JPH0459734A - New anticancer agent - Google Patents

Abstract

PURPOSE:To obtain an anticancer agent containing a cell-bound polypeptide of human fibronectin (FN) and/or heparin-bound polypeptide and having high safety. CONSTITUTION:The aimed anticancer agent containing a compound expressed by the formula (Y is H or cell-bound polypeptide in FN molecule; n is 0 or 1; Z is OH or heparin-bound polypeptide in FN; either one of Y and Z is the above-mentioned polypeptide). The cellbound polypeptide includes a polypeptide contained in cell-bound domain of FN and having cell binding activity, e.g. 274 amino acid residual polypeptide of Pro<1239>-Asp<1512> in FN and the heparin- bound polypeptide includes a polypeptide contained in heparin-bound domain and having heparin-binding activity, e.g. 271 amino acid residual polypeptide of Ala<1690>-Thr<1960> in FN. The above-mentioned compound can be fed in large quantities by genetic engineering technique.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an anticancer agent, and more specifically, the use of a functional domain in a molecule of fibronectin (hereinafter abbreviated as FN), which is an in vivo active polypeptide, as an anticancer agent. Regarding usage.

[Conventional technology]

There are various cancer therapeutic agents such as alkylating agents, anticancer antibiotics, antimetabolites, and alkaloids, and there are currently several dozen types of anticancer agents that can be used clinically.

[Problem to be solved by the invention]

However, these anticancer drugs generally have strong toxicity and side effects, and have not yet achieved sufficient therapeutic effects.

In view of the above-mentioned current situation, the present invention aims to provide a new, safer anticancer agent that utilizes a substance related to the body.

[Means to solve the problem]

To summarize the present invention, the present invention relates to an anticancer agent, and includes the following--
Murder ■: Y − (Met), −Z −−CII (in the formula, Y
is the H- or crepe adhesive polypeptide in the FN molecule, n is 0
or 1, Z represents a heparin-binding polypeptide in the -OH or FN molecule, and at least one of Y and Z is the polypeptide).

The present inventors conducted intensive research on the functional domain of FN, which is an in vivo substance, and found that the polypeptide represented by -killing [1) has a remarkable anticancer effect.
The invention has been completed.

In FN, a polypeptide with a molecular weight of about 250,000 forms a dimer with an S-5 bond near the C-terminus.

The intramolecular amino acid sequence has a repeating structure and consists of ■, ■, ■
Divided into types. Furthermore, it has a domain structure with various functions, and exhibits cell adhesion, binding activity to collagen, heparin, fibrin, etc.

As a result of research into new uses of the functional domain of FN, the present inventors found that cell adhesion polypeptides, chimeric polypeptides of cell adhesion polypeptides and heparin-binding polypeptides, and heparin-binding polypeptides have remarkable anticancer properties. It has been found that it has an effect.

In the present invention, the cell adhesion polypeptide is not particularly limited as long as it is a polypeptide that is contained in the cell adhesion domain of FN and has cell adhesion activity.

Examples of the above-mentioned polypeptides include JP-A-1-180900, JP-A-1206998, and JP-A-2, created by the present inventors.
Examples thereof include cell adhesion polypeptides disclosed in Japanese Patent Application No. 63305820 (274 amino acid residue polypeptide) and Japanese Patent Application No. 1987-97397 (274 amino acid residue polypeptide). These cell adhesion polypeptides can be efficiently produced by genetic engineering techniques.

A 274 amino acid residue polypeptide will be described in detail below as an example.

274 amino acid residue polypeptide (p“01239^s
p + 512) is the following formula ■: Pro Thr
Asp Pro Asp Thr Pro Pro 5er Val Arg Tyr Asp Val Ala Asp Asn Ala Leu Arg Phe Met Arg Val IIe Asp Leu Ser Pro Val Glu Leu 5er Val Val Leu Thr 8sn 1ie Thr Trp Ala Thr Asn Phe Lys Asn Glu lie Sar Pr .

Thr　　Asn　Leu ay Pr.

Leu Glu Sar Leu Pr.

Sar Leu Sar Thr Ala 1e Arg Arg Pr.

Ala Leu Val Thr Ala 1e Pr.

Lys Pr.

Ala 1y Val Arg Pr.

1a Pr.

Arg Pr.

Asn G+y Leu IIe Pr.

Pr.

Pr.

Thr Val Sar 1y Val Thr Tyr G]y Thr Asn Arg lis Arg Sar Thr Asn ly Arg Thr Ala Tyr in Thr Val Thr Glu Glu Arg ay Sar a is Glu 1e u y in Asp Sar Val uy Glu Ala Asp e+y Tyr ln in 1e Phe Thr Pr.

Asp Thr Tyr Arg ln Leu Leu Thr Glu Phe Thr Tyr Arg Val is Lys Asp Thr 1e Glu Arg Leu Val Glu Sar Glu Leu Val Thr Thr 1e Thr uy Val Glu Thr Phe Val Thr IS Val Thr Val Glu Thr Val 1e Arg Ty Val Sar 1e Asp Sar Sar ly Sar lis ly Phe Pr.

Asn Sar Sar Val Val Sar Tyr 1y Pr.

ly Thr Sar Val Thr Leu 8sp Trp Tyr Sar is Leu l5 Pr.

Sar Ala Trp Tyr Asn 1y Leu Val Pr.

Sar Pr.

Asp IIe IIe Arg ly Sar Thr Val Leu Asp Ala Asp Arg Sar Sar Lys Tyr 1a Ser Ser Ser Lys Pro Ile
Ser lie Asn Tyr ArgTh
r Glu lie Asp ・
...CII). The manufacturing method is disclosed in Japanese Patent Application Laid-Open No. 2-93797, and will be specifically explained below.

In this specification, the page numbers assigned to amino acids are from the EMBL Data Bank (εMBL DATA BAN).
The number of amino acid residues from the N-terminus assigned to the amino acids obtained by translation of the FN cDNA sequence of K) is shown.

274 amino acid residue polypeptide (Pro123g^5
p1512) is a 279 amino acid residue polypeptide (prO+239-1etlS+) for which a patent has already been applied.
'? ) (JP-A No. 1-206998) in which the C-terminal 5 amino acid residues are deleted. As a method for genetically preparing the 274 amino acid residue polypeptide (Pr01239 ASp+512), the 274 amino acid residue polypeptide (Pr01239 ASp+512) described above is used.
Amino acid residue polypeptide (pro+238N et
plasmid pTFD7 encoding I517)
It is convenient to use 07. By converting codon AAA of Lys15'3, which is the 5th residue from the C-terminus of the 279 amino acid residue polypeptide, into a stop codon TAA, 2
74 amino acid residue polypeptide (pro+239A S
A plasmid encoding p l S l 2 ) can be prepared. This base conversion can be performed by introducing site-specific mutations.

274 amino acid residue polypeptide (pr, +219A
Escherichia coli JM into which a plasmid expressing sp l S + 2) was introduced
109/pTF 7.221 has been deposited as FERM BP-1915.

Purification of this cell adhesion polypeptide from a recombinant is carried out, for example, as follows. The bacterial pellet is suspended in a buffer and separated into a soluble fraction and an insoluble fraction by sonication. The latter is further solubilized with a buffer containing 7M urea.

The soluble fractions were collected and applied to a column of Sepharose 4B conjugated with an antibody specific for the cell adhesion domain of FN.
Perform affinity purification. A buffer around pH 2 or 3 is used for elution. A cell adhesion polypeptide can be obtained by collecting the target fraction by immunoblotting. If necessary, further purification can be performed by FPLC or HPLC.

Furthermore, in the present invention, heparin-binding polypeptide refers to F
The polypeptide is not particularly limited as long as it is contained in the heparin-binding domain of N and has heparin-binding activity.

An example of the above-mentioned polypeptide is the heparin-binding polypeptide disclosed in Japanese Patent Application No. 1-131453 created by the present inventors. The polypeptides described in this specification can be efficiently produced by genetic engineering techniques.

Below, as an example of a heparin-binding polypeptide, 271
The amino acid residue polypeptide will be explained in detail.

271 amino acid residue polypeptide (A7a18807
hr r 9 # D ) is the following formula m: Ala li
e Pro Ala Pro Thr Asp
Leu Lys PheThr Gln
Val Gln Trp Thr Gly Tyr Arg Lys Thr Gly Ala Pro Asp Gly Leu Met Ser Val Tyr Ser Arg Pr.

Leu Glu Asn Arg Val Thr Thr lie 5er 11e Thr Gly Ala Asn Gly 11e Lys Pr.

Thr Gly Leu 11e Tyr Leu Arg Ser 5er Thr Ala l1e Phe Leu Ala Val　Ser　Trp Thr　Pro　Thr Pro Pro Asn Val　Arg　Val Pro Met Lys Ser　Ser　5er Val Ala Thr Ala　Leu　Lys Ala Gln Gly Val Ser Pr.

Asp Ala Thr Trp Arg Thr Phe Gln Vat Gin Thr Pr.

Asp Val Arg Gln Pro Gly Tyr Thr Leu Pro Val Val Asp Ala Pr.

Thr　Thr　Pr.

Gin Pro Pr.

Ser Leu Ser AlaVal Gi
n Leu ThrThr Pro Lys Glu Ile Asn Val Val V-al Lys Tyr G11l Asp Thr Leu Val Val Thr Pro Arg Arg Glu Thr Thr Thr Lys Thr Glu Asp Ala Val 11e Gin Arg Ser Tyr Thr Thr Thr Asp Tyr Asn Asp Asn 11e Asp Ala Ser Asn Leu Asn Ser Leu Arg Ala Arg Glu Leu Ser Val Thr Thr Ala Ile Thr Pr.

Thr 1e Lys Ala Ser Arg Leu 1e Thr Gly Tyr Ile Ile L
ys Tyr Glu Lys Pr.

GJy Ser Pro Pro Arg G
lu Val Val Pro ArgPro
Arg Pro Gly Val Thr
Glu Ala Thr 1ieThr G
ly Leu Glu Pro Gly Th
r Glu Tyr Thrlle Tyr Va
l Ile Ala Leu Lys Asn
Asn GinLys Ser Glu Pro
Leu Ile Gly Arg Lys
LysThr ・[I[[ ].

The manufacturing method is shown in Japanese Patent Application No. 1-131453, and will be specifically explained below.

cDN encoding the heparin-binding domain of human FN
Plasmid pLF 2'435 containing the ^ fragment was regenerated by ligating the coding regions of pLF 2.4.3 and 5, described in Biochemistry, Vol. 25, pp. 4936-4941 (1986). This is a constructed plasmid.

The necessary cON^ fragment was excised from this plasmid pLF 2435 using a restriction enzyme, and a synthetic NA containing a start codon was ligated to the 5' side, and a synthetic NA containing a stop codon was ligated to the 3' side using DNA ligase. Then, by connecting to an appropriate expression vector, the two heparin-binding domains are isolated.
71 amino acid residue polypeptide (Ala16904hr
A plasmid pH0101 is constructed that expresses 1960).

All existing expression vectors can be used, but for example, pLI[: 11BN/p[1c
11911 [FBBS Let
ters), Vol. 223, pp. 174-180 (198
7), and its derivatives, good results can be obtained. By introducing this plasmid into E. coli, the heparin-binding polypeptide can be expressed and its properties can be investigated.

271 amino acid residue polypeptide (^1 a l 69
E. coli Bscherichia coli HB 101/introduced with a plasmid expressing 0Thr”60)
p) 10101 is Microtechnical Research Institute No. 2264 (FBRM
BP-2264).

Purification of this heparin-binding polypeptide from recombinant
For example, do as follows. After culturing the recombinant Escherichia coli in a medium such as L-gloss and collecting the cells, a bacterial cell disruption solution is obtained by ultrasonication, and this is centrifuged to obtain a supernatant. After dialysis, the supernatant is passed through a DBAE ion exchanger column, and then subjected to affinity chromatography using a CM ion exchanger and/or heparin (iiO agarose, etc.). Through the above operations, the polypeptide of interest can be purified. .

Furthermore, the chimeric polypeptide of a cell adhesion polypeptide and a heparin-binding polypeptide in the present invention refers to a chimeric polypeptide in which the above-described cell adhesion polypeptide of the present invention is bound to the heparin-binding polypeptide of the present invention, either directly or via methionine. It is a peptide.

An example of the above-mentioned polypeptide is the polypeptide (549 amino acid residue polypeptide) disclosed in the aforementioned Japanese Patent Application No. 1-131453 created by the present inventors.
It can be efficiently manufactured using genetic engineering techniques.

The 549 amino acid residue polypeptide will be described in detail below.

549 amino acid residue polypeptide (pro+239S
er ” ”-Met-^Ia1690-ThrI96
°) is the following formula ■: Pro Thr Asp Pro Asp Thr Pro Pro 5er Val Arg Tyr Asp Val Ala Asp Asn Ala Pro Gly Thr Ser Vat Tyr Leu Arg Gly Ser Pro T hr Thr Ala Asn Ala Pro Arg lie Arg His Arg Pro Arg Arg Asn 5er Pro Gly Thr Ala Leu Asn Leu Arg Phe Met Arg Val 11e Asp Leu Ser Pro Val Glu Leu 5er Val Val Val Leu Glu Tyr Val Glu Gin )lis Arg Gin Lys Gly Ile Asp Ser Phe Thr Ala Thr l1e His Pro Glu Glu Asp Arg Ice Thr Leu Glu Tyr Val Gly Arg Glu Thr Asn 1ie Thr, Trp Ala Thr Asn Phe ys Asn (ilu lie Ser Pr.

Thr　　Asn　Leu Val　Ser　Val Glu Ser Thr Thr Gly Leu Phe Ser Asp Val　His　Trp Thr Gly Tyr 1(is　Phe　5er Val Pro! (is Thr　　Asn　Leu Val Ser l1e Glu Ser Pr.

1y Pr.

Lau Glu Ser Lau Ser Pr.

Asp 1e Ile Arg Gay Ser Thr Val Lau Seu Val Thr Ala 1e Pr.

Lys Pr.

81a Ser 11e Gly Pro Arg Pro Thr Pro Ala Thr　Tyr Val　Gln 5er　Thr Gly Val Val Thr Ser　Lys Gln Asp Ser Val ry Glu Ala Asp 1y Pr.

Gin 5er Leu Glu Leu Leu Thr　Val Glu Thr Phe Thr Thr　l1e Tyr Thr Arg　Gly Ice 5er Pro Aha Val Thr Thr Pr.

Arg Val Gay Pr.

Asp Ser Met　Val Tyr Ala Pro Ala Pr.

Pr.

Pr.

Arg Met Ser Ala Seu Gln Thr Thr Asn Val Lys Ser Thr Yes Gay Asp Lau Ser　Leu Val　Gin Thr Pr.

Glu 1ie Val　Val Lys Tyr Asp Thr Val　Val Thr Val 5er Val　Vat　Ala He Ser Trp Arg Tyr Tyr Gay Gly Asn Val Pro Gay Ser　Gly　Leu Ice Thr Val Asp Ser Pr.

11e Asn Tyr Asp Ala Asp Arg Ser Ser Lys Tyr Ala 8rg Lys　Phe Ser　Ala Leu Thr LysGIυ Asn　Leu Val 5er Glu Val seu Thr Thr　Thr Thr Gln Gay Lys Ala Gay Ser Ser Lau Gln Trp Tyr Thr Pr.

Lau Val Arg lu 5n Thr er Ty Ty Pr.

eu eu er 1e la rp yr Pr.

Pr.

eu al al sp rp he in sp in yr Pr.

sp hr in 1e Pr.

uy lu 1e er la rg ln hr al Pr.

hr al la hr Pr.

ie rg al Pr.

^1a Pr.

hr hr al Pr.

rg ly eu al Pr.

Pr.

Pr.

ys lu hr 1y eu Pr.

lu ys sp 1e er hr As mouth 1e er As mouth rg yr al lu hr ys rg hr hr la in yr sp ^sp sp As mouth er la lu al la lu As mouth rg hr lu al rg hr yr As mouth la eu Seu rg ys Pr.

hr yr As mouth la 1e hr Pr.

hr ie ys ^1a er ＾rg eu 1e Pr.

rg 1e hr in rg hr ie la ie hr ie ＾rg hr he al hr 1y Pr.

hr 1e ys al le hr As mouth ys ly yr er la eu er ly er rg ly yr er [■] It is characterized by being represented by the amino acid sequence represented by

The manufacturing method is shown in Japanese Patent Application No. 1-131453, and will be specifically explained below.

279 amino acid residue polypeptide (pr0+239-MetlSlff) described in JP-A-1-206998
A Nco I site was introduced into plasmid pTF 7021 obtained by connecting the DNA encoding %Pro""-3et""-Met to an expression vector to construct plasmid pTF 7520 encoding %Pro""-3et""-Met.

The cDNA fragment was extracted from the aforementioned pH0101 and pTF
By connecting to the 3' terminal Nco I site of the translated region of FN, a recombinant plasmid expressing a 549 amino acid residue polypeptide in which the cell adhesion domain and heparin binding domain of FN are linked can be obtained.

The junction in the plasmid contains a methionine residue derived from the Nco I site as a linker.

The presence or absence of a linker does not affect the effectiveness of the present invention, but if necessary, it can be easily removed by site-specific mutagenesis.

E. coli E! into which a plasmid expressing a 549 amino acid residue polypeptide was introduced. 5cherichia coli
flB 101/pCH101 is Microtechnical Research Institute No. 2799
No. (FBRMBP-2799).

By introducing the obtained plasmid into E. coli and culturing it under appropriate conditions, the target polypeptide is accumulated in E. coli.

Purification of the target polypeptide is performed, for example, as follows. After culturing the recombinant Escherichia coli in a medium such as L-gloss and collecting the cells, a bacterial cell disruption solution is obtained by ultrasonication, and this is centrifuged to obtain a supernatant.

After the supernatant is dialyzed, it is passed through a column of DRAB ion exchanger, followed by affinity chromatography such as CM ion exchanger and/or heparin-agarose. By the above operations, the polypeptide of interest can be purified.

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.

As solid carriers, sugars such as maltose and sucrose, amino acids, cellulose derivatives such as hydroxypropylcellulose, organic acid salts such as magnesium stearate, etc. are used.

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 together with excipients such as sugars such as inositol, mannitol, lactose, and sucrose, and amino acids such as phenylalanine, and is then mixed with an appropriate solvent for injection, such as sterile water, physiological saline, glucose solution, and electrolytes, at the time of administration. It can also be administered by dissolving it in a liquid for intravenous administration, such as a 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 desirable that the active ingredient is normally contained in an amount of 0.1 to 30% by weight, preferably 1 to 10% by weight. In the case of oral administration, tablets,
It is used in the form of capsules, powders, granules, liquids, dry syrups, etc. Capsules, granules, and powders are generally 5
Contains ~100% by weight of active ingredient, preferably 25-98% by weight.

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.
■/kg/day, 5-500■/kg by oral administration
/ day.

[Effect]

Next, the physiological activity of the polypeptide of the present invention will be shown through experimental examples.

Experimental Example 1 Anticancer effect COP, L5178Y-ML on mice (10 mice per group)
25 leukemia cells, or 250 μg of the polypeptide of the present invention and 4 x 10' L5178Y-ML25 cells.
Perform an intravenous injection of the mixture.

The survival rate of the group administered with the polypeptide of the present invention is expressed by the following formula based on the average number of days of survival after intravenous transplantation of leukemia cells in each group of mice and control group of mice.

Table 1 shows the survival days, survival ratio (number of surviving mice on day 26/number of mice used in the group), average survival days, and survival rate of mice in each group on the 26th day after cancer cell transplantation.

As described above, administration of the polypeptide of the present invention exhibits anticancer effects.

Experimental Example 2 Acute Toxicity Test CDF: Various polypeptides of the present invention were intravenously administered to mice. No toxicity was observed at 100 mg/kg administration.

As shown in the above experimental examples, the polypeptide of the present invention has excellent safety and good anticancer effects, and is useful in various cancer treatments.

〔Example〕

EXAMPLES Next, the present invention will be specifically explained with reference to Examples, but the scope of the present invention is not limited to the Examples.

In addition, on each side, parts mean parts by weight.

Formulation Example 1 PBS was added to 30 parts of a 274 amino acid residue polypeptide to make a total amount of 2000 parts, and the solution was dissolved and filtered for sterilization using a Millipore filter GS type. 2 g of this filtrate was placed in a 10-sized vial and lyophilized to obtain a lyophilized injection containing 30 ml of the polypeptide per vial.

Formulation Example 2 PBS was added to 30 parts of the 271 amino acid residue polypeptide to make a total amount of 2000 parts, and the solution was dissolved, followed by sterilization filtration using a Millipore filter - GS type. 2 g of this filtrate was placed in a 10th vial and lyophilized to obtain a lyophilized injection containing 30 μg of the polypeptide per vial.

Formulation Example 3 PBS was added to 30 parts of the 549 amino acid residue polypeptide to make a total amount of 2000 parts, and the solution was dissolved and then sterilized and filtered using a Millipore filter GS type. 10ml of 2g of this filtrate! A lyophilized injection containing 30 mg of the polypeptide per vial was obtained.

Formulation Example 4 50 parts of a 274 amino acid residue polypeptide, 600 parts of lactose, 330 parts of crystalline cellulose and 20 parts of hydroxypropyl cellulose were thoroughly mixed, compressed using a roll compactor, and crushed to give 16 The mixture was sieved to obtain a particle size between 60 mesh and granules.

Formulation Example 5 50 parts of a 271 amino acid residue polypeptide, 600 parts of lactose, 330 parts of crystalline cellulose, and 20 parts of hydroxypropyl cellulose were thoroughly mixed, compressed using a roll compactor, and crushed to give 16 The mixture was sieved to obtain a particle size between 60 mesh and granules.

Formulation Example 6 50 parts of a 549 amino acid residue polypeptide, 600 parts of lactose, 330 parts of crystalline cellulose, and 20 parts of hydroxypropyl cellulose were thoroughly mixed, compressed using a roll compactor, and crushed to give 16 to It was sieved to fit between 60 mesh and made into granules.

〔Effect of the invention〕.

The polypeptide of the present invention is a biologically relevant substance, has high safety, and is useful as an anticancer agent.

It also has a remarkable effect in that it can be supplied in large quantities through genetic engineering.

Procedural amendment (voluntary) August 28, 1990 Toshi Ueki, Commissioner of the Patent Office

Claims (1)

[Claims] 1. The following general formula I: Y-(Met)_n-Z...[I] (wherein, Y is H- or a cell adhesion polypeptide in the fibronectin molecule, and n is 0 or 1 , Z represents -OH or a heparin-binding polypeptide in a fibronectin molecule, and at least one of Y and Z is the polypeptide.