JP2002284798A - Influenza virus/hemagglutinin-binding peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an influenza virus-binding peptide useful for prophylaxis of infection of the influenza virus. SOLUTION: This influenza virus/hemagglutinin-binding peptide is (a) or (b): (a) a peptide having an amino acid sequence selected from either of amino acid sequences represented by sequence numbers 1 to 3 derived from phage library; or (b) a peptide having an amino acid sequence in which one or more amino acids are modified by substitution, deletion or addition in the amino acid sequence denoted in (a) and having a binding ability with the influenza virus/hemagglutinin. Sequence number 1: Ser Arg Ala Phe Asp Leu Leu Asp Gln Pro Leu Ser Asn Ala Arg; sequence number 2: Thr Ser Val Asn Arg Gly Phe Leu Leu Gln Arg Ala Ser His Pro; and sequence number 3: Giy Leu Ala Met Ala Pro Ser Val Gly His Val Arg Gln His Gly.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a peptide having specific binding to influenza virus. More specifically, the present invention relates to peptides that specifically bind to hemagglutinin present on influenza virus membranes.

[0002] Infection of a host with influenza virus is established by the virus binding to a host receptor via hemagglutinin embedded in its surface membrane. The peptide of the present invention prevents binding of influenza virus to a host receptor by binding to hemagglutinin, thereby significantly contributing to prevention of influenza virus infection.

[0003] Therefore, the present invention also relates to the use of the above peptides as inhibitors of influenza virus binding to host receptors via hemagglutinin and as anti-influenza agents.

[0004]

2. Description of the Related Art Two types of spike glycoproteins, such as hemagglutinin (HA) and neuraminidase (NA, sialidase), are present in influenza virus membranes, and play important roles in establishment of virus infection and budding of virus from host cells, respectively. I have. The former hemagglutinin recognizes sialic acid-containing sugar chains that are universally present on the cell membrane of hosts such as humans and other animals (mammals, birds, reptiles, fish, amphibians, etc.) as receptors and specifically binds to them. And lead to endocytosis of the influenza virus into cells. On the other hand, the latter neuraminidase is a receptor-destroying enzyme and plays a role in cleaving sialic acid residues on its own or on the host cell membrane when virus particles germinate or release from host cells.

[0005] Influenza viruses are classified into types A, B and C according to their antigenicity, such as viral nucleoproteins and matrix proteins present inside the virus membrane. In particular, the influenza A virus has been repeatedly spread on a worldwide scale due to a change between subtypes accompanied by a change in antigenicity due to genetic crossing or point mutation.

[0006] Hemagglutinin involved in the first step of the aforementioned influenza virus infection has various subtypes based on the diversity of the amino acid sequences of the antigenic determinants (A to E), which are highly susceptible to mutation. Amino acid sequence differences between hemagglutinin subtypes range from 25-75%, but the so-called receptor binding pocket region that binds to the receptor in the host cell is relatively unmutated and its three-dimensional structure is well conserved. (Y.Suzuki, Prog.Lipid.Res., 33 , 4
29 (1994)) Conventionally, in order to prevent influenza virus infection, the development of a vaccine having an action of specifically binding to hemagglutinin, which contributes to the establishment of infection, and inhibiting the action thereof has been studied.

As an example, based on the recognition and binding of hemagglutinin to a sialic acid-containing sugar chain of a host receptor, various sugar analogs are used to screen for sugars that specifically bind to the binding site of hemagglutinin. To date, various hemagglutinin-binding sugar analogs have been obtained (R. Roy, et al., J. Che.
m. Soc., Chem. Commun., 1869 (1993); M. Mammen, et a
l., J. Med. Chem., 38 , 4179 (1995); T. Sato, et al.,
Chem. Lett., 145 (1999); M.ltzstein, et al., Natur.
e, 363 , 418 (1993)).

In addition, there is a method for preparing an antibody (anti-idiotype antibody) against the antigenicity (idiotype) of the antigen-binding site of the monoclonal antibody against the hemagglutinin receptor sugar chain. In other words, instead of the three-dimensional structure of sialic acid and sialo-glycan that serves as a receptor for hemagglutinin, the amino acid sequence of the hypervariable region of an anti-idiotype antibody having a similar three-dimensional structure to that of the sialic acid and sialo-glycan is created, and the hemagglutinin receptor of the host cell is prepared. The body is simulated (Yasuo Suzuki, "Viral Infection and Sugar Chains", Nikkei Science, Supplement, Sugar Chains and Cells, pp. 89-101, October 1994).

However, none of these is specific for various subtypes of hemagglutinin and the binding constants are not high.

[0010] For this reason, the development of a wide-area vaccine that works on influenza viruses in general regardless of their subtypes is currently awaited.

[0011]

If a substance capable of specifically recognizing and binding to hemagglutinin of an influenza virus, that is, a substance capable of becoming a hemagglutinin receptor instead of a host cell receptor, could be provided, influenza could be transferred to the host via hemagglutinin. It is expected that binding to the receptor can be competitively suppressed or inhibited, and infection of influenza virus can be prevented. In addition, if the substance specifically recognizes and binds to the “receptor binding pocket” of hemagglutinin that is well conserved between subtypes, it can prevent the infection of the influenza virus throughout the influenza virus regardless of the subtype. Is expected. An object of the present invention is to provide an influenza virus hemagglutinin-binding peptide that can exert such effects.

[0012]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems, and found that the peptide selected by using the phage display method specifically recognizes influenza hemagglutinin. And found that the peptide was useful for preventing influenza infection. The present invention has been developed based on such knowledge.

That is, the present invention provides influenza virus hemagglutinin-binding peptides listed in the following (1) to (5): (1). Influenza virus hemagglutinin-binding peptide, which is one of the following (a) or (b): (a) a peptide having an amino acid sequence selected from any of the amino acid sequences shown in SEQ ID NOs: 1 to 3, (b)
A peptide comprising an amino acid sequence in which one or more amino acids have been modified by substitution, deletion or addition in the amino acid sequence shown in the above (a), and having a binding property to influenza virus hemagglutinin. (2). An influenza virus hemagglutinin-binding peptide having the amino acid sequence represented by SEQ ID NO: 2. (3). The influenza virus hemagglutinin-binding peptide according to (1) or (2), wherein the influenza virus is a type A virus. (4). The influenza virus is of the H1 subtype,
The influenza virus hemagglutinin-binding peptide according to (1) or (2). (5). The influenza virus is subtype H3,
Influenza virus according to (1) or (2)
Hemagglutinin binding peptide.

These peptides can be prepared as lipopeptides by alkylation or lipidation (phospholipid). Accordingly, the peptides of the present invention include lipopeptides. The present invention is also a liposome containing the lipopeptide.

Further, the present invention provides an influenza virus hemagglutinin-binding peptide or at least one of these lipopeptides described in the above (1) to (5).
Influenza virus containing seed as an active ingredient
Hemagglutinin binding inhibitor. In addition, the said peptide can also be in the form of a liposome.

Further, the present invention provides an anti-influenza virus containing at least one of the influenza virus / hemagglutinin-binding peptides described in any of the above (1) to (5) as an active ingredient, and further comprising a pharmaceutically acceptable carrier. Influenza drug. Specifically, the anti-fluenza agent is a pharmaceutical composition useful as a prophylactic or therapeutic agent for influenza as listed in (A) to (G) below. (A) A pharmaceutical composition comprising, as an active ingredient, at least one of the influenza virus hemagglutinin-binding peptides according to any of (1) to (5) above and a pharmaceutically acceptable carrier. (B) The pharmaceutical composition according to the above (A), which comprises an influenza virus hemagglutinin-binding peptide as an active ingredient in the form of a lipopeptide. (C) The pharmaceutical composition according to the above (A), comprising the influenza virus hemagglutinin-binding peptide as an active ingredient in the form of a modified liposome. (D) The pharmaceutical composition according to the above (A), wherein the target influenza virus is a type A virus. (E) The pharmaceutical composition according to the above (A), wherein the target influenza virus is an H1 subtype of a type A virus. (F) The pharmaceutical composition according to the above (A), wherein the target influenza virus is an H3 subtype of a type A virus. (G) The pharmaceutical composition according to the above (A), which is used as an agent for preventing or treating influenza.

Hereinafter, amino acids in the present specification,
Abbreviations for peptides, base sequences, nucleic acids, etc.
The PAC and IUB regulations, "Guidelines for the preparation of specifications containing base sequences or amino acid sequences" (edited by the Patent Office) and conventional symbols in the art shall be followed.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION As specific examples of the influenza virus hemagglutinin-binding peptide of the present invention, those having the amino acid sequences shown in SEQ ID NOs: 1 to 3 obtained by the method of the examples described below are exemplified. it can.

Hereinafter, a method for selecting a hemagglutinin-binding peptide (hereinafter, also referred to as an HA-binding peptide) of the present invention, identification of the obtained peptide, and binding properties of the peptide to hemagglutinin will be described.

For the selection and identification of the HA-binding peptide of the present invention, a screening method of a molecular library can be employed. As the library, for example, a phage display library can be preferably used.

As such a library, a commercially available library can be used. According to the random peptide display phage in the library, a peptide that binds to a specific target molecule or a target cell can be screened in vitro using the molecule or cell, and the peptide selected by the screening can be expressed. Therefore, it is useful for selecting and identifying a peptide that specifically binds to a target molecule or cell. Screening using the phage display library is called a phage display method, and has conventionally been used for selecting and identifying ligands and various antibodies that specifically bind to various cell surface receptors. For the method of preparing these phage display libraries and the method of in vitro screening, see the method of Scott and Smith et al. (Scott, JM and Smith, GP, Science,
249 , 386-390 (1990); Smith, GP and Scott, J.
K., Methods in Enzymology, 217 , 228-257 (1993)).

The HA-binding peptide of the present invention can be obtained by screening a peptide binding to hemagglutinin in vitro using the phage display method described above. Specifically, it can be performed by the following method.

That is, first, a random DNA sequence is inserted into a known phage library, and a random peptide display phage constructed to express a peptide having a random amino acid sequence on the outer surface of the phage is used. This is reacted with hemagglutinin of influenza virus previously immobilized on the surface of a solid phase such as a microplate, and a phage that specifically binds to the hemagglutinin is collected (biopanning).

The hemagglutinin of influenza virus immobilized on the microplate is not particularly limited as long as it has at least a “receptor binding pocket” that binds to a receptor of a host cell. The virus may be the influenza virus itself or an influenza virus extract obtained by extracting the influenza virus with various organic solvents such as ether. Also, the type of influenza virus is not particularly limited.
Type, B type or C type, or human isolated type, other mammalian isolated type such as pig and horse, or bird isolated type. Preferably, it is a type A virus or a human isolated virus.

As described above, since the tertiary structure of the “receptor binding pocket” of hemagglutinin is well conserved regardless of the influenza virus subtype, subtypes of influenza virus hemagglutinin immobilized on the microplate are: Although it does not matter at all, H1 and H3 subtypes, which are subtypes of type A that have been preferably isolated from humans for about 10 years can be exemplified.

The recovery of phage which specifically binds to hemagglutinin inhibits the binding of hemagglutinin to the HA receptor substance which has the ability to compete with the receptor of the host cell, or the binding of hemagglutinin to the receptor of the host cell. It can be performed by acting a capable HA inhibitor on immobilized hemagglutinin. That is, the phage that specifically binds to the hemagglutinin immobilized on the microplate can be desorbed and eluted by performing a competitive reaction with the above-mentioned HA receptor substance or HA inhibitor, and can be recovered.

Here, the HA receptor substance is not particularly limited as long as it has an ability to specifically bind to hemagglutinin. To recover phage that specifically bind to the receptor binding pocket of hemagglutinin, use H
It is preferable to use a substance having the ability to specifically bind to the receptor binding pocket of hemagglutinin as the A receptor substance. Such HA receptor substance is not limited, but ganglioside GM3 (Neu5Ac (2 → 3) -Gal-Glc-Ce
r), α (2 → 6) GM3, sialyl Lewis X (Neu5Ac-Ga
l-GlcNAc-Fuc) and the like.

The HA inhibitor is not particularly limited as long as it has an ability to inhibit the binding of hemagglutinin to a host cell receptor. For example, the sialic acid derivative 7-F-
Neu5Ac2en, 2,7-Dideoxy-7-fluoro-2,3-didehydro sial
ic acid, sialic acid dendrimers, sialic acid-containing polymers, and the like.

The phage thus obtained is infected with Escherichia coli, cultured in a large scale, separated and purified, and expressed with a peptide capable of specifically binding to hemagglutinin, preferably a peptide capable of specifically binding to the receptor binding pocket of hemagglutinin. Obtain phage. The phage thus obtained is again subjected to a panning operation for reacting with immobilized hemagglutinin and screening for a phage that specifically binds to hemagglutinin of influenza virus, as described above.

This panning operation is performed several times, preferably four times.
By repeating about 6 times, a phage capable of expressing a peptide that specifically binds to hemagglutinin, preferably a peptide that specifically binds to a receptor binding pocket of hemagglutinin can be selected.

Next, DNA is extracted from the selected phage and its sequence is determined, whereby a peptide expressed by the phage, that is, a peptide that specifically binds to influenza hemagglutinin (HA-binding peptide), preferably hemagglutinin Peptides that specifically bind to the receptor binding pocket can be identified.

The DNA can be easily sequenced by a method known in the art, for example, the dideoxy method [Proc. Natl. Acad. Sci. USA., 74 , 5463-5467 (197)
7)) and the Maxam-Gilbert method [Method in Enzymolog
y, 65 , 499 (1980)]. Such a base sequence can be easily determined even using a commercially available sequence kit or the like.

The phage library used in the phage display method may be any of the known phage libraries usually used in this method. For example, random DNA is inserted into the phage coat protein pIII gene, A random peptide display phage (filamentous phage) constructed so that a peptide having an amino acid sequence consisting of 15 random amino acids can be expressed on the surface of the phage shell can be suitably exemplified (Japanese Patent Application No. 11-118). 000769;
2C103, "Selection of Peptides Binding to Glycolipids by the Phage Display Method" The 3rd Chemical Society of Japan Biotechnology Subcommittee Symposium (1998)).

The affinity (binding ability) of the HA-binding peptide thus identified to hemagglutinin was determined by replacing the random peptide display phage in the above-mentioned method for selecting a phage capable of expressing the HA-binding peptide (panning). Can be confirmed and evaluated in the same manner using the HA-binding peptide to be measured.

As an example of the present invention, the immobilized influenza virus (hemagglutinin) is A / PR / 8/34 (H1N1) which is an H1 subtype of A and A / Wuhan / 35 which is an H3 subtype.
Using an ether extract of 9/95 (H3N2) and an HA receptor substance (GM3) as an eluting substance, a phage expressing a peptide that recognizes and binds both of these hemagglutinins was selected, and the expressed peptide was selected. Is described in Example 1 below.

The peptide identified according to the embodiment of the present invention has any one of the amino acid sequences shown in SEQ ID NOs: 1 to 3. These HA-binding peptides are characterized by having a binding property to at least one of H1 and H3, which are subtypes of hemagglutinin. In particular, an HA-binding peptide having the amino acid sequence shown in SEQ ID NO: 2 is H1 of hemagglutinin.
It has high binding to both subtypes and H3 subtypes. Hemagglutinin H1 and H3 subtypes are known to differ from each other by 75% in their amino acid sequences.
It is considered that the A-binding peptide recognizes a site having high homology between the H1 and H3 subtypes of hemagglutinin and binds to this portion.

In the peptide of the present invention, in addition to the peptide having any one of the amino acid sequences shown in SEQ ID NOs: 1 to 3, one or more amino acids in these amino acid sequences are substituted, deleted or added. And peptides having a binding property to hemagglutinin of influenza virus, particularly hemagglutinin of influenza A virus.

With respect to the peptides shown in SEQ ID NOs: 1 to 3, the degree of amino acid “substitution, deletion or addition” and their positions are determined by the fact that the modified peptide or protein is Influenza virus hemagglutinin as well as a peptide consisting of any of the amino acid sequence shown in the, is not particularly limited as long as it is the same compound having the characteristic of having a binding property to the receptor binding pocket of hemagglutinin, but is preferably Is characterized by having the property of binding to either the H1 or H3 subtype of hemagglutinin, more preferably the property of binding to both the H1 and H3 subtypes. And the same compounds having the following.

Such amino acid sequence modification (mutation)
Can occur due to, for example, mutation or post-translational modification, but can also be performed artificially. The present invention encompasses all modified peptides having the above properties, regardless of the cause and means of such modification / mutation.

The HA-binding peptide of the present invention can be produced by a general chemical synthesis method according to its amino acid sequence. The method includes a conventional liquid phase method and a solid phase method for peptide synthesis. More specifically, such a peptide synthesis method is based on the amino acid sequence information provided in the present invention, a stepwise erosion method in which each amino acid is sequentially bonded one by one to extend the chain, and a fragment comprising several amino acids. Is synthesized in advance, and then a fragment condensation method in which each fragment is subjected to a coupling reaction. The synthesis of the peptide of the present invention can be based on any of them.

The condensation method employed in the above peptide synthesis is also
Various known methods can be followed. Specific examples thereof include azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPPA (diphenylphosphoryl azide) method, and DCC + additives (1-hydroxybenzotriazole, N-hydroxysuccinamide). , N-hydroxy-5-norbornene-2,3-dicarboximide), the Woodward method and the like. The solvent that can be used in each of these methods can be appropriately selected from general solvents that are well known to be used in this kind of peptide condensation reaction. Examples include dimethylformamide (DMF), dimethylsulfoxide (DM
SO), hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and a mixed solvent thereof.

In the above-mentioned peptide synthesis reaction, carboxyl groups in amino acids and peptides not involved in the reaction are generally esterified to form lower alkyl esters such as methyl ester, ethyl ester and tertiary butyl ester such as benzyl ester. , P-methoxybenzyl ester, p-nitrobenzyl ester, aralkyl ester and the like. Further, an amino acid having a functional group in a side chain, for example, a hydroxyl group of Tyr may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, or the like, but such protection is not necessarily required. . Further, for example, Arg
Is a nitro group, a tosyl group, a 2-methoxybenzenesulfonyl group, a methylene-2-sulfonyl group,
It can be protected by a suitable protecting group such as a benzyloxycarbonyl group, an isobornyloxycarbonyl group and an adamantyloxycarbonyl group. Deprotection of these protecting groups in the amino acids and peptides having the above protecting groups and the finally obtained peptide of the present invention can also be carried out by a conventional method such as a catalytic reduction method, liquid ammonia / sodium, hydrogen fluoride, odor, It can be carried out according to a method using hydrogen chloride, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid and the like.

The thus obtained HA-binding polypeptide of the present invention can be obtained by a conventional method, for example, ion-exchange resin, partition chromatography, gel chromatography, affinity chromatography, high performance liquid chromatography (HPLC), countercurrent distribution method. Such purification can be performed appropriately according to a method widely used in the field of peptide chemistry.

The HA-binding peptide of the present invention can be chemically modified as appropriate. For example, the HA-binding peptide can be HA-modified by chemical modification such as alkylation or lipidation (phospholipidation).
Increases the affinity of the binding peptide for cells and tissues,
Alternatively, it can be intended to enhance the pharmacological effect by elongating the half-life in blood.

The alkylation of the HA-binding peptide can be carried out according to a conventional method. For example, it can be easily carried out by an amide bond formation reaction between the fatty acid and the N-terminal amino group of the HA-binding peptide (performed in the same manner as in the above-mentioned peptide synthesis).

As the fatty acid, any fatty acid can be widely exemplified without particular limitation, whether it is linear or branched or saturated or unsaturated. Generally, fatty acids present in the living body can be suitably used, and specifically, saturated fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, and arachiic acid; oleic acid, elaidic acid, linoleic acid,
Fatty acids having about 12 to 20 carbon atoms, such as unsaturated fatty acids such as linolenic acid and arachidonic acid, are exemplified.

In the alkylation, alkylamine and HA
It can also be obtained by an amide bond formation reaction with the C-terminal carboxyl group of the binding peptide (performed in the same manner as in the above peptide synthesis). As the alkyl, various alkylamines can be exemplified as in the case of the above fatty acids, and generally, a fatty chain (having about 12 to 20 carbon atoms) present in a living body can be suitably used.

The lipidation of the HA-binding peptide can be carried out according to a conventional method (New Current, 11 (3), 15-20 (2000); Bi).
ochemica et Biophysica Acta., 1128, 44-49 (1992);
FEBSLetters, 413, 177-180 (1997); J. Biol. Chem., 25
7, 286-288 (1982)). For example, a lipid-modified HA-binding peptide can be prepared via an arbitrary spacer using a 2-position hydroxyl group or a 3-position phosphate group of various phospholipids. Various condensation methods can be used for the reaction. If necessary, an amino acid sequence containing cysteine of any length (usually several) is added to the N-terminal or C-terminal of the HA-binding peptide, and the reaction is used for the condensation. A sex SH group can also be introduced.

The phospholipid is not particularly limited, and phosphatidic acid, phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, etc., composed of the various fatty acids described above, can be used favorably.

The alkylated or lipidated HA-binding peptide (lipopeptide) of the present invention also acts as a lipid component in liposome preparation, and the peptide is presented on the liposome. Are very suitably used in the liposome preparation described below.

The HA-binding peptide of the present invention has an amino acid sequence that specifically recognizes and binds to hemagglutinin of an influenza virus involved in the first step of influenza infection, and has an inv.
In vivo, it can be used as a substance capable of competitively preventing the binding of an influenza virus and a host cell receptor generated via hemagglutinin, that is, as a hemagglutinin binding inhibitor. The hemagglutinin binding inhibitor can be used as a tool for elucidating influenza virus infection generated via hemagglutinin and accompanying various cell functions and life phenomena, and binding of hemagglutinin to host cell receptors. It is expected to be useful as an active ingredient of an anti-influenza agent used to prevent infection of influenza virus caused by the virus or to treat influenza, specifically, an agent for preventing or treating influenza infection.

The influenza virus targeted by the present invention is not particularly limited in its type and origin as described above, and may be of type A, B or C or human-isolated type, such as pig or horse. It can be any other mammal-separable type or bird-separable type. Preferred is influenza A virus. It is also preferably a human isolated or human infectious virus.

When used as a prophylactic or therapeutic agent for influenza virus infection, the HA-binding peptide of the present invention (including modified or modified peptides having HA-binding properties; the same applies hereinafter) is used as such or as a pharmaceutical. Is prepared as a pharmaceutical composition comprising a pharmaceutically acceptable carrier and administered to a subject.

The pharmaceutically acceptable carrier used here can be appropriately selected from carriers commonly used in the art depending on the form of the pharmaceutical composition to be prepared.

For example, when the pharmaceutical composition is prepared in the form of an aqueous solution, purified water (sterile water) or physiological buffer can be used as the carrier. When the composition is prepared in an appropriate solution form, examples of the carrier include injectable organic esters such as glycol, glycerol and olive oil. In addition, these compositions can contain stabilizers, excipients, and the like that are conventionally used in the art, particularly in the field of peptide preparations or protein preparations.

The HA-binding peptide of the present invention can be prepared as a liposome preparation. The liposome preparation can be prepared by retaining the HA-binding peptide of the present invention in a liposome containing an acidic phospholipid as a membrane component, or a liposome containing a neutral phospholipid and an acidic phospholipid as a membrane component.

Here, the acidic phospholipid as a membrane component is defined more narrowly than ordinary acidic phospholipids, and more specifically, dilauroyl phosphatidyl glycerol (DLPG), dimyristoyl phosphatidyl glycerol (DMPG), Natural or synthetic phosphatidyl glycerols (PG) such as palmitoyl phosphatidyl glycerol (DPPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), egg yolk phosphatidyl glycerol (yolk PG), hydrogenated egg yolk phosphatidyl glycerol and the like; Lauroyl phosphatidylinositol (DLPI); dimyristoyl phosphatidylinositol (DMPI), dipalmitoyl phosphatidylinositol (DPI) PI), distearoyl phosphatidylinositol (DSPI), shown dioleoyl phosphatidyl inositol (DOPI), soybean phosphatidylinositol (soybean PI), natural or synthetic phosphatidylinositol such as hydrogenated soybean phosphatidylinositol (PI). These can be used alone or in combination of two or more.

As the neutral phospholipid, soybean phosphatidylcholine, egg yolk phosphatidylcholine, hydrogenated soybean phosphatidylcholine, hydrogenated egg yolk phosphatidylcholine, dimyristoylphosphatidylcholine (DMP)
C), dipalmitoyl phosphatidylcholine (DPP
C), dilauroylphosphatidylcholine (DLP
C), distearoyl phosphatidylcholine (DSP
C), natural or synthetic phosphatidylcholines (PC) such as myristoyl palmitoylphosphatidylcholine (MPPC), palmitoylsteariylphosphatidylcholine (PSPC), dioleoylphosphatidylcholine (DOPC), soybean phosphatidylethanolamine, egg yolk phosphatidylethanolamine, hydrogenated soybean phosphatidyl Ethanolamine, hydrogenated egg yolk phosphatidylethanolamine, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dilauroylphosphatidylethanolamine (DLPE), distearoylphosphatidylethanolamine (DSPE),
Examples include natural or synthetic phosphatidylethanolamines (PE) such as myristoyl palmitoylphosphatidylethanolamine (MPPE), palmitoylstearoylphosphatidylethanolamine (PSPE), and dioleoylphosphatidylethanolamine (DOPE). These can be used alone or in combination of two or more.

The above-mentioned liposome membrane is formed by using the above-mentioned acidic phospholipid alone as a constituent component or by using the above-mentioned neutral phospholipid and acidic phospholipid in combination according to a conventional method. Here, the combined ratio of the acidic phospholipid is about 0.1 to 100 mol%, preferably about 1 to 9 mol%, in the liposome membrane component.
The content is preferably 0 mol%, more preferably about 10 to 50 mol%.

In preparing the liposome, for example, cholesterol or the like can be further added. According to the addition of cholesterol, the fluidity of the phospholipid can be adjusted to make the preparation of the liposome easier. The cholesterol is usually added and blended in an amount equivalent to the phospholipid, preferably in an amount of 0.5 to 1 equivalent.

The mixing ratio of the active ingredient and the acidic phospholipid in the liposome dispersion is such that the acidic phospholipid is about 0.5 to 100 equivalents, preferably about 1 to 60 equivalents, more preferably about 1 equivalent to the active ingredient. It is good to be about 1.5 to 20 equivalents.

The HA of the present invention contained in the liposome
The ratio of the binding peptide can be about several mol% to several tens mol%, preferably about 5 to 10 mol%, and usually about 5 mol% in the total lipid.

Various known methods can be used for producing liposomes using the HA-binding peptide of the present invention as a drug, and the lipopeptide of the present invention can be used as a lipid component in the method. Thus, a desired liposome can be obtained.

For example, a multilamellar liposome (MLV) is prepared as follows. First, after dissolving lipid in an organic solvent (chloroform, ether, etc.), the solution is placed in a round-bottom flask, and the organic solvent is removed under a nitrogen stream or under reduced pressure to form a lipid thin film on the bottom of the flask. In this case, the organic solvent can be completely removed by leaving the mixture in a desiccator under reduced pressure. Next, an emulsion color liposome suspension is obtained by adding a drug solution onto the lipid thin film to hydrate the lipid.

Large unilamellar liposomes (LUV)
Converts phosphatidylserine into small unilamellar liposomes
After adding Ca ²⁺ and fusing to form a cylindrical sheet, a chelating agent EDTA is added to remove Ca ²⁺ (Biochim. Biophys. Acta 394 , 483-491, 1975) or to ether. Inject the dissolved lipid into an aqueous solution at about 60 ° C,
Method for evaporating ether (Biochim. Biophys. Acta 4
43 , 629-634, 1976).

A method for preparing liposomes by the reverse phase method devised by Szoka et al. (Proc. Natl. Acad. Sci. USA 75 ,
4194-4198, 1978) can also be used. According to this method, a drug solution is added to an ether solution of a phospholipid, and the mixture is subjected to ultrasonic treatment to form a W / O emulsion. The W / O emulsion was removed from the W / O emulsion under reduced pressure by an evaporator, and then a buffer solution was added thereto and stirred with a vortex mixer.
A liposome can be prepared by inverting the phase to a W-type emulsion and removing the remaining organic solvent.

In addition to these preparation methods, liposomes having a small particle size can be prepared by a French press method (FEBS lett. 99 , 210-214, 1979). Also, a freeze-drying method with high retention efficiency in liposomes reported by Osawa et al. (Chem. Pharm. Bull. 32 , 2442-2445, 1984)
And freeze-thaw method (Chem. Pharm. Bull. 33 , 2916-2923,198
5) can also be used.

The liposomes prepared in this manner are subjected to a dialysis method (J. Pharm. Sci. 71 , 806-812, 1982) or a filtration method using a polycarbonate membrane (Biochim. Biophys. Acta 55) .
7 , 9-23, 1979; Biochim. Biophys. Acta 601 , 559-57.
1, 1980), the particle diameter can be made constant.
In order to remove the drug not retained in the liposome from the prepared liposome solution, a dialysis method, a gel filtration method, and a centrifugation method can be used (liposome. "Lipid Chemistry" [Japan Biochemical Society] Ed., Biochemistry Experiment Course 3], Tokyo Kagaku Doujin, 1974). It is also possible to concentrate liposomes using a dialysis membrane.

The liposome dispersion thus prepared may contain various additives such as a preservative, an isotonic agent, a buffer, a stabilizer, a solubilizer, an absorption enhancer, etc. as additives necessary for the formulation design. Can be appropriately compounded, and if necessary, can be diluted with a liquid or water containing these additives. Specific examples of the above additives include preservatives such as benzalkonium chloride, benzethonium chloride, chlorohexidine, parabens (methylparaben, ethylparaben, etc.) and antifungal agents such as thimerosal, which are effective against fungi and bacteria, Examples thereof include D-mannitol, D-sorbitol, D-xylitol, glycerin, glucose, maltose, sucrose, polyhydric alcohols such as propylene glycol, and electrolytes such as sodium chloride. Stabilizers include tocopherol and butylhydroxyanisole. , Butylhydroxytoluene, ethylenediaminetetraacetic acid salt (EDTA), cysteine and the like.

Further, the liposome comprising the HA-binding peptide of the present invention can further contain another drug such as an antiviral agent, and can be similarly made into a liposome preparation.

The production of the liposome preparation is specifically described, for example, by Woodre et al. (Long Circulating Liposomes: old drug).
s, New therapeutics., MC Woodle, G. Storm, Eds: Sp.
ringer-Verlag Berlin (1998)) or Namba et al. (Lipos
omal applications to cancer therapy, Y.Namba, N.Ok
u, J. Bioact. Compat. Polymers, 8 , 158-177 (1993)).
Specific examples of the liposome preparation according to the present invention are shown in Examples 4 and 5 below.

The amount of the HA-binding peptide in the pharmaceutical composition of the present invention containing the liposome preparation is not particularly limited and may be appropriately selected from a wide range.
About 002 to 0.2 (w / v%), preferably 0.00
It is good to be about 1 to 0.1 (w / v%).

The influenza virus hemagglutinin binding inhibitor and the anti-influenza virus agent (pharmaceutical composition) of the present invention contain an HA-binding peptide as an active ingredient and, if necessary, a pharmaceutically acceptable carrier. To be administered to a subject infected with or before the influenza virus.

The method of administration of the above-mentioned inhibitor and anti-influenza virus agent (pharmaceutical composition) is not particularly limited, and may be appropriately determined depending on the form of the preparation, the age and sex of the patient, other conditions, the severity of the disease and the like. It is determined. As the preparation form, parenteral administration forms such as injections, drops, nasal drops and inhalants can be preferably mentioned. Particularly, when prepared as injections or drops, glucose and amino acids may be used as needed. And intravenous administration, or intramuscular, intradermal, subcutaneous or intraperitoneal administration.

The daily dose of the influenza virus hemagglutinin binding inhibitor or the anti-influenza virus agent (pharmaceutical composition) of the present invention varies depending on the condition, body weight, age, sex, etc. of the subject, and cannot be determined unconditionally. , In terms of the amount of HA-binding peptide, usually 1 adult
It can be selected from the range of about 0.001 to 100 mg per day. The formulation is not limited to once a day, but can be administered in several divided doses.

[0076]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples, but the present invention is not limited thereto. Reference Example 1 Preparation of phage display library Report from Nishi T., Saya H., et al., FEBS
A phage display library was prepared according to Lett, 399 , 237-240 (1996)) (2.5 × 10 ⁸ clones).
The phage display library specifically comprises N
NK (N represents any one of nucleobases of adenine (A), cytosine (C), guanine (G), and thymine (T);
Represents guanine (G) or thymine (T). ) Is a filamentous phage fd into which DNA containing a sequence repeated 15 times has been inserted by genetic engineering, and a peptide consisting of a random amino acid sequence of 15 residues at the N-terminal portion of the coat protein pIII gene. It has been constructed so that a peptide having a random 15-residue amino acid sequence can be expressed on the outer surface of the phage by inserting the encoding DNA. The phage display library described above is available from Scott et al. (Scott, JKand Smith, GP, Science, 249 , 38).
6-390 (1990)).

Reference Example 2 Immobilization of influenza virus hemagglutinin Influenza virus hemagglutinin was immobilized as follows in order to be used as a target for biopanning.

The immobilized hemagglutinin (HA)
An ether extract of A / PR / 8/34 (H1N1), which is the H1 subtype of type A (hereinafter also simply referred to as HA1 or H1N1), and A / Wuhan / 359, which is the H3 subtype of type A / 95
An ether extract of (H3N2) (hereinafter also simply referred to as HA3 or H3N2) was used.

First, EDC (1-ethyl-3- (3-dimethylamido) was added to each well of a 96-well carboplate (SUMILON).
60 μl of an equal volume mixture of nopropyl) carbodiimide) and N-hydroxysuccinimide was added to activate the carboxyl groups bound to the plate. After 10 minutes, the wells of the plate were washed six times with 50 mM TBS buffer (pH 7.6), and 100 μl of an H1N1 solution (70 μl / ml) or H3N2 solution (70 μl / ml) was added. After leaving for 2 hours, 5
After washing 6 times with 0 mM TBS buffer (pH 7.6), 100 μl of 1 mM ethanolamine aqueous solution was added. After standing for 10 minutes, the plate was washed again with 50 mM TBS buffer (pH 7.6) six times.
Influenza HA was immobilized on the plate (see FIG. 1).

Example 1 Selection and Identification of Influenza HA-Binding Peptide Recognizing H1 and H3 Subtypes (1) Selection of HA-Binding Peptide Display Phage Influenza virus hemagglutinin HA1 (H1N
Biopanning was performed using two types of target, 1, A / PR / 8/34) and HA3 (H3N2, A / Wuhan / 359/95).
Specifically, biopanning was performed using the HA1 immobilized plate and the HA3 immobilized plate prepared according to Reference Example 2 as the HA immobilized plate, and using the GM3 HA receptor substance as the eluting substance, Phages expressing the HA-binding peptide were selected (see FIG. 2).

(I) Biopanning—First time—100 μl of the phage display library solution (6.2 × 10 ¹⁰ TU, in TBS buffer) prepared in Reference Example 1 was injected into the HA1-immobilized plate and the HA3-immobilized plate. And left at room temperature for 4 hours. Thereafter, 200 μl of an elution buffer (50 mM Tris-HCl, 150 mM NaCl) was used for washing 5 times while pipetting well to remove unbound non-specific phage. Next, 100 μl of a 10 mM GM3 solution was added to each well and left at room temperature for 1 hour.

It has been confirmed that GM3 is an HA receptor substance having specific binding property to the hemagglutinin receptor binding pocket. Therefore, by adding the HA receptor substance to the well of the plate to which the phage has been bound, the phage specifically bound to the hemagglutinin receptor binding pocket of the influenza virus can be eluted.

Thus, the HA-binding phage was eluted,
The solution containing the phage was recovered. To the obtained phage solution, 100 μl of the prepared E. coli K91-Kan solution was added, and the mixture was infected for 15 minutes at room temperature. Fifteen minutes later, 0.2 μg / ml tetracycline (Sigma) pre-incubated at 37 ° C
This infection solution was added to 20 ml of NZY medium containing TC (hereinafter referred to as TC) and cultured with shaking at 37 ° C. for 40 minutes.

After 40 minutes, a stock solution of TC (20 mg / ml) 2
0 μl was added (final concentration: 20 μg / ml), followed by shaking culture at 37 ° C. overnight. The resulting culture was centrifuged at 3,000 rpm for 10 minutes to remove E. coli. The supernatant was centrifuged again at 12,000 rpm for 10 minutes to further remove E. coli. PEG / NaCl solution 3m in supernatant
l (0.15 v / v) and gently agitate 100 times, then 4 ° C
For at least 4 hours. The supernatant was removed by centrifugation at 12,000 rpm for 10 minutes, and 1 ml of TB was added to the precipitated phage pellet.
S was added to completely dissolve. The obtained phage solution was used for 1.
The solution was transferred to a 5 ml Eppendorf tube (Ep tube) and centrifuged at 15,000 rpm for 10 minutes to remove insolubles. 150 μl of the PEG / NaCl solution was again added thereto, and the mixture was gently stirred several times, and then allowed to stand at 4 ° C. for 1 hour or more. 15,000
The pellet was collected by centrifugation at rpm for 10 minutes.
Dissolved in N ₃ / TBS (200 μl). Centrifuge at 15,000 rpm for 10 minutes to remove insolubles, and use the supernatant phage solution (200 μl
l) was transferred to a 0.6 ml Ep tube. Half of the phage solution thus obtained was used for the second and subsequent panning, and
l was used for titering and the rest was saved.

(Ii) Biopanning—second round—Phage display library solution amplified above
100 μl was again injected into the HA1-immobilized plate and the HA3-immobilized plate, and left at room temperature for 4 hours. Thereafter, 200 μl of elution buffer (50 mM Tris-HCl, 150 mM NaC
Using l), the wells were washed 10 times with good pipetting to remove unbound non-specific phage. Next, 100 μl of a 10 mM GM3 solution was added to each well, and the mixture was allowed to stand at room temperature for 1 hour to elute phages specifically binding to the binding pocket of influenza HA, and the solution was recovered.

(Iii) Biopanning-3rd time-Phage display library solution amplified above
100 μl was injected again into the H1-immobilized plate and the H3-immobilized plate, and left at room temperature for 1 hour. Then 20
Using 0 μl of elution buffer (50 mM Tris-HCl, 300 mM NaCl), the wells were washed 10 times with good pipetting to remove unbound non-specific phage. Next, 100 μl of a 10 mM GM3 solution was added to each well, and the mixture was allowed to stand at room temperature for 1 hour to elute phages specifically binding to the binding pocket of influenza HA, and the solution was recovered.

(Iv) Biopanning-4th to 5th-The operation described in (iii) is repeated to perform the 4th and 5th
A second biopanning was performed. FIG. 3 shows the phage recovery rate (recovered phage amount (TU) / used phage amount (TU) × 100) by the above-mentioned panning operation (first to fifth times).
In FIG. 3, -−- indicates target HA (plate-bound H
The results when H1N1 was used as A),-□-
This is the result when 3N2 was used. As can be seen from the results, in each case, a remarkable increase in phage was observed from the fourth to the fifth cycles of repeating the panning under the same conditions. This confirmed that phages expressing peptides that specifically bind to influenza virus hemagglutinin (H1N1, H3N2) were recovered.

(2) Determination of Amino Acid Sequence of HA-Binding Peptide In each of the above-mentioned pannings, the amino acid sequence of the expressed peptide was determined using the phage obtained by the fifth panning operation. Specifically, phages that bind to immobilized hemagglutinin H1N1 and are eluted with GM3 (hereinafter, referred to as phages)
The amino acid sequences of the expressed peptides were determined by sequencing for phage which was bound to immobilized hemagglutinin H3N2 and eluted with GM3 (hereinafter referred to as phage H3G: 14 clones).

First, colonies on the plate obtained by titer measurement after the fifth panning were randomly picked up, inoculated again on a new NZY plate, cultured overnight at 37 ° C., and this was mastered. Stored as a plate at 4 ° C. Each colony of the master plate was suspended in a 50 ml centrifuge tube containing 20 ml of NZY medium (containing 20 μg / ml TC) and cultured at 37 ° C. overnight with shaking at 200 rpm. Then 3,00
After centrifugation at 0 rpm for 10 minutes, the supernatant was transferred to an Oak Ridge centrifuge tube, and centrifuged at 12,000 rpm for 10 minutes to eliminate E. coli. Transfer the supernatant to an Oak Ridge centrifuge tube and add 3 ml of polyethylene glycol (PE).
G6000: Nacalai Tesque, Inc.) / NaCl was added, and the mixture was stirred well and allowed to stand at 4 ° C. for 4 hours. The mixture was centrifuged at 12,000 rpm for 10 minutes to precipitate phage. Then, the supernatant was removed, and the precipitated phage was suspended in 1 ml of TBS (Tris buffered saline). Transfer to 1.5ml Eq tube, 15,000r
After centrifugation at pm for 10 minutes to remove insoluble substances, the supernatant was transferred to another Eq tube, 150 μl of polyethylene glycol / NaCl was added, and the mixture was stirred well and allowed to stand at 4 ° C. for 1 hour. Then, the mixture was centrifuged at 15,000 rpm for 10 minutes to reprecipitate the phage. Subsequently, the supernatant was removed and the precipitated phage was resuspended in 200 μl of TBS. After centrifugation at 15,000 rpm for 10 minutes to precipitate insoluble substances, the precipitate was washed with 0.1 ml.
Transferred to a 5 ml Eq tube and stored phage clones at 4 ° C.

D from the phage clone obtained above
For NA extraction, phage clones were placed in 1.5 ml Eq tubes.
100 μl of TBS and 200 μl of TE-saturated phenol (manufactured by Nippon) were added to 100 μl, and the mixture was vigorously stirred for 10 minutes, followed by centrifugation at 15,000 rpm for 10 minutes. Next, 200 μl of TE-saturated phenol and 200 μl of chloroform were added to 200 μl of the supernatant (aqueous phase), and the mixture was vigorously stirred for 10 minutes as described above, followed by centrifugation at 15,000 rpm for 10 minutes. Further, 250 μl of TE, 40 μl of 3M sodium acetate, 1 μl of 20 mg / ml glycogen (manufactured by Boehringer Meinheim) and 1 ml of ethanol were added to 150 μl of the supernatant (aqueous phase) to give 1.
After leaving for 1 hour at -20 ° C in a 5 ml Eq tube,
Centrifuged at 5,000 rpm for 10 minutes. Remove supernatant and 1 ml
80% ethanol (-20 ° C)
The remaining salts were removed by centrifugation at rpm for 10 minutes. After removing the supernatant, the water in the tube is evaporated and the precipitated DN
A was dissolved in 10 μl of sterile distilled water and stored at 4 ° C.
The individual phage DNA thus obtained was used for peptide sequencing.

The sequence of the peptide encoded by the phage DNA was determined by the dideoxy method (Proc. Natl. Acad. Sci.,
USA., 74 , 5463-5467 (1977)) by Amersham.
THERMO Sequence Kit (Amersham Life Sciene, Code; US79
765, Lot number; 201503) according to the instruction manual for the device. The extension reaction of DNA is performed at 96 ° C, 30 seconds, 45 ° C,
The cycle was performed 30 times at a cycle of 15 seconds, 60 ° C. and 4 minutes, and the DNA sequence was determined using a DNA sequencer (ABI PRISMTM377) manufactured by ABI.

The amino acid sequences of the peptides having a binding property to the receptor binding pocket of hemagglutinin determined from each clone of the two types of phages (phage H1G and phage H3G) are shown in Table 1 as one-letter code. Show.

[0093]

[Table 1]

As can be seen from the table, the HA binding peptide determined from phage H3G (H3G, No. 1) is identical to one of the HA binding peptides determined from phage H1G (H1G, No. 4). Met.

Example 2 Binding Property of HA-Binding Peptide to Influenza Hemagglutinin The phage clone obtained in Example 1 (“Phage H1
G-1 "," phage H1G-2 "," phage H1G
-3 "," phage H1G-4 ("phage H3G-
1))), the binding to hemagglutinin (H1 subtype, H3 subtype) was evaluated using ELISA. As a control test, a phage expressing a peptide having a random sequence (control phage) was similarly examined for binding to hemagglutinin (H1 subtype, H3 subtype).

Specifically, hemagglutinin HA1 (H1N
1) and HA3 (H3N2) were respectively fixed to a 96-well carboplate (manufactured by SUMILON), and each phage clone solution was added with 5 × 10 ¹⁰ virions / m1 to give E.
LISA was performed.

The results are shown in Table 2 and FIG. 4 (A in the figure).
Results for (H1N1) and B) for HA3 (H3N2)].

[0098]

[Table 2]

As can be seen from Table 2 and FIG.
The clones “phage H1G-1” and “phage H1G-3” obtained in the above were found to bind to both hemagglutinin H1 and H3 subtypes in common. Also, phage H1G-4 ("phage H3G-1") showed specific binding to hemagglutinin H3 subtype.

It is known that hemagglutinin H1 and H3 subtypes differ by 75% in amino acid sequence.
From this, particularly, the expression peptides of “phage H1G-1” and “phage H1G-3” are hemagglutinin H1
It is considered that the peptide recognizes a site having high homology between both the subtype and the H3 subtype, and specifically binds thereto.

Example 3 Synthesis of Peptide Expressed on Phage The expressed peptides obtained in Example 1 were synthesized as follows. The peptide was synthesized using an automatic peptide synthesizer (ACT357, manufactured by Advanced Chemtech Co., Ltd.) according to the Fmoc / NMP, HOBt method [Fmoc: 9-fluorenylmethoxycarbonyl, NMP:
[N-methylpyrrolidone, HOBt: 1-hydroxybenzotriazole].

Specifically, according to the amino acid sequence of the peptide desired to be synthesized, Fmoc-
An Fmoc-amino acid corresponding to each amino acid from the C-terminus to the amino acid-Alko resin 0.25 mmol was subjected to an extension reaction sequentially according to a synthesis program. The C-terminal amide peptide was subjected to a condensation reaction of 0.25 mmol of Fmoc-NH-FAL resin with the Fmoc-amino acid corresponding to the C-terminal amino acid according to the formation program, and then to the second and subsequent amino acids from the C-terminal. Fmoc-amino acids were sequentially condensed to extend the chain.

After completion of each reaction, the N-terminal Fmoc group was deprotected according to the program. Each peptide resin thus obtained is collected in a polypropylene mini column (manufactured by Assist Co., Ltd.), washed with methanol, dried in vacuum, and subjected to the following operations to cleave the peptide from the resin and perform a side chain deprotection reaction. went. That is, Regent K
(Reagent K, 82.5% TFA, 5% phenol, 5% H
₂ ml of 0.5% thioanisole (2.5% ethanedithiol) was added and reacted in a mini column for 60 minutes. Next, the reaction solution was dropped into 8 ml of cold diethyl ether to stop the reaction, and at the same time, the peptide was precipitated. Further, the mini column was washed with 2 ml of TFA, 5 ml of cold diethyl ether was added, and the mixture was centrifuged. The precipitate was washed four times with 10 ml of diethyl ether, the peptide was solubilized with about 5 ml of 50% acetonitrile, and freeze-dried. Further, the solubilization and freeze-drying operations were repeated twice to obtain a desired crude freeze-dried product.

This was placed on an octadecyl column (diameter 20 × 250).
mm, manufactured by WC Co., Ltd.) to isolate the desired peptide. In addition, the resin and the amino acid derivative used in the above are all manufactured by Watanabe Chemical Industry Co., Ltd. Each of the thus isolated peptides was identified by measuring the molecular weight by amino acid sequence analysis and mass spectrometry.

Example 4 Lipopeptide and Peptide-Modified Liposomes Using the Lipopeptide By binding stearic acid to the N-terminus of the peptide, the HA-binding peptide of the present invention can be introduced into the liposome. As an example, the expression peptide (H1G, N) of the clone “phage H1G-2” obtained in Example 1 is used.
o.2) will be explained.

First, the expressed peptide sequence of the clone “phage H1G-2” (SEQ ID NO: 1) was subjected to an extension reaction in accordance with Example 3 described above. Next, stearic acid is extended to the N-terminus of this peptide according to the same synthesis program, cut out from the resin, deprotected, and subjected to a desired stearylated peptide (“C ₁₈ lipopeptide”).
I got

This lipopeptide, egg yolk lecithin (Mw:
773, Nippon Oil & Fat NC-10S) and cholesterol (Mw: 38)
6.66, Nakarai Chemicals, lot M8A9926), 0.75:
At a molar ratio of 2: 1 (20% lipopeptide), 0.3: 2: 1 (10% lipopeptide) or 0.16: 2: 1 (5% lipopeptide), chloroform /
It was dissolved in 2 ml of a methanol (3/1) solution. The solvent was removed under reduced pressure using a rotary evaporator, and the residue was dried under vacuum overnight. To this, 5 ml of Dulbecco's phosphate buffered saline solution (without Ca and Mg) (pH 7.4) was added, and treated with a bath-type sonicator for 30 minutes to give liposomes (small unilamel).
lar liposome). Lipids that did not form vesicles were subjected to centrifugal ultrafiltration (1,500 × g, 15 minutes: molecular weight cut off 30,0
00, Amiconcentriprep-50).

For the liposomes thus obtained, the content of the peptide present inside was determined using a commercial kit (Bio-Rad Prot
ein Assay Kit), and confirmed that the lipopeptide prepared above was incorporated into the liposome.

Example 5 Peptide-modified liposome DDPE (dipalmitoyl L-α-phosphatidylethanol ami
ne: Sigma) 6.7 mg, NHS-PEG-Mal (poly (e
thylene glycol-α-N-β-maleimide hydroxysuccinimid
(yl propionate: NOF Liposomes Co., Ltd.) 45 mg and triethylamine (980 μl) were dissolved in chloroform (10 ml), and the mixture was stirred at room temperature. The reaction was followed by TLC, and after confirming that the ninhydrin reaction was negative, the solvent was distilled off. 2 ml of distilled water was added to the obtained white viscous material, and the mixture was treated with a bath-type ultrasonic wave.
Micelles formed. This solution was placed in a double volume cellophane tube (fraction molecular weight: 12,000, size: 20/30, manufactured by Sanko Junyaku), and 1 L of distilled water outside the dialysis membrane was replaced as appropriate.
Dialysis was performed for 3 days to remove by-products that did not form micelles. Gel filtration (Sephadex G50 Fine, Amers
The fraction near the void was collected by ham Pharmacia Biotech AB), freeze-dried, and the target DPPE-PEG-Ma as a white powder showing a single band on TLC.
1 was obtained.

A liposome (yolk lecithin / cholesterol = 2/1) prepared in the same manner as in Example 4 and this DPP
Mix E-PEG-Mal and mix 5,10,20 and 100% D
A liposome containing PPE-PEG-Mal was prepared. 1N NaOH aqueous solution is added to the liposome solution to adjust the pH to about 8, and H-Gly-Cys-OH is added to the C-terminal of the expression peptide (H1G, No. 2: SEQ ID NO: 1) of clone "phage H1G-2". The obtained peptides were mixed to a predetermined concentration, gently stirred at room temperature for 6 hours, and subjected to centrifugal ultrafiltration as in Example 3 to obtain a liposome solution. The content of the peptide introduced on the obtained liposome was measured in the same manner as in Example 4.

As described above, DPPE-PEG-M
A modified peptide in which an HA-binding peptide is bonded to the cysteine via the SH group of cysteine via maleimide of al was introduced into the liposome to obtain a peptide-modified liposome.

Example 6 Evaluation of Influenza Virus Infection Inhibitory Activity The HA-binding peptide of the present invention was evaluated for influenza virus infection inhibitory activity.

As cells infected with influenza virus,
MDCK cells (Mardin-Darby canine kidney) were used. Evaluation of influenza virus infection to the cells was carried out by detecting LDH (1actate dehydrogenase) activity using a commercially available kit (LDH-Cytotoxic Test, Wako) (J. Virol. Meth., 51, 185-192). (199
Five)). As influenza viruses, subtypes HA1 (A / PR / 8/34 (H1N1)) and HA3 (A / Victoria /
1/75 (H3N2)) was used.

Specifically, the infectious titer of influenza virus was measured as follows.

That is, 3 × 10 ⁵ cells / ml of MDCK cells cultured in a MEM / 10% FBS medium were seeded at 100 ml on a 96-well microtiter plate under a 5% CO ₂ atmosphere.
Incubated overnight at ° C. After confirming that the cells in all the wells were monolayer-saturated, the wells were washed twice with serum-free MEM (MEM (-)), and the serially diluted virus solution (MEM (-)) was added thereto. Injected 100 μl each. After incubating for 2 hours at 37 ° C. in a 5% CO ₂ atmosphere, the virus solution was removed, washed twice with a medium, and then incubated for 20 hours with the same medium. Take 15 μl of the supernatant in the well, dilute 4-fold with PBS, and add 50 μl
And transfer it to a new plate, and add a coloring solution (nitrobluetetrazolium, diaphorase and NDA + in lit) to each well.
ium lactate PBS) was added. After leaving at room temperature for 20 minutes, 100 ml of a reaction stop solution (0.5 M HCl in PBS) was added to stop the color reaction, and the absorbance at 550 nm was measured using a microplate reader. From the results obtained, I
It was determined the D ₅₀ value.

The influenza virus infection inhibition experiment with the HA-binding peptide was carried out at the ID ₅₀ value obtained above.
The test was performed using the virus amount of double concentration (50 LDH ID ₅₀ ). It was prepared by the method described in Example 4 as a HA-binding peptide "C ₁₈ lipopeptide" ( "H1G, No.1", "H1G,
FIG. 5 shows the results of an influenza virus infection inhibition test performed using liposomes each containing 10% of each of “No. 3” and “H1G, No. 4 (H3G, No. 1)” _C18 lipopeptide). FIG. 5A shows influenza A virus HA1.
(A / PR / 8/34 (H1N1)), and FIG.
Influenza virus HA3 (A / Victoria / 1/75 (H
This is the result of inhibition of infection against 3N2)).

As can be seen from FIG. 5, the peptide “ _C18 lipopeptide” of the present invention effectively inhibits infection of cells with influenza virus, and particularly “H1G, No. 1” and “H1G, No. "3" was found to bind to both influenza hemagglutinin subtypes (HA1, HA3) and inhibit infection by that virus. These facts suggest that the HA-binding peptide of the present invention is useful as an agent for preventing or treating influenza infection.

[0118]

According to the present invention, there is provided a peptide having specific binding to hemagglutinin of influenza virus.

The peptide of the present invention can prevent the binding of the virus to the host receptor by specifically binding to hemagglutinin involved in the first step of influenza virus infection, whereby the prophylactic agent for influenza virus infection can be prevented. As a therapeutic agent for influenza. In particular, preferred peptides of the invention have the property of specifically binding to the receptor binding pocket of hemagglutinin, whose tertiary structure is well preserved between subtypes. It is also stable because of its relatively short peptide, and interacts with the binding site of hemagglutinin due to its side chain diversity. From this, the peptide of the present invention is expected to be applied as a broad-spectrum infection preventive agent and a therapeutic agent effective for all influenza viruses, particularly for all types A, across various subtypes, regardless of subtypes. .

Furthermore, since the present invention is a peptide, it can be easily synthesized and modified in a large amount because it is a peptide as compared with a sugar chain analog which has been conventionally studied and developed as an anti-influenza agent. Useful.

[0121]

[Sequence List] SEQUENCE LISTING <110> Keio University <110> Otsuka Pharmaceutical Co. Ltd. <120> Affinity peptides to Hemagglutinin of Influenza <130> 10201JP <160> 4 <170> <210> 1 <211> 15 <212 > PRT <213> phage library <400> 1 Ser Arg Ala Phe Asp Leu Leu Asp Gln Pro Leu Ser Asn Ala Arg 1 5 10 15 <210> 2 <211> 15 <212> PRT <213> phage library <400> 2 Thr Ser Val Asn Arg Gly Phe Leu Leu Gln Arg Ala Ser His Pro 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> phage library <400> 3 Gly Leu Ala Met Ala Pro Ser Val Gly His Val Arg Gln His Gly 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> phage library <400> 4 Glu Pro Tyr Gly Phe Ile Ala Phe Ser Arg Ala Ala His Ser Pro 1 5 10 15

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a step of immobilizing influenza virus hemagglutinin on a microplate.

FIG. 2 is a schematic diagram showing a method of selecting a phage expressing a desired peptide using a biopanning operation.

FIG. 3 is a diagram showing the yield% of phage recovered by performing biopanning on hemagglutinin HA (H1N1, H3N2) 1 to 5 times (Example 1). -○-shows the result when H1N1 was used as the target HA, and-□-shows the result when H3N2 was used as the target HA.

FIG. 4 shows phage clones obtained in Example 1 (“phage H1G-1” (---), “phage H1G-2” (-△-), “phage H1G-3” (-□-), "Phage H3G-1"("Phage H
1G-4 ") (-×-)) and control phage (control) (-
(-) Shows the results of examining the binding properties of influenza virus hemagglutinin HA1 (H1N1) (FIG. A) and HA3 (H3N2) (FIG. B) (Example 2).

FIG. 5 is a diagram showing the results of examining the influenza virus infection inhibitory activity on cells using the lipopeptide (C ₁₈ -lipopeptide) -modified liposome prepared in Example 4 in Example 5. A) shows the results when H1 subtype (A / PR / 8/34 (H1N1)) was used as the influenza virus, and B) shows the H3 subtype (A / Victoria / 1/75 (H1N1)).
3N2)).

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Riki Tanaka 42-13 Sumiyoshi Chidorigahama, Aizumi-cho, Itano-gun, Tokushima (72) Inventor Koichi Ogino 197-3 Higashihama, Minamihama-cho, Naruto-cho, Naruto-shi, Tokushima (72) Inventor Takao Taki 8-4 F-term (reference) 8-4 Sannomiya, Ejiri-ji, Kitajima-cho, Itano-gun, Tokushima Pref. FA72 FA74 GA26

Claims (5)

[Claims] 1. An influenza virus hemagglutinin-binding peptide which is one of the following (a) or (b): (a) has an amino acid sequence selected from any of the amino acid sequences shown in SEQ ID NOs: 1 to 3 Peptide, (b)
A peptide comprising an amino acid sequence in which one or more amino acids have been modified by substitution, deletion or addition in the amino acid sequence shown in the above (a), and having a binding property to influenza virus hemagglutinin. 2. The method according to claim 1, which is alkylated or lipidated.
2. The influenza virus hemagglutinin-binding peptide according to item 1. 3. The influenza virus according to claim 2,
A liposome containing a hemagglutinin-binding peptide. 4. An influenza virus hemagglutinin binding inhibitor comprising the influenza virus hemagglutinin binding peptide according to claim 1 or 2 as an active ingredient. 5. An anti-influenza agent comprising the influenza virus hemagglutinin-binding peptide according to claim 1 or 2 as an active ingredient together with a pharmaceutically acceptable carrier.