JPH03284700A - Functional polypeptide - Google Patents

Abstract

NEW MATERIAL:A polypeptide obtained by bonding a cell adhesion domain polypeptide of human fibronectin to a region having an adhesion activity to a melanoma cell. USE:An cancer-metastasis suppresser. PREPARATION:Respectively necessary necessary regions are initially separated from a vector (e.g.; manifestation plasmid pTF 7520) containing DNA capable of coding a cell adhesion domain and from a vector (e.g.; manifestation plasmid pHD 102) containing DNA capable of coding a peptide region composed of the N-terminal side 25 amino acids of IIICs containing a region having an adhesion activity to B16-F10 melanoma cell and mutually bonded to obtain a vector capable of manifestation of the objective polypeptide. The resultant vector is then introduced to a colibacillus for transformation and the obtained transformant is cultured, thus producing the objective polypeptide.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a novel polypeptide, and more specifically, a novel artificial functional polypeptide containing a cell adhesion domain polypeptide of human fibronectin, and its use. Regarding usage.

[Conventional technology]

Fibronectin (hereinafter referred to as FN) is a glycoprotein present in plasma and extracellular matrix, and is known to have a variety of functions [Annual Review
Of Biochemistry - (Annual
Review of Biochemistry)
57, pp. 375-413 (1988)). Attempts have been made to use natural FN for wound healing, medicines such as eye drops, and cosmetics, but there are limited supplies due to the fact that it is collected from blood, high cost, and the risk of pathogenic bacteria. It has not been put into practical use due to the possibility of contamination by viruses, etc. Furthermore, extracting and utilizing the functional domain of natural FN has not been put to practical use for the same reason.

Therefore, the present inventors developed a cell adhesion active polypeptide and a method for producing the same by connecting a cDNA fragment encoding the cell adhesion domain of human FN to an expression vector and introducing it into E. coli, and would like to apply for a patent. JP-A-1206
No. 998).

t, the cell adhesion domain of human FN and the heparin-binding domain or the heparin-binding domain followed by <l1lc
We developed a functional polypeptide covalently bound to a fragment containing part of the s region and a genetic engineering method for its production, and filed a patent application (Japanese Patent Application No. 1-131453).

Furthermore, as a result of research into the application of these functional polypeptides to pharmaceuticals, it was discovered that many of them have cancer metastasis suppressive effects, and a patent application was filed (Japanese Patent Application No. 1-2650).
(No. 49) However, these cancer metastasis suppressive activities were not necessarily sufficient. On the other hand, Matsukashi et al. have reported that a 33-kDa fragment containing the heparin-binding domain and part of the llIc5 region of human FN has a cancer metastasis suppressive effect [Journal of National Cancer Institute]
cer Institute) Volume 80, No. 2, No. 1
08 pages (1988)].

[Problem to be solved by the invention]

However, it has not been clarified which region of the 33-kDa fragment has this effect.

The purpose of the present invention is to develop a novel functional polypeptide with a strong cancer metastasis suppressing effect and to provide a method for producing the same.

[Means for Solving the Problems] To summarize the present invention, the first invention of the present invention is an invention relating to an artificial functional polypeptide, and is an invention relating to a human FN cell adhesion domain polypeptide and a melanoma cell. It is characterized in that it is directly or indirectly bonded to an adhesion active site.

Moreover, the second invention of the present invention relates to a cancer metastasis inhibitor, and is characterized in that it contains the functional polypeptide of the first invention.

The third invention of the present invention relates to another cancer metastasis inhibitor, and is characterized in that it contains a functional polypeptide of the active site of adhesion of human FN to melanoma cells.

The present inventors genetically engineered a polypeptide covering the entire heparin-binding domain that does not include the mcs region and examined its cancer metastasis suppressive effect, and found that it had no activity at all.

This finding suggests that the l1lcs region is closely related to cancer metastasis suppressive action. The present inventors B16-
As a result of chemically synthesizing and investigating a peptide consisting of the N-terminal 25 amino acids of mcs (hereinafter referred to as C3I), which is known to have adhesive activity against FIO melanoma cells, we found that C3I has a cancer metastasis suppressive effect. I found it. Furthermore, when we genetically engineered a novel polypeptide in which C3l and a cell adhesion domain were covalently linked and examined its cancer metastasis suppressive effect, we found that the activity was significantly enhanced compared to the cell adhesion domain alone or C3I alone. I found out that it is. The present invention was achieved based on the above findings.

The present invention will be specifically explained below.

The novel polypeptide according to the present invention is a covalent bond of a human FN cell adhesion domain polypeptide and a C3I polypeptide, and can be produced by genetic engineering. For example, by extracting and connecting the necessary regions from a vector containing DNA encoding a cell adhesion domain and a vector containing DNA encoding a C3I region, a vector expressing a polypeptide of interest can be created. . The covalent bond between each region may be a direct bond or an indirect bond, for example, an indirect bond via a spacer. The spacer is an inserted sequence that adjusts the intermolecular distance between the cell adhesion domain and C5I, and any peptide chain can be used, for example, FN
It may also be a flow stop sequence in the C3I region of the molecule. Spacer sequences can be easily introduced by genetic engineering.

A specific example of the novel polypeptide according to the present invention.

Examples include polypeptides represented by the following general formula [1].

That is, the following general formula [■]: C2'r7C5I ・ ・ ・ [1) [In the formula, C2□, hi) Pr of the FN cell adhesion domain.

12395er1515, the following formula [■]: Pro T
hr Pr.

Pr.

Val Asp Asp Pr.

Ser Leu Ser Thr Ala 1e Arg Arg Pr.

Ala Leu Asp Pr.

Arg Val Asn G1. y Va1 Arg Pr.

Ala Pr.

Arg Pr.

Asn Iy Leu 1e Asp Leu Arg Thr Met Arg Ser Ile Asp Tyr Ser Pr.

Ala Glu Leu Ala Val Val Thr Glu Tyr Tyr Glu Gin Gly Arg Gin Thr Gly Ile Asn Ser Phe Arg Ala Thr )1is　　)Iis　Pr.

Arg Glu Asp Ser　Ile　Thr Thr Glu Tyr Asn　Gly　Arg Gly Gin Gin Phe’l’hr Val Thr Leu Thr Val　Lys Ser　l1e Leu Thr Val　Val His Glu Lys Thr ^SP　Phe Thr　Val 11e Thr Glu　His Arg Val Leu Thr Val　Val Glu Glu Ser　Thr Asn rp Asp Asn Ser Asp Ser Ser 1y Ser His Gly Phe Pr.

Asn Ser Ser Va1 1e Ala Phe 1u Pr.

Leu Val Thr Leu Asp rp Tyr Ser His Leu 1e Pr.

Ser 1y Pr.

Seu Glu Ser Leu Ser Pr.

Asp Iis Iis Arg Gly Ser Thr Val Seu Asp Val Thr Ala 1e Pr.

Lys Pr.

Ala Ser Thr Pr.

pr.

pr.

Thr Va1 Ser Gly Val Ser Glu Arg Thr ＾】a Tyr Gin Thr Val Thr Lys 1e Asp Ser Val Gly Glu Ala Asp 1y Pr.

Asp Leu Leu Thr Glu Phe Thr Tyr Arg 1e Lys Glu Leu Val Thr Thr 1e Thr Gly Ser Pr.

Va) val 11e Ser Arg Tyr Gly Gly Val　Pr.

Ser　Gly 11e Thr Asp 5er 11e　Asn Ser Ala rp Tyr As mouth Gly Leu Val Pr.

It has a sequence represented by Tyr Ala Asp Arg Ser Ser Lys Tyr Ala Arg [11], where C3I represents the N-terminal 25 amino acid residues of the cs region of FN, and the following formula [■]: ＾5p -Glu-Leu-Pro-G1n-Leu-V
a to Thr-Leu-Pr.

His-Pro-Asn-Leu-His-Gly-P
ro-Glu-11e-Leu-^5p-Val-Pr
o-3er-Thr...Cm] or a group in which a portion thereof is deleted].

C1-t C3I according to the present invention is pro+2395erIsIs (277
amino acid residues (hereinafter referred to as C2,7) and C of the -\valine binding domain of human FN.
It is a combination of the N-terminal 25 amino acids (C3I) of the Decs region (71 amino acid residues) located on the terminal side.

C2''J'l has been shown to have adhesion spreading activity towards fibroblast cells such as baby hamster kidney cells (BHK) and normal rat kidney cells (NRK).

On the other hand, C8I is known to be a site to which 816-FIO melanoma cells specifically adhere [Journal]
JyCellBiol (JyCellBiol)
Vol. 103, pp. 2637-2647 <1986) and Journal of Biological Chemistry (
J, Biol, Chem, ) Volume 262, No. 6
pp. 886-6892 (1987): l. Furthermore, it is said that the entire C3I region is not necessarily required for melanoma cell adhesion; even a portion of it is said to be active [cell (
Ce1.1), Vol. 60, pp. 53-61 (1990)
:].

C3I according to the present invention does not need to be the entire C81 sequence as long as it has a melanoma cell adhesion activity. C3
I and its related peptides can be easily synthesized using a peptide synthesizer. Examples of polypeptides in which a cell adhesion domain polypeptide and a melanoma cell adhesion sequence are covalently bonded include C, , -[1:
Advantageously, this novel polypeptide is produced by genetic engineering.

The present inventors have already discovered a 296 amino acid residue polypeptide (hereinafter H~29
A plasmid expressing p)I was created, and p)I
[It was named 1102 (Patent Application No. 1131453).

In addition, Escherichia coli HBIOI containing this plasmid was
herichia coli HBIOI/ pH01
02 and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology
10721) ).

On the other hand, the expression plasmid pTF752 encodes the cell adhesion domain polypeptide C2,7 and has introduced a loning site that is advantageous for connecting DNA encoding another polypeptide immediately before the stop codon at its 3' end.
0 (Japanese Patent Application No. 1-131453).

Therefore, by using these plasmids,
[A plasmid expressing l'2tt-C3I can be easily constructed. That is, first, from pHD102, D corresponding to the region containing one ■-type homology (III-16) and C8I on the C-terminal side of the heparin-binding domain.
The NA fragment is removed and connected to the cloning site at the 3' end of the region encoding C21 of pTF7520. A plasmid expressing ['277 C3I can be obtained by removing the DNA corresponding to lll-16 of the heparin-binding domain from the obtained plasmid by site-directed mutagenesis (see FIG. 1).

By introducing the obtained plasmid into E. coli and culturing it under appropriate conditions, the target polypeptide is accumulated in E. coli. Immunoblotting is used to confirm expression. The whole cell protein of recombinant E. coli was 5DS-
After separation by polyacrylamide electrophoresis, the electrophoresis/(turn) is transferred to a nitrocellulose membrane. After being treated with a monoclonal antibody that recognizes the cell adhesion domain of human FN, a band around 33-kDa is isolated using a labeled second antibody. You can see it being dyed.

Purification of the target polypeptide is performed, for example, as follows. Recombinant Escherichia coli cultured in L-broth or the like is disrupted by ultrasonic waves to obtain supernatants.

This is adsorbed onto an anion exchange resin column and fractionated by increasing the salt concentration. By the method described above, a target fraction is detected and adsorbed onto a Sepharose column to which the antibody is bound.

After washing the column with a buffer around neutrality, elute with a buffer around pH 3 and detect the target fraction. Both fractions that give a single electrophoretic band are collected, desalted, and then lyophilized. The obtained polypeptide is confirmed to be the desired one by N-terminal sequence analysis and C-terminal amino acid analysis.

C3 of the C-C5I polypeptide thus obtained
One part is 25 amino acid residues, but as mentioned above [cell no. 60]
Vol., pp. 53-61 (1990)), the minimum unit required for adhesion activity to melanoma cells can be summarized to about 10 C-terminal amino acids.

Therefore, the C31 portion of C-C3I is, for example, C3I
It may also be a polypeptide in which the sequence from the N-terminus to the 15th position of the region is removed. Removal of this sequence corresponds to D
This can be easily achieved by removing the NA sequence by site-directed mutagenesis.

The cell adhesion activity of the obtained polypeptide can be determined, for example, by the method of Ruosiahti et al. [Methods in Enzymology].
ymology), Vol. 82, pp. 803-831 (1
981):]. That is, a suspension of BHK or B16-F10 cells is added to a microtiter plate coated with a sample and blocked with BSA, incubated at 37°C for about 1 hour, and then washed.

Cell adhesion activity can be measured by fixing the adsorbed cells with formalin and measuring the proportion of cells that have spread under a microscope.

In this way, C27, -C3I is more important than BHK than B1.
It was shown to have strong affinity for 6FIO cells.

The cancer metastasis suppressing activity of the polypeptide according to the present invention is measured, for example, as follows. Highly metastatic 816-BL6 melanoma cells are mixed with the sample dissolved in PBS and administered into the tail vein of mice. Colonies that have metastasized to the lungs are counted after about 2 weeks.

The above experiments demonstrate that C3I and C277-C3I have the effect of suppressing cancer metastasis. In addition, the effect is
It is shown that C2, -C31 is stronger than C81.

When the polypeptide of the present invention obtained as described above is used as a medicine, it may be formulated by a conventional method together with a pharmaceutical carrier if necessary, and administered orally or parenterally. A pharmacologically acceptable excipient or carrier is selected, and its type and composition vary depending on the route and method of administration. For example, water, alcohols, animal or vegetable oils such as soybean oil, olive oil, mineral oil, or synthetic oils are used as liquid carriers.

Saccharides such as maltose and sucrose, amino acids, cellulose derivatives such as hydroxypropylcellulose, organic acid salts such as magnesium stearate, and the like are used as solid carriers.

In the case of injections, the solution preferably includes physiological saline, various buffer solutions, saccharide solutions such as glucose, inositol, mannitol, and lactose, and glycols such as ethylene glycol and polyethylene glycol. In addition, it is made into a lyophilized preparation with excipients such as sugars such as inositol, mannitol, lactose, and sucrose, and amino acids such as phenylalanine, and is then administered in a suitable solvent for injection such as sterile water, physiological saline, glucose solution, etc. It can also be administered by dissolving it in a liquid for intravenous administration such as an electrolyte solution or an amino acid solution. The content of the polypeptide of the present invention in a preparation varies depending on the preparation, but is usually 0.1 to 100% by weight, preferably 1 to 98% by weight. For example, in the case of an injection solution, it is usually 0.1 to 30% by weight, preferably 1 to 10% by weight.
It is desirable to contain the active ingredients of When administered orally, it is used in the form of tablets, capsules, powders, granules, liquids, dry syrups, etc. together with the solid carrier or liquid carrier. Capsules, granules and powders generally contain from 5 to 100% by weight of active ingredient, preferably from 25 to 98%.

The dose is determined depending on the patient's age, weight, symptoms, therapeutic purpose, etc., but the therapeutic dose is generally 1 to 100 mg for parenteral administration.
mg/kg/day, 5-500mg/kg orally
/ day.

In a toxicity test of a functional polypeptide using C'57BL/6 mice, 100 mg of this polypeptide
No toxicity was observed after intravenous administration of 100 mg/kg.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The invention is not limited to these examples.

Reference Example I Synthesis of C3I Peptide A C3I peptide consisting of 25 amino acids was chemically synthesized by the Fmoc method. The reagents and equipment were from LKB, and the synthesis was carried out using Q, 2 + nmol nm-. After synthesis, the product was deprotected by a conventional method, and the desired fraction was collected by reverse-phase (IFLC) IFLC and freeze-dried. Yield was 100 mg. The obtained peptides were processed using a protein sequencer (
The correct sequence was confirmed using model 477^/120^ (Applied Biosystems).

Also, the results of the amino acid analysis were as expected.

Example 1 Cell adhesion domain pro12395er15 of human FN
Cloning of a cDNA fragment encoding a fusion protein of 15 (277 amino acid residues) and C3I (see Figure 1) (1-1) p) Introduction of Nco I site into fD102 pi (0102 represents the heparin-binding domain and C51 This is a plasmid that expresses the polypeptide 1l-296 bound to H-296, and is described in Japanese Patent Application No. 1-131453. a I et
An Nco I site was introduced immediately before the sequence corresponding to I. To introduce the Ncol site, use a mutation introduction primer and c! [: pATCAATGGCCATGGTG
GAGGCG) was used, and the site-directed mutagenesis system Mutan - KC Takarashu mun sales] was used as a mutagenesis kit. The experiment was conducted according to the attached protocol, and the desired plasmid was obtained.

(1-2) Preparation of Nco I-H1ncII fragment (
1 μg of the plasmid obtained in 1-1) was injected with Nco I and fl
Digested with incll and separated using agarose electrolysis method.
．． 3 kb Nco I-H1ncII fragment approximately 120n
g was collected.

(1-3) NcoI-HincII fragment pTF7
Cloning pTF7520 into 520 is capable of expressing the FN cell adhesion domain polypeptide C277, and has an Nco I site added to the site corresponding to its C-terminus.
It is an expression plasmid to which DNA encoding other polypeptides can be connected. This plasmid is described in Japanese Patent Application No. 1-131453.

pTF7520 was digested with Nco I and Hloc II and then dephosphorylated. 50ng of this plasmid (1-2)
together with 50 ng of the Nco l-H1ncII fragment obtained in
A μJ solution was prepared, 20 μm of DNA ligation kit [Takara Shuzo Hansho] solution A and 5 μl of solution B were added, and the mixture was incubated at 16° C. for 30 minutes.

E. coli HBIOI was transformed using 10 μm of this reaction solution, and the cell adhesion domain Pro 1239 Se
t”l5 (27? amino acid residue) and Ala of H-296
I 871-Thr I II a S (l l 5
A plasmid expressing a fusion protein (C2tt-Met-Hll,) in which amino acid residues) were linked via Net was obtained.

(1-4) Met and ^l ala'll 'H,
Met and Ala1 were extracted from the plasmid obtained by removing the sequence corresponding to ris6a (1-3).
The sequence corresponding to 871-7hr1860 (9Q amino acid residue) was removed by site-directed mutagenesis. Net
and Ala18'? Removal of the sequence corresponding to +'phr1960 (9Q amino acid residue) was performed using oligonucleotide d [pGGG^AGCTCGTCGGATGGTTT
GTC] was synthesized using the Site-Directed Mutagenesis System Mutan-K (manufactured by Takara Shuzo ■). As a result, the cell adhesion domain Pro
+ 239-3er l S I S (277 amino acid residues) and C3I are directly linked to fusion protein
A plasmid expressing (C2,, -C3I) was obtained, and pC
It was named 325.

In addition, Escherichia coli HBIOI carrying this plasmid is Es
cherichia coli HBIOI/pC32
5 and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology [FERM P1
1339) ].

Example 2 Purification of peptide from recombinant Bscherichia coli HBIOI/p obtained in Example 1
cs25 with 50 Mg/d ampicillin
The culture was carried out in a test tube containing Noshi broth at 37°C overnight with shaking. This was inoculated into 21 Erlenmeyer flasks containing 500 ml of the same medium, culture was continued at 100 rpII, and bacteria were collected after 20 hours. Immunoblotting was performed using a portion of the bacterial cells. In other words, all bacterial protein is converted to 5O3-P.
After separation with AGE and transfer of the migration pattern to a nitrocellulose membrane, a monoclonal antibody PNiO [Zen Shuzo ■] that specifically recognizes the cell adhesion domain of human FN was used.
[Sales]], and then a second antibody recognizing peroxidase A. The peroxidase activity of the w3-conjugated second antibody was developed in the presence of 4-chloro-1-naphthol and hydrogen peroxide, and it was confirmed that the target peptide around 33 kD was produced. Next, add 2 whole bacterial pellets.
0mM) Tris-)ICI (p)17.
5), Suspended in a solution containing 1 mM BDT^, 5 mM mercaptoethanol, and 3 μM baramidinophenylmethanesulfonyl fluoride (p-^PMSF>) and subjected to sonication. Centrifuged at 1200 rpm for 20 minutes, and then 25m7! of supernatant was obtained. This was mixed with 20mM) Lis-HC.
1 (pH 7,5>> buffer) was passed through a D-53 column (40-).
After washing with 20mM) Lys-HCI (pH 7,5) buffer containing [C], 20mM containing 0.15M NaCl
) It was eluted and fractionated with Lis-HCI (pH 7,5) buffer. The eluate was subjected to immunoblotting, and the target fractions were collected.

Next, both aliquots were passed through a Sepharose 4B column (10 mff) to which the monoclonal antibody FNiO was bound. Column 0. After washing with 20mM Tris-HCI (pH 7,5') buffer containing IM NaCl,
．． It was eluted with 1M glycine/HCI (pH 3,0) buffer and fractionated. The target fractions were collected by immunoblotting, desalted, and lyophilized to obtain approximately 7.5 mg of an electrophoretically homogeneous peptide. When the amino acid sequence from the N-terminus of this peptide was examined using ABI's Peptide Sequencer-477^/1208, it was found to match the N-terminal sequence of the target peptide. In addition, by carboxypeptidase P [Zen Shuzo ■] digestion method, the C-terminus is Thr
It was confirmed that

Example 3 Measurement of cell adhesion activity The cell adhesion activity of the C, , -C31 polypeptide obtained in Example 2 to BHK and mouse melanoma cells B16-P10 was measured.

Cell adhesion activity was measured according to the method of Ruoslati et al. [Methods in Enzymology, Vol. 82, pp. 803-831 (1981):]. Distilled water, P
The solution was dissolved in BS (IJ acid buffered saline), etc., and diluted stepwise on a 966 microplate. The sample was incubated at 4° C. for 2 hours to adsorb the sample onto the plate (50 μl/well). 100 μl/well of PBS solution containing 3% BSA was added and incubated at 37° C. for 1 hour to block the plate. After washing the plate with PBS, add Dulbecco's
s) Baby hamster kidney cells (BHK-21) or mouse melanoma cells B16 suspended in Eagle's minimal nutrient medium (DMEM) at 5 x 105 cells/-
- F10 was dispensed at 1 μm/well and incubated at 37° C. for 1 hour). The cells used were those that had been subcultured from a cryopreserved strain and then treated with trypsin (37°C, 5 minutes).

After washing the plate with PBS, cells were fixed on the plate with a 3% formalin solution.

Observe the spread of BHK-21 cells or B16-FIO cells under a microscope, and determine the sample concentration (BO2,) at which the number of spread cells is 50% of the number of spread cells at a high concentration of human FN.
was determined and used as an index of cell adhesion activity.

The results are shown in Table 1.

C,,, -C3148~96 98~24,6 As a result, C2,, -C51 is more B1 than BHKm cell.
It was shown to have strong cell adhesion activity against 6-PLO melanoma cells.

Example 4 Cancer Metastasis Inhibition Test Using chemically synthesized C81 peptide and C277C3I, the effect on experimental lung metastasis was investigated.

Groups of 5 C57BL/6 mice were treated with samples dissolved in PBS and 816~BL6 melanoma cells (3X10'
) was mixed and administered into the tail vein of mice. Two weeks later, the number of colonies that had metastasized to the lungs was counted and compared with the control.

The results are shown in Table 2.

PBS 6
1 ±16 C3I Bebubud 1 ■/mouse 26 ± 2 The above results showed that the C3I peptide has a cancer metastasis suppressive effect. Also, C
It was shown that a fusion peptide of 3I and a cell adhesion domain polypeptide has an even stronger metastasis-inhibiting effect.

Example 5 PBS was added to 30 parts by weight of the polypeptide obtained in Example 2 to give a total amount of 2000 parts by weight, and the solution was dissolved, followed by sterilization filtration using a Millipore filter - GS type. 2g of this filtrate for 10rnp! The polypeptide was placed in a vial and lyophilized to obtain a lyophilized injection containing 30 mg of the polypeptide per vial.

[Effects of the Invention] As described above, the present invention provides novel polypeptides and polypeptides having cancer metastasis suppressing activity! L! method of payment will be provided.

[Brief explanation of drawings]

Figure 1 shows [1:2. , - shows a process diagram for the construction of a plasmid for expressing the C3I polypeptide.

Claims (1)

[Scope of Claims] 1. An artificial functional polypeptide characterized by directly or indirectly binding a cell adhesion domain polypeptide of human fibronectin and an active site for adhesion to melanoma cells. 2. A cancer metastasis inhibitor comprising the functional polypeptide according to claim 1. 3. A cancer metastasis inhibitor characterized by containing a functional polypeptide of the active site of adhesion of human fibronectin to melanoma cells.