JP2995860B2 - New peptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to human ICAM-1 and human lymphocyte function-related antigen-1 by binding to human intercellular adhesion molecule I (hereinafter referred to as ICAM-1). (Hereinafter, referred to as LFA-1). The present peptide is a useful immunosuppressant that can be used as a preventive agent for rejection during organ transplantation and as a therapeutic drug for inflammatory diseases such as allergic diseases and autoimmune diseases.

[Conventional technology]

As the surgical techniques of organ transplantation have significantly improved, the success or failure of organ transplant surgery has been paraphrased on how postoperative graft rejection can be suppressed. In addition, rejection is caused when a living body recognizes a graft as a foreign substance and a series of immune reactions are elicited to eliminate the foreign body. Therefore, administration of so-called immunosuppressants such as steroids, azathioprine, methotrexate, and 6-mercaptopurine have been conventionally performed as anti-rejection drugs. However, the survival rate of the transplanted organs did not improve because of a narrow safety margin or weak effect. However, with the advent of cyclosporin A, which has been recently developed, the engraftment rate has been markedly improved. However, cyclosporin A has been shown to have severe nephrotoxicity, and its use has been restricted. Therefore, the development of safer and more effective immunosuppressants has been desired.

By the way, it is considered that one of the main factors of the immune reaction caused by the rejection of the graft is a reaction in which cytotoxic T lymphocytes recognize the graft as a foreign substance and attack it. These reactions are initiated when the host T lymphocytes specifically recognize the graft and adhere to each other. Therefore, if the adhesion between these cells can be inhibited, it is presumed that rejection can be suppressed. In order for cells to adhere to each other, mediation by binding of glycoproteins called adhesion molecules expressed on the surface of each cell is essential. Various molecules have been discovered as adhesion molecules by recent research, and one of them is known as ICAM-1 and LFA-1 (Cell, 51, 813, 1987). In the case of rejection, it is thought that it is necessary for LFA-1 molecules on cytotoxic T cells to adhere to ICAM-1 molecules on cells of the graft (European Journal of Immunology, 8).
Volume, 637 pages, 1988).

On the other hand, an autoimmune disease in which self destruction is caused by an immune response to its own antigen or an allergic disease that causes injury to itself by an excessive immune response to a foreign antigen, Many are refractory chronic inflammatory diseases. Steroidal anti-inflammatory drugs are currently the most effective treatments, but severe side effects often occur due to their strong nonspecific immunosuppressive effects, which limits their use. Not get. In addition, non-steroidal anti-inflammatory drugs are mostly occupied by inhibitors or antagonists of the production of chemical mediators that mediate the inflammatory response, and many of them have no effect on chronic inflammation. There is a demand for the development of an excellent therapeutic agent. It is unknown to date how these diseases become chronic, but neutrophil / macrophage focusing on inflammatory sites and subsequent lymphocyte focusing may contribute to chronicity. High in nature. In order for these cells circulating in the blood to focus locally, they first adhere to the endothelium of the blood vessel near the site of inflammation, slip through the junction of the endothelial cells of the blood vessel, and furthermore, the basement membrane Is thought to break down and migrate to the site of inflammation.

Therefore, by inhibiting the adhesion of inflammatory cells to vascular endothelial cells, the first step in this series of reactions, suppression of inflammation,
Further, it is considered that chronic inflammation can be suppressed. In the case of adhesion of inflammatory cells to vascular endothelial cells, the binding between LFA-1 molecule expressed on the surface of inflammatory cells and ICAM-1 molecule expressed on the surface of vascular endothelial cells is also observed. It is being considered essential (Clinical Research, 36: 648A, 1988).

That is, it is assumed that if the binding between ICAM-1 and LFA-1 can be inhibited, the adhesion between cells will be inhibited, so that rejection can be prevented or inflammation can be suppressed. However, to date, no substance that selectively inhibits this binding has been known other than an anti-ICAM-1 antibody or an anti-LFA-1 antibody. Also, the only known antibody is a heterologous protein obtained by immunizing xenogeneic animals such as rabbits and mice.When administered to humans as it is, an immune response to the antibody occurs, causing anaphylactic shock. Unfortunately, it is assumed that serious side effects such as illness and serum sickness occur, and that the effect of the antibody is attenuated.

Further, if a binding site of ICAM-1 or LFA-1 is identified and a peptide is designed based on the information, it may be a binding inhibitor, but up to now, ICAM-1 molecule, LFA- Neither of the molecules has a fixed binding site to each other, and that approach is not possible.

[Problems to be solved by the invention]

Accordingly, an object of the present invention is to provide a substance that binds to human ICAM-1 and selectively inhibits the binding between human ICAM-1 and LFA-1. This substance can be expected as an effective immunosuppressant for preventing rejection during organ transplantation and for treating inflammatory diseases such as allergic diseases and autoimmune diseases.

[Means for solving the problem]

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, the following new concepts and methods aim at
The present inventors have found a peptide that selectively inhibits the binding between ICAM-1 and LFA-1, and have completed the present invention.

That is, the present invention is a peptide that inhibits the binding between ICAM-1 and LFA-1. It should be noted that, depending on the number of amino acid residues constituting the peptide chain, a peptide and a polypeptide may be sometimes distinguished from each other.In the present invention, the expression `` peptide '' is used regardless of the number of amino acid residues. I do. Accordingly, strictly speaking, polypeptides classified as polypeptides are all expressed as peptides in the present invention.
Hereinafter, the present invention will be described in detail.

Now, among the antibodies recognizing the ICAM-1 molecule, particularly LFA
It is considered that those having the activity of inhibiting the binding between ICAM-1 and ICAM-1 include those that recognize the binding site itself to the LFA-1 molecule on the ICAM-1 molecule. Can be Furthermore, among the antibodies that recognize the binding site to the LFA-1 molecule on the ICAM-1 molecule, the LFA-1 molecule includes ICAM-1
Like a molecule, it may include a molecule that binds to an ICAM-1 molecule with the same tertiary structure as the ICAM-1 molecule binding site on the LFA-1 molecule. Therefore, LFA
-ICAM- having binding inhibitory activity between ICAM-1 and ICAM-1
A large number of hybridomas producing one antibody are collected, and the amino acid sequence of the variable region (hereinafter abbreviated as V region) of the antibody produced by the hybridoma is determined.
An amino acid sequence identical or similar to the amino acid sequence of the binding site of the M-1 molecule may be obtained. And LFA
When a similar sequence is obtained between the amino acid sequence of -1 molecule and the amino acid sequence of the V region of the antibody, a peptide corresponding to this portion is synthesized, and the peptide is converted to LFA-1 and IFA.
It can be assumed that the binding of CAM-1 can be inhibited. Based on such a concept, the present researchers first produced a hybridoma clone that produces a mouse monoclonal antibody that is specific for ICAM-1 and has an activity of inhibiting the binding of ICAM-1 to LFA-1. A hybridoma clone producing a mouse anti-human ICAM-1 monoclonal antibody is obtained as follows. First, a human colon cancer cell line BM stimulated with γ-interferon and highly expressing an ICAM-1 molecule
Mice are immunized with 314. In this case, human-derived cells expressing the ICAM-1 molecule on the cell surface, such as cells originally expressing the ICAM-1 molecule, cells which induced the expression of the ICAM-1 molecule by various stimuli, etc. Any cell can be used as the immunogen. Next, a hybridoma of myeloma cells and antibody-producing cells present in spleen cells or lymph node cells of the immunized mouse is prepared. The myeloma cells may be derived from any animal species as long as they can be fused, but better results are usually obtained using cells of the same species. The hybridoma obtained in the present invention was obtained by spleen cells of BALB / c mice immunized with γ-interferon-stimulated human colorectal cancer cell line BM314 suspended in physiological saline and BALB /
c This is a hybridoma obtained by fusing X63-Ag8-6.5.3 which is a mouse myeloid cell in the presence of polyethylene glycol. As myeloid cells, in addition to X63-Ag8-6.5.3, P3-X63-Ag8-U1, P3-X63-Ag8, P3-NSI / 1-A
g4-1, MPC11-4.5.6.TG.1.7.SP2 / V-Ag14 (mouse cells), 210.RCY.Ag1.2.3 (rat cells), SK0-007, GH
An 8-azaguanine-resistant cell line such as 15006TG-A12 (or more human cells) may be used. Selection of hybridomas in which antibody-producing cells and myeloid cells in the mouse spleen were fused,
After the cell fusion, the cells can be cultured in a medium containing hypoxanthine, thymidine, and aminopterin (HAT medium). Since not all of the obtained hybridomas produce the desired antibody, a hybridoma producing an antibody against ICAM-1 was selected from the obtained hybridoma clones, and ICAM-1 was further selected from the hybridomas. When
A hybridoma producing an antibody having the activity of inhibiting the binding to LFA-1 must be selected. The selection can be made, for example, using the following method.

That is, human umbilical vein endothelial cells are cultured overnight at 37 ° C. in the presence of γ-interferon and adhered to the surface of a plastic plate. In this case, any cell may be used as the cell to be stuck, as long as it is a human-derived cell expressing an ICAM-1 molecule on the surface. At this time, a hybridoma culture supernatant, which is a sample, is added to each well in advance. HL-60 cells labeled with 2'7'-bis (carboxyethyl) carboxyfluorescein tetraacetoxymethyl ester were added to each well, and the number of HL-60 cells bound to umbilical vein endothelial cells was determined as fluorescence intensity. An antibody against ICAM-1 having the activity of inhibiting the adhesion of HL-60 cells to umbilical vein endothelial cells by the culture supernatant of a sample and inhibiting the adhesion of the desired ICAM-1 molecule to LFA-1 molecule Is obtained. As the cell to be adhered to the umbilical vein endothelial cell for screening, any cell other than HL-60 can be used as long as it is a human-derived cell expressing the LFA-1 molecule on the surface. Labeling of cells may be performed using an isotope, an enzyme, or the like in addition to the fluorescent dye.

Total RNA from the hybridoma clone thus obtained
To obtain a gene (cDNA) encoding the V region of the monoclonal antibody. For the present invention, in order to find a V region having an expected structure, it is necessary to obtain a V region cDNA from many hybridomas and determine the structure.

Thus, the present inventors devised a quicker method and obtained a gene by the following method. That is, first, the heavy chain (H chain) and light chain (L
20) to 30 base sequences (primer DNA) with high commonality at the 5 'end of each V region-encoding gene
Devise. Using a DNA synthesizer, a DNA oligomer having this base sequence (5′-side primer DNA) and a DNA oligomer having a sequence complementary to the constant region mRNA of the antibody (3′-side primer DNA) are synthesized. From the obtained hybridomas, total RNA was extracted according to a conventional method, and a single-stranded cDNA was prepared using reverse transcriptase and an appropriate 3 ′ primer DNA, and 5 ′ primer DNA and 3 ′ primer Using DNA, polymerase chain reaction (PCR) with Taq polymerase (Science, 230
Vol., P. 1350, 1985), selectively amplify and obtain only DNA fragments encoding the V regions of the H and L chains of the antibody. Thereafter, both ends of each of the obtained DNA fragments are blunted by T4 DNA polymerase, and the 5 ′ end is phosphorylated by T4 polynucleotide kinase. This DAN fragment is cut at the SmaI site on the cloning side in advance, ligated with a plasmid vector that has been dephosphorylated with alkaline phosphatase using T4 DNA ligase, and cloned. The sequence of the cloned cDNA was determined by the dideoxy method (Science, 214, 1205, 1981). An amino acid sequence is deduced from the determined base sequences of the V regions of the H and L chains. As the cloning method, any method generally used other than the method described here may be used.

Next, the amino acid sequence deduced from the base sequence of the V region of the H chain and L chain of the obtained anti-human ICAM-1 antibody, and LFA-
A search for homology with that of one molecule was performed. Anti-human ICAM
-1 Monoclonal antibody producing hybridoma, determination of the amino acid sequence of its V region, and homology search with human LFA-1 molecule, a series of steps were repeated for a large number of hybridomas. Six types of peptides having an activity of inhibiting the binding between ICAM-1 and LFA-1 have been found. The specific amino acid sequence of the peptide is as follows.

That is, it has a structure represented by the following amino acid sequences [I] to [VI].

Amino acid sequence [I]: Amino acid sequence [II]: Amino acid sequence [III]: Amino acid sequence [IV]: Amino acid sequence [V]: Amino acid sequence [VI]: Needless to say, the peptide of the present invention that inhibits the binding between ICAM-1 and LFA-1 is not limited to the peptide having the structure shown in the above [I] to [VI], and for example, is shown in the above [I] to [VI]. One having a structure in which one or more amino acids in the amino acid sequence are substituted with another amino acid, and one at the N-terminal and / or C-terminal of the amino acid sequence represented by the above [I] to [VI]. A peptide having a structure in which one or more amino acids are added is also included in the peptide of the present invention as long as it has an activity of inhibiting the binding between ICAM-1 and LFA-1.

Furthermore, the peptides represented by the above-mentioned [I] to [VI] in which the structure is amidated, acetylated or poly (ethylene glycol) -added also have the activity of inhibiting the binding between ICAM-1 and LFA-1. Included in the peptides of the invention.

Now, among the sequences represented by the above [I] to [VI], the formula [I]: Tyr-Ser-Thr-Ser-Asn-Leu-Ala-Ser
The sequence represented by -Gly-Val-Pro-Ala-Arg is particularly ICAM
Strongly inhibits the binding between LFA-1 and LFA-1. The sequences represented by the above [II] to [VI] including this sequence [I] are present in the V region of the antibody derived from the specific hybridoma HA-58 (FERM P-11857), and are very similar to the LFA-1 molecule. This is where the homology is high.

This sequence corresponds to a part of the V region of an antibody, which is called a so-called complementarity determining region (CDR), and is known to be directly involved in binding to an antigen. By the way, the above [I] to [V
The peptide having the sequence represented by I] may be chemically synthesized or may be prepared using a genetic engineering technique. In the case of chemical synthesis, a raw material having a reactive carboxyl group corresponding to one of two types of fragments bisected at an arbitrary position of a peptide bond by a commonly used solid phase method, and a material corresponding to the other fragment When a condensate having a protective amino group is removed by condensing a raw material having a reactive amino group using a dicyclohexylcarbodiimide method and removing the protective group, the compound can be produced (International Journal of Peptide Protein Reserc
h, vol. 30, p. 705, 1987). When using the genetic engineering technique, for example, appropriate restriction enzyme sites are provided at both ends of 5 ′ and 3 ′, and a start codon (AT
G) and subsequently, a DNA chemically synthesized so as to contain a nucleotide sequence corresponding to the present polypeptide, or a cDNA prepared by PCR or the like, is converted into an appropriate expression vector plasmid containing a promoter region, an SD region and a terminator region. Then, Escherichia coli may be transformed, and the desired peptide may be produced in Escherichia coli. The host to be expressed is not limited to Escherichia coli, and any host such as yeast, insect, or animal cell may be used as much as possible. The peptide of the present invention is purified according to a usual method. Specifically, ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography, or the like may be used.

Now, the peptide of the present invention binds to ICAM-1 and binds to ICAM-1.
It has an activity of selectively inhibiting the binding between LFA-1 and LFA-1, and is effective for preventing rejection during organ transplantation. Further, the immunosuppressant according to the present invention contains the peptide in an amount of 0.1% to 100% by weight, preferably 0.5% by weight.
What is necessary is just to contain in the ratio of 70 weight%. Therefore, the peptide of the present invention may be administered as it is, or may be administered in the form of a preparation prepared by mixing with a carrier for preparations. As a pharmaceutical carrier, it is commonly used in the pharmaceutical field,
In addition, a substance that does not react with the polypeptide of the present invention is obtained. In the case of an injection, the polypeptide of the present invention is prepared by dissolving the same in water, but may be dissolved in a physiological saline solution or a glucose solution, if necessary, or may be used as a buffer, a preservative, or a stabilizer. Alternatively, an excipient may be included. These formulations may also contain other components of therapeutic value.

The method of administering the immunosuppressant according to the present invention,
Although any method such as injection and rectum may be used, administration by injection is preferred. The dosage depends on the method of administration,
It depends on the patient's condition, age, etc.
10001000 mg, preferably 0.01-10 mg may be administered 1-3 times per day.

The safety of the peptide according to the present invention has been confirmed.

 Hereinafter, the present invention will be described in more detail based on examples.

Example 1, Preparation of a Peptide Having an Activity to Inhibit the Binding between ICAM-1 and LFA-1 Molecules Human colorectal cancer previously cultured at 37 ° C. for 3 days in the presence of interferon-γ at a concentration of 250 U / ml Cell line BM314
Was suspended in physiological saline and immunized by intraperitoneal administration of 1 × 10 ⁷ female BALB / c mice aged 6 to 8 weeks per mouse. Ten days later, a booster immunization was performed in the same manner,
Five days later, blood was collected from the orbital vein of the mouse, and BM31
The antibody titer to the four cells was measured by a fluorescent staining method using FACScan (manufactured by Becton Dickinson). That is, first, 1 × 10 ⁶ BM314 cells per cell are placed in a tube, centrifuged, and the supernatant is discarded to pellet the cells.
Next, 0.5% bovine serum albumin (BSA), 0.1% NaN ₃ and 0.1%
10 mM phosphate buffer (pH 7.5) containing 5 M NaCl (hereinafter referred to as BSA
-PBS) to 10μ
The reaction was carried out at 4 ° C. for 30 minutes. BSA
-After centrifugal washing three times with PBS, add BSA-PB
Fluorescein isothiocyanate (FITC) -labeled goat anti-mouse immunoglobulin antibody (Tago
Was added and the mixture was reacted at 4 ° C. for 30 minutes.
Further, after washing three times by centrifugation with BSA-PBS, the amount of antibody bound to the cells was measured as fluorescence intensity by FACScan. The antibody titer was measured in this manner, and the mouse having a high antibody titer was finally immunized by the same operation. Three days later, the spleen was removed and spleen cells and mouse myeloma cells (X63-Ag8-6.5.
And 3) were mixed at a ratio of 10: 1 by the number of cells in the presence of 50% polyethylene glycol # 4000 (manufactured by Nacalai Tesque) to cause cell fusion. The fused cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) at 5 ×
The cells were suspended at a density of 10 ⁶ cells / ml and dispensed at 100 μl into a 96-well flat bottom plate (manufactured by Corning) containing 5 × 10 ⁵ mouse thymocytes per well. 1 day, 2 days, 3 days,
Six days later, half of the medium was replaced with a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium).
The same operation was repeated every day. About two weeks after the fusion, the culture supernatant in the well where the fused cells (hybridomas) had proliferated was analyzed for ICAM-1 and LFA- by the method described below.
The activity of inhibiting the binding to No. 1 was measured, and the hybridoma contained in the wells having the inhibitory activity was cloned by the limiting dilution method. Further, the inhibitory activity of each hybridoma clone in the culture supernatant was measured to obtain an anti-ICAM-1 antibody-producing hybridoma.

5 × 10 ⁶ hybridoma HA-58 (FERM P-11857)
After washing with PBS, an RNA extraction buffer (manufactured by Pharmacia) containing guanidine thiocyanate, N-raullisarcosine and EDTA was added and suspended, and an equal volume of a cesium chloride solution (ρ = 1.51 g / ml, Pharmacia) and centrifuged at 125,000 × g for 16 hours. After sucking the supernatant, 10m containing 1mMEDTA
M Tris hydrochloride buffer (pH 7.5) (TE) was added and suspended,
Placed in a new tube and incubated at 65 ° C for 5 minutes. Further, 1/10 volume of 2M potassium acetate (pH 5.0) (manufactured by Pharmacia) and 3 times volume of ethanol (manufactured by Nacalai Tesque) were added, and the mixture was allowed to stand at -20 ° C overnight. 2 at 5,000xg
After centrifugation for 0 minutes and discarding the supernatant, the precipitate was washed by centrifugation with 80% ethanol and the precipitate was dried. Dissolve the precipitate with TE
The RNA fraction was used.

Next, a deoxy-NTP mixed solution, a cDNA synthesis buffer (manufactured by Amersham), an RNase inhibitor (manufactured by Takara Shuzo), and reverse transcriptase (manufactured by Takara Shuzo) are added to the RNA solution, and the mixture is reacted at 42 ° C. for 1 hour. Then, cDNA was synthesized.
Furthermore, a PCR buffer (Cetus), a deoxy NTP mixture, a primer for amplifying mouse immunoglobulin H chain V region cDNA (final concentration of 1 μM each on 5 ′ and 3 ′ side) or a mouse immunoglobulin L chain V region cDNA Amplification primer (same concentration) and Taq polymerase (Takara Shuzo)
PCR with a DNA thermal cycler (Cetus)
Was done. The reaction was denatured for 30 seconds (94 ° C) and annealing for 30 seconds (55
℃), 30 minutes with primer extension 1 minute (72 ℃)
Cycles were performed and the primer extension time was extended by 15 seconds for each cycle. After the reaction, agarose gel electrophoresis was carried out in a 40 mM Tris acetate buffer (pH 8.0) containing 1 mM EDTA, and the corresponding cDNA fragment was cut out and extracted and purified using a GeneClean kit (Bio101). After the purified fragment is blunted at both ends using T4 DNA polymerase (Takara Shuzo),
5 'using polynucleotide kinase (Takara Shuzo)
The ends were phosphorylated, cut in advance with Sma I (Takara Shuzo), and then dephosphorylated with alkaline phosphatase (Takara Shuzo). pUC18 vector (Takara Shuzo) and T4 DNA
Ligation was performed using a ligase (Takara Shuzo). After transformation into Escherichia coli strain JM109 (Takara Shuzo), the clone was inoculated into χ broth, cultured at 37 ° C overnight, and alkaline SDS
The plasmid was extracted and purified by the method.

The nucleotide sequence of the purified plasmid was determined using a sequencing primer M4 or RV (Takara Shuzo) and a 7-DEAZA sequence kit (Takara Shuzo) as follows.

<L chain> <H chain> The amino acid sequence was deduced from the obtained base sequence, and the following sequences [V] and [VI] were obtained.

<L chain: amino acid sequence [V]> <H chain: amino acid sequence [VI]> Preparation of the peptide having the present amino acid sequence requires first
Nco I linkers (Pharmacia) were added to both ends of each cDNA fragment amplified and purified by R.
Nc ligated using ligase (Takara Shuzo)
o Cut with I and pKK233 with Nco I
-2 vector (Pharmacia) was further ligated using T4 DNA ligase. Next, E. coli JM105
After transformation into a strain (Pharmacia), clones were inoculated into L-broth and cultured overnight, and the bacteria were sonicated and purified by reverse-phase liquid chromatography using a C8-bonded silica column (Shiseido). did. In addition, as a result of examining the amino acid sequence near the N-terminus, it was confirmed that peptides having the amino acid sequences V and VI were obtained.

As a result of homology search between the amino acid sequences V and VI and the amino acid sequence of LFA-1, I got Preparation of these peptides was performed as follows. To prepare peptide [I], first, Fmoc-Arg (Mtr) resin (manufactured by Kokusan Chemical Co., Ltd.) was placed in a solid-phase reactor (Kokusan Chemical Co., Ltd.) of Merrifield, and suspended in dimethylformamide (DMF) (manufactured by Kokusan Chemical Co., Ltd.). The solution was turbid and shaken to swell the resin. The resin was shaken in DMF, further shaken in a 50% piperidine-DMF solution, and washed with DMF and isopropanol to remove the Fmoc group. Resuspend in DMF and shake again to swell the resin, Fmo
A DMF solution of c-Ala-OH (manufactured by Kokusan Chemical) was added and shaken. Further, a 1 M dicyclohexylcarbodiimide methylene chloride solution was added, and the mixture was shaken, washed with DMF and isopropanol, and condensed. By repeating such Fmoc group removal and Fmoc-amino acid condensation, Tyr-Ser-Thr-Ser-Asn-Leu
-Ala-Ser-Gly-Val-Pro-Ala-Arg resin was obtained. The resin was detached from the resin by washing with methylene chloride, suspended in a mixed solution of methylene chloride, anisole and thiophenol (15: 3: 1), added with trifluoroacetic acid / methylene chloride (8.6: 1), and shaken. Filtered to a resin, the obtained filtrate was concentrated under reduced pressure, ether was added to the residue, and the obtained powder was further filtered,
Purification was performed by reversed-phase liquid chromatography using a C8-bonded silica column (manufactured by Shiseido Co., Ltd.) to obtain a peptide having the amino acid sequence [I].

In the same manner, the following peptides II, III and IV were synthesized.

(Example 2, Inhibitory effect of peptide I on cell adhesion through ICAM-1 and LFA-1) Human umbilical vein endothelial cells expressing ICAM-1 molecule were cultured at a final concentration of 10 U / ml IL-1 (Genzyme). Peptide I and Peptide prepared at a concentration of 5 × 10 ⁴ cells / ml in a culture medium containing the same (100 μl / well) in a 96-well flat-bottom plate at a concentration of 100 μ (5 × 10 ³ cells) per well and 10 μM. V or control peptide was dispensed in 100 μl aliquots and cultured at 37 ° C. overnight. Separately, 2′7′-bis (carboxy) was added to the HL-60 cell suspension prepared at a concentration of 2 × 10 ⁷ cells / ml in RPMI1640 medium containing 10% FCS so that the final concentration was 10 μM. Ethyl) carboxyfluorocene tetraacetoxymethoxyester (manufactured by Dojin Chemical Co., Ltd.) was added, reacted at 37 ° C. for 1 hour, washed three times with the medium, and then 2 × 10 5
Cells were labeled at a concentration of ⁶ cells / ml. Cultured overnight
After washing the HL-60 cells twice with the medium, the labeled HL-60 cells were washed.
Dispense 100μ (2 × 10 ⁵ ) cells per well,
Incubated at 37 ° C. for 30 minutes. After washing four times with the medium, the cell pellet is solubilized with 1% NP-40, and the amount of labeled cell adhesion is measured using a fluorescence reader (manufactured by Intermed) at an excitation wavelength of 490 nm and a fluorescence wavelength of 530 nm. did. As a result, as shown in Table I, only when the peptide of the present invention was added, the cell adhesion via the ICAM-1 and LFA-1 molecules was significantly inhibited.

<Effect of the Invention> The peptide of the present invention can prevent rejection at the time of organ transplantation,
It can be used as an immunosuppressant with a selective action different from conventional immunosuppressants and few side effects, which is effective as a therapeutic drug for inflammatory diseases such as allergic diseases and autoimmune diseases.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Junji Hamuro 1-1, Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Co., Inc. (72) Inventor Toshiro Shimamura Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa 1-1 Ajinomoto Co., Inc. Examiner at Central Research Laboratory Eiji Takahori (58) Field surveyed (Int. Cl. ⁶ , DB name) C07K 7/08 C07K 16/28 WPI (DIALOG) BIOSIS (DIALOG) CA ( STN) REGISTRY (STN)

Claims (3)

(57) [Claims] 1. A peptide having an activity of inhibiting the binding between human intercellular adhesion molecule-1 and human lymphocyte function-related antigen-1 and represented by the following amino acid sequence [I]. Amino acid sequence [I] 2. A peptide having an activity of inhibiting the binding between human intercellular adhesion molecule-1 and human lymphocyte function-related antigen-1 and represented by the following amino acid sequence [V]. Amino acid sequence [V] 3. The amino acid sequence represented by the amino acid sequence [I] or [V], wherein one or more amino acids in the amino acid sequence are acetylated, amidated or added with polyethylene glycol. Item (1)
Or the peptide according to (2).