JPH08109200A - Anti-c5a receptor antibody, its production and use - Google Patents

Abstract

PURPOSE: To obtain the subject new antibody inhibiting the combination of a C5a receptor to a ligand, not neutralizing a biological activity produced by the combination of the ligand, useful for measuring the receptor of a complement ingredient having a biophylaxis function, etc., and used for researches, etc. CONSTITUTION: The new anti-C5a receptor monoclonal antibody inhibiting the combination of the C5a receptor to the ligand but does not neutralizing a biological activity produced by the combination of the ligand. The anti-C5a receptor antibody is useful for the method for immunologically measuring the receptor of the complement ingredient C5a having a biophylaxis function, and extremely useful on the research of the action of the C5a on various diseases and on the research of the progress on the expression of the C5a receptor. The antibody is obtained by binding a polypeptide having the No. 1-20 amino acid sequence at the N-terminal of the C5a receptor to a carrier protein, administering the obtained hapten in a mouse, collecting the cells of the spleen after finally immunized, fusing the collected splenic cells to myeloma cells, selecting a specific antibody-producing strain, cloning the strain, and subsequently culturing the obtained hybridoma.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunological assay for human C5a receptor expressed on cells, a monoclonal antibody used for the assay, and a hybridoma producing the monoclonal antibody.

[0002]

2. Description of the Related Art In the living body, there are a specific defense function involving antibodies and sensitized lymphocytes and a non-specific defense function not involving immunity as protective functions for preventing the invasion of pathogenic microorganisms and other foreign substances. Complement is an important non-specific defense function. The complement system consists of nine proteins (specifically, about 20 kinds), each of which is produced by hepatocytes and macrophages, and is normally present in the body fluid in an inactive state. However, the antigen-antibody complex, It is activated by the bacterial cell wall and others and functions as a biological defense factor. When the activation of the complement system occurs, the complement system protein in the body fluid is enzymatically degraded to produce fragments having various biological activities. Of these complement protein fragments, three of C3a, C4a, and C5a are collectively called anaphylatoxins, and are chemical media that cause various inflammatory reactions including anaphylactic shock. It has also been clarified that they also have an action as an immunomodulator. Of these three anaphylatoxins, C5a, which is the substance that causes the strongest inflammatory action, is the smaller fragment of the two fragments produced by cleavage of the fifth component of complement C5 by C5 convertase, that is, the N fragment of the C5α chain. It is a glycoprotein with a molecular weight of 11,000 composed of 74 terminal amino acid residues. C
5a has a leukocyte migration action, a vascular permeability enhancing action, a smooth muscle contraction action, and also has an action of regulating cytokines by acting on monocytes to produce cytokines. Therefore, it is a causative agent of many fatal diseases such as sepsis, adult respiratory distress syndrome, asthma, atherosclerosis, myocardial infarction, ischemic diseases such as cerebral infarction, psoriasis, nephritis, and trauma and burns. Has become.

In order for C5a to exhibit these biological activities, it is necessary to bind to its specific receptor, the C5a receptor (C5aR), and transmit the information stimulus into the cell. As a result of gene cloning from cDNAs of myeloid cell lines HL60 and U937, C5aR is a glycoprotein composed of 350 amino acid residues, has a 7-transmembrane structure, and is an intracellular G protein. It has been revealed that it is a member of the rhodopsin receptor that binds to and transduces intracellular signals (Gerard, NP
And Gerard, C., Nature Volume 349: 61.
Page 4, (1991); Boulay, F. et al. Biochemistry (Bio
Chemistry 30: 2993, 1991). An antibody that specifically recognizes C5aR is a monoclonal antibody (Oppermann,
M. et al., The Journal of Immunology
l of Immunology) 151: 3794, 1993) and polyclonal antibodies (Morgan, EL et al., p. 377) were both first produced in 1993. Oppe
Monoclonal antibody S5 / produced by rmann et al.
No. 1 was prepared using a protein obtained by binding a peptide having an amino acid sequence from the N-terminal 1st to 31st C5aR to bovine albumin as an immunogen. This antibody is C
5aR competitively inhibits the binding of ligand C5a and neutralizes the biological activity of neutrophils caused by ligand binding.
It recognizes the site of the th amino acid sequence.

These antibodies inhibited the binding of ligands, and made it possible to perform FACS analysis of the expression of the receptor on the cell surface using a fluorescent antibody. As a result, it was revealed that C5aR was expressed on neutrophils, eosinophils, and monocytes. However, there are some disadvantages due to competitive inhibition with the ligand. That is, whether or not C5aR is present on platelets is controversial in humans, although its expression has been proved in guinea pigs. Since the neutralizing antibody S5 / 1 binds to the same site as the ligand binding site, it cannot be used in an experiment dealing with platelets that are easily activated by a small amount of stimulation. Therefore, for C5aR expression on human platelets,
No conclusion has been reached yet. Furthermore, C5 on cells
The aR expression level is expected to change depending on diseases and the like. At present, no one has succeeded in highly sensitive quantification of C5aR with the existing neutralizing antibody. Furthermore C
It is said that the interaction between 5aR and a ligand involves not only one site but also several sites. Recently, it has been shown that, in addition to the N-terminal, a part called a core surrounded by a transmembrane region is also involved in binding (Siciliano, SJ et al.,
Proceedings of National Academy of
Science (PNAS) Volume 91: 1214, 199
4 years. It has been revealed that a monoclonal antibody recognizing a peptide having an N-terminal 15 to 21 sequence inhibits the binding between C5aR and its ligand C5a and blocks the biological activity of cells caused by the ligand binding. However, a peptide different from this is used as an immunogen to obtain a monoclonal antibody, and whether it inhibits the binding between C5a and the receptor or whether it inhibits the biological activity of cells evoked by C5a binding is determined. It is necessary to analyze the detailed structure of the site and clarify the signal transduction method by the receptor.

[0005]

When inflammation occurs, polymorphonuclear leukocytes such as neutrophils increase the expression of C5aR on the cell surface, accelerate migration to the inflammatory site, and enhance extravasation to the blood vessel. It can be imagined that the body is protected by releasing granules. In addition to these polymorphonuclear leukocytes, platelets release a large amount of granules containing molecules having many biological activities upon stimulation, and are the fastest biological reaction from stimulation. As a result, the platelets themselves aggregate and die. However, in the present situation where quantification of C5aR is not possible, it is not beyond the imagination that cells stimulated by C5a undergo such a phenomenon. If C5aR can be accurately quantified, it is sure to give very useful information for understanding the course of disease and elucidating the etiology and pathological condition.
On the other hand, an antibody that recognizes a ligand-binding site often gives the same effect as a ligand by binding to a receptor, that is, a receptor-mediated signal. As a result, the cells are activated, leading to cell aggregation, internalization of the receptor, and cell death. Therefore, quantification of the receptor using an antibody that does not cause activation of cells by binding of the antibody is desired. Further, as described above, the use of a monoclonal antibody other than the existing neutralizing antibody for identifying the ligand binding site on C5aR and clarifying the receptor structural change following complicated ligand binding and further the signal transduction method. Is desired.

[0006]

Means for Solving the Problems As a result of studies in view of the above circumstances, the present inventors have determined that a polypeptide consisting of amino acids 20 to 20 of the N-terminal of C5aR is designated as a carrier protein keyhole limpet hemocyanin (KLH). A monoclonal antibody was prepared using the conjugated KLH-C5aR _1-20 as an immunogen, and it was made possible to stably quantify C5aR expressed on cells. Based on this finding, further intensive research was conducted to complete the present invention. That is, the present invention provides (1) an anti-C5a receptor monoclonal antibody that inhibits the binding of C5a receptor to a ligand, but does not neutralize the biological activity generated by the ligand binding, (2) the recognition site remains at the N-terminal 20 amino acids of the C5a receptor The antibody according to (1) above, which exists in a region consisting of a group, (3) the antibody according to (1) above, which is a monoclonal antibody MH1 / 20, (4) inhibits the binding of C5a receptor to a ligand, (5) Fusion of mouse spleen cells and mouse myeloma cells immunized with a peptide fragment consisting of the N-terminal 20 amino acid residues of the C5a receptor, which produces an anti-C5a receptor monoclonal antibody that does not neutralize the resulting biological activity (6) The hybridoma according to (4) above, which comprises: Preparation of antibodies as described in (1) above, characterized in that the Ma cultured in a liquid medium or an animal intraperitoneally, and collecting the antibody from the culture supernatant or ascites fluid, (7)
The hybridoma according to (4) above, which is a mouse hybridoma MH1 / 20, (8) a method for measuring C5a receptor, which comprises immunologically reacting the antibody according to (1) with the C5a receptor, (9) flow The measurement method according to (8) above, which is a cytometry method, and (10)
The present invention relates to a C5a receptor assay reagent containing the antibody according to (1) above.

The anti-C5aR monoclonal antibody (mAb) of the present invention can be prepared by a known genetic engineering method, but is usually prepared by a hybridoma method. Production of the antibody-producing hybridoma is carried out according to a conventional method. That is, animals are immunized with C5aR or its partial polypeptide carrier conjugate or C5aR-expressing cells to confirm antibody production. Here, as the immunogen, it is preferable to use a conjugate of a C5aR partial polypeptide, particularly a carrier of a partial polypeptide consisting of 20 amino acid residues at the N-terminal, as long as the desired antibody can be obtained. Not done. Next, antibody-producing cells collected from the immunized animal, such as spleen cells and lymph node cells, are used as myeloma cells (eg, in the case of mice, N
S-1, P3U1, Sp2 / 0), and the resulting hybridomas are screened for cells which specifically bind to the C5aR partial sequence polypeptide conjugate and produce antibodies capable of binding to C5aR-expressing cells. . The C5a receptor fragment peptide carrier protein conjugate used as an antigen in the present invention is prepared by a conventional method. (1) carrier protein solution (prepared to a concentration of 1 mg / ml to 20 mg / ml and having a pH of 7 to 8) and (2) m-maleimidobenzoyl-N-hydroxysuccinimide ester (concentration of 3 mg / ml dimethylforma (Prepared in amide) (3) Prepare an antigen peptide solution (prepared at a concentration of 1 mg / ml to 10 mg / ml), and slowly add (2) to the protein solution (1). After stirring at room temperature for 30 minutes, a protein fraction was collected by a gel filtration column, added to the peptide solution of (3), and further reacted at room temperature for 3 hours. In addition to this method, the peptide-protein conjugate can be prepared by, for example, a polymerization method using glutaraldehyde.

As the immunized animal, for example, rabbit, rat, mouse, hamster, guinea pig and the like are used, but mouse is particularly preferably used. The method of inoculation may be according to the method usually practiced, for example, C5a
In the case of the R partial sequence polypeptide conjugate, 0.05-30 μg, preferably 0.1-5 μ, once per mouse.
In the case of g and C5aR expressing cells, 10 ⁵ -1 was given to the mouse once.
0 ⁷ cell number, was emulsified in complete adjuvant saline and Freund equal volume, the back, a method of inoculating 3-6 times 2-3 weeks every subcutaneously or intraperitoneally abdomen taken. Individuals with a high antibody titer are selected from these immunized animals, for example, mice, and 3 to 5 days after the final immunization, the spleen and / or lymph nodes are collected, and the antibody-producing cells contained therein are fused with myeloma cells. The fusion operation can be performed according to a known method, and examples of the fusion accelerator include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, Sp2 / 0,
Particularly, P3U1 is preferably used. The preferred ratio of spleen cells to myeloma cells is 1: 1-10: 1, to which PEG having an average molecular weight of about 1000-8000 is added to 10-80.
%, And the reaction is allowed to proceed at 20-37 ° C. for 3-10 minutes.

Various methods can be used for screening anti-C5aR mAb-producing hybridomas. For example, C5aR on a microplate or C5a with immunization
A conjugate (for example, BSA-C5aR _1-20 ) of R partial polypeptide and BSA (bovine serum albumin) is immobilized according to a conventional method to prepare an antigen-sensitized plate. Next, the hybridoma culture supernatant is added to this antigen-sensitized plate, and the antibody titer in the culture supernatant is measured by enzyme-linked immunosorbent assay (ELISA), which detects the anti-C5aR antibody bound to the plate. Hybridomas positive for antibody activity, which have been selected and bred in a medium supplemented with HAT (hypoxanthine, aminopterin, thymidine), are immediately subjected to cloning. Usually, this cloning operation is easily carried out by a limiting dilution method or the like. The culture supernatant of the cloned hybridoma is subjected to the above-mentioned ELISA, and among the cells showing positive antibody activity, antibody-producing hybridomas that bind to C5aR-expressing cells (for example, neutrophils) and are detected using a fluorescence-labeled secondary antibody. The target anti-C5a monoclonal antibody-producing hybridoma can be obtained. As an example of the anti-C5aR mAb-producing hybridoma prepared according to the above-mentioned production method, the hybridoma MH1 / 20 shown in Example 1- (3) described below can be mentioned. Cultivation of the above-described hybridoma of the present invention can be carried out in a liquid medium or intraperitoneally in an animal (for example, using a mammal such as mouse) by a known method. The purification of the antibody in the culture medium and the ascites fluid can be performed by combining known biochemical techniques. For example, the cell culture medium or ascites fluid is centrifuged, salted out (usually using ammonium sulfate or sodium sulfate), and the obtained protein precipitate is dissolved in an appropriate solution and dialyzed. Then, the target antibody can be separated and purified by subjecting it to column chromatography (ion exchange column, gel filtration column, protein A column, protein G column, hydroxyapatite column, etc.). By the above separation and purification operation, for example, 9
About 5-20 mg of mAb having a purity of 0% or more can be obtained from 1 L of cell culture supernatant, and about 20-100 mg of 20 ml of ascites fluid.

The mAb obtained as described above is homogeneous as a protein, and by treatment with a proteolytic enzyme,
F (ab ′) ₂ and Fab fragments can be prepared while retaining the binding ability to C5aR, and these are used for the same purpose as the mAb whole IgG molecule of the present invention, and are referred to as “anti-human C5aR” in the present invention. "Monoclonal antibody". In addition, these hybridomas are mouse IgG mA
In the case of producing b, DNA encoding a variable region or hypervariable region including the antigen recognition site of the anti-C5aR mAb was obtained, and the DNA was engineered with a gene manipulation technique (Z. Steplewsk).
i et al., Proc. Natl. Acad. Sc
i.), 85: 4852, 1988; L. Reichmann
Et al., Nature, 332: 323, 198.
8) is used to bind a gene encoding a constant region or / and a variable region framework of human IgG,
Mouse-human chimeric antibody or humanized antibody can also be prepared.

The purified antibody of the present invention obtained as described above is labeled with a radioisotope, an enzyme, a luminescent substance, a fluorescent dye and the like according to a conventional method and used as a reagent for various immunoassays. As the radioisotope, for example, ¹²⁵ I,
¹³¹ I, ³ H, ¹⁴ C, etc. are preferably stable and have a large specific activity. For example, (1) carbohydrase [eg, glycosidase (eg, β-galactosidase,
β-glycosidase, β-glucurosidase, β-fructosidase, α-galactosidase, α-glucosidase, α-mannosidase, amylase (eg, α-amylase, β-amylase, isoamylase, glucoamylase, takaamylase A) , Cellulase, lysozyme)], (2) amylase (eg, urease, asparaginase), (3) esterase [eg, cholinesterase (eg, acetylcholinesterase), phosphatase (eg, alkaline phosphatase), sulfatase, lipase], (4) Nuclease (eg, deoxyribonuclease, ribonuclease), (5) iron / porphyrin enzyme (eg, catalase, peroxidase, cytochrome oxidase), (6) copper enzyme (eg, tyrosinase, ascorbate oxidase), ( ) Dehydrogenase (eg, alcohol dehydrogenase, malate dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase) and the like, luminol as the light-emitting substance, luminol derivatives, luciferin, lucigenin and the like, respectively. As a fluorescent dye,
Fluorescein, rhodamine, phycoerythrin, fluorescamine, fluorescein isothiocyanate and the like can be mentioned. Among the above labeling reagents, a fluorescent reagent is more preferably used. The operation of the immunological assay using the antibody of the present invention for human C5aR expressed on cells will be specifically described below, but it goes without saying that the operation is not limited to this.

1) Indirect staining method 10 ⁵ -10 ⁶ cells were centrifuged to form a cell pellet, and 0.01% sodium azide and 1% BSA were added to the cell pellet.
Alternatively, m diluted with an appropriate buffer containing 1% FCS
Add 20 μl Ab. The concentration of the antibody is 1-100 μ
g / ml, preferably 5-50 μg / ml. The buffer solution is Hanks Balanced Salt Solution (HBSS)
Alternatively, a medium such as RPMI 1640 is used. When using platelets instead of cells, these buffers contained 3.8% of 10% volume of Japanese sodium citrate.
% Aqueous solution is added. The reaction between cells and antibody is about 0 ° C-3
Perform at 20 ° C. for 20 minutes to 1 hour. After the reaction is completed, 1 ml of the buffer solution used for antibody dilution is added to the reaction solution and the mixture is centrifuged. The centrifugation supernatant is removed, and the cell pellet is appropriately diluted with 20 μl of labeled F (ab ′) ₂ anti-mouse IgG antibody. The concentration of the labeled antibody varies depending on the strength of the label or the antibody titer, but is usually 0.1-50 ug / ml. After reacting for 20 minutes to 1 hour under the same reaction conditions, a buffer solution is added to wash. Cells and the like labeled with the labeled antibody may be measured using an appropriate analyzer. For example, fluorescently stained cells or platelets are FAC
Analyze with an analyzer such as Star. 2) Direct staining method This is a method of labeling the anti-C5aR antibody to be reacted in the first step of the method of 1) with a labeling agent such as a fluorescent dye, and the reaction is only in one step. Conditions such as diluent, reaction time, etc.
It is the same as the method of 1).

The present invention will be described in more detail with reference to the following examples, but it goes without saying that these do not limit the scope of the present invention. In addition, the hybridoma MH1 / 20 obtained in the example was received from the Institute for Fermentation and Biological Sciences (IFO) under the accession number IFO from August 5, 1994.
As 50443, since August 23, 1994, Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH)
Deposited under accession number FERM BP-4784. Example 1 Preparation of mouse anti-C5aR mAb producing hybridoma (1) Immunity C5aRN terminal 1-20th sequence (NH ₂ -MNSF)
NYTTPDYGHYDDKDTL-COOH) [SEQ ID NO: 1] -conjugated KLH-C5aR _1-20 conjugated to carrier protein KLH is mixed with an equal amount of Freund's complete adjuvant to emulsify BALB /
The c mouse was immunized intraperitoneally 5 μg once with immunization 5 times at intervals of 3 weeks. Freund's incomplete adjuvant was used except for the first time. The antibody titer in the blood were bled 2 weeks after each immunization, BSA-C5aR _1-20 (C5aRN terminus 1-
The 20th polypeptide was conjugated to the carrier protein BSA) and used as an antigen. That is, PBS containing 20 μg / ml of BSA-C5aR _1-20 and 50 μl of each well, and PBS containing 1% BSA.
Blocked at. There, diluted immune mouse serum 5
0 μl of each was added, and the mixture was reacted at 37 ° C. for 1 hour. After thoroughly washing the wells with PBS containing 0.01% Tween 20,
Horseradish peroxidase-labeled goat anti-mouse IgG antibody was added and reacted for 1 hour. After washing, 0.1 M citrate buffer (pH 4.5) containing o-phenylenediamine and H ₂ O ₂ as an enzyme substrate was added to carry out the enzyme reaction at room temperature. After stopping the reaction with 1N sulfuric acid, the absorbance at 490 nm was measured. KLH-C5aR _1-20 solution was further intravenously administered to the individual having the highest antibody titer in the fifth immunization.

(2) Cell fusion Three days after the final immunization, the spleen was removed and a spleen cell suspension was prepared by a conventional method (about 10 ⁸ cells). Then, mouse myeloma cells P3U1 (2 × 10 ⁷ cells) were added and fused using polyethylene glycol PEG6000 (Kohler and Milstein Nature (Nature) 256: 495,
1975). After completion of the fusion, the cell mixture was suspended in a so-called HAT medium containing hypoxanthine, aminopterin and thymidin and cultured for 10 days. After that, as soon as the selective removal of the parental cell line was completed, H obtained by removing aminopterin from the HAT medium
The culture was continued by changing to T medium. (3) Selection and cloning of hybridoma Since the appearance of hybridoma was observed 10 to 20 days after the fusion, the antibody titer in the hybridoma culture supernatant was determined by ELISA using BSA-C5aR _1-20 as an antigen described in (1). It was measured. Hybridomas showing strong antibody activity were cloned by the limiting dilution method. These hybridomas were assayed for antibody binding to neutrophils isolated from peripheral blood by flow cytometric analysis. That is, the hybridoma culture supernatant was added to neutrophils,
The reaction was carried out at 0 ° C for 30 minutes. Then, after washing once, it was reacted with a FITC-labeled goat anti-mouse IgG antibody as a secondary antibody. After reacting for 30 minutes, it was subjected to FACStar (Becton Dickinson). Only the MH1 / 20 antibody bound to neutrophils. The MH1 / 20 antibody was determined to be of IgG1, κ subclass with the subclass confirmation kit. (4) Purification of MH1 / 20 antibody To BALB / c mice to which 0.5 ml of mineral oil had been intraperitoneally administered, 2 × 10 ⁶ hybridoma cells MH1 / 20 were intraperitoneally administered. After about 10 to 15 days, ascites was collected and the MH1 / 20 antibody was purified by affinity chromatography using protein G according to a conventional method.

Example 2 C5a-induced neutrophil activation inhibition test (1) Differentiation of P39 (+) cells P39 (+) cells were complemented from myeloid cell line P39 cells established from myeloma patient cells. 3 components (C3)
Is a cell line established as a subline that binds to and has the ability to aggregate (Matsumoto, M. et al., European Journal of Immunology (Eur. J. Immunol.) Volume 21:
1787, 1991). When these cells are cultured with addition of dibutyryl cAMP), they have granules in the cytoplasm, polymorphic nucleate, and differentiate into neutrophil-like cells. C5 as one of the indicators of differentiation
When C5a is added to differentiated cells that have aR expression, granules are released into the reaction solution. Therefore, the degree of differentiation can be examined by quantifying one of the enzymes N-acetyl-D-glucosaminidase in the granules. Using this principle, the differentiation of P39 (+) cells was observed. The method is as follows. After washing the differentiated cells twice, 10 ⁷
/ Ml HBSS and final concentration of 5μg / ml Cytochalas
The reaction was carried out in B for 15 minutes. 100μ of this cell suspension
100 μl of C5a diluted to 1 was added and reacted at 37 ° C. for 1 hour. The reaction mixture was centrifuged at 1500 rpm for 10 minutes, 100 μl of the supernatant was transferred to a 96-well plate, and the substrate p was added thereto.
-Nitrophenyl-N-acetyl-β-D-glucosamide was reacted in 50 mM citrate buffer (pH 4.5) for 1 hour and 0.4 M glycine buffer (pH 10.5) was added to stop the reaction, 405 nm. The absorbance was measured with. This result shows that P39 (+) cells (●) are shown in [Fig. 1].
Differentiated in 2 days and then rapidly reached cell death.
As controls, the enzyme activities of dibutyl cAMP non-measurement (□) and C5a-non-added (◯) P39 (+) cells were measured by the same method. (2) Antibody functional inhibition test of C5aR The conditions for P39 (+) cell differentiation shown in Example 1- (1) above, that is, cells differentiated for 2 days in 0.5 mM dibutyryl-cAMP were used to give 0.1. , 0.3, 1 and 3
Enzyme release upon nM C5a stimulation was measured in a similar manner. At this time, there was almost no difference in the amount of released enzyme even when the MH1 / 20 antibody (20 μg / ml) was present [FIG. 2]. In the figure, □ indicates the amount of enzyme in the presence of antibody.
Indicates the amount of enzyme in the absence of antibody (control).

Example 3 C5a Binding Inhibitory Activity Test ¹²⁵ I-C5a was used to bind C5a to C5aR.
It can be quantified by reacting binding to neutrophils, monocytes, or established cells for 30 minutes to 1 hour, removing the unbound portion by centrifugation, and measuring radioactivity. When searching for a substance that inhibits this binding, it is necessary to quantify many samples at the same time. In such a case, use the method of preparing the cell membrane and examining the binding of ¹²⁵ I-C5a to the membrane. You can (1) Preparation of membrane fraction Differentiated P39 (+) cells were washed with HBSS and 0.25M
Sucrose, 0.04M NaCl, 0.1M KCl, 0.00
5M MgCl ₂ , 0.02M Tris-HCl buffer (pH 7.
6) Inhibitor cocktail {10 μM leupeptin, 10 μM pepstatin, 50 μM 0-phenanthroline, 100 KIU aprotinin, 100 μM
p-Amidinophenyl-methanesulfonyl fluoride hydrochloric acid (p-APMSF)} was added to suspend.
Put the cell suspension in a Teflon homogenizer and put it on ice 1
Perform 0-40 stroke crush. It was confirmed with a microscope that the cell membrane was disrupted and the nuclear membrane was retained. The cells were centrifuged at 3000 rpm for 10 minutes to remove the nuclei and unbroken cells, 5 mM EDTA was added to the supernatant, and the precipitate obtained by centrifugation at 100,000 rpm for 20 minutes was added to PBS to which the above inhibitor cocktail and 5 mMEDTA were added. It was suspended in the solution and used as the membrane fraction. (2) C5a binding test ¹²⁵ I-in the equivalent of 10 ⁶ membrane fraction cells prepared in (1) above
C5a (10,000 cpm) was added, the mixture was reacted on ice for 60 minutes, suction filtered on a glass filter (Whatmann GF / B), and washed 3 times. The radioactivity trapped in the filter was measured. To show the specificity of this binding reaction, when radiolabeled cold C5a was added to the reaction system at varying amounts, concentration-dependent inhibition of cold C5a binding was observed, and the binding was receptor / ligand specific. It was shown to be happening [Fig. 3]. When monoclonal antibody MH1 / 20 was added to this reaction system, inhibition of ¹²⁵ I-C5a binding was observed in an antibody concentration-dependent manner (FIG. 4). In the above Example (2), MH1 / 20 was C5a-induced P3.
In addition to not inhibiting the function of 9 (+) cells, C5a
It shows that the binding mode of R and C5a is at two or more positions.

Example 4 Flow cytometry (1) Polynuclear leukocyte cells The MH1 / 20 antibody binding was quantified by flow cytometric analysis for neutrophils isolated from peripheral blood and differentiated P39 (+) cells. FITC with 10 ⁶ cells
Labeled MH1 / 20 antibody (20 μg / ml) and 0.01
The mixture was reacted for 30 minutes on ice in the presence of 1% sodium azide and 1% FCS. It was washed once with the same buffer and subjected to FACStar. As a result, as shown in [FIG. 5] to [FIG. 7], almost all cells of both neutrophils [FIG. 5] P39 (+) [FIG. 6] are strongly stained and a large amount of C5aR is expressed. I understand. On the other hand, undifferentiated P39 (+)
It did not bind the MH1 / 20 antibody at all [Fig. 7].
The dotted line in the figure shows the reaction with the MH1 / 20 antibody, and the straight line shows the reaction with the control OKT3 antibody. (2) Platelet and megakaryoblast MEG01 1. Separation of platelets Since platelets are easily activated and aggregated with a slight stimulus, they were separated by the following method to suppress activation as much as possible. That is, blood was gently collected into 10% volume of citral (3.8% aqueous solution of sodium citrus sodium in the Japanese Pharmacopoeia), and immediately centrifuged at 3000 rpm. Centrifugation was stopped 5 seconds after reaching 3000 rpm, and the supernatant was added to platelet-rich plasma (P
RP). Here, when it was necessary to wash the platelets, Tyrode buffer containing 5 mM EDTA was used and centrifugation was performed at 9000 rpm for 2 seconds. 2. Binding of MH1 / 20 antibody to platelets Even if C5aR is expressed on platelets, it can be expected that the number is small. Therefore, the indirect staining method is used instead of the direct staining method used in (1), and the control antibody is used. The backgrounds were compared more accurately. That is, PRP100
OKT3 antibody (ATCC CR) that reacts only with T cells as MH1 / 20 antibody or control antibody
10 μl of L8001 cell culture supernatant) was added and reacted on ice for 30 minutes. Tyr containing 5 mM EDTA
1 ml of ode buffer was added, and the mixture was centrifuged for 2 seconds after reaching 9000 rotations. The supernatant was removed and the secondary antibody FITC-labeled goat anti-mouse IgG was added to the pellet and reacted on ice for 30 minutes. 1 ml of PBS containing 1% BSA and 5 mM EDTA was added to this reaction mixture, and the mixture was subjected to FACStar. As a result of examining the platelets of 6 normal persons, no significant antibody binding was observed. As a control, strong binding was observed when an antibody (Nichirei) against GIIb / IIIa, which is a platelet surface antigen marker, was used [Fig. 8]. In the figure, the broken line indicates GPIIb / IIIa, the straight line indicates the receptor of C5, and the dotted line indicates the expression of OKT3 antigen. 3. Differentiation of megakaryoblast MEG01 and binding of MH1 / 20 antibody Megakaryoblast cell line MEG0, which is regarded as a precursor of platelets
1 (IFO 50151) differentiates and releases platelet-like particles when given an appropriate stimulus. Therefore,
0.5 mM dibutyryl cAMP was added to MEG01 and the binding properties of the MH1 / 20 antibody were measured with time.
When examined in the same manner as in (1), antibody binding was observed at the peak on the 5th day. Cells in which the antibody was bound were also observed in undifferentiated MEG01 [Fig. 9] to [Fig. 15]. In the figure,
The straight line shows staining with the MH1 / 20 antibody and the dotted line shows staining with the control OKT3 antibody.

[0018]

INDUSTRIAL APPLICABILITY The antibody of the present invention inhibits the binding of C5a receptor to a ligand, but does not neutralize the biological activity caused by the binding of the ligand.
It is extremely useful in investigating the function of 5a or the transition of C5a receptor expression.
It is also clear from this example that the presence or absence of C5a on platelets, for which the conclusion was not obtained in the system using the 5a antibody, was clearly confirmed in this example.

[0019]

[Sequence Listing] SEQ ID NO: 1 Sequence length (SEQUENCE LENGTH): 20 Sequence type (SEQUENCE TYPE): Amino acid Sequence type (MOLECULE TYPE): Peptide Sequence: Met Asn Ser Phe Asn Tyr Thr Thr Pro Asp Tyr Gly His Tyr Asp Asp 1 5 10 15 Lys Asp Tyr Leu 20

[Brief description of drawings]

FIG. 1 shows the differentiation of myeloid cells P39 (+).

FIG. 2 is C5 of differentiated P39 (+) cells in the presence of antibody.
Fig. 3 shows the N-acetyl-β-D-glucosaminidase release reaction by a stimulation.

FIG. 3 shows inhibition of binding of labeled C5a to a differentiated P39 (+) cell membrane fraction by addition of cold C5a.

FIG. 4: Differentiation P39 (+) by addition of MH1 / 20 antibody
3 shows inhibition of C5a binding to cell membrane fraction.

FIG. 5: C5 on the surface of polynuclear leukocytes separated from peripheral blood
Analysis of aR expression.

FIG. 6: Analysis of C5aR expression in differentiated P39 (+) D cells.

FIG. 7: Analysis of C5aR expression in undifferentiated P39 (+) cells.

FIG. 8: Analysis of C5aR expression on human normal platelets.

FIG. 9: Changes in the expression of C5aR accompanying the differentiation of megakaryocyte cells. After addition of dibutyl cAMP (0 days)

FIG. 10: Transition of C5aR expression accompanying the differentiation of megakaryocyte cells. After addition of dibutyl cAMP (2 days)

FIG. 11: Changes in the expression of C5aR accompanying the differentiation of megakaryocyte cells. After addition of dibutyl cAMP (3 days)

FIG. 12: Changes in the expression of C5aR accompanying the differentiation of megakaryoblasts. After addition of dibutyl cAMP (4 days)

FIG. 13: Changes in the expression of C5aR accompanying the differentiation of megakaryoblasts. After addition of dibutyl cAMP (5 days)

FIG. 14: Changes in the expression of C5aR accompanying the differentiation of megakaryoblasts. After addition of dibutyl cAMP (7 days)

FIG. 15: Changes in the expression of C5aR accompanying the differentiation of megakaryocyte cells. After addition of dibutyl cAMP (9 days)

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Claims (10)

[Claims] 1. An anti-C5a receptor monoclonal antibody which inhibits the binding of C5a receptor to a ligand but does not neutralize the biological activity produced by the ligand binding. 2. The recognition site is the N-terminal 20 of the C5a receptor.
The antibody according to claim 1, which is present in a region consisting of amino acid residues. 3. The antibody according to claim 1, which is the monoclonal antibody MH1 / 20. 4. A hybridoma that produces an anti-C5a receptor monoclonal antibody that inhibits binding of C5a receptor to a ligand but does not neutralize biological activity caused by ligand binding. 5. The hybridoma according to claim 4, which is obtained by fusing mouse spleen cells and mouse myeloma cells immunized with a peptide fragment consisting of the N-terminal 20 amino acid residues of the C5a receptor. 6. The method for producing an antibody according to claim 1, wherein the hybridoma according to claim 4 is cultured in a liquid medium or an abdominal cavity of an animal, and the antibody is collected from the culture supernatant or ascites. 7. The hybridoma according to claim 4, which is a mouse hybridoma MH1 / 20. 8. A method for measuring a C5a receptor, which comprises immunologically reacting the antibody according to claim 1 with the C5a receptor. 9. The measuring method according to claim 8, which is a flow cytometry method. 10. A reagent for measuring C5a receptor, which comprises the antibody according to claim 1.