JP3344609B2 - Mutant human tumor necrosis factor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a mutant human tumor necrosis factor gene, a mutant human tumor necrosis factor, a method for producing the same and a use thereof.

[0002]

BACKGROUND OF THE INVENTION Tumor necrosis factor is a bioactive factor that directly kills tumor cells. Tumor necrosis factor has the strongest killing power among bioactive factors discovered to date, and has an antitumor effect by acting together with other biological factors, for example, cytokines such as interferon. And the range exerted by it can be expanded. As human-derived tumor necrosis factor,
Recombinant human tumor necrosis factor-α is known, its amino acid sequence and base sequence have been elucidated, and its development as a pharmaceutical for cancer patients has been attempted. However, when human tumor necrosis factor-α is used in a safe use amount, an effective antitumor effect cannot be obtained. On the other hand, in order to obtain an effective antitumor effect, the maximum allowable dose of 5
2525 times the amount must be administered. Therefore, it is difficult to clinically use the conventional human tumor necrosis factor-α as it is.

[0003]

DISCLOSURE OF THE INVENTION The present invention relates to a mutant of human tumor necrosis factor-α, which has an effective antitumor effect of human tumor necrosis factor-α and has low toxicity (side effect) to cancer patients. An object of the present invention is to provide a method for producing the same and an anticancer agent using the same.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies based on the above-mentioned problems, and as a result, by substituting the amino acid sequence at the N-terminal second position in the amino acid sequence of human tumor necrosis factor, The present inventors have found a mutant human tumor necrosis factor having excellent antitumor effect and safety, and have completed the present invention. That is, the present invention relates to a mutant human tumor necrosis factor comprising the amino acid sequence represented by SEQ ID NO: 1 in which the second arginine in the amino acid sequence of human tumor necrosis factor represented by SEQ ID NO: 3 has been substituted with lysine. It is.

[0005] The present invention also relates to a mutant human tumor necrosis factor gene comprising a DNA encoding the amino acid sequence of the mutant human tumor necrosis factor. Here, examples of the mutant human tumor necrosis factor gene include those represented by SEQ ID NO: 2.

Further, the present invention is a recombinant DNA having the mutant human tumor necrosis factor gene incorporated therein.

[0007] Further, the present invention is a transformant transformed with the recombinant DNA.

Further, the present invention is a method for producing a mutant human tumor necrosis factor, which comprises culturing the transformant and isolating the mutant human tumor necrosis factor from the obtained culture.

Further, the present invention is an anticancer agent comprising the above mutant human tumor necrosis factor as an active ingredient.

Hereinafter, the present invention will be described in detail. (1) DNA Synthesis and Plasmid Construction First, the mutant human tumor necrosis factor (hereinafter referred to as “mutant hTNF-α”) gene of the present invention can be obtained by, for example, synthesizing using a DNA synthesizer. That is, in the 157 amino acid sequence of natural human tumor necrosis factor-α (hereinafter referred to as “hTNF-α”) (SEQ ID NO: 3),
The codon corresponding to arginine ("CGT") corresponding to the second amino acid residue is replaced with the codon ("AA
A "or" AAG ").

[0011] The obtained DNA fragment was subjected to PCR.
The DNA can also be amplified by the (polymerase chain reaction) method. The desired DNA
Can be confirmed by, for example, agarose gel electrophoresis, polyacrylamide gel electrophoresis, or the like.

Next, D which encodes the mutant hTNF-α
All or part of NA may be replaced with an appropriate restriction enzyme (for example, Nco
I, BamHI, etc.), ligate it downstream of an appropriate promoter, enhancer, etc., and introduce it into a transformable host for transformation. As the vector DNA used herein, a plasmid vector or the like is used. Escherichia coli,
For example, Escherichia coli 71/18 strain and the like can be mentioned.

(2) Purification of mutant hTNF-α In order to obtain the mutant hTNF-α of the present invention, a transformant obtained by transformation is cultured in a commonly used medium, and the resulting culture is cultured. And are purified by conventional methods. Examples of the medium include an LB medium (for culturing Escherichia coli) and the like, and the culture is usually performed at 37 ° C. for about 16 to 20 hours. After the culture, centrifugation, ultrasonic crushing and the like are performed to remove the precipitate, and then a culture supernatant is obtained.

Mutant hTNF from the obtained culture supernatant
The separation and purification of -α may be performed according to a generally known protein purification method. For example, salting out, centrifugation, dialysis,
Purification is performed by appropriately combining various types of chromatography, electrophoresis, and the like. Examples of various types of chromatography include gel filtration, ion exchange chromatography, reverse phase chromatography, affinity chromatography and the like. The purity and the approximate molecular weight of the purified product are confirmed by SDS (sodium lauryl sulfate) polyvinylamide gel electrophoresis (for example, SDS-polyacrylamide gel electrophoresis).

(3) Anti-cancer agent The administration of the mutant hTNF-α and TNF-α of the present invention as an anti-cancer agent is not particularly limited. The method of administering the mutant hTNF-α of the present invention may be oral or parenteral, and oral administration includes sublingual administration. In the case of parenteral administration, it includes injection, for example, subcutaneous, intraperitoneal, intramuscular, intravenous injection, infusion and suppository. The dose varies depending on the age of the subject, the route of administration, and the number of administrations, and can be varied over a wide range. In the case of parenteral administration, it contains additives such as a stabilizer, a buffer, a preservative, and an isotonic agent, and is usually provided in the form of a unit dose ampule or a multi-dose container. The compositions described above may be in powder form for reconstitution with a suitable carrier, eg, sterile pyrogen-free water, for use.

In the case of oral administration, tablets, granules, fine granules, powders and the like may be used. Granules, fine granules and powders may be used in capsules, if necessary. Dosage forms can be provided. These solid preparations for oral administration usually contain additives such as binders, inclusion agents, excipients, lubricants, disintegrants, wetting agents and the like which are generally used in pharmaceutical preparations in their compositions. In addition, the liquid preparation for oral use may be in any form such as a solution for internal use, a suspension, an emulsion, a syrup and the like, or may be a dry product to be redissolved when used. Further, the composition may contain either additives or preservatives.

Here, the mutant hTN of the present invention is described below.
An antitumor effect (anticancer activity) using the present compound will be described with reference to pharmacological test examples of F-α. [Test Example 1] Antitumor activity against various tumor cells (1) Specific activity of mutant hTNF-α and hTNF-α 2 × 10 5 cells / well in a 96-well culture plate
Maki ^four L929 cells, 37 ° C., of under 5% CO _2,
The cells were cultured overnight in an incubator. Next, a mutant hTNF-alpha or hTNF-alpha diluted to each stage was added to each well and, after addition of further Senkinmoto D (final concentration 1μg / ml), 37 ℃, 5% CO 2 conditions For 16 hours.
After the culture, the absorbance (570 nm) of the culture solution was measured. L929
The dilution of the mutant hTNF-α or hTNF-α at the time of killing the cells by 50% was defined as one activity unit. as a result,
The specific activity of the mutant hTNF-α is 6.9 (± 0.5) × 10 ⁷ U / mg
And the specific activity of hTNF-α was 1.5 (± 0.3) × 10 ⁷ U / mg. From this, the specific activity of the mutant hTNF-α is h
It was found that the specific activity was at least 4 times higher than the specific activity of TNF-α.

(2) Anticancer activity against human tumor cell lines In this test, M7609 (human colon cancer cell line), OS (human osteosarcoma cell line), LAX (human lung adenocarcinoma cell line) were used as tumor cell lines. , MGC (human gastric cancer cell line) and SPC (human small cell lung cancer cell line). Each of the above tumor cell lines was spread on a 96-well culture plate at a concentration of 1 × 10 ⁴ cells / well, and different concentrations of the mutant hTNF-α or hTNF were used.
-Α was added. After culturing at 37 ° C. and further culturing for 24 hours in a 5% CO ₂ incubator, 0.5 μCi of ³ H-TdR was added.
/ Well, cells were collected using a multi-head cell collector, and CPM values were measured as radioactivity. Based on the CPM value, the tumor cell growth inhibition rate was calculated by the following equation. Tumor cell growth inhibition rate (%) = [(CPM value of control group−CPM value of experimental group) / CPM value of control group] × 100 As a result, the mutant hTNF-α has M
The inhibitory effects on GC and LAX cells are different and 2 × 10
Only at ⁴ U / mg a partial inhibitory effect was seen.

(3) Growth inhibitory effect on mouse tumor cell line In this test, mouse leukemia L1210 cells, mouse melanoma B16 cells, mouse YAC-1 cells, mouse leukemia P388 cells, mouse leukemia Ehclich (E
C) Ascites cancer cells were used. In the same manner as in (2) above, a test was performed on the effect of mutant hTNF-α or hTNF-α on the growth suppression of mouse tumor cells. Table 1 shows the results.

[0020]

[Table 1]

As apparent from Table 1, the mutant hTNF-α of the present invention (in Table 1, denoted as “[Lys ² ] hTNF-α”)
The above various mouse tumor cells P388, L1210, B16 and Y
AC-1 had a certain growth inhibitory effect. On the other hand, hTNF-α is a mutant hTNF-α ([L
ys ² ] hTNF-α) had a weaker tumor cell growth inhibitory effect.

(4) Mutant hTNF-α and hTNF-
Proliferation inhibitory effect of α on mouse transplantable tumor P388 leukemia cells P388 ascites carcinoma cells were collected from growing mice, diluted 1: 3 in physiological saline, and 0.2 ml / mouse in DSA / 2 mice one by one. Inoculated. Once a day from the day after tumor inoculation, a total of 5
Mutant hTNF-α or hTNF-
α was administered. The number of days after the administration was recorded, and the survival rate of the mice was calculated. As a control, mutant hTNF was used.
A group to which neither -α nor hTNF-α was administered (only saline was administered) was used. The survival extension rate was determined by the following equation. Survival prolongation rate (%) = [(average surviving days of administration group−average surviving days of control group) / average surviving days of control group] × 100 The results are shown in Table 2.

[0023]

[Table 2] As is clear from this experiment, the mutant hTNF-α
When administered at a dose of × 10 ⁴ U / animal, a certain therapeutic effect on the mouse transplantable tumor P388 leukemia cells was observed. hTN
In the case of the group to which F-α was administered, all the mice died three days after the administration. This indicates that the side effect of hTNF-α is strong or hTNF-α
It is considered that NF-α promotes the spread of tumor P388 leukemia cells.

[0024]

[Example 1]

(1) Synthesis of DNA, Construction of Expression Plasmid and Transformation of Escherichia coli Synthetic DNA for introducing mutation was synthesized using an automatic DNA synthesizer. The cloning site between the promoter and the terminator of the expression vector pSB-92 was cut with restriction enzymes NcoI and BamHI, and the DNA was recovered. Next, the mutant hTNF-
and alpha, and pSB-92 treated as described above were combined using T4DNA ligase to obtain an expression plasmid pSB-TNF-K _2. This was transformed into E. coli 71/18.

(2) Purification of mutant hTNF-α The transformant obtained in the above (1) is cultured at 37 ° C. for 16 to 20 hours. After the culture, the cells are suspended in a Tris buffer and the cells are suspended. A liquid was prepared. Subsequently, the mixture was subjected to ultrasonic crushing and centrifugation to remove the precipitate, thereby obtaining a supernatant. To this, 40% ammonia sulfate was added and centrifuged to remove the precipitate. To the obtained supernatant, 60% ammonium sulfate was added and centrifuged to obtain a precipitate. Next, after dialysis using a phosphate buffer (pH 6.9), the obtained inner dialysis solution was subjected to ion exchange chromatography using a CM-sepharose column with a NaCl concentration gradient of 0 to 0.5 M, and TNF fractionation was performed. Was recovered. It was dialyzed against TE buffer (pH 7.8), and the TNF fraction was collected by ion exchange chromatography with a NaCl concentration gradient of 0-0.5 M. After dialysis using PBS, Sephacry
The mutant hTNF-α was purified by gel chromatography using a l 200 column. Table 3 shows the purification results.

[0026]

[Table 3]

(3) SDS-polyvinylamide gel electrophoresis The purified mutant hTNF-α was subjected to SDS-polyvinylamide gel electrophoresis. The results are shown in FIG.
In the figure, lanes 1, 2, 3 and 4 show the electrophoretic diagrams of the molecular weight marker, the culture after cell culture, the fraction after ion exchange chromatography, and the fraction after gel chromatography, respectively.

[0028]

According to the present invention, a mutant human tumor necrosis factor which is effective on various tumor cells and has low toxicity can be obtained.

[0029]

[Sequence list]

SEQ ID NO: 1 Sequence length: 157 Sequence type: Number of amino acid chains: Single chain Topology: Linear Sequence type: Protein Sequence: Val Lys Ser Ser Serg Arg Thr Pro Ser Asp Lys Pro 1 5 10 Val Ala His Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln 15 20 25 Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu 30 35 40 Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu Tyr Leu 45 50 55 Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro Ser Thr 60 65 70 His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr 75 80 85 Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln 90 95 100 Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro 105 110 115 Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 120 125 130 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser 135 140 145 Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 150 155

SEQ ID NO: 2 Sequence length: 471 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: A GCC ATG GTA AAA TCT AGC TCT CGC ACT CCA TCT GAC AAA CCA Met Val Lys Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro 1 5 10 GTT GCT CAT GTT GTT GCT AAC CCA CAG GCT GAA GGT CAG CTG CAA Val Ala His Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln 15 20 25 TGG CTG AAC CGT CGT GCT AAC GCT CTG CTG GCT AAC GGT GTT GAA Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu 30 35 40 CTG CGT GAC AAC CAG CTT GTG GTA CCG TCT GAA GGT CTG TAC CTG Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu Tyr Leu 45 50 55 ATC TAC TCC CAG GTT CTT TTC AAA GGT CAG GGT TGC CCA TCT ACA Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro Ser Thr 60 65 70 CAC GTT CTG CTT ACC CAC ACT ATC TCT CGT ATT GCT GTT TCC TAC His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr 75 80 85 CAG ACT AAA GTT AAC CTG CTG TCT GCG ATC AAA TCT CCG TGC CAG Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln 90 95 10 CGT GAA ACC CCA GAA GGT GCT GAA GCT AAA CCA TGG TAT GAA CCG Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro 105 110 115 ATT TAC CTT GGT GGT GTT TTC CAA CTG GAG AAG GGT GAC CGT CTG Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 120 125 130 TCT GCT GAA ATC AAC CGT CCA GAC TAC CTT GAC TTC GCT GAA TCT Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser 135 140 145 GGT CAG GTA TAC TTC GGT ATT ATC GCT CTG TGA Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 150 155

SEQ ID NO: 3 Sequence length: 157 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: protein Sequence: Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro 1 5 10 Val Ala His Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln 15 20 25 Trp Leu Asn Arg Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu 30 35 40 Leu Arg Asp Asn Gln Leu Val Val Pro Ser Glu Gly Leu Tyr Leu 45 50 55 Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys Pro Ser Thr 60 65 70 His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala Val Ser Tyr 75 80 85 Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys Gln 90 95 100 Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro 105 110 115 Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 120 125 130 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser 135 140 145 Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 150 155

[Brief description of the drawings]

FIG. 1 shows the results of SDS-polyvinylamide gel electrophoresis.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI C12P 21/02 C12P 21/02 // (C12N 1/21 A61K 37/02 C12R 1:19) C12N 15/00 ZNAA (C12P 21 / 02 C12R 1:19) (56) References JP-A-3-297388 (JP, A) US Patent 5,891,679 (US, A) US Patent 5,587,457 (US, A) (58) Fields studied (Int. Cl. ⁷ , DB name) C07K 14/525 C12N 15/00-15/90 C12P 21/00-21/02 PubMed SwissProt / PIR / GeneSeq GenBank / EMBL / DDBJ / GeneSeq

Claims (7)

(57) [Claims] 1. A mutant human tumor necrosis factor comprising the amino acid sequence represented by SEQ ID NO: 1. 2. A mutant human tumor necrosis factor gene comprising a DNA encoding the amino acid sequence of the mutant human tumor necrosis factor according to claim 1. 3. The mutant human tumor necrosis factor gene according to claim 2, wherein the DNA encoding the amino acid sequence is represented by SEQ ID NO: 2. 4. A recombinant DNA having the mutant human tumor necrosis factor gene according to claim 2 or 3 incorporated therein. A transformant transformed with the recombinant DNA according to claim 4. 6. A method for producing a mutant human tumor necrosis factor, which comprises culturing the transformant according to claim 5, and isolating the mutant human tumor necrosis factor from the resulting culture. 7. An anticancer agent comprising the mutant human tumor necrosis factor according to claim 1 as an active ingredient.