WO2001083548A1

WO2001083548A1 - Novel derivative of cell chemotactic factor

Info

Publication number: WO2001083548A1
Application number: PCT/JP2001/003727
Authority: WO
Inventors: Shiro Kanegasaki; Hiroshi Maeda; Kouji Matsushima
Original assignee: Effector Cell Institute
Priority date: 2000-04-28
Filing date: 2001-04-27
Publication date: 2001-11-08
Also published as: AU2001252650A1

Abstract

It is found out that a peptidyl cell chemotactic factor chemically modified with an amphipathic polymer specifically attains target cells such as tumor cells and, therefore, is effective in treating diseases.

Description

Description Novel derivatives of cell chemotactic factors

The present invention relates to a novel cell chemotactic factor derivative useful as a medicament, particularly to a peptide cell chemotactic factor chemically modified with an amphipathic high molecule. Background art

Neocarzinostin, chemically modified with a partially butyl esterified styrene-maleic acid copolymer, which is a type of amphiphilic polymer, is already known as an anticancer drug (generic name: dinostin chinstimarama). No. 1-333119). It is known to exhibit a so-called EPR effect that when administered to the blood, it almost selectively accumulates in solid tumors and is maintained in the tumor for a long period of time. It is used as a target-type anticancer drug. It is also known that xanthine oxidase similarly modified with polyethylene glycol belonging to an amphiphilic polymer also exhibits an EPR effect on tumor cells (Japanese Patent Application Laid-Open No. 11-060949). No.). All of these achieve an antitumor effect by using a substance having direct aggressiveness against tumor cells. The aim is to reduce the effects on sensitive cells and tissues.

It is known that modification of a protein with polyethylene glycol delays in vivo clearance or reduces antigenicity (Yoshimoto et al., Jpn. J. Cancer Res., 77, 1264 ( 1986), Abuchowski et al., Cancer Biochem. Biophys., 7, 175 (1984), JP-A-61-178926, JP-A-62-115280, JP-T-W096 / 28475, JP-T Hei 10—513187, JP-A-11-310600, JP-T-2000-517304,). Further, interleukin-1, interleukin-16, inferon-feron and the like modified with polyethylene glycol are known (JP-A-5-117300, JP-A-6-256394, JP-A-9-91). No. 25298). Disclosure of the invention

The present inventors have proposed a type of disease treatment system in which target cells such as tumor cells are specifically targeted, instead of the conventional technical means of directly targeting an aggressive substance directly to target cells. The aim was to use a combination of substances and defense functions that exist in living organisms. Such a disease treatment system has never been attempted.

First, there are cells in the body that fight infectious microorganisms such as tumor cells, virus-infected cells, bacteria, and parasites. These cells are powerful effect substances (active oxygen, hypochlorite derived from active oxygen). Acids, hypobromite, basic peptides, hydrolases, etc.) to attack malignant neoplasms and invading foreign bodies. Examples of such aggressive cells include granulocytes such as neutrophils and eosinophils, and mononuclear phagocytes such as monocytes and macrophages, and the use of these aggressiveness destroys tumor cells and the like. However, these cells, on the other hand, attack not only the target diseased area but also their own healthy cells, causing tissue damage and allergic symptoms such as atopic dermatitis and bronchial asthma. Is likely to worsen. Therefore, if these aggressive cells can be controlled and accumulated in a target diseased site such as a tumor cell, it is possible to construct a system that intensively attacks and cures only the target diseased site without damaging healthy cells. It is considered possible.

The present inventors have focused on peptide cell chemotactic factors present in vivo in order to construct a system for controlling the behavior of such aggressive cells in vivo. In other words, it utilizes the fact that peptide cell chemotactic factors impart chemotaxis to these aggressive cells. To control the biodistribution of aggressive cells and thereby to utilize the aggressive properties of the cells, while at the same time aiming to build a system that can treat diseases while minimizing side effects. . Specifically, granulocytes such as neutrophils and eosinophils, which are a kind of aggressive cells, and mononuclear phagocytes such as monocytes and macrophages, are not only aggressive against invading foreign substances but also aggressive against cancer cells. Neutrophils and eosinophils by inducing neutrophil and eosinophil chemotactic factors or mononuclear phagocyte chemotactic factors to target diseased sites such as solid tumors Or, a mononuclear phagocyte was accumulated in a target diseased site such as a solid cancer, and a system capable of treating a disease such as cancer was aimed at.

In addition, by inducing peptide cell chemotactic factors to the target cell site and utilizing the strong aggressive power of neutrophils, eosinophils, mononuclear phagocytes, etc., invading foreign substances and tumor cells Are expected to be destroyed, and the resulting fragments of target cells, such as tumor cells, are expected to act as antigens to induce the immune function of T cells, and it is also expected that the defense mechanism in vivo will be synergistically exerted.

However, a derivative of a cell chemotactic factor suitable for such purpose has not yet been known, and a first object of the present invention is to provide a derivative of a peptide cell chemotactic factor capable of accumulating in a specific target cell or target lesion. Is to provide.

Various compounds are known as cell chemotactic factors. Among them, proteins that mediate cell-cell interaction are used to control aggressive cells, and in vivo peptide-like cell chemotactic factors and A mutant thereof or a peptidic cell chemotactic factor chemically synthesized or produced by genetic engineering is used.

However, these peptide chemotactic factors are generally susceptible to degradation in vivo and have a short duration. Therefore, if the peptidic cell chemotactic factor can be sustained, a sufficient therapeutic effect can be achieved with a small number of administrations. A second object of the present invention is to provide such a derivative of a peptide-type cell chemotactic factor which can exist stably in a living body and has a long-lasting property. For example, interleukin-8 (IL-8) imparts chemotaxis to neutrophils, plays an important role in the initial host defense response in microbial infection, and has antitumor activity and various other physiological activities. Have been reported. Galectin-9 (The Journal of Biological Chemistry Vol.272, No.9, pp.6078-6086, 1997 and Vol.273, No.27, pp.16976-16984, 1998) is an eosinophil. It has been confirmed by the present inventors that chemotaxis is imparted. Eotaxin (The Journal of Biologica 1 Chemistry Vol.271, No.13, pp.7725 -7730, 1996, Nat.Med.Vol.2, No.4, pp.49 -456, 1996, J. Exp. Med. Vol.1833, No.5, pp.2349-2354, 1996) is also known to impart chemotaxis to eosinophils. It has also been reported that MCP-1 induces chemotaxis of mononuclear phagocytes such as macrophages (The Journal of Biological Chemistry Vol.273, No.27, pp.16976-16984, 1998). In using such aggressive properties of neutrophils, eosinophils, mononuclear phagocytes, etc. in the treatment with IL-8, galectin-9, eotaxin, MCP-1, etc., in order to enhance the therapeutic effect, After administration of these cell chemotactic factors into the body, it is preferable to rapidly accumulate them in a target diseased site such as a local part of a cancer and to exert their effects over a long period of time. Accordingly, the present invention provides a derivative such as IL-8, galectin-19, eauxin or MCP-1, which selectively accumulates in a target diseased site such as a tumor and maintains its activity for a long time. One of the purposes.

The present inventors have conceived of imparting an EPR function to a cell chemotactic factor, and a peptide-type cell chemotactic factor chemically modified with an amphiphilic polymer can be used in vivo without impairing its biological activity. In addition, it is clarified that it behaves as a macromolecular substance and becomes soluble in oils, and as a result, selectively accumulates in a target diseased site such as a solid tumor. The present inventors have found that they show sustainability when administered to a living body, and have completed the present invention.

That is, the present invention relates to a peptide cell chemotactic factor chemically modified with an amphiphilic polymer, and a novel peptide cell capable of controlling the in vivo behavior of aggressive cells. Related to derivatives of chemotactic factors. In order to accumulate aggressive cells in the target affected area, instead of the conventional idea of accumulating a substance having an aggressive activity itself such as an anticancer substance in the target affected area, the present invention firstly requires a cell chemotactic factor. The main point is to provide the function of getting a target all night. More specifically,

[1] a peptide-type chemotactic factor chemically modified with an amphiphilic polymer,

[2] the amphiphilic polymer is a partially alkyl esterified styrene-maleic acid copolymer, [1] the peptide cell chemotactic factor according to [1], [3] a partially alkyl esterified styrene The alkyl moiety of the maleic acid copolymer is an alkyl group having 1 to 5 carbon atoms which may be linear or branched, or the alkyl group is substituted with a lower alkoxy group. The peptide cell chemotactic factor according to (2),

[4] the partially alkylesterified styrene-maleic acid copolymer is a partially butyl esterified styrene-maleic acid copolymer, [3] the peptide-type cell chemotactic factor described in [3],

[5] the amphiphilic polymer is a polyethylene glycol derivative,

(1) the peptide cell chemotactic factor according to

[6] the polyethylene glycol derivative is a compound which binds to an amino group of a peptide chain of a cell chemotactic factor, [5] the peptide cell chemotactic factor according to [5],

(7) the peptide glycolipid cell chemotactic factor according to (5), wherein the polyethylene glycol derivative is a compound that binds to a carboxyl group of a peptide chain of the cell chemotactic factor;

(8) a peptide cell chemotactic factor according to any one of (1) to (7), wherein the peptide cell chemotactic factor is a biologically-derived factor;

(9) the peptide chemotactic factor according to any of (1) to (7), wherein the cell chemotactic factor is a leukocyte chemotactic factor; [10] the leukocyte chemotactic factor is a phagocytic chemotactic factor, [9] the peptide cell chemotactic factor described above,

[11] the phagocyte chemotactic factor is a granulocyte chemotactic factor, [10] the peptide cell chemotactic factor described in the above,

(12) the granulocyte chemotactic factor is a neutrophil chemotactic factor or an eosinophil chemotactic factor, wherein the peptide cell chemotactic factor according to (11),

[13] the peptide cell chemotactic factor of [12], wherein the neutrophil chemotactic factor is IL-8;

(14) the eosinophil chemotactic factor is galectin-19, (12) the peptide cell chemotactic factor according to the above,

[15] The present invention provides the chemically modified peptidic cell chemotactic factor according to [10], wherein the phagocytic chemotactic factor is a mononuclear phagocyte chemotactic factor.

Preferred examples of the amphiphilic polymer used for chemically modifying the peptidic cell chemotactic factor in the present invention include a partially alkyl esterified styrene-maleic acid copolymer. Examples of the moiety include an alkyl group which may be linear or branched and has 1 to 5 carbon atoms, and these alkyl groups may be substituted with a lower alkoxy group. More specifically, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 2 , 3-Dimethyl-1-propyl, 2-pentyl, 3-methyl-2-butyl, 3-pentyl, 2-methyl-2-butyl, methylacetosolp, ethylcellosolve and the like. In this specification, a partially alkylated styrene-maleic acid copolymer is a copolymer of styrene and maleic acid in which a part of maleic acid, which is a component of the copolymer, forms a half ester. Say. An example of a preferable partially alkyl esterified styrene-maleic acid copolymer is a partially butyl esterified styrene-maleic acid copolymer described in JP-B-1-33119, which has an average molecular weight of 1,000. Those having a polymerization degree of 1 to 100, preferably 3 to 35, are mainly used.

Another preferred example of the amphiphilic polymer used for chemically modifying the peptidic cell motility factor in the present invention is a derivative of polyethylene glycol (hereinafter sometimes referred to as PEG). . Here, the polyethylene glycol derivative refers to a PEG moiety represented by 10— (CH ₂ CH ₂₀ ) n— (n is an integer of 20 to 280) and a peptide chain of a cell motility factor. Means a compound having a binding moiety of Examples of the polyethylene glycol derivative include a compound that binds to an amino group of a peptide chain (an N-terminal amino group or an amino group of a lysine residue), for example, a compound represented by Formula 1 (wherein R ¹ and : ² is the same or different lower alkyl group, p and q are the same or different and represent an integer of 20 to 280, t represents 0 or an integer of 1 to 5) or a compound represented by the formula 2 ( Wherein R ³ represents a lower alkyl group; r represents an integer of 20 to 280, s represents 0 or an integer of 1 to 5), and _c (Formula 1)

R ¹ 0 (CH ₂ CH ₂ 0) p

R ² 0 (CH ₂ CH ₂ 0) q (CH ',) t CO.H

(Equation 2)

R ³⁰ (CH ₂ CH ₂₀ ) r—CO— (CH ₂ ) s CO. 2H In addition to the above, use any known compounds that can introduce PEG into the amino group of a peptide chain. It is possible. With other examples of polyethylene glycol derivatives And a compound that binds to a carboxyl group of the peptide chain (C-terminal carboxyl group, aspartic acid residue, carboxyl group of glutamic acid residue), for example, a compound represented by Formula 3 (where R ⁴ is a lower alkyl group) And w represents an integer of from 20 to 280). In addition, any compound known as a compound capable of introducing PEG into the carboxyl group of a peptide chain can be used.

(Equation 3)

^{_{_{R 4 0 (CH 2 CH 2}}} 0) w- CH 2 CH 2 NH 2 Further, as another example of the amphipathic polymer to be used in the present invention, may be mentioned polyvinyl pyrrolidone others.

In the present invention, a cell chemotactic factor is a factor that imparts chemotaxis to target cells such as leukocytes, for example, a factor that imparts chemotaxis to lymphocytes, monocytes, granulocytes, and the like. Means child. Among them, the group of granulocyte chemotactic factors related to neutrophils and eosinophils and the group of mononuclear phagocyte chemotactic factors related to monocytes, macrophages, etc. are worthy of note in treating various diseases.

The peptide-type cell chemotactic factor used in the present invention can be selected from various cell-mediated peptide-mediated peptides, and the peptide-type cell chemotactic factor derived from a living body and its mutant or chemical Peptidic cell chemotactic factors which are synthetically synthesized or produced by genetic engineering are used.

More specifically, interleukin 8 (IL-8) can be mentioned as the neutrophil chemotactic factor, and galectin-19, eotaxin and the like can be mentioned as the eosinophil chemotactic factor. In addition, galectin-9 has an S form consisting of 311 amino acids (US Patent No. 6027916 (2000)) and an M form consisting of 323 amino acids (also called ekalectin. Matsumoto et al., JBC, 273, 16976). (1998)), and three L-forms composed of 35 amino acids (Suk K. et al., J. Clin. Immunol. 19, 158 (1999)) are known. , Any of them can be used You. MCP-1 can be cited as a chemotactic factor for mononuclear phagocytes such as monocytes and macrophages.

The peptide chemotactic factor chemically modified with the amphiphilic polymer according to the present invention is a chemical bond between the amphiphilic polymer and the peptide cell chemotactic factor, optionally via a linker arm. And can be obtained by partial purification. That is, the amphiphilic polymer is reacted with the peptide cell chemotactic factor in a buffer, and then purified using column chromatography, and the eluted fractions are separated.

Taking the case where the amphiphilic polymer is a partially alkyl esterified styrene-maleic acid copolymer as an example, more specifically, first, styrene-maleene obtained by radical copolymerization of styrene and maleic anhydride is used. The acid anhydride of the acid copolymer (SMA) is dissolved in an organic solvent such as cumene, and n-butanol is added dropwise to this solution with stirring to allow the reaction to occur. By doing so, a partially butylesterified styrene-maleic acid copolymer (Bu-SMA) can be obtained. The bond between Bu-SMA and IL-8 thus obtained can be reacted with each other in a 0.3 M solution of sodium hydrogen bicarbonate at pH 8.5 to form a bond between the acid anhydride ring in Bu-SMA and the amino group in IL-8. It can be carried out by forming an amide bond between them. The number of Bu-SMA bonds to IL-8 may be 1 or more, preferably 3 to 10, and more preferably 6 to 8.

The Bu-SMA-bound IL-8 thus obtained (Bu-SMA-IL-8) can be used in the form of a water-soluble or oily injection. Water-soluble injections are mainly used for intravenous administration. The oil-based injection is administered to a target diseased site such as a tumor tissue through a catheter in which Bu-SMA-IL-8, which is uniformly dispersed in an oil such as riviodol, is fixed upstream of a tumor-feeding artery. Bu-SMA-bound IL-8 (Bu-SMA-IL-8) binds to albumin in blood and behaves as a polymer compound, and Bu-SMA-IL-8 becomes soluble in oils Have been found by the present inventors, and as a result, oil-based Bu-SMA— If IL-8 is injected into a local artery, it is expected that it will selectively accumulate in the target affected area such as a solid tumor due to the so-called EPR effect.

As an amphiphilic polymer: When PEG is used to modify the amino group of the peptide chain of a cell chemotactic factor, a functional group capable of reacting with an amino group, such as a carboxyl group, is introduced into the PEG terminal. Is preferred. Typical examples include PEG derivatives represented by Formulas 1 and 2. In order to bond to the amino group in the peptide, it is preferable to convert the functional group into a reactive group. In the case of a carboxyl group, an active ester method, a mixed acid anhydride method and the like are preferable examples.

Specifically, in the case of an active ester, phenyl esters such as p-nitrophenyl ester and pen-fluorophenyl ester, and dicarboxylic acids such as N-hydroxyphthalimidester and N-hydroxysuccinimide ester And hydroxyl-based active esters such as imidester and N-hydroxypiperidine ester. The preparation of these active esters can be carried out according to a conventional method. For example, the carboxyl group of a PEG derivative and the alcohol compound corresponding to the above-mentioned active ester are combined with dicyclohexylcarbodiimide {1-ethyl-3- The reaction can be carried out by using a condensing agent such as (3-dimethylaminopropyl) carbodiimide at a temperature of −20 ° C. to room temperature for 1 to 24 hours. Alternatively, by reacting the carboxyl group of the PEG derivative with a halide corresponding to the active ester at 0 ° C. to 80 ° C. for 1 to 72 hours in the presence of a base such as triethylamine. Can also be prepared.

In the case of the mixed acid anhydride method, in the presence of a base such as N-methylmorpholine or N-ethylpiperidine, isoptyl chloroformate, ethyl chloroformate, isovaleryl chloride, etc. at −20 ° C. to 0 ° C. For 1 to 30 minutes.

The PEG derivative can be directly introduced into the peptide chain of the cell chemotactic factor, or can be introduced via a linker arm. For example, short amino acids containing lysine The amino acid sequence is introduced into the desired peptide chain, and the lysine amino group is modified with a PEG derivative. In the modification reaction, it is preferable to use a PEG derivative having a molar ratio of 10 to 30 times the number of amino groups contained in the peptide chain to be modified.

When using PEG as the amphiphilic polymer to modify the carboxyl group of the peptide chain of the cell chemotactic factor, a functional group capable of reacting with a carboxyl group, for example, an amino group, is introduced into the terminal of the PEG. Is preferred. As typical examples, a PEG derivative represented by Formula 3 and others are known.

The peptide chemotactic factor chemically modified with PEG thus obtained is expected to selectively accumulate in a target diseased site such as a solid tumor by the so-called EPR effect. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing an elution pattern of a reaction product of Bu-SMA and IL-8 from a gel filtration column. The vertical axis indicates UV absorbance at 260 nm. Bu-SMA and insulin were used as controls. The horizontal axis is the test tube number.

FIG. 2 is a view showing UV absorption curves of Bu-SMA, Bu-SMA-IL-8 and IL-8. FIG. 3 shows neutrophil chemotactic activity of Bu-SMA-IL-8 and unmodified IL-8. The vertical axis represents the number of cells, and the horizontal axis represents the concentration.

FIG. 4 is a diagram showing the amino acid sequence of the linker arm at the N-terminal of the GSK-galectin-19S form.

FIG. 5 is a photograph showing SDS-PAGE during the purification process of GSK-galectin-9S form. Each lane is as follows.

Lane 1: Molecular weight marker (unit: K Da)

Lane 2: E. coli BL21 total protein

Lane 3: E. coli total protein after IPTG induction

Lane 4: supernatant protein after sonication in lane 3

Lane 5: Precipitated protein after sonication in Lane 4 Lane 6: Lactose-saga mouth-non-adsorbed fraction

Lane 7: GSK-G-9S protein eluted after lactose agarose adsorption

FIG. 6 is a diagram showing the chemotactic activity of GSK-galectin-9S body.

FIG. 7 is a photograph showing an SDS-PAGE of a reaction product of a GSK-galectin-19S form and a PEG derivative. Each lane is as follows.

Lane 1: Molecular weight marker (unit: KDa)

Lane 2: GSK-G-9S (without PEG)

Lane 3: 2x PEG (ratio to amino group)

Lane 4: 5-fold amount PEG (ratio to amino group)

Lane 5: 10 times PEG (ratio to amino group)

Lane 6: 20 times PEG (ratio to amino group)

Lane 7: 30 times PEG (ratio to amino group)

FIG. 8 is a view showing chromatography in the purification of PEG-GSK-galectin-19S form. The horizontal axis represents the test tube number, and the vertical axis represents the absorbance.

FIG. 9 is a photograph showing SDS-PAGE analysis of PEG-GSK-galectin-9S form. FIG. 10 shows the chemotactic activity of PEG-GSK-galectin-9S. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] Synthesis of Bu-SMA-bound IL-8

IL-8 was dissolved in a 0.3 M sodium bicarbonate buffer solution (pH 8.5) to a concentration of 0.5 mg / ml. To 2 ml of this solution, Bu-SMA (9 mg) was added and reacted at 37 ° C. for 2 hours. After completion of the reaction, the reaction mixture was subjected to gel filtration chromatography (filler: Sephadex G-50 manufactured by Pharmacia, mobile phase: 10 mM ammonium bicarbonate solution (pH 8.5), column size: 2x90 c m) and partially purified. The eluate from the column was separated into 65 test tubes for every 100 drops (5.5 ml) using a reservoir (manufactured by Isco) (see Fig. 1). Then, based on the absorbance at 260 nm, the test tubes were collected from fraction No. 1 to No. 7, and each was concentrated by freeze-drying. The fraction (No. 3) eluted in 72-88 ml (corresponding to test tube numbers 13-18) was lyophilized to obtain white powdery Bu-SA-IL-8.

FIG. 2 shows UV absorption curves of the obtained Bu-SMA-IL-8 and the raw materials Bu-SMA and IL-8. As apparent from the figure, Bu-SMA-IL-8 has a different peak from any of the starting compounds.

The UV absorption at 260 nm and 280 nm of Bu-SMA-IL-8 of fraction No. 3 and that of unreacted Bu-SMA and IL-8 were quantified and compared. Approximately 7 molecules of Bu—SMA were found to bind to 8c (Table 1)

UV absorbance at 260 nm and 280 nm

[Example 2] Neutrophil chemotactic activity of Bu-SMA—IL-8

Neutrophils were separated from peripheral blood of healthy subjects using a monopoly separation solution (manufactured by Dainippon Pharmaceutical Co., Ltd.), and the cell count was 5 x 10 ⁵ / m in R PMI 1640 medium containing 0.5% ゥ serum albumin. A cell suspension was prepared to be 1. Adjust 12 microliters of the solution containing fraction No.3 Bu-SMA-IL-8 to each dilution and add to the Boyden Chamber. Add 0.115 ml of each, and cover the sample with a chemotaxis-measuring membrane (Neucribore, 5 μm pore size, manufactured by Nomura Microscience) containing no polyvinylpyrrolidone. 5 ml was added. The chamber was allowed to stand overnight in an incube filled with 95% normal air and 5% carbon dioxide, and cultured at 37 ° C; for 90 minutes.

Thereafter, the chemotaxis measurement membrane was immersed in Diff Quick Staining Solution (manufactured by Kokusai Reagent Co., Ltd.) to stain neutrophils that appeared on the back surface through the membrane. The cell number was counted under a microscope (at a magnification of 400). Unmodified IL-18 was used as a control. Figure 3 shows the results. Bu-SMA-IL-8 in fraction No. 3 showed neutrophil chemotactic activity depending on its concentration, and its activity was comparable to that of unmodified IL-8. That is, it was shown that the activity of IL-8 was maintained in Bu-SMA-IL-8.

[Example 3] Pharmacokinetics of Bu-SMA-IL-8 and comparative experiment with IL-8

0.1 ml of Bu-SMA-IL-8 (two-fold dilution of the solution of fraction No. 3) or 50 μg of unmodified IL-8 was administered from the tail vein of the ddY mouse for 5 minutes. Later, venous blood was collected from the mesenteric vein. The obtained venous blood was centrifuged to collect serum from which blood cell components had been removed. The IL-8 activity contained in the serum was quantified by the same method as described above. Further, in an experimental system using ddY mice prepared independently of the above experiment, venous blood 10 minutes later was collected and IL-8 activity was measured.

The results are shown in the table below. In the case of venous blood collected after 5 minutes, when the serum was diluted 10-fold, the activity of unmodified IL-8 was below the detection limit, whereas that of Bu-SMA-IL-8 was still remarkable. showed that. In a separate experiment, venous blood collected 10 minutes later showed that Bu-SMA-IL-8 was 5-6 times more active than unmodified IL-8 (using undiluted serum).

(Table 2) (1) Number of permeated cells after 5 minutes

Serum dilution ratio I L-1 8 Bu-SMA-IL-8

10 Below detection limit 4.5 Sat 1.1

100 0 7.8 1.9 1.9 0 5.2 Sat 1.1

(2) Number of transmembrane cells after 10 minutes

Serum dilution ratio I L-1 8 Bu-SMA-IL-8

1 3.8 Sat 0.8 23 J 3.2

[Example 4] Synthesis of PEG-modified galectin-19S form

1) Construction of GSK-galectin-19S (abbreviation: GSK-G-9S) with a linker arm containing a lysine residue at the N-terminus

The HisTag-added galectin-9S form has a total of eight amino groups in the molecule: seven lysine residues and one N-terminal methionine. When this amino group was reacted with a PEG derivative, it was found that there was no reaction at all.

On the other hand, the N-terminal HisTag is already known to be adsorbed to Ni-sepharose, and it is thought that the introduction of a new lysine residue at this position will enhance the reactivity with the PEG derivative. The sequence was removed, and a galectin-19S form was newly constructed with a linker and one arm containing a lysine residue.

The histidine tag sequence of galectin-9S was added to the N-terminal side of the 9S-form, and the histidine tag sequence of pTrcHicB-gal9s was cleaved off using two types of restriction enzymes Ncol and Xhol. There sequence that is newly complementary synthetic DNA to ^5, - CATGGGGGGTTCTAAAGGGTCTGGGTCTG GGTCTAAAGGGTCTGGGTCTGGGTCTAAAGGGTCTGGGAGCTCGAGTGGT -3 5 (SEQ ID NO: 1) and SEQ 5, - CCCCCCAAGATTTCCCAGACCCAGACCCAGATTTCCCAGACCCAGACCCAGATTTCCCAGACC CTCGAGCTCACCA -3 '(SEQ ID NO: 2), 70 ° C ;, After treatment for 2 minutes, the mixture was gradually cooled to a double strand, and inserted into restriction sites Ncol and Xhol. The nucleic acid sequence used was high in E. coli. In order to make it easy to react with the PEG derivative, a glycine-serine sequence was used three times between lysine residues (Fig. 4). The thus constructed expression plasmid vector is called pTrc-GSK-gal9s.

This expression vector is transformed into E. coli BL21 strain, pre-cultured in 40 ml of LB medium containing 2% glucose, and then 1/50 inoculated into 2 XYT medium containing 2 L of 2% glucose at 30 ° C. At A600 = 0.7, O. lmM IPTG was induced. The cells were further cultured for 3 hours, centrifuged, and the cells were stored at -80 ° C. Purification was performed according to the method for purifying wild-type ecarelectin.

Add a Sonication buffer (50 mM HEPES (pH 7.5), 0.5 M NaCl ヽ ImM DTT ヽ ImM PMSF) to the cells at 10 times the wet weight of the cells, and sonicate (200 W, 15 min ₃₀ ° C) I did. Triton X100 was added to the resulting solution to a final concentration of 1%, and the mixture was stirred at 4 ° C for 30 minutes. After centrifugation (2000G, 4 ° C, 30 minutes), 3 ml of lacto-sugarose (Seikagaku Corporation) was added to the soluble fraction, and the mixture was stirred at 4 ° C for 3 hours by a batch method to adsorb. The resin is packed in a column, washed with 100 ml of Wash buffer (25 mM HEPES (pH 7.5), 0.15 M NaCl _N 0.03% CHAPS ヽ O.lmM DTT), and washed with Elution buffer (25 mM HEPES (pH 7.5), 0.15 M NaCl, 0.2M Lactose, ImM DTT). Perform gel filtration (Bio Had 10DG, PBS (-) 0.1 lmMDTT) to remove excess lactate from the eluate, collect the peak fraction, add 0.1% human serum albumin, and dialyze (PBS, 0.05 mM 2-mercaptoethanol. The recovery was almost the same as that of wild type ecarelectin. Approximately 1 mg / L culture was obtained by 1-step purification using lactose agarose.When lactose was removed by gel filtration and dialysis, most of the precipitate was precipitated, and the final recovery was about 0.3 mg / L culture. Was. Figure 5 shows the analysis of the purification process by SDS-PAGE. This protein is called "GSK-Galectin-1 9S" (abbreviation: GSK-G-9S). The number of amino groups in the molecule is 1 N-terminal methionine and 9 lysines, for a total of 10 amino acids, which means that there are two more amino groups than the processed calectin with HisTag. 2) Confirmation of human eosinophil migration activity of GS1 (— G-9S)

When the chemotactic activity of this eosinophil was examined by the following method, it was confirmed that the activity of galectin-9S was maintained (FIG. 6).

2- 1) Preparation of eosinophil suspension

Peripheral blood of a healthy person was treated with heparin (10 U / ml), and then an equal amount of 1-1.25% dextran was added, and the mixture was allowed to stand to separate a plasma layer. The plasma layer was overlaid on a discontinuous density gradient Percoll solution (61% and 66%), the lymphocyte layer was removed by centrifugation, and the pellet of the granulocyte layer was collected. This is a magnetic bead to which anti-CD16 antibody is bound.

After removal of the (Mirteny Biotec Ltd. GmbH) and treated with neutrophils, 0.1 to 1% © shea fetal blood Kiyoshin containing RPM 11640 medium, cell suspension as the cell number is lx 10 ⁶ / m 1 Was prepared (eosinophil purity 90% or more).

2-2) Chemotactic activity of GSK-G-9S on eosinophils

GS -G-9S a 0.07x10- ⁷ M solution 26～28〃1 placed in Boyden chamber one lower chamber containing poly force one Boneitofiru coater (pore size 8 micrometer one Torr containing no polyvinylpyrrolidone thereon, Neuro (Produced by Probe Co., Ltd.), and 50 μl (0.5 × 10 ⁵ cells) of an eosinophil suspension was further added thereto. This chamber was left standing overnight in an incubator filled with 95% normal air and 5% carbon dioxide, and cultured at 37 ° C for 90 to 120 minutes. Thereafter, the filter was taken out, immersed in a diff quick fixative and a staining solution (manufactured by Kokusai Reagent Co., Ltd.), and the eosinophils that appeared on the back surface through the membrane were stained. The cell number was counted under a microscope (400 times). Was used as control ^{His- G- 9S (0.1xl (T 7} M) and Eotaxin (10_ ⁹ M). The results are shown in Figure 6. In addition,. Leftmost bar graph vertical axis representing the number of cells in FIG. Indicates that the sample amount is zero, that is, the background (the same applies hereinafter).

From FIG. 6, it is clear that GSK-G-9S exhibits eosinophil chemotactic activity similarly to His-G-9S and Eotaxin, and it is apparent that GSK-G-9S has a GSK sequence added to the N-terminal. Thus, it was shown that the original activity was maintained. 8

3) Chemical modification of GSK-G-9S with PEG derivative

3- 1) Examination of reaction ratio

The chemical modification of ekalectin by an amino group-reactive PEG derivative represented by the formula 4 Succinimidyl Succinate PEG (M.W.5000, n = 108 in the formula 4) (Shearwater Polymers, Inc.) was examined. To the 1 ml of GSK-G-9S solution (pH 7.5, concentration 0.3 mg / ml 総 total protein 0.3 mg), add the above PEG derivative at a ratio of 0, 2, 5, 10, 20, or 30 times the ratio of amino groups. The reaction was carried out at 4 ° C for 30 minutes. The reaction product was subjected to SDS-PAGE (15 ° / ° and silver-stained. As a result, it was found that almost all of the ekalectin protein reacted with the PEG derivative at a ratio of 20 times or more (Fig. 7). A major band shift was observed around 46 kDa, suggesting that about 2 (5000 x 2 = 10,000) PEGs were added to the GSK-G-9S1 molecule (this is called PEG-GSK — G— 9S.)

3-2) Synthesis and purification of PEG-GSK-G-9S

0.287mg / ml GSK- G-9S solution (elution buffer containing lactose, pH7.5) PEG derivative represented by formula 4 (n = 108) in 17.5ml (total protein 5.02mg, 6L culture) Was added and reacted at 4 ° C. for 30 minutes.

(Equation 4)

CH ₃ 0 (CH ₂ CH ₂ 0) n— CO— (CH ₂ ) ₂ CO-0

Ll Of this, ll ml was applied to a Sephadex G-75 gel filtration column (3 cm × 60 cm), and PBS (-) was used as a buffer, and 5.5 ml fractions were collected. Purified brophy Figure 8 shows the result. Furthermore, 0.3 ml was collected from each fraction, 1.2 ml of cold acetone was added thereto, and the mixture was allowed to stand at -20 ° C. The mixture was centrifuged (14K rpm, 10 minutes) at 4 ° C, 10 l of PBS (-) was added to the precipitate, and the whole was subjected to 15% SDS-PAGE and analyzed. In each peak fraction, a smear band of 40 Kda or more, which is considered to be PEG-GSK-G-9S, was confirmed (FIG. 9).

[Example 5] Eosinophil chemotactic activity of PE G-modified galectin-9 (S)

1) Eosinophils were prepared as described in Example 4, 2-1).

2) Chemotactic activity of PEG-GSK-G-9S

Example 4, 2 -2 definitive when a similar manner to the) measuring the activity of the purified samples, confirmed that there is a chemotactic activity in fractions 17 of FIG. 8 (first peak, the concentration is from about 10- ⁸ M) Was done. That is, prepared from Fraction 17, PEG- GSK- G- concentration 9S is 0 · 024χ10- ^{7 Μ,} the solution is 0.24χ10- ⁷ Μ and ^{2.4χ10- 7 Μ, His- G- 9S (} 0.4xlO - a ⁷ M) and Eotaxin (10- ⁹ M) results measured as a control is shown in FIG. 10. From FIG. 10, it is clear that PEG-GSK-G-9S has eosinophil chemotaxis. The vertical axis in the figure represents the number of cells. The bar graph at the left end shows that the sample amount is zero, that is, the background. Industrial applicability

As a result of using the above method, fraction No. 3 (FIG. 1) in which Bu-SMA-IL-8 was chemically bonded was obtained, and neutrophil chemotactic activity was recognized (FIG. 3). This indicates that a peptide cell chemotactic factor derivative that can be specifically targeted to a target lesion such as a tumor cell has been synthesized. According to the present invention, it has been revealed that IL-8, which is a neutrophil chemotactic factor, can be modified with Bu-SMA without losing its activity. It can be used as a medicine. That is, when Bu-SMA-IL-8 was intravenously administered to a carrier cancer mouse, it was confirmed that neutrophils were accumulated at the tumor site.

It has been shown that galectin-19 can be modified with PEG without losing its activity, and it is expected that it can be used as a cancer therapeutic drug that specifically accumulates in cancer cells. Furthermore, similar results are expected when eotaxin, MCP-1 or the like is used as a material.

In the disease treatment system according to the present invention, the peptide chemotactic factors and cells used, ie, IL-8, galectin-9, eotaxin, MCP-1 and the like, and neutrophils and eosinophils are all originally used. Since it is of human origin, the problem of anaphylaxis shock can be avoided, and superior disease treatment that has not existed before can be achieved.

Claims

The scope of the claims

1. A peptide-type chemotactic factor chemically modified with an amphiphilic polymer.

2. The peptide cell chemotactic factor according to claim 1, wherein the amphiphilic polymer is a partially alkyl esterified styrene-maleic acid copolymer.

3. The alkyl portion of the partially alkylated styrene-maleic acid copolymer is an alkyl group having 1 to 5 carbon atoms which may be linear or branched, or the alkyl group is a lower alkyl group. The peptide cell chemotactic factor according to claim 2, wherein the peptide cell chemotactic factor is substituted with an alkoxy group.

4. The peptide cell chemotactic factor according to claim 3, wherein the partially alkyl esterified styrene-maleic acid copolymer is a partially butyl esterified styrene-maleic acid copolymer.

5. The peptide cell chemotactic factor according to claim 1, wherein the amphiphilic polymer is a polyethylene glycol derivative.

6. The peptide cell chemotactic factor according to claim 5, wherein the polyethylene glycol derivative is a compound that binds to the amino group of the peptide chain of the cell chemotactic factor.

7. The peptide cell chemotactic factor according to claim 5, wherein the polyethylene glycol derivative is a compound that binds to a carboxyl group of a peptide chain of the cell chemotactic factor.

8. The peptide cell chemotactic factor according to any one of claims 1 to 7, wherein the peptidic cell chemotactic factor is a biologically-derived factor.

9. The peptide cell chemotactic factor according to any one of claims 1 to 7, wherein the cell chemotactic factor is a leukocyte chemotactic factor.

10. The peptide cell chemotactic factor according to claim 9, wherein the leukocyte chemotactic factor is a phagocyte chemotactic factor.

11. The peptide cell chemotactic factor according to claim 10, wherein the phagocyte chemotactic factor is a granulocyte chemotactic factor.

12. The peptidic cell chemotactic factor according to claim 11, wherein the granulocyte chemotactic factor is a neutrophil chemotactic factor or an eosinophil chemotactic factor.

13. The peptide chemotactic factor according to claim 12, wherein the neutrophil chemotactic factor is IL-8. 13.

14. The peptide cell chemotactic factor according to claim 12, wherein the eosinophil chemotactic factor is galectin-19.

15. The chemically modified peptide cell chemotactic factor according to claim 10, wherein the phagocyte chemotactic factor is a mononuclear phagocyte chemotactic factor.