JP4203082B2 - Peptides with affinity for gp120 - Google Patents

Description

  The present invention relates to a peptide having affinity for the gp120 molecule constituting the outermost shell of human immunodeficiency virus (HIV).

As a treatment method for HIV infection, chemotherapy is mainly used as represented by 3'-azido-2 ', 3'-dideoxythymidine (AZT) which is a nucleoside derivative. Although the above-described treatment with AZT or a protease inhibitor developed thereafter has been effective in prolonging the lives of HIV-infected persons, various problems caused by chemotherapy itself have not been avoided.

  Such problems are as follows: first, chronic toxicity appears after long-term administration; second, drug-resistant HIV strains appear during treatment; and third, malignant tumors occur frequently in patients who have seen life-prolonging effects. Fourthly, the recovery of the immune response that is the final goal of treatment cannot be obtained; and fifthly, there is no method for monitoring the therapeutic effect. Thus, since chemotherapy cannot be a therapeutic method that can fundamentally treat HIV infection, development of a vaccine is expected.

  Generally speaking, vaccines are inactivated by chemical treatment of viruses and other microorganisms without changing their structure (inactivated vaccines); cowpox viruses against attenuated strains and smallpox viruses that have lost their pathogenicity, etc. Thus, similar strains (live vaccines) that do not have lethal effects on humans are used. However, although HIV itself is originally an attenuated strain, it is known that once it enters a host cell, it can stay for a long period of time, and gradually destroys the function of the host cell. Are mainly lymphocytes that are responsible for immune function; considering that HIV has spread to hemophilia patients via freeze-dried blood products, it is either inactivated or attenuated. Regardless of the choice, using HIV itself as a vaccine is problematic in terms of safety.

  Therefore, in developing an HIV vaccine, it is ideal to prevent infection by preparing a peptide vaccine using a portion of the outermost shell of the virus.

  From this point of view, many researchers have conducted epitope analysis of the gp120 molecule that constitutes the outermost shell of the virus, and focused on the V3 region (3rd hypervariable region) as an epitope of the gp120 molecule. The region was highly mutated [Palker TJ, et al., Proc. Natl. Acad. Sci. USA 85: 2709-2713, 1988; Rusche JR, et al., Ibid 85: 3198-3202, 1988; Gouddsmit J., etal., Ibid 85: 4478-4482.1988; Matsushita S., et al., J Virol. 62: 2107-2114,1988]. Subsequently, a peptide antigen was prepared using a part of this region, and an HIV infection prevention experiment using monkeys [Emini EA, et al., Nature 355: 728-730, 1992] was conducted. Results have not yet been reported.

  In addition, a device for enhancing the immunogenicity of the peptide antigen has also been devised (Tam et al., JP-T-3-503539), but most of the V region suitable as an epitope such as the V3 region is mutated or missing. Since loss frequently occurs, the desired vaccine has not yet been obtained.

  Furthermore, so-called neutralizing antibodies have also been developed with the aim of preventing HIV lymphocyte infection by using an antibody prepared by using a part of the V3 region as an antigen. For example, in Japanese Patent Application No. 63-171385, a partial antibody in the above region is used as an antigen, a monoclonal antibody is produced in a mouse, and its Fab ′ is bound at the protein level or by genetic engineering techniques. A method for producing an anti-HIV chimeric antibody in which a human antibody molecule and a mouse antibody molecule are hybridized has been reported. However, even with such a neutralizing antibody, the ability of HIV to inhibit infection of lymphocytes is only a laboratory level, and no practically useful neutralizing antibody has yet been obtained. .

  On the other hand, as described above, since chemotherapy has problems such as drug resistance and side effects, there is an idea of removing HIV from a living body by plasma exchange therapy without such problems. Specifically, a method of removing the HIV by reducing the pore size of the filtration membrane used for plasma exchange can be considered, but since the HIV is as small as about 70 nm, it is difficult to make the pore size uniform. In addition, there is a possibility that clogging may occur during plasma filtration, and as a result, there are many technical problems to be solved, such as deterioration of pressure resistance of the filtration membrane. Therefore, a method of using CD4 protein derived from lymphocytes having an affinity specific for HIV as an adsorption carrier for plasma exchange is also conceivable. However, CD4 is denatured by high-pressure sterilization and loses affinity. Cannot be used. In addition to the above-mentioned CD4, there is a method of using a high-molecular-weight polymer having an affinity for HIV or a high-pressure sterilization-resistant one such as a dye ligand, but since these are originally not specific for HIV, HIV is adsorbed. The blood component is adsorbed nonspecifically to the polymer or the like before it is used, and the adsorption of HIV is inhibited, so it cannot be used.

  As described above, research for producing vaccines and neutralizing antibodies has been actively conducted for the purpose of developing an HIV therapeutic agent, but a useful therapeutic agent has not yet been obtained.

  Focusing on such a current situation, the present inventors have developed a peptide having a specificity specific to gp120 that is equivalent to or higher than that of an antibody and excellent in high-pressure sterilization resistance, and has already filed an application. (Japanese Patent Application No. 8-351474 and Japanese Patent Application No. 8-351475). This peptide basically consists of a three amino acid sequence, but subsequent studies have revealed that the affinity of the peptide for gp120 decreases with the type and number of amino acids linked to it. Therefore, it is desired to provide a peptide with more stability.

  The present invention has been made by paying attention to the above circumstances, and the object thereof is a novel peptide having affinity for the gp120 molecule constituting the outermost shell of HIV and having excellent stability. And providing various utilization modes using the peptide.

The peptide having affinity for the gp120 molecule according to the present invention that has solved the above problems is H-A1-A2-A3-A4-A5-R (wherein H represents a hydrogen atom, A1 is a residue of aspartic acid, lysine, valine, glutamic acid, glycine, asparagine, or tyrosine, A2 is a residue of valine, aspartic acid, tryptophan, lysine, phenylalanine, isoleucine, leucine, or tyrosine, A3 is a lysine , Valine, aspartic acid, arginine, alanine or tryptophan residue, A4 is alanine, tryptophan, glycine residue, A5 is glycine, alanine, valine, leucine, isoleucine, serine, threonine, methionine, asparagine, glutamine Histidine, lysine, arginine, Phenylalanine, tryptophan, proline or residues tyrosine,, R is a peptide represented by a carboxyl group which is NH ₂ from OH or acid amide groups derived from), the peptide of Table 1 No. 1-19, 22-27, No. 2 in Table 2. 28 to 40, No. 4 in Table 4. A peptide consisting of the amino acid sequence described in 21. Specifically, it is a peptide having the amino acid sequence shown by SEQ ID NO: 1, and the peptide has a gist in the place consisting of the amino acid sequence shown by SEQ ID NO: 2-40.

  The peptide of the present invention is a peptide having 5 amino acid sequences consisting of A1, A2, A3, A4 and A5 as a basic component, and A1′-A2-A3-A4-A5-R (wherein A1 ′ Is a residue of aspartic acid, lysine, valine, glutamic acid, glycine, asparagine, or tyrosine, or a polypeptide residue in which any amino acid is arranged on the N-terminal side of the amino acid, A2, A3, A4 , A5 and R have the same meaning as before, Peptides having the amino acid sequences described in 20, 21 are also an embodiment of the present invention. Specifically, a peptide having the amino acid sequence represented by SEQ ID NO: 41, wherein the peptide has the amino acid sequence represented by SEQ ID NOs: 42 to 43, is also an embodiment of the present invention.

  In addition, a compound in which a polymer compound having a functional group and / or a pharmaceutically active substance is bound to the above-mentioned peptide or a pharmaceutically acceptable salt thereof is also included in the scope of the present invention.

  In addition, what contains these peptide compounds and a compound can be called the affinity agent with respect to gp120 in other words.

  Furthermore, a composition containing the peptide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or pharmaceutically active substance is also included within the scope of the present invention. In addition, various forms of detecting, diagnosing, removing, etc., viruses such as HIV using the above peptides (for example, HIV diagnostic test agents or test kits containing the test agents, and HIV adsorption removal agents) And other aspects such as use in plasma exchange therapy are also included within the scope of the present invention.

  The “peptide” used in the present invention includes those in which the C-terminus of the peptide is COOH, as well as those in the form of acid amides and esters, etc. Unless otherwise indicated, oligopeptides having 10 or fewer amino acids to higher polypeptides are included.

  In addition, amino acids constituting the peptide include amino acid derivatives in which a functional group is protected with a protecting group. As such amino acid derivatives, protected amino acid derivatives corresponding to various amino acids such as those in which the functional group of the side chain is substituted or modified without changing the peptide backbone itself; those in which the chain length of the carbon chain is changed are commercially available. However, in the present invention, these various amino acids can be used. For example, as a derivative of tyrosine, 2,6-dichloro-L-tyrosine having a chloro group in the side chain, p-Nitro-L-phenylalanine in which the hydroxyl group at the p-position of the phenyl group in the side chain of phenylalanine is substituted with a nitro group, Examples include 4-chloro-L-phenylalanine substituted with a chloro group at the hydroxyl group, and derivatives of valine include Norvaline: N-α-L-norvaline, MeVal: N-α-methyl-L-valine, etc. Is mentioned.

  As described above, the peptide of the present invention has a stable and excellent affinity for gp120, and has an aggregation ability as an anti-HIV agent having neutralizing activity comparable to that of conventional antibody molecules. The HIV diagnostic agent used is extremely useful as a medical device such as a device for removing HIV that requires high-pressure sterilization and utilizes physical stability not found in antibody molecules.

  As a reason why the present inventors have not used any conventional HIV therapeutic agent to obtain a practical level of results, whether it is a vaccine or a neutralizing antibody, the living body can recognize an HIV region as an antigen. However, based on the viewpoint that there is the biggest problem in the V region of the gp120 that is intensely mutated that constitutes the outermost shell, a peptide having affinity for gp120 is obtained instead of the antibody produced by the living body. We paid attention and have been studying earnestly. As a result, we have developed a peptide that has a high specificity for gp120, which is equivalent to or higher than that of an antibody, and also has excellent high-pressure sterilization resistance, and has already been filed (see Japanese Patent Application No. 8-351474 and Application No. 8-351475).

  However, subsequent studies have found that the affinity of the above peptide for gp120 decreases with the type and number of amino acids linked to it. Thus, as a result of further studies to provide a peptide with more stability, the present invention has been completed.

  The “affinity” in the present invention represents a specific strong bond in which weak interactions other than a covalent bond such as an electrostatic force, a hydrogen bond, a Van der Waals force, and a hydrophobic bond are combined.

  The peptide of the present invention is configured as described above. Basically, H-A1-A2-A3-A4-A5-R (wherein A1, A2, A3, A4, A5 and R are the same as the former). The same meaning) and a peptide consisting of 5 amino acid residues (see SEQ ID NO: 1). These peptides may be such independent molecules, or the amino acid sequences of A1′-A2-A3-A4-A5 are arranged in this order from the N-terminal side in the polypeptide, respectively. It doesn't matter.

The peptide of the present invention can be produced by public methods such as solid phase synthesis. For example, when synthesizing a peptide comprising A1-A2-A3-A4-A5, when A5 is a glycine residue, the insoluble in which a functional group capable of binding the carboxyl group of N-protected glycine to the carboxyl group is coupled After binding to a resin-like carrier, each protected amino acid from A2 to A5 is sequentially bound by a solid phase synthesis method, and then the insoluble resin and the protecting group of the amino acid are removed, thereby removing the desired peptide. Obtainable. The terminal of the carboxyl group of the amino acid residue of A5 may be free (that is, R corresponds to —OH) or converted to an acid amide (that is, R corresponds to —NH ₂ ). Also good. In addition, the carboxyl group end of A5 is bonded to a commonly used polymer compound having a functional group such as a synthetic polymer or a biopolymer, together with a carboxyl group of a spacer bonded to the carboxyl group as necessary. Is also good (described later). The amino acids used in the solid phase synthesis method may be L-forms in common or D-forms in common. More preferably, it is L-form.

  In the above case, the carrier used in the solid-phase synthesis method can be bonded to the carboxyl group of the C-terminal N-protected glycine via the amino group or to the carboxyl group, and can be detached after the binding. For example, chloromethyl resin (chloromethylated styrene-divinylbenzene copolymer and the like), oxymethyl resin (oxymethylstyrene-divinylbenzene copolymer and the like) and the like can be mentioned. In addition, a resin such as 4- (oxymethyl) phenylacetamidomethyl resin bonded to an insoluble resin having an amino group via a functional group capable of binding to a carboxyl group and a spacer having the carboxyl group, benzyloxybenzyl Examples include alcohol resins, benzhydrylamine resins that are insoluble resins having amino groups, methylbenzhydroxylylamine resins, aminomethylphenoxymethyl resins, dimethoxybenzhydrylamine (DMBHA) resins, and derivatives thereof. Of these, benzhydrylamine resin, methylbenzhydroxylylamine resin, aminomethylphenoxymethyl resin, and DMBHA resin can be directly cleaved after bonding to directly obtain an acid amide. From the viewpoint of yield, it is preferable to use an aminomethyl resin.

  Examples of the functional group capable of binding to the carboxyl group and the spacer having the carboxyl group include those capable of converting the carboxyl group of glycine into p-carboxymethylbenzyl ester.

  The “protected amino acid” means an amino acid having a functional group protected with a protecting group by a known method, and various protected amino acids are commercially available. In order to synthesize the peptide of the present invention, it is preferable to use any of the protecting groups shown below.

For example, the protecting group for the α-amino group of an amino acid is Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenomethyloxycarbonyl); the protecting group for the ξ-amino group of lysine is Z (benzyloxycarbonyl). ), Cl.Z (2-chlorobenzyloxycarbonyl), Boc, Npys (3-nitro-2-pyridinesulfenyl); As a hydroxyl-protecting group of tyrosine, Bzl (benzyl), Cl ₂ .Bzl (2, 6-dichlorobenzyl) or t-Bu (t-butyl), but the hydroxyl group of the tyrosine may not be protected with the above protecting group; the protecting group for the guanidino group of arginine includes Tos (tosyl) , NO ₂ (nitro), Mtr (4-methoxy-2,3,6) or pmc ( , 2,5,7,8-pentamethylchroman-6-sulfonyl); As a protecting group for the carboxyl group of glutamic acid, Bzl ester, t-Bu ester, cHx (ester cyclohexyl ester); Protecting group for the amide group of glutamine May include Trt (trityl), but may not be protected with the protecting group; the protecting group for the indole group of tryptophan includes formyl group or Boc, but is protected with the protecting group. It is not necessary. These protecting groups can be appropriately selected and used according to the peptide synthesis conditions.

Conjugation of protected amino acids can be accomplished by conventional condensation methods such as DCC (dichlorohexacarbodiimide) method (RB Merrifield: Biochemistry, 3,1385, 1964), DICDI (diisopropylcarbodiimide) method (D. Sarantakis, et al: Biochem. Biophys Commun., 73 , 336, 1976), active ester method (F. Weygand, et al: Z. Naturforsch., B, 21 , 1141, 1966), mixed or symmetric anhydride method (D. Yamashiro, et. al: Proc. Natl. Acad. Sci. USA, 71 , 4945, 1945), carbonyldiimidazole method, DCC-HOBt (1-hydroxybenzotriazole) method (Keonig, W., et al .; Chem. Ber , 103 : 788, 1970), diphenylphosphoryl azide method, etc., among which DCC method, DCC-HOBt method, DICDI-HOBt method, and symmetric acid anhydride method are preferably used. These condensation reactions are usually performed in an organic solvent such as dichloromethane or dimethylformamide, or a mixture thereof.

In addition, as the elimination reagent for the protective group of the α-amino group, trifluoroacetic acid / dichloromethane, HCl / dioxane, piperidine / dimethylformamide and the like are used, and can be appropriately selected depending on the kind of the protective group to be used. The degree of progress of the condensation reaction at each stage of the synthesis can be confirmed by the ninhydrin reaction method (E. Kaiser, et al, Anal. Biocehm., 34 : 595, 1970).

Thus, after obtaining the protected peptide resin having the amino acid sequence represented by the above formula, the desired peptide can be obtained by removing the insoluble resin and the amino acid protecting group. Specifically, for example, when a chloromethyl resin derivative is used as the insoluble resin, anisole may be added and treated with hydrogen fluoride. Also, when benzyloxybenzyl alcohol resin, benzhydrylamine resin, or DMBHA resin (Funakoshi, S., J. Chem. Soc., Chem. Commun., 198 : 382, 1988) is used as the insoluble resin, By treating with hydrogen fluoride, TFMSA (trifluoromethanesulfonic acid), TMSOTF (trimethylsilyltrifluorate), TMSBr (trimethylsilylbromide) or the like, the resin and the protecting group can be eliminated simultaneously.

  The peptide thus obtained can be isolated and purified by known means such as various types of chromatography (gel filtration, ion exchange, distribution, adsorption, reverse phase), electrophoresis, ultrafiltration and the like.

  In the present invention, a peptide obtained by substituting the above peptide with a similar protein (active center or binding domain of antibody, receptor, enzyme, etc.) obtained by gene recombination can also be used as the peptide of the present invention. For example, when a human anti-gp120 antibody is produced by genetic recombination, it is related to epitope recognition in the V gene region of human immunoglobulin according to the method described in US Pat. No. 114632. The three hypervariable groups (VH31 to 35) of CDRs (complementarity determination region) -1, CDR-2 VH50 to 52 and / or CDR-2 VH58 to 60 Hypervariable cluster) amino acids (Ohno, S., Mori, N. & Matunaga, T .; Proc. Nat. Acad. Sci. USA, 82, 2945, 1985). Good.

  Thus, by replacing the peptide of the present invention with a gene recombination method according to the purpose, a gp120-binding protein can be prepared.

  Specific examples of the peptide of the present invention include those shown in Tables 1-2. In addition, * in a table | surface means that aggregation or neutralization was seen when the aggregation test and the neutralization test were implemented based on the method as described in the Example mentioned later.

Each amino acid symbol in the formula represents an internationally recognized amino acid residue represented by three letters, and details thereof are as follows.
Tyr: tyrosine Lys: lysine Trp: tryptophan Arg: arginine Glu: glutamic acid Gln: glutamine His: histidine Ala: alanine Phe: phenylalanine Gly: glycine Met: methionine Asp: aspartic acid Asn: asparagine Val: valine Ser: cysteine Thr: cysteine Thr : Threonine Ile: Isoleucine Leu: Leucine Pro: Proline

  A peptide having such an amino acid sequence has excellent affinity for gp120, and can be effectively used as an anti-HIV agent by taking the form of the following compound or composition.

  The compound of the present invention is a compound in which a high molecular compound having a functional group and / or a pharmaceutically active substance is bound to the above peptide, and pharmaceutically acceptable salts thereof are also included in the present invention.

  Here, examples of the “pharmaceutically acceptable salts” include the following commonly used non-toxic salts.

  (A) As a salt with a base such as an inorganic base, an alkali metal salt (for example, sodium salt, potassium salt, etc.), an alkaline earth metal salt (for example, calcium salt, magnesium salt, etc.), an ammonium salt; (b) an organic base salt Organic amine salts (for example, triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N, N′-dibenzylethylenediamine salt, etc.); As salts with acids such as inorganic acids, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc .; (d) As salts with acids such as organic acids, organic carboxylic acids (acetic acid, propionic acid, maleic acid, Succinic acid, malic acid, citric acid, tartaric acid, salicylic acid, etc.), organic sulfonic acid (methanesulfonic acid, p-toluenesulfonic acid, etc.), acidic sugar (glucuron) , Galactonic acid, gluconic acid, ascorbic acid).

  The “polymer compound having a functional group” used in the present invention is not particularly limited as long as it has a functional group capable of binding to the peptide of the present invention, and examples thereof include the following.

(1) Synthetic polymer compound As said polymer compound, arbitrary things, such as a linear polymer, branched polymer, and cyclic polymer, are used, for example, amino acid homopolymers, such as polylysine and polyglutamic acid, or cyclic polyamine, cyclodextrin In addition to cyclic peptides, insoluble solid phase carriers such as polystyrene, polypropylene, nylon, silica gel, polyethylene glycol, cellulose, and polyacrylamide can be used.

  Among these, the branched polymer has a functional group concentration per unit higher than that of a normal linear polymer because a part of the molecules in the polymer is branched. For example, a polymer based on two or more identical molecular chains derived from a core molecule having at least two or more functional groups such as the lysine core disclosed by Denkewalter (US Pat. No. 4,289,872), or The same molecule proposed by Tomalia et al. May be a starburst dendrimer with a strict regularity in polymer size due to continuous reaction, or the same / different It may be a molecule whose size is irregularly formed by the molecules reacting discontinuously. Further, the linear / branched polymer does not need to be a carrier molecule having a sufficient size, and includes those containing about three monomers that are not normally recognized as a core. It is not limited by the number of introductions. However, when a large number of peptides of the above formula are introduced, it is recommended to use a polymer having a large number of branches regardless of the polymer. In binding the peptide of the present invention to the above-mentioned polymer, the peptide may be synthesized and extended directly or indirectly directly from the branched functional group, or a newly synthesized peptide may be separately synthesized. It may be conjugated directly / indirectly to the functional group of the polymer.

  In addition, when binding a cyclic polymer such as cyclic polyamine, cyclodextrin, cyclic peptide, etc., the above formula peptide may be directly synthesized from the same functional group and extended, or a newly synthesized peptide may be separately synthesized. Further, it may be bonded directly / indirectly to the functional group of the cyclic polymer. In addition, when an insoluble carrier such as silica gel is bound, the same functional group may be introduced into the carrier in advance, and then the above formula peptide may be directly synthesized from the functional group and extended. The synthesized peptide may be conjugated directly / indirectly to the functional group of the insoluble carrier. The size and shape of the carrier having the same functional group is not particularly limited, and it may be appropriately selected and used depending on the purpose of use, such as a spherical shape, a hollow fiber shape, and a fibrous shape. The number of functional groups is not limited at all.

(2) Biopolymer Examples of the biopolymer include linear polysaccharides such as heparin, hyaluronic acid, chitosan, and chitin; proteoglycans, peptide hormones; proteins such as gelatin, albumin, antibodies, and antibody fragments. It is done.

  Of these, the size of the linear polymer may be appropriately selected according to the purpose of use, and includes those containing about three monomers that are not normally recognized as polymers, including the size and the number of functional groups. It is not limited at all. In binding the peptide of the above formula to the linear polymer, the peptide may be directly synthesized from the same functional group and extended, or a newly synthesized peptide may be synthesized with the linear polymer. It may be conjugated directly or indirectly to the functional group.

  When peptide hormones or proteins are bound, cysteine is bound to either end of the peptide of the above formula, and S—S bond is established with the cysteine residue in the peptide hormone / protein. The functional group of the peptide may be directly / indirectly conjugated with the functional group in the peptide hormone / protein. As described above, these binding methods can be appropriately selected according to the purpose of use, and the same applies to the type and the number of introduced peptides of the above formula.

  The pharmaceutically active substance used in the present invention is, for example, a nucleoside derivative AZT known as an anti-HIV inhibitor, 3,4-Dihydroxy-2,5-di [N -methyl- (2-pyridylmethyl) carbamoyl] valylamino] -1,6-diphenylhexane and the like. These pharmaceutically active substances can be directly / indirectly conjugated avoiding the active site of the peptide of the present invention, so that preparations specific to HIV can be obtained without side effects. Therefore, such a preparation is useful as a therapeutic agent capable of specifically curing HIV.

  Furthermore, compositions containing the above-described peptides of the present invention or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers and / or pharmaceutically active substances are also included within the scope of the present invention.

  As the above-mentioned “pharmaceutically acceptable carrier”, excipients (disintegrants, lubricants, bulking agents, etc.), coloring agents, flavoring agents, preservatives, stabilizers, and other conventional carriers are appropriately used. be able to. Specific examples include crystalline cellulose, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, ethylcellulose, magnesium stearate, talc, light anhydrous silicic acid, food coloring, aromatic essential oils and the like.

  The present invention is described in detail below based on examples. However, the following examples are not intended to limit the present invention, and all modifications made without departing from the spirit of the preceding and following descriptions are included in the technical scope of the present invention.

Synthesis Example 1: Compound obtained by binding polyethylene glycol to the peptide of the present invention After introducing a carboxyl group by reacting succinic anhydride with the hydroxyl group of polyethylene glycol (MW.20,000), MBS (m-maleimidobenzoyl-N -Methylated polyethylene glycol was synthesized by reacting with -hydroxysuccinimide).

  In addition, No. 1 in Table 1 above. The peptide-polyethylene glycol binding compound was obtained by peptide-binding a peptide having cysteine introduced at the C-terminal of 1 peptide. The obtained compound was suspended in a phosphate buffer and purified by affinity with gp120-binding carrier, gel chromatography, and the like.

Synthesis Example 2: Compound obtained by binding a branched polymer to the peptide of the present invention The N-terminal amino acid side of MAPs (Multiple antigenic peptide) is represented by No. 1 in Table 1 above. A compound that was elongated with 1 peptide and bound to a branched polymer was obtained. The obtained compound was suspended in a phosphate buffer, and purified by affinity chromatography using gp120 binding carrier, gel chromatography, and the like.

Synthesis Example 3: Adsorption removal carrier obtained by binding the peptide of the present invention to Sephadex 6MB No. 1 in Table 1 above One peptide was covalently bound to Sephadex 4B pre-activated through a spacer to produce peptide / Sepharose 6MB (1 μmol of gp120 / ml in bed volum). The unreacted peptide was removed by centrifuging for 10 minutes (12,000 rpm) at room temperature using a phosphate buffer and repeating the operation of removing the supernatant by suction.

Example 1 Measurement of Neutralization Activity In this example, various peptides shown in Table 3 were used to examine the neutralization activity against HIV-1.

Specifically, 50 μL of the above peptide in a 96-well microplate; as virus solution, 200 TCID ₅₀ HTLV-III B (laboratory strain), 200 TCID ₅₀ KK-1 strain (fresh isolate, Toru Ohtake et al. , Journal of Infectious Diseases, 64, 1284-1294, 1990); and 50 μL of the above-mentioned peptide, which was diluted 2-fold accurately in stages, was added and mixed. In addition, AZT was used as a positive control.

After reacting at 37 ° C. for 30 minutes, another 100 μL of 3 × 10 ⁴ MT-4 cell suspensions were added and cultured at 37 ° C. for 6 days in the presence of 98% humidity and 5% CO ₂ . After culturing, when cytopathic effect (CPE) due to the proliferation of HIV-1, that is, the drug is diluted in stages and the infected cells gather to form islands (focus formation), The previous stage of the dilution factor was determined as the neutralizing activity amount (infection inhibitory concentration). These results are also shown in Table 3.

  As is apparent from the results in Table 3, none of the peptides that satisfy the requirements of the present invention show neutralizing activity against the HIV-1 strain, whereas any of the peptides that satisfy the requirements of the present invention It was found that the neutralizing activity was excellent against the HIV-1 strain. In addition, when these peptides of the present invention are used, excellent neutralizing activity is recognized not only in the case of laboratory strains but also in the case of using fresh isolates. Therefore, the peptides of the present invention are practically used beyond the laboratory level. It is suggested that the level is extremely useful.

Example 2: Aggregation test In this example, the peptides shown in Tables 4 to 5 were used, and the affinity for gp120 was evaluated by an aggregation test.

  1% activated latex beads (Polyscience, particle size 0.2 mm) suspension and avidin (10 mg / mL) were mixed in equal amounts and reacted at 37 ° C. for 1 hour. After completion of the reaction, bovine serum albumin (BSA, 1 mg / mL) was added, and unreacted active sites were blocked at 37 ° C. for 30 minutes. Then, after removing unreacted substances by repeating the centrifugation, each biotinylated peptide (10 mg / mL phosphate buffer, pH 7.5) was added and reacted at 37 ° C. for 1 hour. An anti-gp120 agglutination test reagent was obtained.

  As a positive control, a gold colloid labeled with recombinant gp120 expressed by baculovirus (Immuno Diagnostics, Inc., particle size: 30 nm) was used, while as a negative control, the relevant recombinant gp120 was phosphate-buffered. What was melt | dissolved in the liquid was used.

  The agglutination plate was added with each 20 μg of the agglutination test reagent and the positive control, mixed and allowed to stand for 10 minutes, and then the presence or absence of aggregation was judged with the naked eye. These results are also shown in Tables 4-5. In this example, it was confirmed that no aggregation was observed when a negative control was used instead of the positive control.

  From the results of Tables 4 to 5, it was confirmed that all of the peptides of the present invention have excellent affinity for gp120.

Example 3: Influence on aggregation ability of A4 and A5 in the peptide of the present invention The effect on the aggregation ability was examined by changing the chain length of one peptide. The aggregation ability was measured in the same manner as in Example 2. These results are also shown in Table 6. In the table, ± means “aggregation to the extent of traces”.

  From Table 6, No. 3 which has only 3 amino acids. No. 3 (no amino acids A4 and A5) and No. 4 having only 4 amino acids. No. 2 (there is no A5 amino acid) is No. 5 having 5 amino acids. It can be seen that the neutralization activity is significantly reduced compared to 1. That is, the neutralization activity tends to decrease due to the decrease in the number of amino acids. In 3, the neutralizing activity disappeared completely.

Example 4: Effect on aggregation ability of A4 and A5 in the peptide of the present invention. No. 1 peptide was used as a positive control, and the peptide in which the amino acid type of A4 was replaced with proline, which is the same hydrophobic amino acid (No. 2), or was replaced with aspartic acid, which was an acidic amino acid (No. 1). 3); Similarly, using the peptide in which the amino acid type of A5 is replaced with proline (No. 4) or the peptide in which glutamic acid is replaced (No. 5), the effect on the aggregation ability is the same as in Example 2. And measured. These results are also shown in Table 7. In the table, ± means “about the trace”.

  From Table 7, No. It can be seen that when the amino acid types A4 and A5 are substituted with different numbers of amino acids in 1, the aggregation ability is negative or almost reduced to a trace. These results suggest that the types of amino acids A4 and A5 have an important influence on the aggregation ability.

Example 5: Affinity for gp120 The horseradish peroxidase (HRP) -labeled HIV-1-gp120 (Immuno Diagnostic) and the enzyme-unlabeled HIV-, which were prepared by adjusting the peptide-bound Sephadex 6MB prepared in Synthesis Example 3 to various concentrations in advance. The dissociation constant (kd) of the peptide of the present invention was determined by adding 1-gp120 and creating a Schacherd Plot, and kd = 2.14 × 10 ⁻¹⁰ M.

  From this result, it is clear that the peptide of the present invention has an affinity equal to or higher than that of an antibody.

Example 6: Recognition site of gp120 In order to confirm the specificity of the peptide of the present invention, No. “Peptide-labeled latex beads” were prepared by labeling 1 peptide with red latex beads (Polyscience, diameter 200 nm). When the same amount of pseudo HIV-1 (gp120-labeled gold colloid) solution was added to 20 μl of the latex bead solution, a red aggregated image that was immediately and easily observable with the naked eye was generated. On the other hand, since the unlabeled gp120 solution (1 μg protein / ml) not labeled with the gold colloid does not aggregate at all, it is speculated that the peptide of the present invention binds to only one site of gp120. It was. These results are shown in Table 8.

Example 7: Specificity test The presence of non-specific adsorption to other pathogenic viruses was examined using the latex agglutination test reagent "peptide labeled latex beads" prepared in Example 6 (No. 1 in Table 1). . Viruses used in this example are serum, laboratory strains, fresh isolates, etc. collected from patients with exacerbated C or B hepatitis. Further, the virus lysate was used by labeling in advance with a gold colloid. Table 9 shows the infectious titer or number of viruses used and the presence or absence of aggregation.

  Table 9 shows that the affinity for gp120 by the peptides of the present invention is specific for HIV-1.

Example 8: Removal of HIV in serum by HIV adsorption column Using the cellulosic carrier labeled with the peptide of the present invention prepared in Synthesis Example 3, the adsorption removal of HIV-1 was examined. Here, the amount of the peptide of the present invention labeled on this carrier was about 5 mg per mL of bed volume.

First, 100 μl (1 volume) of the adsorbent previously suspended in a PBS buffer solution (pH 7.2) was placed in a test tube, and then autoclaved at 121 ° C. for 30 minutes and naturally decompressed to obtain a test column. . On the other hand, the HIV-1 virus solution used was a kk-1 strain that was freshly isolated and cryopreserved by Otake et al. (Infectious Diseases Journal, 64: 1284-1294, 1990) from domestic AIDS patients. This kk-strain (1 × 10 ⁵ TCID ₅₀ ) was rapidly thawed and then subjected to ultracentrifugation and suspended in 100% human serum for culture that had been immobilized in advance as a test virus solution. Next, 24 volumes of the test virus solution was passed through the column with respect to 1 volume of the carrier in the column. After completion of the addition, in order to wash the unreacted virus remaining on the column, 5 volumes of normal human serum was poured. The amount of virus was measured using p24 Antigen ELISA Kit (Cellular products Inc.), the cut off value was determined by the calculation formula attached to the kit, and S / CO was calculated. The measured sample is a test sample (virus solution), a passing sample, a washing solution, and a solution solubilized and extracted from the carrier (column adsorbed fraction). It was set as the fraction containing. These results are shown in Table 10.

  From Table 10, it can be seen that a little less than 10% of the test virus amount was adsorbed on the column.

  In the present example, the reason why the above-described method for measuring the amount of p24 was used to evaluate the amount of virus will be described.

  In this example, a fresh isolate KK-1 strain suspended in 100% human serum that seems to be the closest to the patient's condition was used as the virus solution, but it is extremely difficult to accurately evaluate the amount. . The gp120 ELISA Kit can be used for laboratory strains but not for fresh isolates such as HIV-1. Rather, this is also the reason for using this virus stock. Therefore, it is conceivable that the amount of p24, which is a core protein having a consensus sequence on the primary structure, is measured by the EIA method or assumed by the RT-PCR method, but these methods also include degradation products. There is a disadvantage that it will be measured by. As mentioned above, HIV itself is extremely unstable and breaks down in minutes even during operation. Therefore, not only the virus degradation product is present in the virus solution used in the experiment, but also the degradation product is not present. Since it is considered that it is increasing more and more during the operation, if these methods are employed and the amount of decomposition products increases more than the amount of adsorption, the result cannot be understood. Therefore, in order to accurately determine the amount of virus, determination by an infection experiment is desirable. However, the fresh isolate KK-1 used in this example is not as easily infected as laboratory strains such as HIV-1III B. Considering the necessity of a virus solution with a very high concentration, it is extremely difficult to conduct an experiment. Therefore, since it is difficult to determine the amount of adsorption unless the virus solution is diluted to some extent, in this example, the amount of p24 was measured, and S / CO was calculated so that it could be compared with other data. is there.

Example 9: Binding of HIV by latex labeled peptide 2 virus / No. The state of HIV aggregation by the peptide-latex beads 1 was photographed with an electron microscope. Specifically, the agglutination test agent was added to the laboratory strain HIV-1 LAV1 for aggregation, and then allowed to stand at 4 ° C. for 6 hours. Next, the aggregated liquid was placed on an electron microscope support film, negatively stained with uranyl acetate, and then observed. For reference, this electron micrograph is shown in FIG.

  From FIG. 1, it can be seen that the HIV virus is firmly bound to latex beads labeled with the HIV-gp120 affinity peptide, and these beads are firmly bound to each other to aggregate.

10 is an electron micrograph showing the results of Example 9.

Claims (5)

A peptide having the amino acid sequence represented by SEQ ID NO: 1,
The peptide having affinity for gp120 consisting of the amino acid sequence represented by any of SEQ ID NOs: 2 to 40. A peptide having the amino acid sequence represented by SEQ ID NO: 41,
The peptide having affinity for gp120 consisting of the amino acid sequence represented by SEQ ID NO: 42 or 43.   A compound in which a peptide having a functional group and / or a pharmaceutically active substance is bound to the peptide according to claim 1 or 2.   The compound according to claim 3, which is used as a carrier for adsorption removal.   A virus aggregation diagnostic test using the peptide according to claim 1 or 2, or a kit containing the diagnostic test.