Anti-T cell receptor gamma-chain monoclonal antibody

Abstract: Disclosed are T cell receptor γ-chain specific monoclonal antibodies, a method for the production thereof, and hybridoma cell lines capable of producing them.

Description: The present invention relates to anti-T cell receptor γ-chain monoclonal antibodies which are useful as a reagent for investigating functions of T cell receptor γ-chains. T cells play an important role in the control of immune response and cellular immunity of the immune system. On its surface membrane, a T cell has T cell receptors, which participate in performing the functions of the T cell. Studies of the structure of the receptor using a monoclonal antibody obtained from a mouse immunized with cloned T cells have revealed that the receptor consists of two polypeptides, identified as the α- and β-chains. The sequence of the genes that encode these chains was determined by analysis based on molecular genetics. Additionally Tonegawa et al. have discovered a gene which encodes for a T cell receptor γ-chain (hereinafter referred to as the γ-chain) as well as a gene coding for the α- and β-chains, but the role played by the γ-chain in the function of the T cell still remains uncertain. In addition, the presence of the δ-chain linked to the γ-chain has been reported in Nature, 322 , 145-149 (1986). A γ-chain monoclonal antibody has been reported in WO-A-88/00209, which forms part of the state of the art by virtue of Art. 54(3)EPC, as well as monoclonal antibodies to γ,δ-complexes. Also a mouse-derived rabbit anti-γ-chain antiserum was reported [Nature, 325 , 720-723 (1987)]. However, in order to perform a comprehensive and detailed investigation of the structure and role of T cell receptor γ-chain, a monoclonal antibody of high purity and high specificity is still in demand. In accordance with this invention three novel anti-T-cell receptor γ-chain monoclonal antibodies are provided having the following features: (i) Class: IgG₁; Reactivity (Western blot): reactive with E. coli produced T-cell γ-chain, but not with murine fetal thymocytes; Antigen-specifity (partially purified): specific to E. coli produced T-cell γ-chain:binding activity (OD 415nm ) at 10µg/ml 1.550; to BSA 0.005; Reactivity (immuno fluorescence): No reaction with murine T-cell hybridoma cell lines KN6, KN3 or T-cell leukaemia cell line BW5147. Source: obtainable from hybridoma cell line KM-367 (ECACC Deposit No. 87041604). (ii) Class: IgG 2b : Reactivity (Western blot): reactive with E. coli produced T-cell γ-chain and murine fetal thymocytes, but not T-cell δ-chain. Antigen specificity (partially purified): specific to E. coli produced T-cell γ-chain, binding activity (OD 415nm ) at 10 µg/ml 1.780; BSA 0.005; Reactivity (immuno fluorescence): No reaction with murine T-cell hybridoma cell lines KN6, KN3 or T-cell leukaemia cell line BW5147; Source: obtainable from hybridoma cell line KM-365 (ECACC Deposit No. 87052101) and: (iii) Class IgG₁: Reactivity (Western blot): reactive with E. coli produced T-cell γ-chain and murine fetal thymocytes but not T-cell δ-chain Antigen specifity (partially purified): specific to E. coli produced T-cell γ-chain, binding activity (OD 415nm ) at 10µg/ml 1.993; BSA 0.000; Reactivity (Immuno fluorescence): reactive with murine T-cell hybridoma cell line KN6, but not with T-cell hybridoma cell line KN3 or T-cell leukaemia cell line BW5147; Source: obtainable from hybridoma cell line KM-369 (ECACC Deposit No. 87080601). These monoclonal antibodies have been obtained by immunizing a mouse with a γ-chain antigen, fusing spleen cells from that immunized mouse with a murine myeloma cell line to produce a series of hybridomas, selecting from among the hybridomas obtained one or more hybridoma clones capable of producing γ-chain specific monoclonal antibody, and cultivating the selected hybridoma or hybridoma(s), either in a suitable culture medium, or by administering the selected hybridoma(s) to mice thereby to cause hybridoma cell propagation in the ascitic fluid, followed by separation, from the culture medium or the ascitic fluid as the case may be, of the antibody. In particular the γ-chain monoclonal antibody producing hybridomas are obtained by fusing spleen cells of a DBA/2 mouse immunized with the γ-chain of BALB/c mouse origin, and which has been expressed in Escherichia coli by recombinant DNA techniques. A detailed protocol for the production of monoclonal antibodies according to the present invention is as follows: (1) Immunization of animal and preparation of antibody-producing cells DBA/2 mice (purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals) are immunized with γ-chain to generate antibody producing cells in spleen, lymph node and peripheral blood. As the γ-chain, one produced in Escherichia coli by recombinant DNA technique based on the cDNA sequence cloned from T cells derived from BALB/c mice is used [Nature, 313 , 752-755 (1985)]. The immunization is performed by administering the γ-chain (10-100 µg per animal) together with an appropriate adjuvant (e.g. Freund's complete adjuvant or aluminium hydroxide gel plus B . pertussis vaccine) to 8- to 10-week old DBA/2 mice subcutaneously, intravenously or intraperitoneally. Thereafter, administration of the γ-chain is repeated two to ten times at 1- to 2-week intervals. One week after each immunization, the blood is sampled from the eyeground venous plexus and the serum of each sample is tested as to the anti-γ-chain antibody titer by the solid-phase enzyme immunoassay technique given below [Enzyme-linked Immunosorbent Assay (ELISA), published by Igaku Shoin, Tokyo 1976]. Enzyme Immunoassay Technique : The specific antigens are distributed in suspension into wells of a 96-well EIA plate (product of Flow Laboratories) in an amount of 100 µg per well. For this purpose the antigens are suspended in phosphate-buffered saline (PBS) containing 1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate and 7.65 g of sodium chloride in 1 liter of distilled water (pH 7.2) at a γ-chain antigen concentration of 10 µg/ml. Alternatively solutions containing other types of antigen in place of γ-chain, or PBS solution containing 1% BSA (BSA/PBS) can be used for the study of cross reactions. After allowing the plate to stand overnight at 4°C to ensure coating of the wells with the antigens, the supernatant is removed from the plate and then the plate is washed well with deionized water or PBS. Then BSA/PBS solution is distributed into the wells (200 µl per well), and the protein-binding sites remaining on each well are blocked by allowing the plate to stand overnight at 4°C. After discarding the BSA/PBS solution, the wells are washed well with PBS. Serial dilutions of samples (mouse antisera, hybridoma culture supernatants or purified antibodies) are distributed into the wells (50 µl per well) as the first antibody, followed by overnight-standing at 4°C or by three to four hour-standing at room temperature. After washing the wells six times with PBS, a 400-fold dilution of peroxidase-conjugated rabbit immunoglobulins to mouse immunoglobulins (product of DAKO and distributed by Kyowa Medex; used as the second antibody) is distributed into the wells (100 µl per well). The plate is then allowed to stand at room temperature for two hours. After washing with PBS, 100 µl of an ABTS substrate solution [prepared by dissolving 500 mg of diammonium 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) in 1 liter of 0.1 M citrate buffer (pH 4.2) and adding, just prior to use, hydrogen peroxide to a concentration of 1 µℓ/mℓ] is applied and the color developed is measured in terms of absorbance OD 415 nm . Those mice showing an anti-γ-chain titer higher than 10³ times as much as normal mice serum (OD 415nm ) are used as sources of supply of antibody-producing cells for hybridoma production. For submitting to cell fusion, γ-chains are intraperitoneally administered to the immunized mice in a dose of 10 to 100 µg per animal three to four days prior to the cell fusion treatment. The spleen is extirpated, cut into fragments in MEM (product of Nissui Pharmaceutical), loosened up with a pair of forceps, and centrifuged at 1200 rpm for 5 minutes. The supernatant is discarded, and the sediment is deprived of erythrocytes by treatment with Tris-ammonium chloride buffer (pH 7.65) for 1-2 minutes, washed three times with MEM and used as the spleen cells for fusion. (2) Preparation of myeloma cells A mouse-derived established myeloma cell line is preferably used. Usable examples of such cell lines include the 8-azaguanine resistant murine (BALB/c-derived) myeloma cell lines P3-X63-Ag8-Ul (P3-Ul) [Current Topics in Microbiology and Immunology, 81 , 1-7 (1978)], P3-NSI/1-Ag4.1 (NS-1)[European J. Immunology, 6 , 511-519 (1976)], SP2/0-Ag14 (SP-2) [Nature, 276 , 269-270 (1978)], P3-X63-Ag8.653 (653) [J. Immunology, 123 , 1548-1550 (1979)] and P3-X63-Ag8 (X63) [Nature, 256 , 495-497 (1975)]. The initial culture of these cell lines is performed in 8-azaguanine medium [normal medium prepared by adding, to RPMI-1640 medium, glutamine (1.5mM), 2-mercaptoethanol (5 x 10⁻⁵M), gentamycin (10 µg/mℓ) and fetal calf serum (FCS: product of CSL, 10%), with further supplementation with 8-azaguanine (15 µg/mℓ)]. The cell line selected for cell fusion is transferred to normal medium three to four days before fusion to ensure the cell count of not less than 2 x 10⁷ on the day of cell fusion. (3) Cell fusion The spleen cells prepared in (1) and the myeloma cells obtained in (2) are washed well with MEM or PBS, and mixed in a cell number ratio of spleen cells : myeloma cells = 5 to 10:1 and then subjected to centrifugation (1200 rpm, 5 minutes). The supernatant is discarded and the cell sediment is loosened up. With stirring at 37°C, a mixture of 1 to 4 g of polyethylene glycol (PEG-1000 - 4000), 1 to 4 mℓ of MEM and 0.5 to 1.0 mℓ of dimethyl sulfoxide is added in an amount of 0.1 to 1.0 mℓ per 10⁸ spleen cells, and 0.5 to 3 mℓ of MEM is further added after 0.5 to 10 minutes. After several additions of 0.5 to 3 mℓ of MEM at 0.5- to 2-minute intervals, 30 to 60 mℓ of MEM is added. After centrifugation (900 rpm, 5 minutes), the supernatant is discarded and the cell sediment is loosened gently. To the cells is added 50 to 200 mℓ of the normal medium [RPMI-1640 medium with 10% FCS, glutamine (1.5mM), 2-mercaptoethanol (5x10⁻⁵M) and gentamycin (10 µg/mℓ)], and the cells are gently suspended in the medium by a measuring pipette. The suspension obtained is distributed into the wells of an incubation plate (half the volume of each well). Incubation is carried out in 3 to 7% CO₂ incubator at 35 to 40°C for 10 to 30 hours. HAT medium [normal medium supplemented with hypoxanthine (10⁻⁵ to 10⁻³M), thymidine (10⁻⁶ to 10⁻⁴M) and aminopterine (10⁻⁸ to 10⁻⁷M)] is added to the incubation plate (half the volume of each well), and incubation is conducted for a further 10 to 30 hours. Thereafter, half the volume of supernatant is discarded and the same volume of fresh HAT medium is added at 10- to 30-hour intervals for one to three days. Incubation in the CO₂ incubator at 35 to 40°C is continued for 10 to 14 days. From those wells in which fused, colony-forming grown cells are found, half the volume of supernatant is discarded and the same volume of HT medium (HAT medium minus aminopterine) is added, followed by medium replacement with fresh portions of HT medium at 10- to 30-hour intervals for one to three days. After three to four days of cultivation in HT medium, a portion of the supernatant is collected and anti-γ-chain antibody titer is assayed by the above-mentioned enzyme immunoassay technique. For the wells showing a certain antibody titer, cloning is repeated two to four times by the limiting dilution technique. In this way, those clones for which high antibody titer values are stably obtainable are selected as anti-γ-chain monoclonal antibody-producing hybridoma cell lines. (4) Preparation of monoclonal antibodies Eight- to ten-week-old female BALB/c nu/nu nude mice treated with pristane [intraperitoneally administered with 0.5 mℓ of 2,6,10,14-tetramethylpentadecane (pristane) and fed for one to two weeks] are intraperitoneally injected with the anti-γ-chain monoclonal antibody-producing hybridoma obtained in procedure (3) at a dose of 2-4 x 10⁶ cells per animal. Within 10-21 days, the hybridoma cells produce ascites carcinoma in the mice. The ascitic fluid is collected from such mice, centrifuged (3000 rpm, 5 minutes) to remove solids, subjected to salting out with 50% and 40% ammonium sulfate, and dialyzed against PBS (pH 7.2) for one to two days. The dialyzate is collected as a partially purified monoclonal antibody, which may be used for quantitative analysis. When further purification is needed, the partially purified monoclonal antibody is passed through a DEAE-Sepharose column or protein-A-Sepharose column, and the IgG fraction is collected. The isotype and subclass of the antibody is determined by the Ouchterlony's method (double immunodiffusion) as described in [Seibutsukagaku Jikkenho (Methods of Experimental Biochemistry) vol. 15, Introduction to Experimental Immunology, p.74, Gakkai Shuppan Center, 1981]. The quantity of protein is estimated by Folin's method, followed by calculation based on the absorbance at 280 nm [1.4 (OD₂₈₀) approximately corresponds to 1 mg of immunoglobulin per mℓ]. It was determined that the monoclonal antibodies referred to herein as KM-367, KM-365 and KM-369 obtained from hybridoma cell lines named KM-367, KM-365 and KM-369 belong to IgG₁, IgG 2b and IgG₁ respectively. The monoclonal antibodies KM-367, KM-365 and KM-369 have specificity to γ-chain. Therefore, the monoclonal antibodies KM-367, KM-365 and KM-369 are useful as a reagent for investigating functions of T cell receptor γ-chain. The monoclonal antibodies KM-365 and KM-369 have reactivity with γ-chain in cell-disrupted suspension of thymocyte which expresses γ-and δ-chains thereon, and the monoclonal antibody KM-369 has reactivity with γ-chain of ethanol-fixed murine T cell hybridoma cell line which expresses γ- and δ-chains thereon. The Western blotting technique used in the Examples is a modified one of the method of Towbine et al . [Proc. Natl. Acad. Sci., 76 , 4354 (1979)]. Proteins are subjected to SDS-polyacrylamide gel electrophoresis, and the gel is placed on a sheet of nitrocellulose. Blotting is carried out at 40V, 600mA at 10°C or lower in a buffer solution [containing 3 g/ℓ Tris(hydroxymethyl)aminomethane, 14.4 g/ℓ glycine and 20% methanol; pH 8.3] using an electrophoretic transfer blotting apparatus (product of ATTO), thereby transferring the proteins to the nitrocellulose sheet. The nitrocellulose sheet is air-dried, blocked with 3% gelatin solution, washed with water, and soaked in anti-γ-chain hybridoma culture supernatant at room temperature for two hours to complete the reaction. The sheet is then soaked in a solution of peroxidase-conjugated rabbit immunoglobulins to mouse immunoglobulins (product of DAKO and distributed by Kyowa Medex) at room temperature for two hours and washed with PBS containing 0.05% Tween 20 (product of Wako Pure Chemicals). It is finally soaked in a color developer to develop color and the reaction is terminated with cold water. The color developer is prepared by mixing at room temperature, (A)a solution of 100 mℓ of Tris-HCl buffered saline (TBS) [2.42 g Tris(hydroxymethyl)aminomethane and 29.24 g of sodium chloride in 1 liter of distilled water; pH 7.5] and 60 µℓ of 30% hydrogen peroxide; with (B)a solution obtained by adding 20 mℓ of ice-cold methanol to 60 mg of HRP color development reagent (product of BIO-RAD) in the dark at room temperature. The electrophoresis for immune precipitate is carried out according to the method of Nakanishi, et al . [Nature, 325 , 720 (1987)]. Thymocytes of fetal BALB/c mouse or C57BL/6 mouse on the 17th day of fetal life are labeled with ¹²⁵I by the lactoperoxidase method [Methods of Immunology, 178 (1977)]. In case of immunoprecipitation under non-reducing condition, the cell are washed three times with PBS, suspended in ice-cold NP40 buffer [10mM Tris-HCl buffer (pH 7.5) with 1mM phenylmethylsulfonyl fluoride and 10% Nonidet 40 (product of Particle Data Laboratories) added thereto], and disrupted by vigorous shaking. Each of the monoclonal antibodies KM-367, KM-365 and KM-369 is added to the resulting cell-disrupted suspension to effect immune precipitation. In case of immunoprecipitation under reducing condition, the cells are washed with PBS, suspended in ice-cold NP40 buffer supplemented with 2mM dithiothreitol and disrupted by vigorous shaking. Each of the monoclonal antibodies KM-367, KM-365 and KM-369 is added to the resulting cell-disrupted suspension to effect immune precipitation. When the one-dimensional electrophoresis is carried out, the immunoprecipitates under reducing condition is subjected to electrophoresis using 12% polyacrylamide gel according to the method of Laemmli [Nature, 227 , 680(1970)]. When the two-dimensional electrophoresis is carried out, the immunoprecipitates under non-reducing condition is subjected to electrophoresis in the first dimension using 10% polyacrylamide gel prepared inside a glass tube 1.5 mm in internal diameter. After the electrophoresis, the gel is taken out from the glass tube, held in a buffer solution containing 5% 2-mercaptoethanol at room temperature for 30 minutes, and then placed on a 12% slab gel to be subjected to electrophoresis in the second dimension. Immunofluorescence staining of cells treated with ethanol is carried out according to the method of Brenner, et al . [Nature, 322 , 145 (1986)]. Murine T cell hybridoma cell line KN6, which expresses γ- and δ-chains on the membrane surface, murine T cell hybridoma cell line KN3, which expresses α- and β-chains but not γ- and δ-chains on the membrane surface and murine T cell leukemia cell line BW5147 [J. Immunol. 110 , 1470 (1973)], which is host cell line of KN6 and KN3, not expressing any of α-, β-, γ- and δ-chains, and used as the control, are suspended in PBS (pH7.2), respectively and distributed, in 180 µℓ-portions, into an Eppendorf tube in amount of 10⁷ cells. 140 µℓ of 95% ethanol-PBS solution is added to the tube at -20°C to ensure ethanol final concentration of 70%, and incubation is carried out for 10 minutes at 4°C. The cells are then washed three times with PBS prior to the addition thereto of a culture supernatant of each of the monoclonal antibodies KM-367, KM-365 and KM-369. Incubation is carried out at 4°C for 30 minutes. Then, the cells are washed three times with PBS supplemented with 0.1% BSA and 0.1% sodium azide and 30µℓ of a 15-fold dilution of biotin-labeled goat anti-mouse immunoglobulin antiserum (product of Kirkgaard Terry) is added thereto, and incubation is carried out at 4°C for 30 minutes. After washing three times with PBS supplemented with 0.1% BSA and 0.1% sodium azide, 20 µℓ of Phycoerythrine-streptavidin (product of Beckton-Dickinson) is added to the cells, and incubation is carried out at 4°C for 30 minutes. After washing three times with PBS supplemented. with a 0.1% BSA and 0.1% sodium azide, the cells are suspended in 200 µl of PBS supplemented with 0.1% sodium azide. Immunofluorescence intensity is detected by the cell sorter (FCS-1, product of Japan Spectroscopic Co., Ltd.) to measure the titer of antibodies. Murine T cell hybridomas KN3 and KN6 have been deposited under the Budapest Treaty with Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, on 14th April, 1988 as FERM BP-1854 and FERM BP-1853, respectively. Certain specific embodiments of the present invention are illustrated by the following examples and with reference to the accompanying drawings, in which: Figure 1 shows the assay results of the specificity of KM-367 by Western blotting in Example 2, in which (A) gives staining with Amide Black and (B) gives immunostaining with KM-367. In (A) and (B), 1 ○ shows molecular weight markers (14.4 kd: Iysozyme, 21.5 kd: soybean trypsin inhibitor, 31 kd: carbonic anhydrolase, 45 kd: ovalbumin, 66.2 kd: bovine serum albumin (BSA), and 92.5 kd: phosphorylase b), 2 ○ shows γ-chain and 3 ○ shows foreign proteins derived from host cells of a microorganism belonging to Escherichia coli . Figure 2 shows the result of two-dimensional electrophoresis of immunoprecipitates in Example 3. The electrophoresis was performed under non-reducing conditions in the first dimension (horizontal direction) and under reducing conditions in the second dimension (vertical direction). Molecular weight markers are shown in the left lane (14 kd: Iysozyme, 18 kd: β-lactoglobulin, 26 kd: α-chymotrypsinogen, 43 kd: ovalbumin, 69 kd: BSA, 92 kd: phosphorylase b, and 200 kd: myosin heavy chain). Figure 3 shows the result of immunofluorescence staining in Example 4, in which (a) gives the reactivity of KM-369 with BW5147, (b) gives the reactivity of KM-369 with KN3 and (c) gives the reactivity of KM-369 with KN6. Example 1 (1) Preparation of antibody-producing cells Five 8-week old female DBA/2 mice (purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals) were intraperitoneally administered with γ-chain as antigen (100 µg per animal) together with aluminium hydroxide gel (2 mg per animal) and killed B. pertussis vaccine (1 x 10⁹ cells per animal, product of Chiba Serum Research Institute) as adjuvant. Thereafter, immunization was repeated with said γ-chain without adjuvant at a dose of 100 µg per animal at 2-week intervals. From the third immunization on, the blood was sampled from the eyeground venous plexus five to seven days after each immunization, and the serum of each sample was tested as to the anti-γ-chain antibody titer by the above-mentioned solid-phase enzyme immunoassay. Antibody titer was observed with all the test animals from the third immunization on, but immunization was repeated five times to ensure efficient production of anti-γ-chain monoclonal antibodies. After the fifth immunization, the γ-chain was again intraperitoneally administered at a dose of 100 µg per animal, and the spleen cells were prepared from such mice three days later and submitted to cell fusion. (2) Preparation of myeloma cells The 8-azaguanine-resistant mouse myeloma cell line P3-Ul was cultivated in a normal medium (RPMI-1640 medium supplemented with 1.5 mM glutamine, 5 x 10⁻⁵M 2-mercaptoethanol, 10 µg/mℓ gentamycin and 10% FCS) to thereby secure not less than 2 x 10⁷ cells after four days. (3) Hybridoma production The spleen cells (1 x 10⁸ cells) and murine myeloma cells (2 x 10⁷ cells) obtained in (1) and (2) respectively were washed well with MEM and mixed and then the mixture was subjected to centrifugation (1200 rpm, 5 minutes). The supernatant was discarded and the cell sediment was loosened up. With stirring at 37°C, a mixture of 2 g polyethylene glycol-1000 (PEG-1000), 2 mℓ of MEM and 0.7 mℓ of dimethyl sulfoxide was added in amount of 0.5 mℓ per 10⁸ spleen cells, and 1 mℓ of MEM was further added one minute later. After 5-additions of 1 mℓ of MEM at 1-minute intervals, MEM was added until the whole volume was made 50 mℓ. After centrifugation at 900 rpm for 5 minutes, the supernatant was discarded and the cell sediment was loosened gently. To the loosened cells were was added 100 mℓ of normal medium. The cells were gently suspended in the medium by a 10 mℓ-measuring pipette. The suspension obtained was distributed in 1 mℓ-portions into the wells of a 24-well incubation plate (product of Flow Laboratories). Incubation was carried out in a 5% CO₂ incubator at 37°C for 24 hours. HAT medium [the above-mentioned normal medium supplemented with hypoxanthine (10⁻⁴M), thymidine (1.5 x 10⁻⁵M) and aminopterine (4 x 10⁻⁷M)] was added to the incubation plate (1 mℓ per well) and incubation was conducted for a further 24 hours. Thereafter, 1 mℓ of the culture supernatant was discarded and the same volume of fresh HAT medium was added, and incubation in the CO₂ incubator at 37°C was continued for 24 hours. Thereafter, 1 mℓ of culture supernatant was discarded and the same volume of fresh HAT medium was added. Incubation was further continued at 37°C for 10 to 14 days. From those wells in which fused, colony-forming grown cells were found, 1 mℓ of the supernatant was discarded and the same volume of HT medium was added. Incubation was continued at 37°C. The medium replacement with fresh HT medium was repeated in the same way for two days, followed by incubation for four days. A portion of culture supernatant was collected and assayed for anti-γ-chain antibody titer by the above-mentioned solid-phase enzyme immunoassay technique. For the wells showing a certain antibody titer, cloning was repeated twice by the limiting dilution technique. In this way, three clones for which high antibody titer values were stably obtainable, KM-367, KM-365 and KM-369 were selected as anti-γ-chain monoclonal antibody-producing hybridoma cell lines. Hybridoma cell lines KM-367, KM-365 and KM-369 have been deposited under the Budapest Treaty with European Collection of Animal Cell Cultures, Great Britain, on 16th April, 21st May, and 6th August, 1987 as ECACC Deposit No. 87041604, No. 87052101 and No. 87080601, respectively. (4) Partial purification of antibodies Pristane-treated 8-week old female BALB/c nu/nu nude mice were intraperitoneally injected with each of the hybridoma cell lines obtained in (3) at a dose of 4 x 10⁶ cells per animal. In 10-21 days, the hybridoma cells produced ascites carcinoma. The ascitic fluid was collected from such mice (4-10 mℓ per animal), deprived of solids by centrifugation (3000 rpm, 5 minutes), subjected to salting out with 50% and with 40% ammonium sulfate, and dialyzed against PBS (pH 7.2) for two days, giving partially purified monoclonal antibodies KM-367, KM-365 and KM-369. (5) Antigen-specificity of partially purified monoclonal antibodies. Antigen-specificity of the thus-obtained partially purified monoclonal antibodies was examined by the solid-phase enzyme immunoassay using, as antigen, γ-chain produced in Escherichia coli , other proteins derived from host cells of Escherichia coli and bovine serum albumin (BSA) (product of Seikagaku Kogyo Co., Ltd.). The result is summarized in Table 1. Antibody Concentration or dilution Binding activity (OD 415nm ) γ-chain BSA Other proteins derived from host cells Normal mouse serum x 10⁻² 0.001 0.000 0.130 γ-chain-immunized mouse serum x 10⁻² 1.750 0.005 0.340 Partially purified KM-367 10 µg/mℓ 1.550 0.005 0.025 Partially purified KM-365 10 µg/mℓ 1.780 0.005 0.001 Partially purified KM-369 10 µg/mℓ 1.993 0.000 0.000 Partially purified monoclonal antibodies were passed through a DEAE Sepharose (product of Whatman) column (bed volume 1 mℓ). A non-adsorbed IgG fraction was collected and used as highly-purified antibody. (6) Classification of monoclonal antibodies Isotype and subclass of monoclonal antibodies KM-367, KM-365 and KM-369 was determined by Ouchterlony's method to be IgG₁, IgG 2b and IgG₁, respectively. Example 2 Reactivity of KM-367 with γ-chain produced in Escherichia coli was examined by the Western blotting technique described above. The result is shown in Figure 1. It was demonstrated that KM-367 specifically reacts with γ-chain. A similar result was obtained also with KM-365 and KM-369. Example 3 Reactivity of KM-365 with thymocytes having γ-chain expressed thereon from fetal C57BL/6 mice on the 17th day of fetal life was examined by the one-dimensional electrophoresis and two-dimensional electrophoresis for immunoprecipitates. The result of the two-dimensional electrophoresis is shown in Figure 2. It was demonstrated that precipitates by KM-365 contained γ-chain (35 kd) and δ-chain (45 kd). The one-dimensional electrophoresis shows KM-365 immunoprecipitates γ-chain but not δ-chain under reducing condition. These results shows that KM-365 has reactivity with γ-chain but not δ-chain and that δ-chain is linked to γ-chain through the disulfide bond. A similar result was obtained also with fetal thymocytes from a BALB/c mouse. A similar result was obtained with KM-369, but not with KM-367. Example 4 According to the above-mentioned immunofluorescence staining procedure, the reactivities of KM-369 to murine T cell hybridoma cell line KN6, which expressed γ- and δ-chains on the cell surface, murine T cell hybridoma cell line KN3 which expressed α- and β-chains but not γ- and δ-chains on the cell surface, and murine T cell leukemia BW5147 which was host cell line, not expressing any of α-, β-, γ- and δ-chains, and used as control, were examined after the cell lines were fixed with treatment of ethanol. The results are shown in Figure 3. The relative fluorescence intensity to KN3 coincide with that to BW5147. The peak position of the relative fluorescence intensity to KN6 was moved to the right direction (high area) compared with that to BW5147. It is shown that the monoclonal antibody KM-369 reacted with KN6. As the results, it was proved that the monoclonal antibody KM-369 which was shown to be capable of reacting with γ-chain in the cell-disrupted suspension in Example 3, was capable of reacting with γ-chain on the ethanol-fixed cell membrane. Using KM-367 and KM-365 in place of KM-369, the immunofluorescence staining procedure was repeated, but no reactivity to any of KN6, KN3 and BW5147 was observed. The invention also extends to hybridoma cell lines having the ability to produce T cell receptor γ-chain specific monoclonal antibodies, in particular the cell lines KM-367 ECACC No. 87041604, KM-365 ECACC No. 87052101 and KM-369 ECACC No. 87080601, as well as to mutants and transformants thereof, i.e. cell lines transformed using genetic material therefrom, and having the ability to produce T cell receptor γ-chain specific monoclonal antibody, and the use thereof in the production of T cell receptor γ-chain specific monoclonal antibody.

Claims: A T-cell receptor, γ-chain specific, monoclonal antibody characterised by the following: Class: IgG₁; Reactivity (Western blot): reactive with E. coli produced T-cell γ-chain, but not with murine fetal thymocytes; Antigen-specificity (partially purified): specific to E. coli produced T-cell γ-chain: binding activity (OD 415nm ) at 10 µg/ml 1.550; to BSA 0.005; Reactivity (immuno fluorescence): No reaction with murine T-cell hybridoma cell lines KN6, KN3 or T-cell leukaemia cell line BW5147. Source: obtainable from hybridoma cell line KM-367 (ECACC Deposit No. 87041604). A T-cell receptor, γ-chain specific, monoclonal antibody characterised by the following: Class: IgG 2b Reactivity (Western blot): reactive with E. coli produced T-cell γ-chain and murine fetal thymocytes, but not T-cell δ-chain. Antigen specificity (partially purified): specific to E. coli produced T-cell γ-chain, binding activity (OD 415nm ) at 10µg/ml 1.780; BSA 0.005; Reactivity (immuno fluorescence): No reaction with murine T-cell hybridoma cell lines KN6, KN3 or T-cell leukaemia cell line BW5147; Source: obtainable from hybridoma cell line KM-365 (ECACC Deposit No. 87052101). A T-cell receptor, γ-chain specific, monoclonal antibody characterised by the following: Class: IgG₁: Reactivity (Western blot): reactive with E. coli produced T-cell γ-chain and murine fetal thymocytes but not T-cell δ-chain Antigen specificity (partially purified): specific to E. coli produced T-cell γ-chain, binding activity (OD 415nm ) at 10µg/ml 1.993; BSA 0.000; Reactivity (Immuno fluorescence): reactive with murine T-cell hybridoma cell line KN6, but not with T-cell hybridoma cell line KN3 or T-cell leukaemia all line BW5147; Source: obtainable from hybridoma cell line KM-369 (ECACC Deposit No. 87080601). A monoclonal antibody according to claim 1, obtained from the hybridoma cell line KM-367 (ECACC Deposit No. 87041604). A monoclonal antibody according to claim 2, obtained from the hybridoma cell line KM-365 (ECACC Deposit No. 87052101). A monoclonal antibody according to claim 3, obtained from the hybridoma cell line KM-369 (ECACC Deposit No. 87080601). A method of producing a T-cell receptor γ-chain specific monoclonal antibody as claimed in claim 1, 2 or 3 which comprises cultivating cells from the appropriate hybridoma cell line, or a mutant or transformant thereof which retains the ability to produce that monoclonal antibody when cultivated, in a culture medium, or intraperitoneally administering those cells, or said mutant or transformant, to a mammalian animal, thereby to cause propagation of that cell line or that mutant in said culture medium or in the ascitic fluid of said mammal, and recovering product monoclonal antibody from the culture medium or from said ascitic fluid. A method of producing a T-cell receptor γ-chain specific monoclonal antibody according to claim 1, 2 or 3 which comprises immunizing a mammalian animal with a T-cell receptor γ-chain antigen, fusing spleen cells from the immunized mammal with mammalian myeloma cells, selecting from the hybridoma cell lines thus obtained a hybridoma cell line capable of producing the required antibody, cultivating the selected hybridoma cell line in a culture medium, or intraperitoneally administering the selected cell line to a mammalian animal, thereby to cause hybridoma cell line propagation in said culture medium or in the ascitic fluid of said mammal, and recovering product monoclonal antibody from the culture medium or from said ascitic fluid. The hybridoma cell lines KM-367, ECACC No. 87041604, KM-365, ECACC No. 87052101, and KM-369, ECACC No. 87080601, and mutants and transformants thereof which have the ability of the parent strain to produce a T-cell receptor γ-chain specific monoclonal antibody.

Inventors: SHITARA KENYA C O PATENT DEPAR, YOSHIDA HAJIME C O PATENT DEPA, SHITARA, KENYA C/O PATENT DEPARTMENT, YOSHIDA, HAJIME C/O PATENT DEPARTMENT
Applicants: KYOWA HAKKO KOGYO KK, KYOWA HAKKO KOGYO CO., LTD.
Application year: 1988
Main classification: C12P21
IPC codes: C07K16