WO1998015643A1

WO1998015643A1 - Fragments of plasminogen effective in inhibiting tumor metastasis and growth and process for preparing the same

Info

Publication number: WO1998015643A1
Application number: PCT/JP1997/003635
Authority: WO
Inventors: Wataru Morikawa; Seiji Miyamoto
Original assignee: Juridical Foundation The Chemo-Sero-Therapeutic Research Institute
Priority date: 1996-10-09
Filing date: 1997-10-09
Publication date: 1998-04-16
Also published as: AU4571497A; JPH10114796A; JP3806471B2

Abstract

Fragments of a plasminogen effective in inhibiting tumor metastasis and growth, a process for preparing the fragments, and a tumor metastasis and growth inhibitor containing the fragments as the active ingredient. The fragments are obtained from the elastase-induced hydrolysis product of Lys-plasminogen obtained by treating a plasminogen with plasmin and preferably have a potent heparin-binding activity. The inhibitor is useful for clinical therapy of solid cancers typified by lung and colon cancers.

Description

Specification

FIELD OF THE INVENTION

The present invention relates to a novel biologically active plasma protein fragment and a method for preparing the protein fragment. More specifically, the present invention relates to an elastomer of rice plasminogen (Lys-Plasminogen; hereinafter, sometimes referred to as "Lys-P1g."), Which is a kind of N-terminal modified plasminogen. A plasminogen fragment having an inhibitory effect on tumor metastasis and proliferation, which is preferably a fragment having a high heparin binding property, which is a digestion product, and a tumor metastasis and growth inhibitor comprising the plasminogen fragment as a main component And a method for preparing the plasminogen fragment. The elastase degradation product of Lys-P 1 g., Particularly the Lys-P 1 g. Fragment exhibiting high heparin binding, according to the present invention is represented in the fields of biochemistry and medicine, such as lung cancer and colon cancer. It is useful in the field of clinical treatment of solid cancer and the like.

Background art

Plasminogen is a plasma protein with a molecular weight of 800,000, and is a precursor to the enzyme plasmin involved in the blood coagulation and fibrinolysis systems. Plasminogen is a glycoprotein, and it is known that there are about 10 components having different isoelectric points due to differences in the sugar chain structure. Plasminogen itself has no enzymatic activity, but undergoes limited degradation by plasmin, perokinase, or plasminogen activator to be converted to the active form of plasmin, which breaks down fibrin clots It becomes more active. Plasminogen contains five kringle moieties (kringle 1 to kringle 5) and a serine protease domain with an active center (see FIGS. 1 and 2). Plasminogen also Since it can bind to lysine depending on the properties of the kringle portion, it can be specifically prepared using a lysine-binding carrier.

As shown in Figure 1, plasminogen is restricted by elastase to three sites in the serine protease domain, including the amino acid sequence 79Tyr to Kringle 3, the Kringle 4 site, and the Kringle 5 site. Generate Each of these three sites is a lysine binding site I

(Lysine Binding Site I; hereinafter may be referred to as “LBS-I”), lysine binding site Π (hereinafter sometimes referred to as “LBS-I”), and miniplasminogen ( mini—

Plasminogen (hereinafter sometimes referred to as “mini P 1 g.”) (Davidson JF et al., “Primary structure of human plasminogen: limited by elastase-catalyzed specific proteolysis. Isolation of lysine-binding fragment and "mini-one" plasminogen (Mw 38,000) (The primary structure of human plasminogen:

isolation of two lysine-binding fragments and "mini-" plasminogen

(MW: 38,000) by elastase-catalyzed-specific limited proteolysis), Raven Press), New York, vol. 3, 191-209, 1978).

Plasminogen includes glulusminogen having a glutamic acid residue at the N-terminus (Glu-Plasminogen; hereinafter sometimes referred to as “G1u-P1g.”) And rice plasminogen having a lysine residue ( Lys—

Plasminogen; Lys-P 1 g.) Is known. The former is an intact type of plasminogen, while the latter, as shown in Fig. 2, shows that Lys in the N-terminal region of G1 uP 1 g. s 76-Ly s 77 You. In addition, in what is known as Lys-P1g., In addition to those in which Lys is an N-terminal amino acid, those in which Va1 and Met are N-terminal amino acids are also detected. When these Lys-Plg and Glu-Pig. Are compared, Lys-P1g. Has a much higher activation rate by perokinase than G1u-P1g. Lys-P1 g. Has several different properties, such as greater fibrin-binding ability than G1u-P1 g. It is thought to be due to the difference in the higher-order structure of the gen molecule, and in particular to the change in the spatial arrangement of kringle 1 in its molecular structure (Lerch PG et al., Human Plasminogen Localization of individual lysine-binding regions in human plasminogen and investigations on their complex-forming

properties), Eur. J. Biochem. 107: 7-13, 1980). Recently, O'Reilly and colleagues found an anti-angiogenic activity in LBS-I in the degradation product of human G 1u-P 1 g., And reported that this substance suppressed the growth of cancer after metastasis. 〇 'Reilly MS, et al., Angiostatin: a novel angiogenesis inhibitor that mediates suppression of metastasis by Lewis lung cancer

Angiostatin: a navel angiogenesis inhibitor that mediates the suppression of metastases by a lewis lung carcinoma) ヽ Cell 79: 315-328, 1994). The present inventors have proposed a plasminogen lysine binding site I (hereinafter referred to as rice-type lysine binding site I (Lys-LBS-I) obtained by treating G 1 u-P 1 g. To distinguish them, they may be referred to as “G1u-LBS-I”), and reproduction experiments were performed in accordance with their experiments. Some difference, but significant difference compared to control group I didn't do it.

In view of the above-mentioned problems, the present inventors have found that if plasminogen is treated with a certain substance (enzyme) before it is directly treated with elastase to obtain G1u-LBS-I, We hypothesized that it would exhibit its activity. Based on this hypothesis, plasmin is digested with plasmin before elastase degradation to prepare a molecule whose N-terminal starts with a lysine residue, that is, Lys-P 1 g. The rice-type lysine-binding site I (Lys-LBS-I) having a different N-terminal amino acid residue from the lysine-binding site I (G1u—LBS—I) of Oleilly et al. And examined the effect of this molecule on cancer metastasis and proliferation. C As a result, surprisingly, elastase treatment was performed using Lys—P 1 g, a degradation product of plasminogen degraded with plasmin, as a starting material. It was found that Lys-LBS-I obtained as described above had a strong tumor metastasis growth inhibitory effect. Furthermore, as a result of examining the substance of the substance exhibiting the tumor metastasis growth inhibitory effect, it was found that Lys-LBS-I was bound to the heparin carrier at a low ion intensity which is not observed in G1u-LBS-I. After G1u-P1g. Was converted to the Lys Form (plasminogen whose N-terminal is a lysine residue) by the action of plasmin, It has been found that Lys-LBS-I, which is produced by elastase degradation, has the effect of inhibiting the growth of tumor metastasis for the first time. Furthermore, they have found that a fragment having an angiogenesis inhibitory effect can be efficiently prepared using a carrier to which heparin is bound by utilizing the property of binding to heparin, and have completed the present invention.

Disclosure of the invention

The present invention relates to an elastase degradation product of Lys-P 1 g. It is intended to provide a plasminogen fragment having a tumor metastasis growth inhibitory effect, in which a fragment exhibiting particularly high heparin binding properties is a preferred embodiment.

The present invention also provides a method for preparing a plasminogen fragment having the tumor metastasis growth inhibitory effect. The preparation method according to the present invention comprises the following steps: 1) Plasmin or the like is allowed to act on plasminogen to produce Lys-P 1 g .; 2) Lys-P 1 g. To obtain a fraction of kringle 3-containing fragment (Lys-LBS-I) from kringle 1; ③ of the obtained fractions, select a portion having strong binding to heparin, and obtain a desired tumor. A plasminogen fragment having a metastatic growth inhibitory effect is obtained. The present invention further provides a tumor metastasis growth inhibitor comprising, as an active ingredient, a plasminogen fragment having the tumor metastasis growth inhibitory effect.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram showing a product obtained by elastase digestion of plasminogen.

FIG. 2 is a schematic diagram showing degradation products when plasminogen is subjected to elastase treatment after limited degradation with plasmin.

FIG. 3 is a graph showing the elution pattern of Lys-LBS-I by immunoaffinity mouth chromatography using heparin as a ligand.

FIG. 4 is a graph showing an elution pattern of G1u-LBS-I by immunoaffinity chromatography using heparin as a ligand.

FIG. 5 is a diagram showing the results of analysis of the heparin high-binding fraction of Lys-LBS-I by SDS-PAGE (gel electrophoresis).

FIG. 6 is a graph showing the heparin binding of Lys-LBS-I at various ρΗ.

Figure 7 shows suppression of lung metastasis growth of Lewis lung cancer using C57BL6ZJ mice 4 is a graph showing the effect of Lys-LBS-I in a test in comparison with G1u-LBS-I.

FIG. 8 is a graph showing the effect of Lys-LBS-I as compared to G1u-LBS-I in a test for inhibiting the growth of lung metastasis of Lewis lung cancer using skid (SCID) mice.

FIG. 9 is a graph showing the effect of Lys-LBS-I on inhibiting the tumor metastasis and growth of the fraction showing high heparin binding, as compared to the fraction not binding to heparin.

BEST MODE FOR CARRYING OUT THE INVENTION

FIG. 1 shows a degradation product obtained by elastase digestion of plasminogen, and FIG. 2 shows a degradation product obtained by digesting plasminogen according to the present invention with plasmin and then with elastase.

As shown in Fig. 1, when plasminogen is directly degraded by elastase, the fragment (G1u) that is cleaved at 78 Va1-79Tyr and contains kringles 1-3 with N-terminal Tyr — LB SI) is released. In contrast, when plasminogen is limitedly degraded by plasmin, it is limited by 76Lys-77Lys, and plasminogen (Lys-Pig.) Whose N-terminal starts with Lys is prepared. Next, when this is cleaved with elastase, a fragment (Lys-LBS-I) containing Kringles 1-3 of Lys at the N-terminal residue is released (Fig. 2). In the case of plasminogen (Lys-P1 g.) Whose N-terminal starts with Lys, LBS-I whose N-terminal starts with Lys without being cleaved at the 79Tyr is obtained by Eras This is probably because the steric configuration of the amino acid subjected to the decomposition action of Yuze was not established on the N-terminal side. In any case, it is interesting to note that a difference of only one or two amino acid residues at the N-terminus greatly changes the tumor metastasis growth inhibitory effect and the binding to heparin. The plasminogen fragment having a tumor metastasis growth inhibitory effect of the present invention is Ly s-LBS-I obtained by elastase decomposition of Lys-P 1 g. Among them, a particularly strong tumor metastasis growth inhibitory effect is exhibited. Indicates high heparin binding. Lys-LBS-I is a protein that is composed of kringles 1 to 3 whose N-terminal amino acid starts with 77 Lys and has a sugar chain-free molecular weight of 38 Kda on SDS polyacrylamide gel electrophoresis (PAGE). This shows stronger heparin binding as compared to other sugar chain-containing isoforms. The fragment cannot bind to heparin at physiological ion concentrations (physiological conditions) in the neutral region, but has the characteristic that it can bind to heparin at physiological ion concentrations when the environment is acidic.

Heparin has an action of suppressing blood coagulation by binding to antithrombin m which is a plasma component. Heparin or heparin-like substances are widely distributed on vascular endothelial cells occupying the vascular lumen, and suppress extra coagulation in the blood vessel. The heparin-binding property of the plasminogen fragment of the present invention is determined by the binding mode exemplified by the binding with lysine, which is required when plasminogen (plasmin) binds to fibrin or the like, which has been conventionally considered. In addition, it has the potential to directly bind to the vascular endothelial cells and exert some effect. In order for the distant metastatic lesion of the tumor to grow, blood vessels that transport nutrients and waste to the tumor cells are necessary. In the process of tumor growth, the phenomenon of "forming new blood vessels and drawing them into the tumor (angiogenesis)" is observed. New blood vessels are formed through endothelial cells of existing blood vessels, which receive signals from tumor cells, destroying existing blood vessels, and undergoing stages of “proliferation”, “elongation”, and “lumen formation” toward the tumor cells. You.

Therefore, by suppressing the action of such vascular endothelial cells, Since it is possible to suppress the growth of distant metastatic foci, the heparin-binding property of the plasminogen fragment of the present invention must satisfy the necessary conditions for exerting an inhibitory effect on the function of vascular endothelial cells. I can say.

However, since all blood vessels, including existing blood vessels, are occupied by vascular endothelial cells, it is necessary to suppress only the growth of distant metastatic lesions of tumor cells without affecting normal vascular endothelial cells. Requires selective binding of the agent only to neovascularization of tumor cells. (J ain, RK) reported that blood vessels in tumors were extremely polarized, blood flow was congested, and the environment was inclined to acidic due to hypoxia. Barriers to drug delivery in solid tumors, ci. Am. Vol. 27 1 (1) 58—5, 199 4). As described above, the plasminogen fragment of the present invention cannot bind to heparin or a heparin-like substance under physiological conditions (isotonic state), but has non-physiological conditions in which the environmental pH is reduced. Underneath, it has the property of acquiring heparin binding. Therefore, as reported by Jane et al., In an acidic environment such as in a tumor, the plasminogen fragment of the present invention binds to heparin or heparin-like substance in the tumor, and as a result, It seems to act specifically on the ulcer.

The mechanism by which the plasminogen fragment of the present invention suppresses the growth of tumor cells is unknown, but considering its structure, it may be due to competitive inhibition of plasmin action.

As described above, Olairy et al. Report that plasminogen fragments directly inhibit the proliferation of vascular endothelial cells. They found that tumor-bearing animals transplanted with Lewis lung cancer produced potent angiogenesis inhibitors in their blood and urine, purified this substance and named it angiostatin. did. The angiostatin showed extremely high homology with the internal fragment of plasminogen, and it was reported that purified human LBS-I (G1u-LBS-I) showed similar activity. The human LBS-I is a substance having three isoforms having a molecular weight of 38 Kda to 42.5 Kda, and is noted for showing high homology with the plasminogen fragment of the present invention.

At this stage, it is not possible to clearly determine whether the plasminogen fragment of the present invention and angiostatin are identical as a mechanism of action and a substance.However, the plasminogen fragment of the present invention and angiostatin do not ,

(1) The plasminogen fragment of the present invention does not contain a sugar chain, and significantly inhibits the growth of distant metastatic tumor cells as compared with other isoforms containing a sugar chain.

(2) It is also a fact that the plasminogen fragment of the present invention has a clear difference in physical properties and biological activity such as not exhibiting the effect of suppressing the growth of vascular endothelial cells as much as angiostin.

It is undeniable that the high inhibitory effect of the plasminogen fragment produced by the present invention on the metastasis of tumor metastasis may be attributable to the aforementioned difference from angiostatin.

The method for producing the plasminogen fragment of the present invention is not particularly limited, and includes, for example, a method comprising the following steps: (1) Lys-P 1 g. The solution containing 1 g of Lys-P is treated with elastase to obtain a fraction of a fragment containing kringle 3 from kringle 1 (Lys-LBS-I); ③ of the obtained fractions, A portion having a strong binding property to heparin is selected to obtain a plasminogen fragment having a desired tumor metastasis growth inhibitory effect.

Specifically, first, plasminogen is separated from a blood sample, and Lys-Pig. Is prepared from the obtained plasmaminogen. Blood-derived plasmino The following method is mentioned as a method for producing one gene. For example, as a typical production method, a method of Deutsch (DG) purifying by affinity chromatography using a lysine carrier (Deutsch, DG, Science 170: 1095, 1970) and a modified method thereof (Blockway ( Brockway.W.J.) Et al., Arch. Biochem. Biophys. 151: 194, 1972). That is, aprotinin (2 OUZm 1) and EDTA (2.5 mM) are added to fresh plasma and mixed, then bound to a lysine carrier, and a buffer solution containing 0.1 M Na C 1 /2.5 mM EDTAZ20U aprotinin / m 1, Further, after washing with the same buffer solution containing a surfactant, elution with 6-aminohexanoic acid makes it possible to prepare high-purity plasminogen. Finally, purification and concentration are performed using an ultrafiltration membrane (for example, YM10: manufactured by Amicon).

Most plasminogen in its full molecular form is present in the blood, and there is little Lys-P1 g. Whose N-terminal residue is lysine. Therefore, for the purpose of the present invention, a step of converting the complete molecular form of plasminogen to Lys-P 1 g. Is required. As a method for this conversion, a method of directly acting plasminogen on plasminogen is used. (Ruzy Jungberg

(Ljungberg, J.) et al., Thromb. Res. 53: 569—576, 1989), a method of directly acting plasmin on plasminogen (Castellino).

(Castelino, F.S.) et al. Methods in Enzymology (Academic Press). New York, vol. 80, 365, 1981) or a method by incubating plasminogen for a long time (Max.

(Markus, G.) et al., J. Biol. Chem., Vol. 254, 1211-1216, 1979). In the present invention, a preferable method is to incubate plasminogen in the presence of tranexamic acid, autolyze it, and prepare Lys-P The method of preparing 1 g. Is recommended.

Then, 1 g of Lys-P obtained is digested with elastase, and a molecule (Lys-LBS-I) containing 1 to 3 kringles of Lys-P is recovered from the resulting fragment. . In this case, good preparation of Lys-LBS-I is achieved, for example, by a gel filtration method using Sephadex G-75 and a subsequent lysine-affinity chromatography. Subsequently, the obtained Lys-LBS-I is contacted with a resin using heparin as a ligand to obtain a bound fraction, whereby a fraction that strongly binds to heparin can be specifically prepared.

The plasminogen fragment of the present invention, Lys-LBS-I, can also be produced directly based on gene recombination techniques. That is, Lys-plasminogen-producing cells are constructed by gene recombination technology, and Lys-plasminogen thus prepared is fragmented with elastase, or the plasminogen fragment (Ly- s-LBS-I) is directly introduced into a suitable host cell such as a eukaryotic cell, a mammalian cell or an insect cell using a suitable vector or the like to constantly produce a desired plasminogen fragment. As a result, Lys-LBSI-I can be prepared.

The plasminogen fragment of the present invention prepared by the above-mentioned method should be used fresh to maintain the activity to the maximum, or within 5 words after storage when stored at 4 ° C. It is preferred to use The plasminogen fragment of the present invention can also be stored in lyophilized or liquid form with suitable stabilizers such as human albumin, gelatin, salts, sugars or amino acids, and furthermore, plasminogen. It is also possible to freeze and store the gene fragment solution. In order to inactivate infectious contaminating viruses, Heat treatment of lyophilized or liquid state under specified conditions, for example, lyophilized state at 65 ° C for 96 hours, and liquid state at 60 ° C for 10 hours, is required from the viewpoint of drug safety. This is a very preferred embodiment.

The plasminogen fragment of the present invention can be used as a tumor metastasis growth inhibitor by using the fragment as an active ingredient and combining it with a known suitable excipient.

The effective dose of the tumor metastasis / proliferation inhibitor comprising the plasminogen fragment of the present invention as an active ingredient varies depending on various factors, for example, the age, symptoms, and severity of the administration subject. Depending on the judgment, it may be generally in the range of 50 to 50 mg / day for an adult, preferably 100 to 300 mg / day in 1 to 2 divided doses. The best mode of administration is a single large dose (bolus) or intravenous drip. It is also possible to use the antitumor agent in combination with the antitumor agent provided by the present invention, which is a preferred embodiment.

Hereinafter, the present invention will be described with reference to examples for better understanding of the present invention, but the present invention is not limited to these examples. In addition, blood-derived plasminogen fragments used in the following Examples were used in single intravenous toxicity studies in mice, repeated intravenous toxicity studies, and general pharmacology studies (respiratory circulatory system using beagle dogs). Its safety has been confirmed by virus inactivation tests and the like.

Example 1

(Preparation of plasminogen)

20 mM benzamidine, ImM PMSF, 10 OU / ml aprotinin (tradiol; manufactured by Bayer) were added to 10 fresh frozen pool plasma, and the mixture was cooled and thawed at room temperature. Then, the suspended matter is separated into a high-speed centrifuge (RS-20IV; The mixture was centrifuged at 8,000 rpm and 4 ° C for 20 minutes with a centrifuge at 8,000 rpm to obtain a supernatant. The supernatant was washed with 50 mM Tris 0.5 M NaC 1

The solution was passed through a lysine-sepharose 4B column (inner diameter 5.0 × 30 cm; manufactured by Pharmacia) at a flow rate of 1.0 m1 equilibrated with (pH 7.5), and further washed with 5 volumes of the same buffer. Thereafter, the buffer was replaced with the same buffer containing 1 OmM aminohexanoic acid to perform elution. The eluate was dialyzed overnight at 4 ° C against 0.1 M ammonium carbonate buffer.

Example 2

(Preparation of Lys-P 1 g.)

After the eluate obtained by the chromatographic operation of Example 1 was concentrated, it was dialyzed overnight against 5 OmM Tris / ^ 2 OmM citrate buffer (pH 6.5), and ImM tranexamic acid was added to the concentrate. Incubated overnight at 30 ° C. Example 3

Preparation of (Elastase-Sepharose)

Dissolve 50 mg of elastase (type IV from pig glands; Sigma) in a 0.1 M sodium bicarbonate solution containing 0.5 M NaC1, then add the buffer to the same buffer at 140 ° C. Dialyzed. The gel for immobilizing elastase was prepared using CNB r-activated Sepharose 4 Fast Flow (Pharmacia), and the binding was determined according to the attached instruction manual. This was performed on a 5 mg elastase Zm1 gel. Example 4

(Preparation of elastase hydrolyzate of plasminogen and Lys-P 1 g.) 1 g of G 1 uP and 1 g of Lys-P prepared in Examples 1 and 2 were obtained according to the method of Davidson et al. (See above), decompose with the elastase prepared in Example 3, and separate the elastase degradation products of both plasminogens. Released. That is, 10 OU / ml of aprotinin (tradiol; manufactured by Bayer) was added to 1 g of purified G1υ-Ρ1 g. Or Lys-Ρ1 g.1 OmgZml, and dissolved in a 0.1 M ammonium carbonate solution. To this, elastase-sepharose was added so that the enzyme-substrate ratio was 1: 100.

The reaction was carried out while stirring at 5 ° C overnight. After completion of the reaction, the reaction solution was filtered with a glass filter, and the filtrate was passed through resin-sepharose (Pharmacia) equilibrated with 0.1 M, and then washed with the same buffer. The lysine-cepharose-bound fraction was eluted with the same buffer containing 2 OmM aminohexanoic acid. The eluate was concentrated on an ultrafiltration membrane (YM-10; Amicon) and equilibrated with 0.1 M ammonium carbonate buffer (Sephadex G-75 column (5.0 x 40 cm id; Pharmacia)) And G 1 u—resin binding site I

(Glu-LBS-I) and Lys-lysine binding site I (Lys-LBS-I) were prepared. Both LBS-I were lyophilized and stored at 4 ° C until use.

Example 5

(Coupling with heparin)

The G 1 u-LB S-I and Lys-LB S-I obtained by the method described in Example 4 were used for immunoaffinity chromatography using heparin as a ligand (high trap heparin (Hi trap trap Heparin) (trade name; manufactured by Pharmacia), and the protein in the heparin-bound fraction obtained by performing a concentration gradient elution based on the salt concentration is monitored by absorbance, and the heparin affinity and amount are measured. The ratios were compared.

That is, G1u—LBS—I (lmgZm1) and Lys—LB dissolved in Imno affinity resin lm1 equilibrated with Tris buffer (pH 7.2) containing 5 OmM NaC1 S—I (lmg / m 1) After washing with 10 ml of the same buffer at a flow rate of 0.5 m 1 Z, add 10 ml of 50 mM NaCl / Tris buffer, and 1 Oml of 1 M NaC1 Tris buffer (pH 7.2). Gluted out.

The results of heparin affinity are shown in FIGS. 3 and 4. As shown in Figure 3, Lys-LBS-I is divided into a non-heparin-binding fraction, a medium-binding fraction, and a high-binding fraction, while G1u-LBS-I is a high-binding fraction. No part corresponding to was found (Fig. 4). In addition, SDS-PAGE of 12.5% of the high binding fraction of heparin showed that its molecular weight was around 38 kda, which was consistent with the molecular weight of LBS-I containing no sugar chain (FIG. 5). The plasminogen used as the raw material did not dissolve in the equilibration buffer composition, and the same operation could not be performed.

Example 6

(Relationship with pH of heparin binding)

Lys-LBS-I obtained by the method shown in Example 4 was subjected to immunoaffinity chromatography using heparin as a ligand at a physiological salt concentration in the range of pH 5.0 to 7.2 (high trap heparin). Pharmacia) to determine the binding to heparin. That is, Lys-LBS-I (lmg / ml) dissolved in the chromatresin lm1 equilibrated with a citrate buffer (pH 5.0 to 7.2) containing 15 OmM NaC1 with the same buffer Was washed with 1 Oml of the same buffer under the conditions of a flow rate of 0.5 m1Z, and eluted with 10 ml of 1 M NaC1 / citrate buffer (pH 5.0 to 7.2).

FIG. 6 shows the relationship between the heparin binding property of Lys-LBS-I and pH. As shown in the figure, Lys-LBS-I cannot bind to heparin near neutral under isotonic conditions, but as pH decreases, heparin's binding property increases. All the fractions bound to heparin under the condition of pH 5.0.

Example 7

(Preparation of human plasminogen heparin-binding fragment)

G1u-LBS-I and Lys-LBS-I obtained in Example 4 were dialyzed overnight against Tris buffer (pH 7.2) containing 50 mM NaCl, and then the heparin affinity gel of Example 5 was used. A chromatographic operation was performed to prepare a heparin-binding fraction at a concentration of 1 Omg / ml. Also, 3 ml of the above-mentioned elastase fragment (1 Omg / m1) was brought into contact with 5 ml of the parin to the high trap, washed at a flow rate of 2.5 ml / min for 40 minutes, and then washed with 1 M NaCl / Tris buffer (pH 7.2) Gradient elution was performed at 25 ml, and the eluted fraction was collected using a fraction collector (Ladylac; manufactured by Pharmacia). The heparin-bound fraction was pooled, dialyzed against a 0.1 M ammonium carbonate buffer, subjected to aseptic filtration, freeze-dried, and subjected to animal experiments.

Example 8

(Lung metastasis growth inhibition test)

Cancer cells are from Lewis Lung Cancer LL 2 (Bertram, JS) and others. Establishment of a cloned line of Lewis Lung uarcinoma cells adapted to cell culture: Cancer Lett. Vol.11, 63-73, 1980) was purchased from Dainippon Pharmaceutical Co., Ltd. and used after continuous cultivation and passage in high-concentration glucose-DMEM medium Zl 0% FCS.

30 mice 6 weeks old male mice (C57BL 6ZJ), 100 1 Lewis lung carcinoma ¹⁰⁷ cells Zm 1 were implanted subcutaneously on the back, were housed 15 to 18 days. Thereafter, the formed primary lesion was removed by a surgical operation, and the skin was sutured. Mice are divided into three groups and bred for 14 days, taking into account body weight and primary tumor weight After that, in each group, Lys-LBS-I 0.5 mg / kg prepared in Example 4 and G1υ-LBS-I 0.5 mg / kg, and saline 1001 as a control group daily for 10 days It was administered intraperitoneally. After the administration, the lungs of the mice were excised and their weights were compared. The statistical processing used nonparametric analysis. FIG. 7 shows the effects of Lys-LBS-I and G1u-LBS-I on metastatic growth of tumors. The lung weight of the control group (physiological saline) -administered group was 0.705 ± 0.411 g, whereas the weight of the Lys-LBS-I-administered group was 0.247 ± 0.05 g, and Lys- LBS-I Significantly inhibited metastatic growth of cancer. On the other hand, the G 1 u—LB S—I administration group weighed 0.406 ± 0.186 g, and no significant difference was observed.

Example 9

(Lung metastasis growth inhibition test using immunodeficient animals)

For the test of Example 8, the mice were replaced with immunodeficient animal skid (SCID) mice, and lung metastatic growth was similarly evaluated.

FIG. 8 shows the results of the model of the present example in which the effect of immunity due to continuous administration of a heterologous protein was considered. Lung weights of the control group, saline administration group, Lys-LBS-I administration group and G1u-LBS-I administration group were 0.522 ± 0.232g, 0.217 ± 0.019g and 0.324 ± 0.152g, respectively. And the same results as in Example 8 were obtained.

Example 10

Effect of tumor-bound fraction on tumor metastasis growth)

FIG. 9 shows the results of evaluating the tumor metastasis growth inhibitory effect of the heparin-binding fraction and the heparin non-binding fraction prepared by the method of Example 7 in the same manner as in Example 8. The lung weight of the control group (physiological saline) administration group was 0.689 ± 0.250 g, while the weight of the heparin-binding fraction administration group was 0.248 ± 0.05 g. Yes, the heparin-bound fraction significantly suppressed metastatic growth of cancer. On the other hand, in the group to which the heparin non-binding fraction was administered, the value was 0.515 ± 0.208 g, and no significant difference was observed.

Example 11

(Determination of N-terminal amino acid sequence of heparin-binding fraction)

After performing 12.5% SDS-PAGE on the heparin-bound fraction prepared by the method of Example 7, transplotting to a membrane is performed according to a standard method, and the resulting band is cut out, and the N-terminal amino acid sequence analysis is performed. The N-terminal amino acid residue was measured using a device (Bio Applied). As a result, the substance was found to be a mixture of two proteins having Lys and Va1 at the N-terminus, and the ratio was 1.56 to 2.3: 1.

Claims

The scope of the claims

Lysine binding site I.

2. The leistipridin binding site I according to claim 1, which is obtained by elastase degradation of rice plasminogen, exhibits heparin binding, and has an effect of inhibiting tumor metastasis and growth.

3. The rice-type lysine binding site I according to claim 2, which does not contain a sugar chain and exhibits strong heparin binding properties.

4. A rice-type lysine binding site, comprising preparing rice plasminogen from plasminogen, treating the rice plasminogen with elastase, and recovering a fragment exhibiting heparin binding from the obtained fragment. I manufacturing method.

5. The method according to claim 4, wherein the preparation of rice plasminogen from plasminogen is performed by adding plasmin to the plasminogen-containing solution or by naturally digesting plasminogen in the presence of tranexamic acid. .

6. The recovery of fragments exhibiting heparin-binding properties is carried out by passing a solution containing rice plasminogen degradation product of elastase into a carrier using heparin as a ligand, and adsorbing and eluting the solution. The method described in 4 or 5.

7. A tumor metastasis growth inhibitor comprising the rice-type lysine binding site I according to claim 1, 2 or 3 as an active ingredient.