JP2979321B2 - New recombinant lymphotoxin - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel bioactive polypeptide useful as an anticancer agent and a polydeoxyribonucleic acid (hereinafter abbreviated as DNA) having its genetic information.

The present invention also relates to a replicable recombinant DNA containing the DNA, a microorganism or a cell transformed with the replicable recombinant DNA, and a novel physiologically active polysaccharide obtained by expressing the genetic information of the DNA. The present invention relates to a peptide and a method for producing the peptide.

Amino acids and peptides herein are IUPAC-I
It is indicated by the abbreviation adopted by the UB Biochemical Nomenclature Committee (CBN). For example, the following abbreviations are used. In addition, when there is an optical isomer with respect to an amino acid or the like, the L-form is indicated unless otherwise specified.

Gln: Glutamine residue Asp: Aspartic acid residue Pro: Proline residue Tyr: Tyrosine residue Val: Valine residue Lys: Lysine residue Glu: Glutamic acid residue Ala: Alanine residue Asn: Asparagine residue Leu: Leucine residue Group Phe: phenylalanine residue Gly: glycine residue His: histidine residue Ser: serine residue Thr: threonine residue Ile: isoleucine residue Trp: tryptophan residue Arg: arginine residue Met: methionine residue Cys: Cysteine residue In the present specification, a polymer or oligomer of DNA is represented by the following abbreviation sequence.

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

[Conventional technology]

Lymphotoxin (hereinafter abbreviated as LT) is a protein having an antitumor activity derived from lymphocytes, and is expected to have clinical applications together with macrophage-derived cancer necrosis factor (hereinafter abbreviated as TNF). These LT and TNF were obtained by adding and activating endotoxin and phorbol ester to lymphocytes and macrophages, respectively. The use of the encoded gene makes it possible to produce the protein in a microorganism or cell culture. Gray et al. [Nature, 312 (1984) 72]
1) and Pennica et al. [Neture, 312 (19
(1984), p. 724], respectively, succeeded in cloning using this gene manipulation technique.

[Problems to be solved by the invention]

With the advancement of these genetic technologies, LT and TNF are now
Has become possible for mass production, and has been used in clinical applications. However, the results of many clinical trials obtained to date, especially the clinical data on TNF, are not always good [Tetsuo Taguchi: Cancer and Chemotherapy, 13 , 3491 (1986), Ichiro Urushizaki et al .: Oncologia] , 20 , 105 (1987), A.Kl
ansne: BioTech., 5 , 335 (198
7)]. The biggest problem is the occurrence of serious side effects, and the administration of TNF has the disadvantage that fever, chills, shivering, blood pressure decrease, etc., and therefore, it is not possible to stop administration and prevent tumor growth . The cause of this is that TNF is not as excellent as the tumor selectivity was initially thought to be, and TNF receptors are actually found on many normal cells and fibroblasts [Yojiro Niitsu: Terapoitic・ Research (Therapeu.Res.), 7 , 275 (1987)], one that responds to TNF has been found.

Although clinical results on LT have not been as extensive as those on TNF, they have a very similar chemical structure to TNA and also bind to a common receptor on cells (BYRubin, et al .: Journal of Experimental Medicine (J.Exp.Med.), 16
2 , 1099 (1985)] also shows non-specific cytotoxicity to non-tumor organs, tissues, and cells, and it is possible that the same side effect as TNF is caused.

As described above, it is difficult to obtain clinical effects as expected from TNF and LT itself, and application to new formulations containing new modifications and combination therapy is awaited.

On the other hand, as a drug that selectively kills tumor cells, an antitumor immune complex in which an anticancer antibody is bound to a chemotherapeutic agent or a biotoxin, a so-called “missile therapeutic agent” has been disclosed. They have the property of recognizing and binding to tumor-specific antigens or tumor-associated antigens on tumor cells, and stopping the DNA or protein synthesis of these tumor cells, resulting in death. Therefore, there are already clinically applied antibody-drug or antibody-toxin conjugates which are specific to tumor organs / tissues / cells and have few side effects on normal cells. It is the current situation.

Under these technical backgrounds, the present inventors have developed a novel immunotoxin by binding to an anticancer antibody to make LT, which is a human-derived toxin having a strong cytotoxic effect, more tumor-specific. This research was advanced for the purpose of development. There were two major problems in producing such immunotoxins. One is that in the case of conventional LT or LT mutin, there is a large possibility that the antibody binds to the active site on the molecule, and during the binding process, the biological activity of LT itself is impaired. Even if the immunotoxin is synthesized in some retained form, the toxin moiety, that is, the LT molecule, is not released from the antibody after being taken up into the tumor cells, and is therefore transferred to the intracellular target site. Is difficult, and its activity expression is expected to be extremely small.

[Means for solving the problem]

In order to solve the above problems, research on a novel recombinant LT mutein that binds to the antibody at the inactive site of the LT molecule and easily releases the LT molecule from the antibody after being taken into tumor cells will be conducted. As a result, a novel LT mutein in which an amino acid or a peptide having a functional group for antibody binding and a lysozol enzyme-sensitive linker peptide are combined at the N-terminal of the N-terminal-deleted LT was prepared, and the present invention was developed. It is completed.

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

Wherein R ₁ is Csy, Lys, Ser, Cys-Ser or Lys-Ser, R ₂
Is Ala-Leu-Ala, Leu-Ala-Leu, Leu-Ala-Leu-Thr, A
la-Leu-Ala-Leu, Ala-Leu-Ala-Leu-Ser-Asp-Ly
s-Pro, Ala-Leu-Ala-Leu-Ser-Asp, Gly-Phe-Leu
-Gly-Ser, Gly-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-Leu-Ala-His
It represents a peptide represented by -Ser-Thr-Leu or a part thereof, wherein m represents 0 or 1, and n represents 0 or 1. In the above formula, R ₁ is an amino acid residue or a peptide chain having a functional group for antibody binding, R ₂ is a lysosomal-sensitive linker peptide chain, and R ₃ is an N-terminal portion of the N-terminal-deleted LT. , Or all or part of the amino acids No. 10 to No. 27 of LT represented by

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

The present invention relates to a replicable recombinant DNA capable of expressing a polypeptide containing the above amino acid sequence in a transformed microorganism or cell. As such recombinant DNA,
For example, pTB773, pTB775, pTB858, pTB860, pTB864, pTB1
004, pTB1005, pTB1006, pTB1007 and the like.

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

Further, the present invention provides a method comprising expressing a gene encoding a novel LT mutein gene in a microorganism or a cell.
Pertaining to the method of manufacturing. For more information, see Replicatable recombinant DNA
The present invention relates to a method for producing the polypeptide, which comprises growing a microorganism or a cell transformed by the method described above and recovering the peptide efficiently.

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

1. m-RN from human peripheral lymphocytes induced by LT synthesis with 12-O-tetradecanoylphorbol-13-acetate (TPA) and concanavalin A (ConA) in a known manner.
A can be collected, and then about 5 × 10 ⁵
A cDNA library can be created.

2. Usually 10-mer to 5 which encodes a partial peptide chain of LT
A 0-mer oligonucleotide is synthesized and used as a probe to screen LT cDNA. For example, C
When using a terminal 18-mer (TCCAAAGAAGACAGTACT) synthetic nucleotide, about 50 clones can be obtained.

3. Isolate the plasmid from the resulting LT cDNA clone and determine the nucleotide sequence. A plasmid encoding the previously reported LT amino acid sequence is selected, cut with an appropriate restriction enzyme, and appropriately introduced into an expression vector to prepare a recombinant DNA containing the DNA.

4. Transformation of various hosts, for example, Escherichia coli, using the vector prepared in 3. can obtain a strain having a DNA encoding LT.

5. After culturing the transformant in step 4 and isolating the plasmid, the DNA encoding the novel LT mutein is
Can be prepared.

In the case of the human LT gene, a restriction enzyme is located in the region encoding methionine-histidine at positions 20-21 from the N-terminal side.
There is an Nsi I recognition site. Therefore, a DNA fragment encoding the N-terminal LT can be obtained by cutting the LT gene with NsiI. To this fragment, the following adapter sequence and an amino acid or a peptide for antibody binding and a DNA encoding an amino acid sequence containing a linker peptide were ligated to prepare a DNA encoding the novel LT mutein,
Insert into the appropriate vector.

Similarly, in the case of the human LT gene, 9-terminal from the N-terminal side
Restriction enzyme at the 10th serine-alanine coding region
There is a Pvu II recognition site. Then, the LT gene can be cut with Pvu II to obtain a DNA fragment encoding an N-terminal deleted LT. This fragment is ligated with an adapter sequence and a DNA encoding an amino acid sequence containing an amino acid or peptide for antibody binding and a linker peptide to prepare a DNA encoding the novel LT mutein, and insert an appropriate vector. .

The DNA fragment encoding the N-terminal deletion LT after digestion with Pvu II or Nsi I is further subjected to an exonuclease, for example, a nuclease BAL31 to remove a region encoding 1 to 18 amino acids, an adapter sequence therefor, and antibody binding. The amino acid or peptide and the DNA encoding the amino acid sequence containing the linker peptide were ligated. By selecting a gene in which the reading frame of the gene is properly maintained, LT (x-171), an amino acid or peptide for antibody binding and a linker peptide linked to the N-terminal part of x = 10-28 are selected. DNA encoding a new LT mutein
Can be prepared.

In addition, site-directed mutagen
esis) [Smith, M. and Gillam, S, "Genetic Engineering", 3 , 1 (198
1)] can also be applied. That is, Pvu II
The DNA fragment encoding the N-terminal-deleted LT after cleavage was inserted into
13 and inserted into E. coli JM103 (Pharmacia P-L Bioc).
hemicals). After growth, the M13 phage released into the broth was precipitated with polyethylene glycol,
Then, M13 phage single-stranded DNA can be obtained by phenol treatment.

Next, a part of the amino acid chain or peptide in the peptide chain from the 10th alanine to the 27th leucine on the N-terminal side of LT is deleted, and an amino acid or peptide for antibody binding and a linker peptide are added to the N-terminal thereof. A DNA encoding the polypeptide having the polypeptide can be prepared by chemical synthesis and used as a primer. This primer and the previously prepared M13 phage DNA are mixed, and double-stranded by the action of DNA polymerase I large fragment.
It can be circularized by the action of T4 DNA ligase. After this circular DNA was introduced into E. coli JM103, and the released come M13 phage DNA was transferred to a filter, ³² P
Hybridization [Maniatis, T. et al., Molecular Cloning, Laboratory Manual (Molecu
lar Cloning, A Laboratory Manual), Cold Spring H
arbor Laboratory, P.312 (1982)]. A DNA encoding a novel LT mutein modified by preparing DNA from a phage in which a strong signal is detected, cutting out the DNA with an appropriate restriction enzyme, and incorporating this DNA fragment into a plasmid.
Is obtained.

Plasmid DN containing all or part of human LT gene
A, cosmid DNA, phage DNA and the like can be used as template DNA for specific site-directed mutation. M13 phage and φX174 phage are more preferable as template DNA because single-stranded DNA can be easily prepared. For example, when using M13 phage or φX174 phage in which the whole or part of the human LT gene is incorporated, the phage particles present in the broth are precipitated with polyethylene glycol, deproteinized by phenol treatment, and then ethanol. Precipitation is performed to obtain single-stranded DNA in the phage particles. In addition, double-stranded plasmid DNA or cosmid D
When NA is used, the double-stranded DNA can be heat-treated at 100 ° C. for 1 to 10 minutes, preferably 3 to 5 minutes, and then rapidly cooled with ice water to denature the single-stranded DNA before use.

Primers for site-directed mutations have the DNA sequence to be converted and hybridize with the template DNA.
Any DNA sequence may be used as long as it can function as a primer during DNA synthesis. The primer may be prepared by any method, but it is preferable to prepare a single-stranded DNA having an appropriate sequence by chemical synthesis. Mixing such primers with the previously prepared single-stranded DNA, DNA
2 by the action of polymerase I large fragment
After the DNA is repaired, the DNA can be circularized by the action of T4 DNA ligase. After introducing this circular DNA into Escherichia coli, plaque hybridization (Ma) was performed using a primer labeled with a radioisotope as a probe.
niatis, T. et al., Molecular Cloning, A Labora
tory Mannual) ", Cold Spring Harbor Laboratory, P.3
12 (1982)] or colony hybridization [p. 326], and the desired mutant can be selected. Phage DNA and plasmid DNA are prepared from the plaques and colonies thus obtained, and a novel LT mutein gene can be prepared using the DNA.

Code the new LT mutein obtained in this way
By inserting DNA into the 3 'end of the promoter region that functions in various hosts (eg, Escherichia coli, Bacillus subtilis, yeast, animal cells), it is possible to construct a recombinant DNA capable of expressing a novel LT mutein-encoding DNA. it can.

The promoter region may be any region containing a site necessary for initiating mRNA synthesis by binding of RNA polymerase.

For example, when Escherichia coli is used as a host, a recombinant DNA capable of expressing a novel LT mutein can be constructed by inserting a DNA encoding a novel LT mutein into the 3 'end of a promoter region capable of functioning in Escherichia coli. In addition, pB is used as a vector when E. coli is used as a host.
R322, pBR325, ptrp 781, pUC8, pUC9, pUC19, pJB8, etc. are used, and the DNA encoding the novel LT mutein is added to T4 DNA
Inserted by the action of ligase. Using this reaction solution,
E. coli (eg, C600, MM294, DH1, W3110, RR1, P
R13 strain etc.) by a known method [Cohen, SN et al., "Proceding of National Academy of Science (Proc. Natl. Acad. Sci. USA)" 69 , 2110 (197)
2)] or by a method analogous thereto.

The promoter used is the trp promoter (trp-
need not be limited to p), for example, recA promoter [Japanese Patent 59-65099], lac promoter, and the like may be used .lambda.P _L promoter.

The transformant carrying the novel recombinant plasmid DNA containing the DNA encoding the novel LT mutein obtained as described above can be selected as a phenotype, for example, ampicillin resistance, tetracycline resistance, or both drug resistances.

The above transformant is cultured in a medium known per se.
As the medium, for example, L-broth, Penassay
M-9 medium containing broth, glucose and cadamino acid [Miller, J., "Experiments in Molecular Genetic"
s) ", 431-433 (Cold Spring Harbor Laboratory, New
York, 1972)]. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently.

The culture of the transformant is usually performed at 15 to 43 ° C, preferably at 28 to 43 ° C.
The reaction is carried out at 40 ° C. for 2 to 24 hours, preferably 4 to 16 hours, and if necessary, ventilation or stirring can be applied.

For example, when an animal cell is used as a host, a DNA encoding a novel LT mutein is inserted into the 3 ′ end of a region of a promoter (eg, SV40 promoter) capable of functioning in the animal cell, and the method is carried out by a method known per se. By transforming a host with the recombinant DNA and culturing the transformant, a novel LT mutein can be produced.

For example, when Bacillus subtilis or yeast is used as a host, a DNA encoding a novel LT mutein is inserted into the 3 'end of a promoter region capable of functioning in Bacillus subtilis or yeast, and the recombinant DNA is prepared by a method known per se. By transforming a host and culturing the transformant, a novel LT mutein can be produced.

 Among the above hosts, Escherichia coli is more preferred.

After the culture, the cells are collected by a known method, and in the case of a transformant of Escherichia coli, the cells are suspended in an appropriate buffer, for example, Tris-HCl buffer (pH 7.5), sonicated, and treated with lysozyme and lysozyme. A method of obtaining a supernatant containing a novel LT mutein by centrifugation after disrupting cells by freeze / thaw and / or freeze-thawing may be used as appropriate. Preferably, the cells are collected, suspended in a buffer, lysozyme is added thereto, and the mixture is incubated at 0 to 10 ° C for 10 minutes to 3 hours, and after sonication at 0 to 10 ° C for 30 seconds to 5 minutes,
A method of obtaining a supernatant by centrifugation is used.

Separation and purification of the novel LT mutein from the extract include gel filtration, hydroxyapatite column chromatography, ion exchange column chromatography, ultracentrifugation,
It can be performed by affinity chromatography using a human LT antibody.

As the amino acid or peptide having a functional group for antibody binding contained in the N-terminal of the novel LT mutein of the present invention, Cys, Lys, Ser, Cys-Ser or Lys-Ser is used. In the case of Cys and Cys-Ser, it is bound to the antibody via the sulfhydryl group contained. Especially human LT
In the case of (1), since Cys is not contained in the molecule, the antibody can be very specifically bound to the antibody via the N-terminal portion which is an inactive site of LT. In the case of Lys and Lys-Ser, it can be bound to the antibody via the contained α- and ε-amino groups. In the case of Ser, Cys-Ser and Lys-Ser, they can be bound to the antibody through the contained hydroxyl group.

Various known methods can be used for binding of these antibodies to human LT [VP Butler: Pharmacological Review (Pharmacol. Rev.), 29 , 103 (1978), Kitagawa Tsunehiro: Synthetic Organic Chemistry, 42 , 283 (1984)] For example, after an anti-cancer antibody is modified with N-succimidyl-pyridyl-dithiopropionate (hereinafter abbreviated as SPDP), it is added to LT mutein containing Cys at the N-terminus of the present invention, and a thiol exchange reaction is performed. The anticancer antibody is maleimidated with N- (γ-maleimidobutylyloxy) -succinimide (hereinafter abbreviated as GMBS), and then added to LT mutein containing Cys at the N-terminus of the present invention to form a thioether. For LT muteins containing a Lys at the N-terminus to form a complex by bonding
After modification with PDP, reduce and add to maleimidated anti-cancer antibody to form a complex with thioether bond. Similarly, LT mutein containing Lys at the N-terminus is modified and reduced with SPDP, SPD
It is conceivable to add a P-modified anticancer antibody to form a complex by a thiol exchange reaction. In these methods, the antibody fragment F (a
b ′) ₂ , Fab ′ or Fab can also be used. It is also possible to form a complex by completely reversing the treatment of the anti-cancer antibody and the LT mutein.

Next, as the lysozole enzyme-sensitive linker peptide contained in the N-terminal of the novel LT mutein of the present invention, Ala-L
eu-Ala, Leu-Ala-Leu, Leu-Ala-Leu-Thr, Ala-Leu
-Ala-Leu, Ala-Leu-Ala-Leu-Ser-Asp-Lys-Pro
And Gly-Phe-Leu-Gly and the like, and any known lysosomal enzyme-sensitive peptide chain may be used. In particular, the above-mentioned Ala-Leu-Ala, Leu-Ala-Leu, Al
A linker containing a-Leu-Ala-Leu or Gly-Phe-Leu-Gly is effective and preferably used for release into a drug after being taken into cells (HB.Rihova et al .: Clinical. Immunology and Immunopathology (Cli
n.Immunol.Immunopathol.), 46 , 100 (1988), A. Trouet
et al .: Proceedings of National Academy of Science (Proc.Natl.Acod.Sci.US
A), 79 , 626 (1982)].

As described above, the present inventors have obtained a novel LT mutein gene, and have described a method for producing a novel LT mutein using this gene. However, the present invention is not limited thereto.

In the present invention, a part or all of the base sequence is replaced with an organically synthesized artificial DNA without changing the amino acid sequence, for example, because the frequency of use of codons (genetic code) corresponding to each amino acid is different. Is also possible.

The novel LT mutein of the present invention has an amino acid or peptide at the N-terminal portion not involved in the activity that can be used for binding to an antibody, and further includes a lysosomal enzyme-sensitive linker peptide following the sequence. Therefore, upon binding to an anti-cancer antibody, it is possible to form an immune complex without impairing the LT's natural biological activity, and after being taken up by tumor cells, the LT molecule is affected by intracellular lysosomal enzymes. Is expected to be released from the antibody and exhibit its cytotoxicity effectively. These properties are extremely advantageous for preparing a tumor-selective anticancer drug, and are effective for enhancing the action of LT and reducing side effects.

Hereinafter, the present invention will be described in detail with reference to Reference Examples and Examples, but needless to say, these do not limit the scope of the present invention.

In the practice of the present invention, the production of recombinant DNA and the introduction of the recombinant into microorganisms were carried out according to the following experiments unless otherwise specified.

(1) T. Maniatis, EFFritsch, J. Sambrook, "Molecular Cloning", Cold Spr
(2) Yasutaka Takagi, edited by Yasutaka Takagi, “Gene manipulation experiment method”, published by Kodansha Reference Example 1. Evaluation of cytotoxicity of L929 cells The cytotoxicity of LT was measured using L929 cells [Journal of Immunology J. Immunol.), 126 (1981) 235
Page] or [J. Immunol. Methods, 70 (1984) p. 257]
The measurement was carried out according to the above method. That is, using a 96-well tissue culture microplate (Flow Laboratories), the above medium containing 4 μg / m of mitomycin C in 50 μl of a sample that was serially diluted 2-fold with RPMI 1640 medium containing 10% fetal calf serum (FCS). L929 cells at a concentration of 4 × 10 ⁵ cells / m
And cultured in 5% carbon dioxide gas at 37 ° C. for 48 hours. After completion of the culture, live cells are stained with dimethylthiazoyl / diphenyltetrazolium bromide (MTT), and 10% SDS-0.01N
After dissolving with HCl, the absorbance at 590 nm was measured by Titertec Multiscan (Flow Laboratories). The resulting absorbance is proportional to the number of viable cells. The amount of biological activity required to kill 50% of L929 cells was defined as 1 unit / m and the biological activity of the sample was expressed in units / m.

Reference Example 2. Preparation of Escherichia coli Strain for Transformation A colony of Escherichia coli DH1 was transferred to an SOB medium [Experiment (1) 69
And the culture was cultured until the absorbance at 550 nM reached 0.5.
Collect the culture _{30m, 0.1M RbCl-10mM CaCl 2} -5 of 12m
0.2 M acetate buffer containing 0 mM MnCl ₂ -15% glycerol (p
H5.8), centrifuged for 5 minutes on ice, and then 10 mM RbCl
Resuspended in 10 mM MOPS buffer (pH 6.5) containing -75 mM CaCl ₂ -15% glycerol. After ice cooling for 15 minutes, the mixture was rapidly cooled with dry ice-ethanol and stored at -70 ° C.

Reference Example 3. Preparation of Escherichia coli Strains for Transformation
Culture was performed using 10 m until the absorbance at 550 nm reached 0.3. After cooling on ice, the cells obtained by centrifugation were centrifuged at 5m and 10mM NaCl.
And washed. Resuspend cells in 5m 50mM CaCl ₂ and cool on ice
Left for 15 minutes. After centrifugation, the cells were suspended in 0.5 mM 50 mM CaCl ₂ and used immediately.

Example 1. Preparation of cDNA library using mRNA derived from human lymphocytes TPA (15 ng / m) was prepared from lymphocytes prepared from human peripheral blood.
1640 medium containing 10% FCS and ConA (40 μg / m)
) At 37 ° C to induce LT. Twenty-four hours later, the induced 1 × 10 ¹⁰ human lymphocytes were treated with 5 M guanidine thiocyanate, 5% mercaptoethanol, 50 mM
After destruction and denaturation with a Teflon homogenizer in Tris · HCl pH 7.6, 10 mM EDTA solution, sodium N-lauroyl sarcosinate was added to 4%, and the homogenized mixture was added to a 5.7 M cesium chloride solution (5.7 M cesium chloride solution). , 0.1M
EDTA) was overlaid on 6 m, and centrifuged at 15 ° C. at 24,000 rpm for 48 hours using a Beckman SW28 rotor to obtain an RNA precipitate. This RNA precipitate was dissolved in a 0.25% sodium N-lauroyl sarcosinate solution and precipitated with ethanol to obtain 10 mg of RNA. Transfer this RNA to a high salt solution (0.5M NaCl,
The oligo (dT) cellulose column is adsorbed in 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3% SDS), and the mRNA containing poly (A) is reduced to a low salt solution (10 mM Tris-HCl pH 7.6, 1 mM EDTA).
By eluted with 0.3% SDS), 300 μg of mRNA containing poly (A) was collected.

This mRNA was further precipitated with ethanol, dissolved in a 0.2 mM solution (10 mM Tris · HCl pH 7.6, 2 mM EDTA 0.3% SDS), treated at 65 ° C. for 2 minutes, and centrifuged at 10-35% sucrose gradient (Beckman). 20 ° C, 25,00 using SW28 rotor
(Centrifugation at 0 for 21 hours). A portion of the NRA for each of these fractions was injected into Xenopus oocytes, the LT activity in the synthesized protein was measured, and the LT activity was detected in the fraction corresponding to a precipitation constant around 16S. . The LT mRNA in this fraction was about 25 μg.

Using this poly (A) RNA as a template, a cDNA library
The method of kayama and Berg [Molecular and Cellular Biology (Mol. Cell. Biol.), 2 (1982) 161]
P.D., p.3 (1983), p. 280] using the pcDV1 vector and pL1 linker. The vector plasmid containing the circularized cDNA was infected with E. coli DH1 and
Starting from g of poly (A) RNA, a cDNA library comprising about 5 × 10 ⁵ clones and using Escherichia coli DH1 as a host was obtained.

Example 2 Isolation of Plasmid Containing Human LTcDNA and Determination of Its Base Sequence The above human cDNA library using Escherichia coli DH1 was placed on a nitrocellulose filter (Millipore, HATF filter) at about 3 × 10 ⁴ clones / filter. To be 1
0 sheets were sowed, and a total of 20 replica filters were prepared, each set including two filters each having the filter as a master filter. 0.5N E. coli on this replica filter
Plasmid DNA denatured by exposure to NaOH solution was dried and fixed on a filter [Grunstein, M, & Hogness, D, S.,
Proc. Of National Academy of Science, Proc. Natl. Acad. Sci. USA, 72 (1975) p. 3961].

On the other hand, the already reported base sequence of LT gene [Gra
y, et al., Nature, 312 (1984) 721
Page] (the gene portion corresponding to amino acids NO. 162-167) Was synthesized and used as a screening probe for human LTcDNA.

The 5 'end of these oligonucleotide probes was converted to ³² P using T4 polynucleotide kinase, [γ- ³² P] ATP.
Labeled with.

Labeled probes were separately associated with replica filters on which DNA was immobilized. The association reaction was performed using 5 × SSC (0.15 M NaCl, 0.015 M sodium citrat) containing 10 μCi of a labeled probe.
e), 5 × Denhardt's, 0.1% SDS, 100 μg / m denatured salmon sperm D
The reaction was performed in 10m of NA solution at 40 ° C for 16 hours. After the reaction, the filter was washed three times with a 6xSSC, 0.1% SDS solution at room temperature for 30 minutes each, and twice at 43 ° C for 60 minutes. [Experiment book (1) 309
page〕. Radioautograms were taken from the washed filters, and strains that reacted with the probe were searched for by superposing radioautograms of one set of two replica filters. According to this method, 50 E. coli DH1 strains which react with the probe were obtained from about 3 × 10 ⁵ colonies.

Plasmid DNA was isolated from these strains by the alkaline method [Birnb
oim, HC & Doly, J., “Nucleic Acids Res., 11 (1979), p. 1513]
Was extracted and purified. After cutting the DNA with the restriction enzyme BamHI (Takara Shuzo) and fractionating by agarose gel electrophoresis,
The DNA fragment was transferred from the agarose gel onto a nitrocellulose filter (S & S, BA85) [Southern blotting method, Experimental Manual (1), p. 382]. When this filter was associated with the above-mentioned oligonucleotide probe, the plasmid DNA fragment reacted with the probe.

Therefore, one strain E. coli having a plasmid in which the largest BamH I DNA fragment (cDNA portion) was generated was used. coli K12 DH1
/ pTB618 was selected. The nucleotide sequence of the cDNA portion of this plasmid DNA was determined by the dideoxynucleotide synthesis chain termination method (J. Me.
ssing et al., “Nucleic Acids Research (Nucleic
ic Acids Res.), 9 (1981), p. 309].

As a result, it was found that the LT gene contained in the plasmid pTB618 was not complete but contained the 3rd terminal untranslated portion up to the third C of the codon CCC of Pro, which is the amino acid of No. 18 on the upstream side.

Example 3 Construction of Human LT (1-171) Expression Vector pTB69 Step 1 (Preparation of pTB693 Plasmid DNA) As shown in FIG.
The digestion was carried out at 37 ° C. for 6 hours using 6 units of Bal I (Takara Shuzo).

On the other hand, Bgl II linker 2 represented by 8-mer of CAGATCTG
after phosphorylation with 0.5 mM ATP and 25 units of T4 polynucleotide kinase, the phosphorylated linker 0.2 μg
Was added to 1.6 μg of the above Bal I digested pTB618 and 35 units of
The reaction was performed overnight at 14 ° C. in the presence of T4 DNA ligase. After inactivation at 65 ° C. for 5 minutes, trimming was performed using 30 units of Bal II, and subjected to 1.2% agarose gel electrophoresis.

The main band corresponding to 4.3 Kbp was cut out, extracted with a Tris / hydrochloric acid buffer, and then purified with an RDP mini column (Bio-Rad). To 100 ng of the linear DNA was added 10 units of T4 DNA ligase to obtain pTB603 plasmid-containing DNA, which was then transformed into Escherichia coli DH1 according to a conventional method. Specifically, the DH1 strain for transformation prepared in Reference Example 3 and cryopreserved at -70 ° C was gradually thawed under ice-cooling, and 30 ng of DNA containing pTB693 was added to 100 µ of the suspension. After reacting for 30 minutes under ice-cooling,
A heat shock was applied for 2 seconds and ice-cooled for 1-2 minutes. 0.2m
Of SOB medium containing 20 mM glucose was added, and the mixture was cultured at 37 ° C. for 1 hour. The suspension was treated with L containing 35 μg / m ampicillin.
The seeds were spread on a B agar plate and cultured overnight at 37 ° C. As a result, colonies of ampicillin-resistant transformants could be obtained from the plasmid pTB693.

The above Escherichia coli DH1 strain containing the plasmid pTB693 was
LB medium containing μg / m ampicillin [Experiment (1) 68
After culturing in 250 m, the plasmid was isolated according to the method described in Experimental Manual (1), p. 88, to obtain about 300 μg of pTBN693.

Step 2 (Preparation of human LT-cDNA) 50 μg of the pTB693 plasmid was added to 100 units of NsiI and 120
It was digested with a unit Bgl II (Takara Shuzo) at 37 ° C. for 1 hour, and subjected to 2% agarose gel electrophoresis. A 0.56 Kbp band corresponding to the Nsi I-Bgl II fragment containing LT-cDNA was cut out and purified using the RDP mini column described in Step 1.

On the other hand, the following six oligonucleotide chains encoding the N-terminal peptide (1-20) of LT were used in Applied Biosystems
Chemical synthesis using Model 380A-DMA synthesizer (US) [[Tetrahedron Lett.
t.) ", 21 (1980), p. 3243], and adding 1 μg of each mixture to 12.5 units of T4 polynucleotide kinase and 1 mM.
ATP was added and phosphorylated at 37 ° C. for 1 hour.

After inactivation at 70 ° C. for 5 minutes, further react with 350 units of T4 DNA ligase at 14 ° C. overnight, then inactivate at 65 ° C. for 5 minutes and after completion of the reaction (containing about 3.5 μg of DNA), EcoRI (Takara Shuzo) 45 units and 35 units of NsiI were added, digested at 37 ° C. for 2 hours, and subjected to 10% polyacrylamide gel electrophoresis.

A band corresponding to about 70 bp was cut out, and the RDP mini column was purified.

30 ng of the above 70 bp EcoR I-Nsi I fragment was added with 0.56 Kbp of Nsi.
110 ng of the I-Bgl II fragment was added and reacted at 14 ° C. for 2 hours in the presence of 35 units of T4 DNA ligase. The reaction completed solution was digested with 6 units of Bgl II and 9 units of EcoRI at 37 ° C. for 1 hour to prepare a cDNA encoding the entire amino acid sequence of human LT (1-171) of 0.63 Kbp. .

Step 3 (Preparation of pTB692 Plasmid DNA) 2 μg of the plasmid ptrp781 was digested with 32 units of Pst I (Takara Shuzo) at 37 ° C. for 1 hour. After completion of the reaction, a TNE buffer (Experimental Manual (1), p. 448) and SDS having a final concentration of 0.2% were added, and the mixture was extracted and purified with phenol-chloroform.

To 1 μg of the PstI digested ptrp781 was added 0.1 mM XTP and 4 units of T4 DNA polymerase. After reaction at 37 ° C. for 5 minutes, a TNE buffer and SDS were added, followed by extraction and purification with phenol-chloroform.

Next, 0.2 μg of the phosphorylated Bgl II linker described in Step 1
And 35 units of T4 DNA ligase and ptrp781 DNA
The reaction was carried out overnight at 14 ° C. in addition to 0.8 μg. After inactivation at 65 ° C. for 5 minutes, phenol was trimmed with 30 units of Bgl II.
Extracted with chloroform and further purified on a Sepharose 4B column. Thereafter, 10 units of T4 DNA ligase was added to obtain pTB692-containing DNA, and then transformed into Escherichia coli DH1 according to the usual method described in Step 1. However, using an LB agar plate containing 10 μg / m tetracycline instead of 35 μg / m ampicillin, a colony of a tetracycline-resistant transformant was obtained. The colonies were cultured in an LB medium containing 10 μg / m tetracycline, and the pTB692 plasmid was obtained according to the method in Step 1.

Step 4 (Preparation of pTB694 Plasmid DNA) To 10 μg of pTB692 plasmid DNA described in Step 3, add 54 units of EcoR I and 30 units of Bgl II, react at 5 ° C. overnight, and purify by 1% agarose gel electrophoresis to obtain 3.3 Kbp DNA.
The band was cut out. To 36 ng of the 3.3 Kbp DNA, 15 ng of the 0.63 Kbp DNA described in Step 2 was added, and a pTB694-containing DNA was obtained with 10 units of T4 DNA ligase. A tetracycline-resistant transformant was prepared according to the method described in Step 3, and human LT (1-17
1) The expression plasmid pTB694 was obtained.

Example 4 Construction of Human LT [Lys-Leu-Ala-Leu-Thr- (20-171)] Expression Vector pTB773 As shown in FIG. 2, plasmid pT described in Example 3
B694 was digested with restriction enzymes EcoR I and Nsi I to remove the DNA fragment encoding the N-terminal part of LT. The large fragment then encodes Lys-Leu-Ala-Leu-Thr and A
Oligonucleotide containing EcoRI linker with TG
Attached by A ligase. After inactivation at 65 ° C for 5 minutes, E
It was trimmed with coRI and subjected to 0.8% agarose gel electrophoresis.

After excising the main band corresponding to 3.8 Kbp and purifying using an RDP mini column, T4 DNA ligase was added to add pTB773-containing DNA.
I got Then, Escherichia coli DH1 strain was transformed according to the usual method described in Example 3, Step 3, and the transformant was used to transform pTB77.
Three plasmids were obtained.

Example 5 Construction of Human LT [Ser-Leu-Ala-Leu (19-171)] Expression Vector pTB775 As in Example 4, pTB694 was replaced with restriction enzymes EcoR I and Nsi I.
And Ser-Leu-Ala was added to the large fragment.
Oligonucleotides encoding Leu and containing an EcoRI linker with ATG were ligated by T4 DNA ligase. Hereinafter, the pTB775 plasmid could be obtained in the same manner as in Example 4.

Example 6 Human LT [Lys-Ser-Ala-Leu-Ala-Leu-Ser-Asp-Ly
s-Pro- (10-171)] Construction of Expression Vector pTB858 As shown in FIG. 3, pTB694 was ligated with restriction enzymes EcoRI and Pvu.
II and the large fragment was added to Lys-Ser-
Oligonucleotides encoding Ala-Leu-Ala-Leu-Ser-Asp-Lys-Pro and containing an EcoRI linker with ATG
Ligation was performed with 4 DNA ligase. Hereinafter, the pTB858 plasmid could be obtained in the same manner as in Example 4.

Example 7 Human LT [Lys-Ser-Ala-Leu-Ala-Leu-Ser-Asp-Ly
s-Pro- (10-171)] Construction of Expression Vector pTB864 As shown in FIG. 4, after pTB694 was digested with the restriction enzyme BglI, T4 DNA polymerase was reacted, and the trp-promoter and 1.2 Kbp of human LT (1 A DNA fragment encoding the entire amino acid sequence of -171) was prepared. Then the plasmid pUC19
(Manufactured by Takara Shuzo) was digested with Pvu II, and the above 1.2 Kbp DNA fragment and T4 DNA ligase were added to obtain pTB867.

On the other hand, pTB858 prepared in Example 6 was digested with EcoRI and BglII, and a 0.6 Kbp DNA fragment was isolated. Also pTB867
Decompose with EcoR I and Bgl II, add the above 0.6 Kbp DNA fragment and T4 DNA ligase to this large fragment and add pTB
864 plasmid could be obtained.

Example 8 Construction of Human LT [Cys-Ser-Ala-Leu-Ala- (22-171)] Expression Vector pTB860 As in Example 4, pTB694 was cut with a restriction enzyme NsiI, and
4 DNA polymerase was reacted. Then, after digestion with EcoRI, the large fragment was added to CyS-Ser-Ala-Leu.
Oligonucleotides encoding Ala and containing an EcoRI linker with ATG were ligated by T4 DNA ligase.
Thereafter, the pTB860 plasmid was obtained in the same manner as in Example 4.

Example 9.Human LT [Cys-Ser-Ala-Leu-Ala-Leu-Ser-Asp-Ly
s-Pro- (10-171)] Construction of Expression Vector pTB865 The pTB864 obtained in Example 7 was digested with EcoRI and Eco47III, and the large fragment thereof was added to Cys-Ser-Ala-Leu.
Oligonucleotides encoding Ala and containing an EcoRI linker with ATG were ligated by T4 DNA ligase. Thereafter, the pTB865 plasmid was obtained in the same manner as in Example 4.

Example 10 Construction of Human LT [Lys-Ser-Ala-Leu-Ala- (22-171)] Expression Vector pTB866 After pTB864 was digested with NsiI, T4 DNA polymerase was reacted. Next, after digestion with Eco 47 III to remove small fragments, T4 DNA ligase was allowed to act to close the circle. Thereafter, the pTB866 plasmid was obtained in the same manner as in Example 4.

Example 11. Expression of human LT in E. coli strain Plasmid pTB694, p prepared in Examples 1 and 4-10
The E. coli DH1 strain described in Reference Example 3 was transformed using TB773, pTB775, pTB858, pTB864, pTB860, pTB865, and pTB866.

The obtained transformant was transformed into an M9-CA medium [Experimental Manual (1) 69)
Page] Incubate at 37 ° C for 4 hours in 4m, and indole acrylic acid
25 μg / m was added, and the culture was continued for another 4 hours. After collection, the cells were suspended in 0.3 m of Tris / hydrochloric acid buffer (pH 7.5) containing 0.01% lysozyme and 10% sucrose, reacted at 5 ° C. for 1 hour, and then subjected to ultrasonic treatment under ice-cooling for 45 seconds.

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

Example 12. Purification of a novel human LT mutein Plasmid pTB8 prepared as described in Example 9.
The E. coli strain DH1 for transformation described in Reference Example 3 was transformed using 65. The obtained transformant was cultured by the method described in Example 11, and then disrupted by lysozyme and ultrasonic treatment to obtain a cell extract containing human LT.

Next, DEAE-Sepharose CL-6B (Pharmacia) was prepared by equilibrating the extract with a 20 mM phosphate buffer (pH 8.0).
Was washed with the same buffer, and eluted with the same buffer containing 0.1 M NaCl to obtain a crude purified solution.

After adjusting the crude solution to pH 6.0 using hydrochloric acid, 01M NaC
l to a Blue Sepharose CL-6B column equilibrated with 20 mM phosphate buffer (pH 6.0), washed thoroughly, and
Elution was performed with a 20 mM phosphate buffer (pH 8.0) containing aCl.

Further, the eluate was subjected to gel filtration with a Sephacryl S-200 column equilibrated with a 20 mM phosphate buffer (pH 7.3) to obtain a human LT preparation having a specific activity of 5.7 × 10 ⁶ U / mg.

Example 13 Construction of Human LT [Phe- (25-171)] Expression Vector pTB953 (1) Production of Rabbit Anti-human LT-C-terminal Peptide Antibody A peptide synthesizer (Applied System, Modem 430A) by a known solid phase synthesis method. LT-C created using
A 10 mg / 5 m aqueous solution of the terminal peptide (152-171) was added to BTG 40 mg / 5
2% GLA solution 1m
The mixture was dropped gently under ice-cooling and reacted for 5 hours. After dialysis three times with physiological saline (3 × 3), the solution was cryopreserved and used as an immunogen.

An equal volume of Freund's complete adjuvant was added to a peptide-BTG complex 4 mg / 1.5 m physiological saline solution, and rabbits (ウ サ ギ, n
(= 3.1.3 mg / 1 m / rabbit) on the back and in the subcutaneous palm. Booster immunizations were performed by adding an equal volume of incomplete Freund's adjuvant to the immunogen and inoculating 5 times every 4 weeks.

Seven to ten days after the final immunization, blood was collected from the ear vein and centrifuged to obtain an antiserum.

For production of a specific antibody, the above serum was subjected to salting out and column chromatography in accordance with a known method, and the obtained antibody IgG fraction was purified by affinity chromatography on an insolubilized human LT-Cellulofine column. That is, a rabbit anti-human LT-IgG fraction is added to a human LT binding column equilibrated with a 0.02 M borate buffer (pH 8.0) containing 0.15 M NaCl, and the column is sufficiently washed. (P
By eluting with H 2.3), neutralizing specific antibodies LT-R1, LT-R2 and LT-R3 having high affinity for human LT were obtained.

(2) Construction of pTB953 As shown in FIG. 5, pTB864 obtained in Example 7 was replaced with Nsi
After cleavage with I, Bal 31 (manufactured by Takara Shuzo) was added to decompose and remove the terminal orionucleotide. Then, T4 DNA polymerase was reacted to blunt the ends, and then cut with EcoRI. Phe-Ser-Thr-Leu was added to the resulting large DNA fragment.
Oligonucleotides encoding Lys-Pro and containing an EcoRI linker with ATG were ligated by T4 DNA ligase.

After transforming Escherichia coli DH1 strain using the obtained plasmid, a cell extract was prepared according to Example 11. Next, LT-expressing strains were selected by immunoblotting using a rabbit anti-human LT-C-terminal peptide antibody LT-R2, and the plasmid DNA was prepared from those expression-positive strains and the nucleotide sequence was confirmed. Phe- (25-17
1)] can be obtained plasmid pTB953.

Example 14 Human LT [Lys-Ser-Ala-Leu-Ala-Leu-Ser-Asp-
(28-171)] Construction of Expression Vector pTB1004 The pTB953 obtained in Example 13 was cleaved and digested with EcoR I and Pvu II.
Oligonucleotides encoding ys-Ser-Ala-Leu-Ala-Leu-Ser-Asp-Lys-Pro and containing an EcoRI linker with ATG were ligated by T4 DNA ligase. Thereafter, a plasmid was prepared in the same manner as in Example 4, and the presence of Nhe I and Eco47 III cleavage sites derived from the synthetic oligonucleotide was confirmed as shown in FIG. 6 to obtain the desired pTB1004 plasmid. Was completed.

Example 15 Human LT [Cys-Ser-Ala-Leu-Ala-Leu-Ser-Asp-
(28-171)] Construction of Expression Vector pTB1005 pTB1004 obtained in Example 14 was cleaved and decomposed with EcoRI and Eco47III, and the resulting large DNA fragment was encoded with Cys-Ser-Ala-Leu. EcoR I with ATG
The oligonucleotide containing the linker was ligated with T4 DNA ligase. Thereafter, a plasmid was prepared in the same manner as in Example 4, and as shown in FIG. 7, the desired pTB containing the Eco47 III cleavage site without the Nhe I cleavage site (see FIG. 6).
1005 plasmid could be obtained.

Example 16 Human LT [Cys-Ser-Gly-Phe-Leu-Gly-Ser- (27-1
71)] Construction of Expression Vector pTB1006 pTB953 obtained in Example 13 was cleaved and digested with EcoRI and PvuII, and the resulting large DNA fragment
Oligonucleotides encoding ys-Ser-Gly-Phe-Leu-Gly-Ser-Leu-Lys-Pro and containing an EcoRI linker with ATG were ligated by T4 DNA ligase. Thereafter, a plasmid was prepared in the same manner as in Example 4 to obtain a target pTB1006 plasmid containing an Acc III cleavage site as shown in FIG.

Example 17 Human LT [Cys-Ser-Gly-Phe-Leu-Gly-Ser-Leu-Ly
s-Pro (10-171)] Construction of Expression Vector pTB1007 The pTB867 obtained in Example 7 was cleaved and digested with EcoR I and Pvu II, and the obtained DNA large fragment was further digested with C
Oligonucleotides encoding ys-Ser-Gly-Phe-Leu-Gly-Ser-Leu-Lys-Pro and containing an EcoRI linker with ATG were ligated by T4 DNA ligase. Thereafter, a plasmid was prepared in the same manner as in Example 4 to obtain a target pTB1007 plasmid containing an Acc III cleavage site as shown in FIG.

Example 18 Expression of human LT in E. coli strains Plasmids pTB1004, pTB100 created in Examples 14-17
Using pTB1006 and pTB1007, an extract of Escherichia coli transformant was prepared by the method described in Example 11. Next, an L929 cytotoxicity test was carried out by the method described in Reference Example 1, and the results shown in Table 2 were obtained.

Example 19 Purification of New Human LT Mutein (1) Construction of Human LT (21-171) Expression Vector pTB622 The plasmid pTB618 prepared above was replaced with the restriction enzyme NsiI.
(Manufactured by Takara Shuzo) and BamHI to isolate a DNA fragment of 1.1 kilobase pairs (hereinafter abbreviated as Kbp) containing the LT gene. The DNA was reacted with T4 DNA polymerase (manufactured by PL) to blunt the ends, and then 16-mer
EcoRI linker with ATG (AACATGAATTCATGTT) to T4DNA
It was bound by ligase. T4 DNA ligase at 65 ° C 10
After inactivation by heat treatment for more
The fragment was cut with oRI, and a 0.6 Kbp DNA fragment containing the LT gene to which the linker was bound was separated by agarose electrophoresis.

On the other hand, the plasmid ptrp781 described in [Kurokawa, T. et al., “Nucl. Acids Res”, Vol. 11, (1983), p. 3077] was digested with a restriction enzyme EcoRI.
The 5 'terminal phosphate was removed by alkaline phosphatase treatment. This DNA is mixed with a 0.6 Kbp LT DNA fragment to which an EcoRI linker with ATG is bound, and the DNA is ligated with TA DNA ligase to cause a downstream of the tryptophan promoter.
Escherichia coli human LT expression vector pT into which the LT gene has been inserted
B622 was built.

Escherichia coli DH1 was transformed using the plasmid pTB622 prepared as described above.

The obtained transformant was transformed into an M9-CA medium [Experimental Manual (1) 69)
Page] Incubation in 2.5 ° C at 37 ° C for 4 hours, addition of 25 µg / m of indoleacrylic acid, and continued culturing for 4 hours.
After collection, the cells were suspended in 100 m of Tris / hydrochloric acid buffer (pH 7.5) containing 0.01% lysozyme and 10% sucrose, reacted at 5 ° C. for 1 hour, and then subjected to ultrasonic treatment under ice cooling for 5 minutes.

Then, after centrifuging the extract, 5 mM phosphate buffer (pH
8.0) and added to a column of DEAE-Sepharose CL-6B (Pharmacia) which had been smoothed in 8.0). 0.1M after washing with the same buffer
Elution was performed with the same buffer solution containing NaCl to obtain a crude purified solution having a specific activity of 7.8 × 10 ⁵ U / mg.

The crude solution was adjusted to pH 6.0 with hydrochloric acid, then 0.1M Na
After adding to a Blue Sepharose CL-6B column smoothed with 5 mM phosphate buffer (pH 6.0) containing Cl and washing well,
and eluted with 5 mM phosphate buffer (pH 8.0). The specific activity of the eluate was 7.4 × 10 ⁶ U / mg.

Further, the eluate was subjected to gel filtration with a Sephacryl S-200 column smoothed with 5 mM phosphate buffer (pH 7.3),
A purified solution having a specific activity of 1.6 × 10 ⁷ U / mg was obtained.

(2) Production of Mouse Anti-Human LT Monoclonal Antibody After adding an equal amount of Freund's complete adjuvant to the above-mentioned purified human LT 200 μg / m physiological saline solution and sufficiently emulsifying,
BALB / c mice (♀, n = 10: 20 μg / 0.2 m / mouse) were intraperitoneally and subcutaneously administered to the back and boosted at three-week intervals. After the four booster immunizations, the same LT antigen solution (50 μg / 0.1m saline / mouse) was intravenously administered to the individual showing the maximum serum antibody titer at 2 weeks.

The spleen was excised 3 days after the last immunization, were prepared by a conventional method Spleen cell suspension (about 10 ^8). Then, 2 × 10 ⁷ mouse myeloma cells (P3U1) were added, and the method of Koehler and Milstein [Nature, 256 ,
495 (1975)].

After the completion of the fusion, the cell mixture was suspended in a so-called HAT medium containing hypoxanthine / aminopterin and thymidine and cultured for 10 days. After that, as soon as the selection of parent cells is completed, HA
The culture was continued by replacing the T medium with the HT medium from which aminopterin was removed.

The antibody titer of the hybridoma culture supernatant was measured by an ELISA method using a microplate having purified human LT adsorbed on a solid phase. 10 to 20 days after the fusion, the appearance of hybridomas was observed, and the appearance of antibodies binding to human LT was observed. Particularly, hybridomas having a strong binding activity were subjected to cloning by the limiting dilution method.

The culture supernatant of the cloned hybridoma is
It was subjected to screening by the ISA method, and one having strong human LT binding ability was selected. For these, the ability to neutralize human LT activity was further measured using the L929 cytotoxicity test described in Reference Example 1. That is, human LT100 unit / m
An equivalent amount of the hybridoma culture supernatant was added thereto, and the mixture was reacted at 37 ° C. for 1 hour, and then subjected to an L929 cytotoxicity test.

As a result, MoAb-producing hybridomas LT3-11 and L3 that bind to human LT and neutralize their cytotoxic activity
T3-135 was obtained. These immunoglobulin classes
Subclass as determined by the Ouchterlony method, were respectively IgG ₂ b and IgG ₂ a.

The cloned hybridoma was cultured at 37 ° C. in 5% CO 2 in an Iscove-ham mixed medium (I-H medium) containing 10% FCS.
_The cells were cultured using a _two- concentration incubator, and an antibody was obtained from the supernatant.

On the other hand, in order to obtain a large amount of antibodies, 5 × 10 ⁶ hybridomas were intraperitoneally inoculated into BALB / c mice to which 0.5 m mineral oil had been administered intraperitoneally in advance. Approximately 10-15 days later, ascites accumulation was seen.

Purification of the antibody was carried out by fractionation with 45-50% saturated ammonium sulfate, followed by DEAE-cellulose and protein A column chromatography by a conventional method.

(3) Preparation of Cell Extract A transformant of Escherichia coli was prepared using pTB860 obtained in Example 8 by the method described in Example 11, and a cell extract containing human LT was obtained. Next, the crude extract was subjected to salting out treatment with 40% saturated ammonium sulfate, and then dialyzed using 20 mM phosphate buffer-1 mM dithiothreitol (pH 8.0).

(4) Purification by antibody column chromatography An immunoaffinity column obtained by binding mouse anti-human LT monoclonal antibody LT-311 to formyl cell fine (manufactured by Seikagaku Corporation) was converted to a 20 mM phosphate buffer-0.15 mM NaCl- After equilibration with 5 mM EDTA (pH 8.0), the bacterial cell extract obtained in (3) was added, and the cells were sufficiently washed with the same buffer. The human LT adsorbed on the column was dialyzed against 20 mM phosphate buffer-0.15 mM NaCl-5 mM EDTA (pH 8.0).

(5) Purification by gel filtration column kalmatography After concentration of the human LT-containing solution eluted from the above antibody column by ultrafiltration, 20 mM phosphate buffer-0.15 mM NaCl-5
Purification and separation were performed on a Sephacryl S-200 column equilibrated with mM EDTA (pH 8.0).

All the operations (3) to (5) were performed at a low temperature (5 to 10 ° C). 3.3 mg of purified human LT preparation was obtained from 42 g of wet cells.

Example 20 Preparation of human LT-human transferrin (hTf) complex (1) Preparation of maleimidated hTf To 5 mg of commercially available hTf, 8-fold molar GMBS dissolved in dimethylformamide (DMF) was added and reacted at 30 ° C. for 1 hour. . Next, the reaction mixture was applied to a Sephadex G-25 column equilibrated with a 0.1 M phosphate buffer (pH 6.5) to remove unreacted maleimidation reagent. The resulting maleimidated hTf is hTf
4.7 maleimide groups were introduced per molecule.

(2) Preparation of human LT-hTf complex The human LT obtained in Example 19 [Cys-Ser-Ala-Leu-Al
a- (22-171)] 1 mg was added to 1 ml of 0.1 M phosphate buffer-5 mM ED.
It was dissolved in TA (pH 6.5), and then 1 mg of maleimidated hTf obtained in (1) was added. After overnight reaction at 4 ° C., 15 mg of a human LT-hTf complex purified and separated by a Sephacryl S-200 column equilibrated with a 10 mM phosphate buffer was obtained.

(3) Cytotoxic activity of human LT-hTF complex Using human 96-well microplate, various concentrations of human LT-hT
f Add 1.0 × 10 ⁴ tumor cells to 50 μl of the complex-containing solution, and add 3
The cells were cultured at 7 ° C for 3 days. After completion of the culture, the number of living cells was measured by the method described in Reference Example 1 to evaluate the cytotoxic activity of the composite.

The results were as shown in Table 3. The human LT-hTf conjugate was cytotoxic to human LT-resistant, human transferrin receptor (hTfR) positive tumor cells.

Example 21 Preparation of human LT-anti-hTfR antibody complex (1) Purification of hTfR 1.5 kg of human placental tissue was finely cut, blended in PBS (PH7.5), and then centrifuged. 4% of the obtained sediment
After homogenizing in Triton X-100-containing PBS, the mixture was further sonicated and then centrifuged again. Then about 100m of supernatant
After adding 32 g of ammonium sulfate and salting out, the mixture was applied to an anti-hTf antibody binding column, and sufficiently washed with PB (pH 7.5) containing 0.5 M NaCl. The hTfR fraction eluted with a 0.02 M glycine buffer (PH 10.0) containing 0.5 M NaCl and 0.5% Triton X-100 was further purified.
After applying to the hTf binding column and washing with PB containing 1M NaCl, 1M NaCl
And 1.5% hTfR purified sample by elution with 0.05M glycine buffer (pH 10.0) containing 1% Triton X-100.
I got

(2) Immunization and cell fusion This was performed according to the method described in Example 19- (2).

(3) Selection and cloning of hybridoma A commercially available rabbit anti-mouse IgG antibody solution (20 μg / m) was dispensed at 100 μl into a 96-well microplate and left at 4 ° C. overnight.
Further, 2% BSA-containing PBS (pH 7.3) was added to prepare a sensitized plate. The purified hTfR sample obtained in (1) was labeled with HRP according to a conventional method and then used for ELISA [Tonehiro Kitagawa: Synthetic Organic Chemistry, 42 , 283 (1984)]. That is, the hybridoma culture supernatant was added to the second antibody-sensitized plate, reacted at room temperature for 2 hours, and washed with PBS. Then, HRP-labeled hTfR was added, and further reacted at room temperature for 2 hours.

After washing the plate, a 0.1 M citrate buffer containing orthophenylenediamine and H ₂ O ₂ as enzyme substrates was added, and the enzyme reaction was performed at room temperature. After stopping the reaction with 1N sulfuric acid,
The amount of coloring dye was measured at a wavelength of 492 nm using a multiscan (manufactured by Flow).

Particularly, a hybridoma having a strong binding ability was cloned by the limiting dilution method to obtain an anti-hTfR antibody-producing hybridoma 22C6. Subclass of the antibody is IgG _1, it showed a high affinity for human tumor cell lines K562.

(4) Preparation of maleimidated antibody Mouse anti-hTfR monoclonal antibody 22C6 was prepared in Example 20-
(1) Maleimidation was carried out according to the method described. A complex having about 7.7 maleimide groups introduced per antibody molecule was obtained.

(5) Preparation of human LT-anti-hTfR antibody conjugate According to the method described in Example 20- (2), 1.5 mg of a maleimidated antibody was reacted with 1 mg of human LT, and the mixture was applied to a Sephacryl S-200 column and applied to a human LT-anti-hTfR antibody. 1.9 mg of the complex was purified and isolated.

(6) Cytotoxic activity of human LT-anti-hTfR antibody conjugate According to the method described in Example 20- (3), human LT-anti-hTfR
The cytotoxic activity of the antibody conjugate was measured.

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

The plasmids pTB618, pTB622, ptrp781,
pTB773, pTB775, pTB858, pTB860, pTB864, pTB865 and pTB
DH1 strain or C600 strain retaining 866 ( E. coli DH1 / pTB61
8, E.coli C600 / pTB622, E.coli DH1 / ptrp781, E.coli DH1
/ pTB773, E.coli DH1 / pTB775, E.coli DH1 / pTB858, E.coli
DH1 / pTB860, E.coli DH1 / pTB864 , E.coli DH1 / pTB865, E.
coli DH1 / pTB866, E.coli DH1 / pTB953, E.coli DH1 / pTB10
04, E.coli DH1 / pTB1005, E.coli DH1 / PTB1006, E.coli DH
1 / pTB1007) and animal cells (mouse-mouse hybridoma LT3-11 and mouse-mouse hybridoma 22C)
The accession number of the depositary institution 6) is as shown in Table 4.

[Brief description of the drawings]

FIG. 1 shows the recombinant plasmid pTB 694, FIG. 2 shows the recombinant plasmid pTB 773, and FIG.
858, FIG. 4 is a schematic diagram showing the assembly of the recombinant plasmid pTB864. FIGS. 5, 6, 7, 8 and 9 each show plasmid pTB95
3, 5'-terminal DNA sequence encoding human LT mutein retained in pTB1004, pTB1005, pTB1006 and pTB1007;
And the amino acid sequence deduced from them. The abbreviations in each figure and their meanings are as follows. B: Bal I Ba: Bam HI Bg: Bgl II C: Cla IE: Eco RIH: Hind III N: Nsi IP or Ps: Pst I Pv: Pvu II S: Sal IX: Xho IBI: Bgl I E47: Eco 47 III

Continuation of the front page (51) Int.Cl. ⁶ identification number FI C12R 1:19) (C12P 21/02 C12R 1:19) Accession number of microorganism FERM BP-1836 Accession number of microorganism FERM BP-1837 Accession number of microorganism FERM BP-1836 Accession number of microorganism FERM BP-2282 Accession number of microorganism FERM BP-2283 Accession number of microorganism FERM BP-2284 Accession number of microorganism FERM BP-2285 Accession number of microorganism FERM BP-2286 Accession number of microorganism FERF BP -1813 Accession number of microorganism FERM BP-2054 (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 14/52 C12P 21/00-21/06 C12N 1/21 BIOSIS (DIALOG) WPI (DIALOG) CA (STN) REGISTRY (STN)

Claims (6)

(57) [Claims] 1. A protein having the following amino acid sequence: Wherein R ₁ is Lys, R ₂ is Leu-Ala-Leu-Thr, m is 1, R ₃ is Met-His-Leu-Ala-His-Ser-Thr-Leu,
R ₁ is Ser, R ₂ is Leu-Ala-Leu, m is 1, R ₃ is Lys-Met-
His-Leu-Ala-His- Ser-Thr-Leu, R 1 is Cys-Se
r, R ₂ is Ala-Leu-Ala, m is 1, R ₃ is Leu-Ala-His-S
er-Thr-Leu, R ₁ is Cys-Ser, R ₂ is Ala-Leu-Ala-L
eu-Ser-Asp-Lys-Pro, m is 1, R ₃ is Ala-Ala-Gln
-Thr-Ala-Arg-Gln-His-Pro-Lys-Met-His-Leu
-Ala-His-Ser-Thr- Leu, R 1 is Lys-Ser, R ₂ is Ala
-Leu-Ala-Leu-Ser-Asp-Lys-Pro, m is 1, R ₃ is A
la-Ala-Gln-Thr-Ala-Arg-Gln-His-Pro-Lys-M
et-His-Leu-Ala- His-Ser-Thr-Leu, R 1 is Lys-
Ser and R ₂ are Ala-Leu-Ala, m is 1, R ₃ is Leu-Ala-His
-Ser-Thr-Leu, R ₁ is Lsy-Ser, R ₂ is Ala-Leu-Ala
-Leu-Ser-Asp, m is 0, R ₁ is Cys-Ser, R ₂ is Ala-
Leu-Ala-Leu-Ser-Asp, m is 0, R ₁ is Cys-Ser, R
₂ is Gly-Phe-Leu-Gly-Ser, m is 1, R ₃ is Leu, or 10 R ₁ is Cys-Ser, R ₂ is Gly-Phe-Leu-Gly-Ser-Le.
u-Lys-Pro, m is 1, R ₃ is Ala-Ala-Gln-Thr- Ala-
Arg-Gln-His-Pro-Lys-Met-His-Leu-Ala-His-
Represents Ser-Thr-Leu, and n represents 0 or 1. ] 2. A polydeoxyribonucleic acid comprising a base sequence encoding the following amino acid sequence and a polydeoxyribonucleic acid comprising a sequence complementary thereto. Wherein R ₁ is Lys, R ₂ is Leu-Ala-Leu-Thr, m is 1, R ₃ is Met-His-Leu-Ala-His-Ser-Thr-Leu,
R ₁ is Ser, R ₂ is Leu-Ala-Leu, m is 1, R ₃ is Lys-Met-
His-Leu-Ala-His- Ser-Thr-Leu, R 1 is Cys-Se
r, R ₂ is Ala-Leu-Ala, m is 1, R ₃ is Leu-Ala-His-S
er-Thr-Leu, R ₁ is Cys-Ser, R ₂ is Ala-Leu-Ala-L
eu-Ser-Asp-Lys-Pro, m is 1, R ₃ is Ala-Ala-Gln
-Thr-Ala-Arg-Gln-His-Pro-Lys-Met-His-Leu
-Ala-His-Ser-Thr- Leu, R 1 is Lys-Ser, R ₂ is Ala
-Leu-Ala-Leu-Ser-Asp-Lys-Pro, m is 1, R ₃ is A
la-Ala-Gln-Thr-Ala-Arg-Gln-His-Pro-Lys-M
et-His-Leu-Ala- His-Ser-Thr-Leu, R 1 is Lys-
Ser and R ₂ are Ala-Leu-Ala, m is 1, R ₃ is Leu-Ala-His
-Ser-Thr-Leu, R ₁ is Lsy-Ser, R ₂ is Ala-Leu-Ala
-Leu-Ser-Asp, m is 0, R ₁ is Cys-Ser, R ₂ is Ala-
Leu-Ala-Leu-Ser-Asp, m is 0, R ₁ is Cys-Ser, R
₂ is Gly-Phe-Leu-Gly- Ser, m is 1, R ₃ is Leu or R ₁ is Cys-Ser, R ₂ is Gly-Phe-Leu-Gly- Ser-Leu,
-Lys-Pro, m is 1, R ₃ is Ala-Ala-Gln-Thr- Ala-A
rg-Gln-His-Pro-Lys-Met-His-Leu-Ala-His-S
er-Thr-Leu, and n represents 0 or 1. ] 3. A polydeoxyribonucleic acid obtained by substituting one or more 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, or 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. A polydeoxyribonucleic acid comprising the polydeoxyribonucleic acid according to claim 2 and a sequence complementary thereto, or one of the polydeoxyribonucleic acids according to claim 3 incorporated into a replicable vector. A replicable recombinant DN containing the polydeoxyribonucleic acid
A, transforming a microorganism or a cell with the obtained recombinant DNA to obtain a transformant containing the recombinant DNA, culturing the obtained transformant, and obtaining genetic information of the polydeoxyribonucleic acid. 2. The method for producing a protein according to claim 1, comprising expressing.