JPH02157295A - New antitumorous polypeptide - Google Patents

Abstract

NEW MATERIAL:A polypeptide having an amino acid sequence expressed by the formula. USE:An antitumor agent. PREPARATION:For example, plaque hybridization is carried out from a rat chromosomic genetic library using a rabbit tumor necrosis factor(TNF)cDNA Pst I fragment as a probe to select a clone having a DNA capable of coding the rat TNF. The resultant phage DNA is cut out with a suitable restriction enzyme to provide a plasmid having a DNA fragment, capable of coding the TNF and integrated thereinto. The obtained recombinant plasmid is then inserted into a host, such as Escherichia coli. After transforming the host, the transformed host microorganism is subsequently cultured to separate the resultant product from the culture. Thereby, the polypeptide expressed by the formula is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to new antitumor polypeptides, DNA encoding the polypeptides, methods for producing them, and pharmaceutical compositions containing the polypeptides.

(Prior Art) Tumor necrosis factor (TNF) is a physiologically active substance released from macrophages, and its characteristics are (i) when administered to salt cancer animals, it causes necrosis and treatment of certain types of cancer; (ii) in It is known that it damages mouse cells in vitro, and (iii) its activity has little species specificity. Because of these characteristics, TNF is expected to be clinically applied as a new type of anticancer agent.

The current name, Hi-TNF, was obtained by the cloning of the gene by the present inventors and other groups ((S
hirai et al., Nature 313
+803 (1935) ), (Pennica
et al,, Nature 3￥2゜724
(1984)), (Hang et al.
5cicne 228.

149 (1985))), and the rabbit TNF gene has also been cloned by the present inventors ((It
o etal, +DNA 5, 149 (1986)
), (Ito et al, DNA 5.1
57 (1986) )).

(Problems to be Solved by the Invention) The purpose of the present invention is to solve the problem of anti-11! I! The object of the present invention is to provide a novel polypeptide that has tumor activity and is effective for pharmaceutical use.

(Means for Solving the Problems) As a result of extensive research, the present inventors succeeded in obtaining a novel polypeptide with cancer cytotoxic activity from a rat chromosomal gene library using genetic recombination techniques. and completed the present invention.

The present invention provides a polypeptide having the following amino acid sequence: L R55SQNSSDK PVAIIVVANH
Q AEEQLEWLSQ RANALLANGM
DLKDNQLVVP ADGLYLIYSQ V
LFKGQGCPD YVLLTIITVSRFAI
SYQEXVS LLSAIKSPCP KDTP
EGAELK PWYEPMYLGGVFQLEKG
DLL 5AEVNLPKYL DITESGOV
YI? In addition to GVIAL, polypeptides provided by the present invention include those in which methionine is added to the amino terminus of the above-mentioned polypeptides, or those in which the two amino acids (leucine-arginine) are deleted from the amino terminus.

Next, the present invention provides DNA having a base sequence encoding the above polypeptide.

Furthermore, the present invention provides DNA having the following base sequence: CTCAGATCATCTTCTCAAAACTCGA
GTGACAAGCCCGTAGCCCCAGTCGT
AGCAAACCACCAAAGCGGAGGAGCAG
CTGGAGTGGCTG8GCCAGCGTGCCA
ACGCCCTCCTGGCCAATGGCATGGA
TCTCAAAGACAACCAACTGGTGGTA
CCAGCAGATGGGCTGTACCTTATCT
ACTCCCAGGTTCTCTTCAAGGGACA
4GGCTGCCCCGACTACGTGCTCCTC
ACCCACACCGTCAGCCGGTTTGCCA
TTTCATACCAGGAGAAAGTCAGCCT
CCTCTCTGCCATCAAGAGCCCTTGC
CCTA4GGACACCCCTGAGGGAGCTG
AGCTCAAGCCCTGGTATGAGCCCAT
GTACCTGGGAGGGAGTCTTCCAGCTG
GAGA4GGGGGACCTGCTCAGCGCTG
AGGTCAAACCTGCCCAAGTACTTAGA
CATCACGGAGTCCGGGCAGGTCTC
TTTGGAGTCATTGCTCTG
TCCCCLl, 2) and mouse Me
It is evaluated by tumor necrosis activity against th A sarcoma. Incidentally, the crude extract of the recombinant microbial cells described below can also be used as it is for activity measurement.

Furthermore, the present invention provides pharmaceutical compositions containing the above-described polypeptides. In order to use the polypeptide of the present invention as a medicine, it is desirable to administer it in a form suitable for exerting antitumor effects.

The polypeptide of the invention may be administered alone or may be diluted with a pharmaceutically acceptable diluent.

Such diluents include, for example, mannitol, human serum albumin, glycine, and the like. The dose of the polypeptide of the present invention is preferably 1 μg or more per day for adult patients, but this dose may vary depending on race, sex, age, body weight, administration method, interval, physical condition, type of preparation, etc. may also be less.

The novel polypeptide and DNA of the present invention can be produced as follows.

(1) Cloning of rat TNF t'' color from the rat chromosomal gene library, rabbit TN
FcDNA Pst I fragment (rto et
at.

DNA□5, 149 (1986)] was used as a probe to perform plaque hybridization and screen for a clone of interest. By excising the cloned phage DNA with an appropriate restriction enzyme, a plasmid incorporating a DNA fragment encoding TNF can be obtained. The DNA fragment thus obtained was subjected to the method of Metzing et al. (Nucleic Ac1d Res,
9 +309 (1981)) and compared with mouse TNF (Shirai et al.
, DNA Yu, 193 (198B)), the amino acid sequence of the N-terminal portion was determined (Figure 1).

Of course, in cloning the chromosomal gene encoding rat TNF, we selected parts with high homology in the amino acid sequences of human, mouse, and rabbit TNF, and synthesized NA.
Screening may be performed using Also, it is not a chromosomal gene, but similar to rabbits (It.

et al., DNA 5.149 (1986)
), mRNA was obtained from macrophages and cDNA was obtained.
It's good as well. In selecting cDNA, synthetic NA, human, mouse, and rabbit TNF cDNA or chromosomal genes can be used as probes, but activity-based screening using Xenopus oocytes can be used. You can also get

In the case of a chromosomal gene, it may be made into a cDNA type by deleting an intron using synthetic NA, or it may be made into a cDNA type using a chromosomal DNA fragment and synthetic NA.

(2) The first part of the DNA encoding the polypeptide
In the base sequence shown in the figure, intron 3 (1743rd to 2026th), which is unnecessary for expression in E. coli, is synthesized and NA (ME34 (5'-CTTGGTGGTTTG
CTACGACGTGGGC-3'))
193 (1988)), which joins exon 3 and exon 4 necessary for expression. Restriction enzyme Taql, Bam1
After excising with ll, the amino terminal portion is closed with synthetic NA and connected downstream of the tac promoter to prepare an expression vector. The thus obtained plasmid pRtacl
was used to transform E. coli strain JM105, and target E. coli was selected from ampicillin-resistant colonies.

Examples of promoters for expressing the novel polypeptide of the present invention in E. coli include lactose promoter system, tryptophan promoter system, E. coli alkaline phosphatase promoter system, etc.
Any promoter that can be expressed in E. coli can be used.

In addition, when the novel polypeptide of the present invention is expressed in yeast, the yeast vector is a replication origin from a 2 micron yeast plasmid, an autonomously replicating sequence, a promoter, a DNA encoding the polypeptide of the present invention, and translation if necessary. Those with sequences for initiation and termination, as well as sequences for polyadenylation and transcription termination, and a selection gene sequence are used.

Furthermore, animal cells can also be used as hosts.

Examples of host cells include CHO, CV-1, CO3-1,
Examples include CO5-7 and C127. Examples of promoters include SV40 early promoter and SV40 late promoter. Selectable markers include neomycin resistance gene (Neo) and dehydrofolate reductase (
DIIPR), etc. When using animal cells, it is also possible to connect a so-called signal sequence to the amino terminus of 1) NA encoding the polypeptide of the present invention, and secrete it to the outside of the cell.

(3) Large polypeptide 1111'pRtacl
After culturing E. coffii, the cells are collected, crushed, and centrifuged to obtain a crude extract containing the polypeptide of the present invention.

Purification of the polypeptide of the present invention produced by such a method is achieved by combining known methods for purifying polypeptides and proteins.

Examples of such methods include high performance liquid chromatography, gel filtration chromatography, ion exchange chromatography, affinity chromatography,
Polyacrylamide gel electrophoresis, isoelectric focusing, salting out, ammonium sulfate precipitation, etc. can be appropriately combined.

Note that the following abbreviations are used in the present invention.

In the base sequence, A: adenine, C: cytosine, G
; Represents guanine, T:, thymine.

In the amino acid sequence, A: alanine, R: arginine, N: asparagine, D: aspartic acid, C nigisteine, Q: glutamine, E: glutamic acid, G nigericin, H: histidine, ■: isoleucine, L: leucine, K: Lysine, M: methionine, F: phenylalanine, P nibroline, S: serine, T nisthreonine, W:
Triphytophan, Y: tyrosine, ■: valine.

The present invention will be explained below using examples.

(Example) Runt chromosomal gene (Clontic) 2 x 106 plaques were injected into rabbit TNF cDNA pB2-7 P
stI fragment [Ito et al., DNA
5.149-156 (1986) E as a probe, hybridization was performed and three positive clones were obtained. DNA was obtained from one of these clones and analyzed by Southern analysis.
J, Mol, Biol, 98+503-51
7 (1975)), IEcoRI-Bamll
A 3.5 kb fragment was found to encode rat TNF. This 3.5 kb fragment was made blunt-ended using the usual method, and then transformed into Sma I of M13mp18.
The site was subcloned and named mplB-REB.

[Construction of recombinant plasmid pRtacl] As shown in FIG. 2, single-stranded DNA of mplB-RUB was prepared and used for deletion of intron 3. Synthetic NA (ME34 (
5'-CTTGGTGGTTTGCTACGACGTG
GGC-3')) was used (Gene 57.11-
19. (1987)) intron 3.
mplB-RHD was created in which mplB-RHD was deleted.

Excise the double-stranded DNA of mplB-RI+D with old ndll and subclon the approximately 840 bp fragment into pUc113 to create plasmid pR). pRH is cut with Taql and Bamll to excise a approximately 720 bp fragment. Two Synthetic NA (MO-1: GATCC
ATGCTCAGATCATCTTCTCAAAATT
, MU-1:GTACGAGTCTAGTAGAA
GAGTTTTAAGC), and MO-1 is phosphorylated with T4 polynucleotide kinase in the presence of ATP. Phosphorylated MO-1, MU-1, a fragment of about 720 bp, and BamHr-digested plJc18 are reacted in the presence of ligase to obtain plasmid pRI3.

Plasmid pRB was cut with BamHI to approximately 760 bp.
Obtain fragments of. On the other hand, plasmid pGtacllIO(S
Hiraie et al., Nature '313.
803-806. (1985) ) to Bgf[, Bam
Cut at H and take a portion of the vector. A part of this vector was reacted with a fragment of about 760 bp in the presence of ligase,
Obtain the desired plasmid pRtacl.

[Expression of novel polypeptide in E. coli] pRtacl obtained in Step 1 of Example 2 is introduced into E. coli strain D1210, and precultured overnight in I7 medium containing 50 μg of ampicillin, 7 ml. This was placed in a new medium so that the volume was 1/100 of that of the medium, and cultured at 37°C until the absorbance at a wavelength of 600 nm became 0.2 to 0.3. IPTG (isopropyl-β-D-thio-galactoside) was added to a final concentration of 1 mM, and the cells were further cultured until the absorbance (600 nm) reached 5. Centrifuge the culture solution to collect bacteria, and store at 0°C.
It was crushed by ultrasonication. The resulting crushed solution is centrifuged to obtain its supernatant.

Step 1 - [Purification of novel polypeptide] Add amphibian to the supernatant obtained in step 2) (p13-10
) to a final concentration of 2%, and make up to 50ml with distilled water. The sample is filled in a Rotophor (Bio-Rad, preparative liquid isoelectric focusing device) and electrophoresed for about 4 hours at a constant power of 12W. After separating the sample, SO
3-Collect the fractions in which the rat TNF band is visible by polyacrylamide gel electrophoresis and fill them into the rotophore again. After electrophoresis in the same manner for about 3 hours, the sample is separated.

This is purified by IIPLC (high performance liquid chromatography). 20 with DEAE-5PW (manufactured by Tosoh)
Under a solution of mMTris-11Cffi (pH 8), N
A gradient is applied to aCe from 0.011 to 0.3 M in 1 hour, the resulting peak is identified by the L cell cytotoxic activity, and the peak showing the cytotoxic activity is concentrated using Centricon 10 (Amicon).

Next, using a 5IIODEX Its-803F column (manufactured by Showa Denko), 20mM Tris-11cjl! (
+) 118%o), 150mM NaCI! The product is subjected to gel filtration using , and the portion showing harmful activity to L cells is condensed to obtain a purified product.

When the amino acid sequence of this purified product was examined using a gas phase amino acid sequencer (manufactured by Applied Biosystems), the amino acid sequence from the N-terminus was found to be Leu-A
rg-3er-3er-3er-G In-Asn-. Furthermore, when the molecular weight of the novel polypeptide obtained by the present invention is measured by SDS polyacrylamide electrophoresis, it is approximately 17,000.

The required activity is defined as 1 unit/relative, and 50% of the absorbance of the control.
The dilution rate of the sample corresponding to the value was taken as the titer.

The specific activity of the novel polypeptide obtained in Example 2 is approximately 4
X 10”u/mg.

Yamakyo et al. (Japan, J, Med, Sci, B
Iol 29+ 105118 (1986)).
Antitumor activity against CCL1, 2) was measured. 100μi of the sample diluted with Eagle's Minimum Essential Medium (MEM) containing 1% fetal bovine serum and L-
100 μr of a suspension of M cells 105118 was added to a 96-well microplate (Flow Laboratory).

This was heated at 37°C, 15% CO2, and 100% humidity.
Incubate for hours. Thereafter, 20 μl of 25% glutaraldehyde solution was added to fix the living cells. After washing, the fixed cells were stained with 100μi of 0.05% methylene blue solution, and after washing, they were extracted with 200μi of 0.33N hydrochloric acid.
The absorbance at nm was measured. In order to kill 50% of L-M cells, 2XIO was added to the abdomen of BALB/C mice.
' implant female A sarcoma cells. After 7 days, a mouse was selected in which the transplanted tumor had a diameter of 7 to 8 [lll11] and had no hemorrhagic necrosis and had good blood circulation, and the mouse was diluted with physiological saline from the tail vein. The novel polypeptide sample of the present invention obtained in
The injection amount was 10'u, and 24 hours later, evaluation was made according to the following criteria. The results are shown in Table 1.

Margin below Table 1 and the amino acid sequence of rat TNF precursor.

FIG. 2 shows a conceptual diagram of the construction of plasmid pRtacl.

Judgment criteria: (-): No change +): Faint hemorrhagic necrosis (+): Moderate hemorrhagic necrosis (necrosis extending over 50% or more from the center of the graft surface) (4H-), marked hemorrhagic necrosis (Severe necrosis in the center of the transplanted cancer, with a small amount of surrounding cancer tissue remaining)

Claims (6)

[Claims] (1) The following amino acid sequence: [There is a gene sequence] polypeptide with (2) Antitumor pharmaceutical composition containing the polypeptide according to claim 1 as an active ingredient (3) DNA having a base sequence encoding the polypeptide according to claim 1 (4) The following base sequence: [There is a gene sequence] DNA with (5) A method for producing the polypeptide according to claim 1 by culturing a microorganism having the DNA according to claim 3. (6) A method for producing the DNA according to claim 4 by obtaining it from a chromosomal gene library.