nl solution with a specific activity of 7.8X]O'tJ/■.

After adjusting the above crude purified liquid to pH 6.0 using hydrochloric acid,
The column was added to a Blue Sepharose CL-6B column equilibrated with 5mM phosphate buffer (pH 6,0) containing IMNaCQ, washed thoroughly, and eluted with 5mM phosphate buffer (pH 8,0) containing 0.5M NaCQ. The specific activity of the same eluate was 7.4 x 10' U/mg.

Furthermore, this eluate was added to 5mM phosphate buffer (PH7,3
) to obtain a purified solution with a specific activity of 1.6 x 10'U/■.

(2) Production of mouse anti-human LT monoclonal antibody Add an equal amount of complete Freund's adjuvant to the 200 μg/m Q physiological saline solution of the above human LT purified sample, thoroughly emulsify it, and prepare for BALB/c mice (♀, n= 10:
After 64 booster immunizations, the same LT antigen was administered intraperitoneally and dorsally to 20ALg10.2 m Q/mouse), and boosters were given at 3-week intervals. The solution (50 μg 10.1 mQ physiological saline/mouse) was administered intravenously.

Three days after the final immunization, the spleen was removed, and a suspension of malignant cells was prepared using a conventional method (approximately 10" cells). Then, 2X 10" mouse myeloma cells (P3Ul) were added, and PEG600 was added.
Köhler and Milstein's method using 0 [Nature, 256.495 (+975)
) was used for cell fusion.

After the fusion was completed, the cell mixture was suspended in a so-called HAT medium containing hypoxanthine, aminopterin, and thymidine, and cultured for 10 days. Thereafter, as soon as the selection of parent cells was completed, the HAT medium was replaced with HT medium in which aminopterin was removed and culture was continued.

The antibody titer of the hybridoma culture supernatant was measured by ELISA using a microplate in which purified human LT was adsorbed onto a solid phase. 10 to 20 days after the fusion, the appearance of hybridoma Q 1 tg and antibodies that bind to human LT were observed. Hybridomas with particularly strong binding activity were subjected to cloning using the limiting dilution method.

Similarly, the culture supernatant of the cloned hybridoma was subjected to EL.
They were subjected to screening using the ISA method and those with strong 2-human LT binding ability were selected. Regarding these, please refer to Reference Example 1
The ability to neutralize the activity of human LT was measured using the L929 cytotoxic activity test described above. That is, human L T
An equal amount of hybridoma culture supernatant was added to 100 units/mQ, and after reacting at 37°C for 1 hour, it was subjected to an L929 cytotoxic activity test.

As a result, Mo Ab-producing hybridoma LT that binds to human LT and neutralizes its cytotoxic activity
3-11 and LT3-135 were obtained. These immunoglobulin classes and subclasses were determined to be IgG2b and IgG2a, respectively, by the Aucteronie method.

The cloned hybridomas were incubated at 37°C in Iscove-Ham mixed medium (I-H medium) containing 10% FC8.
The cells were cultured using an incubator at % CO□S, and antibodies were obtained from the supernatant.

On the other hand, in order to obtain a large amount of antibodies, 0.5+o9 mineral oil was administered intraperitoneally to BALB/c mice.
×10′ hybridomas were inoculated intraperitoneally. about 10
- Ascites accumulation was observed after 15 days.

The antibody was purified by a conventional method by fractionation with 45-50% saturated ammonium sulfate, followed by DEAE-cellulose and protein A column chromatography.

(3) An E. coli transformant was prepared by the method described in Example 11 using pT B g60 obtained in Example 8.

Furthermore, a bacterial cell extract containing human LT was obtained. Next, this crude extract was salted out with 40% saturated ammonium sulfate, and then
0mM phosphate buffer - 1mM dithiothreitol (pH
8,0).

(4) Immunoaffinity lamb obtained by binding the mouse anti-human LT monoclonal antibody LT3-11 to formylcellulofine (Seikagaku Corporation) by column chromatography was mixed with 20mM phosphorus buffer-0.15mM Na C1-5mME DT
After equilibration with A (p I 48.0), the bacterial cell extract obtained in (3) was added and thoroughly washed with the same slow TL solution. The human L'F adsorbed on the column was dissolved in 20mM phosphate buffer-0,15mM NaCl-5mME DT.
Dialyzed with A (PI(8,0)).

(5) Symptoms by gel filtration column chromatography After concentrating the human LT-containing solution eluted from the above antibody column by ultrafiltration, 20d phosphate buffer -〇, I5mM
It was purified and separated using a Sephacryl S-200 column equilibrated with NaCl-5dE DTA (pH 8,0).

All operations (3) to (5) were performed at low temperatures (5 to 10°C). 3.3 μ of purified human LT preparation was obtained from bacterial cells weighing 142 g wet.

Example 20 Human LT-Human Transferrin H Dingf Compound (1) Maleimidized hTf 8 times the mole of GMBS dissolved in dimethylformamide (DMF) was added to 5 mg of commercially available hTf and reacted at 30°C for 1 hour. Ta. The reaction mixture was then diluted with O, 1M phosphate buffer (
The mixture was applied to a Sephadex G-25 column equilibrated at pH 6.5) to remove unreacted maleimidation reagent. The resulting maleimidized hTf contained 4.7 molecules of maleimide groups per molecule of hTf.

(2) Human LT-hTf compound obtained in Example 19 (Cys-5er-
Ala-Leu-Ala-(22-171)) l m
g to 1 ml of 0.1M phosphate buffer - 5mM EDTA
(pH 6.5), and then 1 mg of maleimidized hTf obtained in (1) was added. After reacting at 4°C overnight, human LT-hTf was purified and separated using a Sephacryl S-200 column equilibrated with io+nM phosphoric acid solution.
1.5 μ of composite was obtained.

(3) Human LT-hTf A at various concentrations using 966 microplates.
Add 1.0 x 109 tumor cells to 50 μl of hTf complex-containing solution.
and cultured at 37°C for 3 days. After completion of the culture, the number of viable cells was measured by the method described in Reference Example 1 to evaluate the cytotoxic activity of the composite.6 The results were as shown in Table 3. Human L T-hT
Compound f1 exhibited cytotoxicity against human LT-resistant, human transferrin receptor (hTfR)-positive tumor cells.

Example 21 Human LT-hTfR' complex Δ (1) Box rfRO and layer weight.

A 41.5 kg human placenta was cut into small pieces and placed in PBS (PH7).
, 5) and then centrifuged. The obtained precipitate was homogenized in PBS containing 4% Triton x-ioo, and further centrifuged after sonication. Next, approximately 32 g of ammonium sulfate was added per supernatant Loom Q, and after salting out, it was applied to an anti-hTf antibody binding column at 0.5M.
It was thoroughly washed with NaCl-containing PB (pH 7.5). 0.5M N a C1 and 0.5% I Noton X
-100 containing 0.02M glycine buffer (Pi(]0.
The hTfR fraction eluted with 0) was further applied to a binding column, washed with PB containing 1M NaCl, and then subjected to IMNaC.
h by elution with a 0.05M glycine equilibration solution (PH1O,0) containing 1% l- and 1% l-lydone X-100.
Approximately 1.5 μ of a purified TfR sample was obtained.

(2) i-mutual hair rat rattan steep hill It was carried out according to the method described in Example 19-(2).

(3) Hybridoma selection and cloning 20 μg/mQ of a commercially available rabbit anti-mouse rgG antibody solution was dispensed into 96-well microplates in 100 μα portions, left overnight at 4°C, and further added to PBS containing 2% BSA (pH 7).
, 3) was added to prepare a sensitized plate. Also (1)
The hTfR purified sample obtained in
Used for ISA [Tsunehiro Kitayo: Organic Synthetic Chemistry, 42, 28
3 (1984)). That is, hybridoma culture supernatant was added to the second antibody-sensitized plate, and after reacting at room temperature for 2 hours, the plate was washed with PBS. Then I(RP mark i hTfR
was added, and the reaction was further allowed to proceed at room temperature for 2 hours.

After washing the plate, an O, 1M citric acid buffer containing orthophenylenediamine and ■(yo02) as enzyme substrates was added, and the enzyme reaction was carried out at room temperature.

After stopping the reaction with IN sulfuric acid, multiscan (manufactured by Flow)
The amount of coloring dye was measured at a wavelength of 492 nm.

Hybridomas with particularly strong binding ability were cloned by limiting dilution method, and anti-hTfR antibody-producing hybridoma 2206 was obtained. The subclass of this antibody is rgG
562 showed high affinity for human tumor cell lines.

(4) Maleimidized antibody Mouse anti-hTfR monoclonal antibody 2206 manufactured by f was maleimidized according to the method described in Example 2O-(1). A composite in which approximately 7.7 maleimide groups were introduced per antibody molecule was obtained.

(5) Preparation of human LT-hTfR body Δ product Example 2O-
(2) According to the method described, 1.5 mg of maleimidized antibody
Sephacryl S-20 was reacted with human I, T 1■.
human LT-anti-hTfR antibody complex],
, 9+ng was purified and isolated.

(6) Detailed investigation of human LT-hTfRA According to the method described in Example 2O-(3), human LT-hTfRA
The cytotoxic activity of the T f R antibody conjugate was measured.

The results were as shown in Table 3. human LT-anti-hT
The fR antibody conjugate was cytotoxic to LT-resistant, hTfR-positive tumor cells.

pTB1006 LT (Cys-5er-Gly-P
he-Leu-Gly-5er-pTBl(107LT(Cys-5er-Gly-Ph
e-Lpu-Gly-5er- Leu-Lys-Majesty 1.1XIO'1.2X10'1.2X10' +0001.4
X10' 1.0X104 1.2XlO'1.3XlO' 1081.6
×10' TB694 LT (1-171) pTB1004 LT (Lys-5er-Ala-L
au-A 1a-173u-5er-Asr(28-1
71)] pTBIO05LT (Cys-5er-Ala-1iu
-Ala-IJ3u-5er- Asp-(28-171)) 1.1X10"1.2X]0" 1.2X]021 1.3χ102 1.6X10' 1.6X10S1.7X10514172.0X10S 2.1χ10' 2. 4X1032.5XIO" 213.0XI
O' human LTL) 0.8 ng/ml 3.1 ng/ml 12.5 ng/ml 2, Ong/ml 8, Or+g/ml Th7fR antibody 5.0 ng/ml 22C62 20. Ong/ml Tf human LT-hTf i), 8 ng,/ml complex 3.1 ng/ml +2.5 ng/ml human IT-anti-OJ n1ml hTfR antibody 3.1 nF,/ml complex 12
．． 5 ng/ml Cys-5er-Ala-Leu-Al
a-(22-171)): Described in Example 19.

') Described in Example 21.

Converted to Lihito L T amount.

') K562: human leukemia cells (hTfR positive)
, LU99: human lung cancer cells (hTfR positive), P
388: Mouse leukemia cells (hTfR negative).

Plasmids described herein ρTB61g, pTB
622. p tr p781. pTB773.
pTB775. pTB858. pTB+360.
pTB864. pTB865 and PTB86
DHI strain or C600 strain (E, col
i D H1/ p T B 618. E,coLi
C600/ p T B622. E. coli D
H1/ptrp781. E, coliDHl/p
TB773. E. coli DHL/p TE10
1°E, coli DHI/pTB858. E,co
li DHL/pTB860. E. coli DH
] / p T B864. E. coli DH1/
pTB865. E. coli DHI/pTB865
．． E.

coli DHI/pTB953. E.coli
DH1/pTB1004. E. coli DHL/
pTE1005. E. coli DH1/p T
E1006. E. coli DHI/pTBlo07
) and animal cells (mouse-mouse hybridoma LT
3-11 and mouse-mouse hybridoma 22C6
) are listed in Table 4.

Microorganisms and animal cells E, coli DHI/pTB618 E, coLi C600/p TB6221 Shioki DH
I/Pt So 781 E, coli DHI/pTB773 臥9其DHI/PTB775 臥其DH1,/pTB858 Table 4 IFOFERM (IFONo,) (FERM No,) 14542
BP-1587 14544BP-1589 14546BP-1591 14732BP-1832 14733BP-1833 14734BP-1834 E, coli I) Hl/pTB860E, coli
D Hl/p T B 864E, colj D
H1/P TE86513, coli DH1/p
TB8f56E, coLi DH1/p TE953
B, coLi DH1/P TB1004E, col
i DHI/pTB1005E, coli D Hl
/ p T B 1006E, coli DHI/pT
B1007'louse-Mouse hybrid
ow LT3-11Mouse-Mouse hybrid
idoia 22C61li 737 1,4738 BP-1835 BP-1836 BP-1837 BP-1,838 BP-2282 BP-2283 BP-2284 BP-2285 BP-2286 BP-1813 BP-2054 TFO: Foundation Fermentation Research Institute (Osaka) ) FRI: Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Microbial Technology Research Institute

[Brief explanation of the drawing]

Figure 1 shows recombinant plasmid pTB694, and Figure 2 shows recombinant plasmid pTB773. Figure 3 shows recombinant plasmid pTB858, Figure 4 shows recombinant plasmid PT.
Figures 5.6, 7.8 and 9 are schematic diagrams showing the assembly of B864, respectively.
B 953, p TB 1004, p TB 1
005, pTB1006 and pTB 1.007
The 5'-terminal DNA sequence encoding the human LT mutin carried by the LT mutin and the amino acid sequence derived therefrom are shown. The abbreviations in each figure and their meanings are as follows. B: BaQI Ba: Bam HI Bg: BgQ 11 C: CQaT E : Eco RI H: HindlU N: N5il P or Ps: Pst I Pv: Pvu II S: 5aQI X: Xhol BI: BgQ I E47: Ec. ■

Claims (1)

[Scope of Claims] (1) A protein having the following amino acid sequence or a part of its active portion. [There is a gene sequence. ] [In the formula, R_1 is Cys, Lys, Ser, Cys-S
er or Lys-Ser, R_2 is Ala-Leu-A
la, Leu-Ala-Leu, Leu-Ala-Le
u-Thr, Ala-Leu-Ala-Leu, Ala
-Leu-Ala-Leu-Ser-Asp-Lys-
Pro, Ala-Leu-Ala-Leu-Ser-A
sp, Gly-Phe-Leu-Gly-Ser, Gl
y-Phe-Leu-Gly-Ser-Leu-Lys
-Pro or Gly-Phe-Leu-Gly, R_3
is Ala-Ala-Gln-Thr-Ala-Arg-
Gln-His-Pro-Lys-Met-His-L
It represents a peptide represented by eu-Ala-His-Ser-Thr-Leu or a part thereof, m represents 0 or 1, and n represents 0 or 1. (2) A polydeoxyribonucleic acid characterized by containing a base sequence encoding the following amino acid sequence and a polydeoxyribonucleic acid containing a sequence complementary thereto. H-(Met)n-R_1-R_2-(R_3)m-L
ys-Pro-Ala- [Gene sequence is available. ] [In the formula, R_1 is Cys, Lys, Ser, Cys-S
er or Lys-Ser, R_2 is Ala-Leu-A
la, Leu-Ala-Leu, Leu-Ala-Le
u-Thr, Ala-Leu-Ala-Leu, AIa
-Leu-Ala-Leu-Ser-Asp-Lys-
Pro, Ala-Leu-Ala-Leu-Ser-A
sp, Gly-Phe-Leu-Gly-Ser, Gl
y-Phe-Leu-Gly-Ser-Leu-Lys
-Pro or Gly-Phe-Leu-Gly, R_3
is Ala-Ala-Gln-Thr-Ala-Arg-
Gln-His-Pro-Lys-Met-His-L
It represents a peptide represented by eu-Ala-His-Ser-Thr-Leu or a part thereof, m represents 0 or 1, and n represents 0 or 1. (3) A polydeoxyribonucleic acid obtained by substituting one or more of the bases according to claim 2 without changing the amino acid sequence according to claim 1 based on the degeneracy of the genetic code. (4) A polydeoxyribonucleic acid comprising the polydeoxyribonucleic acid according to claim 2 and a sequence complementary thereto. Alternatively, a replicable recombinant DNA comprising the polydeoxyribonucleic acid according to claim 3 and a replicable vector. (5) A microorganism or cell transformed with the replicable recombinant DNA according to claim 4. (6) The polydeoxyribonucleic acid according to claim 2 and the polydeoxyribonucleic acid comprising a sequence complementary thereto, or the polydeoxyribonucleic acid according to claim 3, is incorporated into a replicable vector, and the polydeoxyribonucleic acid is Obtaining a replicable recombinant DNA containing deoxyribonucleic acid, transforming microorganisms or cells with the obtained recombinant DNA to obtain a transformant containing the recombinant DNA, and culturing the obtained transformant. 2. A method for producing the protein or a part of its active portion according to claim 1, which comprises expressing genetic information possessed by the polydeoxyribonucleic acid.